EGERTON JOURNAL OF SCIENCE AND TECHNOLOGY. ISSN Volume 13 (2013) Published by Egerton University

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1 EGERTON JOURNAL OF SCIENCE AND TECHNOLOGY ISSN Volume 13 (2013) Published by Egerton University

2 EGERTON JOURNAL OF SCIENCE AND TECHNOLOGY ISSN Volume 13 (2013) EDITORIAL BOARD Chief Editor: Editor: Associate Editors: Prof. J. G. Mwangi, PhD S. O. Oduor, Ph.D. A. C. Kibor, Ph.D I.N. Wagara Ph.D R. Mulwa Ph.D M. Njue Ph.D E. Mbuthia Ph.D B. Bebe Ph.D M. Odhiambo Ph.D L. Orawo Ph.D ADVISORY BOARD Professor E.R. Øskov Professor J.K. Tuitoek Professor S.A. Abdulrazak Professor E.H. Ayiemba Macaulay Institute, Aberdeen, Scotland, UK. Egerton University, Njoro, Kenya. National Council for Science and Technology, Nairobi, Kenya. University of Nairobi, Nairobi, Kenya. Professor V.G. Masanja National University of Rwanda, Butare, Rwanda. Dr. W. Songa Dr. Z.R. Khan Dr. J.K. Bakari Ministry of Agriculture, Nairobi, Kenya. International Centre of Insect Physiology and Ecology, Nairobi, Kenya. Open University of Tanzania, Dar es Salaam, Tanzania.

3 Egerton University, 2013 All rights reserved. No part of this publication may be reproduced, transmitted in any form or by any means, electronic or mechanical, including photocopying, recording of any information, storage or retrieval system, without permission from the publisher. Egerton Journal of Science and Technology (ISSN ) is published twice annually by Egerton University, P. O. Box , Egerton, Kenya. Circulation and Sales Cover Price: Per Copy Kenya K. Sh. 400 Rest of Africa US$15 Rest of the World US$25 Text Layout and Design: Printed by: Susan A. I. E. Mbanda-Obura Egerton University Press

4 EGERTON JOURNAL OF SCIENCE AND TECHNOLOGY ISSN: Volume 13 (2013) i Editorial 1-10 Inhibitory Action of some Essential Oils on Growth of Various Moulds Isolated from Dried Maize Grains Nyukuri N. J., Wagara N. I., Matasyoh C. J. and Nakavuma L. J Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding on Dumpsite Waste in Urban Slums of Industrial Towns in Western Kenya Muleke I. C., Maina, J.N., Osuga M. I., Mutai, J. K.O., Karubiu N., and Bebe B Management of stem borers using selected botanical pesticides in a maize-bean cropping system Ogendo, J. O., Deng, A. L., Omolo, E.O., Matasyoh J. C., Tuey, R.K., Khan, Z. R Screening for host plant resistance to Helicoverpa armigera (Lepidoptera: Noctuidae) in selected chickpea (Cicer arientinum L.) genotypes in Kenya Ruttoh E. K., Mulwa R.M.S., Ngode L., Gohole L., Towett, B., Njogu N., Silim, S., Rao G. V. R, and Kimurto P. K The prevalence and effects of P. falciparum, S. mansoni and soiltransmitted helminth co-infections on haemoglobin concentration amongst school-aged children in Kisumu Municipality, Kenya Ngetich C. E., Odhiambo R., Mwandawiro C., Kihara J.

5 72-94 Creating a grading scheme for fresh ice-stored White snapper (Lutjanidae spp.) and Rabbit fish (Siganus sutor) using quality indices Lokuruka, M. N. I., Muyela, B., Okeyo, G. O. and Anakalo, A Histopathological effects of Trypanosoma brucei brucei infection on the master circadian rhythm pacemaker in rats Maina C. I., Ng wena G. M., Mwaniki D. M., Oucho A. O Growth Performance, Haematological Indices and Carcass Characteristics of Broilers Fed Diet Containing Different levels of Chromolaena odorata Leaf Meal Bonsu F. R. K., Kagya-Agyemang J. K., Kwenin W. K. J., Hope K. N. and Sekyere F. O Free surface Flow in Open Channels over Complex Shapes Gathia P.W. and Lonyangapuo, J. K Influence of Feeding Fat during Lactation on Growth Performance of Pups and Maternal Organ Morphology in Laboratory Mice Kagya-Agyemang, J. K., Król E. and Speakman J. R Faecal bacterial contamination of borehole water between points-ofaccess and points-of-use in Naivasha, Kenya; Public health implication Donde O. O., Wairimu A. M., Shivoga A. W., Trick G. C. and Creed F. I Improved Estimators in Adaptive Cluster Sampling with a Data Driven Stopping Rule Salim E. M., Wachira J. M., Islam S. A Instructions to Contributors and Reviewers

6 1 Nyukuri, et al., (2013) Inhibitory Action of some Essential Oils on Growth of Various Moulds Isolated from Dried Maize Grains Nyukuri N. J. 1, Wagara N. I. 1, Matasyoh C. J. 2 and Nakavuma L. J. 3 1 Department of Biological Sciences, Laikipia University, P.O Box , Egerton, Kenya 2 Department of Chemistry, Egerton University, P.O BOX 536, Egerton Kenya 3 College of Veterinary Medicine, Animal Resources and Bio-security, Makerere University, P.O Box 7062, Kampala, Uganda Submitted 7 th January 2013; Reviewed 19 th March 2013; Accepted 20 th May 2013 Abstract Moulds destroy more than 30% of crop yields in developing countries and produce poisonous mycotoxins like aflatoxins, fumonisins, ochratoxins, deoxynivalenol and zearalenones. The aim of this study was to identify aromatic plants with essential oils that can control the growth of moulds in maize and, therefore inhibit the production of associated mycotoxins in the grains. The assayed mould strains were Fusarium spp and Aspergillus spp. Five essential oils were extracted from aromatic plants (Tarchonanthus camphoratus, Artemisia vulgare, Piper capense, Foeniculium vulgare and Rosmarinus officinalis) using hydro-distillation and screened for antifungal activity against the moulds using the paper disc diffusion inhibition test. The antifungal activity tests showed that all the essential oils screened were active against 14 Aspergillus and 13 Fusarium species. These results show that the essential oils screened have antifungal activities against A. parasiticus, A. flavus, A. wentii, A. ustus, A. nidulans, A. ochraceus, A. tamarii, A. niger, A. versicolor, A. flavipes, A. terreus, A. fumigatus, F. solani, F. proliferatum, F. sporotrichioides, F. moniliforme, F. scirpi, F. chlamydosporum, F. trincinctum, F. oxysporum, F. subglutinans, F. merismoides, F. semitectum, F. nivale, F. avenaceum, F. graminearum, F. culmorum and F. crookwellence species that are the producers of aflatoxins, fumonisins, ochratoxins, deoxynivalenol and zearalenones in maize. These oils can be used in food preservation systems to inhibit the growth of moulds because the inhibitory action involves cytoplasm granulation, cytoplasmic membrane rupturing and inactivation and/or synthesis inhibition of intercellular and extracellular enzymes. This in turn will retard subsequent mycotoxin production. Keywords: Antimould activity, Aspergillus, Fusarium, Inhibition, Mycotoxins. Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

7 2 Nyukuri, et al., (2013) Introduction Moulds are opportunistic biological agents of ubiquitous nature (Ryan and Ray, 2004). Because of their powerful arsenal of hydrolytic enzymes, these microorganisms can cause a high degree of deterioration when present in foods and are responsible for considerable economic losses (Souza et al., 2005). Furthermore, they can act as potential producers of toxic metabolites, named mycotoxins, which are potentially poisonous to consumers health. The most common moulds that develop on foods and feeds include those from the genera Aspergillus, Penicillium, Fusarium, Rhizopus and Mucor. One of the most destructive activities of moulds in foodstuffs occurs in stored seeds and grains. Mycotoxins contaminate 25% of agricultural crops worldwide and are a source of morbidity and mortality throughout Africa, Asia and Latin America (Smith et al., 1994). They cause diseases referred to as mycotoxicoses in animals and humans (Agrios, 1997). Most mycotoxicoses are caused by the common and widespread moulds Aspergillus, Penicillium and Fusarium. Aspergillus and Penicillium produce their toxins mostly in stored seeds but also on commercially processed foods and feeds whereas Fusarium produces its toxins primarily on maize or other grains infected in the field or in storage (Agrios, 1997). Some of the toxins for example ochratoxins can persist in meat of animals fed on contaminated feed and can be transmitted to humans through the food chain. There is, therefore, urgent need to control moulds and their associated mycotoxins in fields and also in storage. The wide and indiscriminate use of chemical preservatives has been the cause of the appearance of resistant micro-organisms, leading to occurrence of emerging food borne diseases (Gibbons, 1992; Kaur and Arora, 1999; Akinpelu, 2001). Due to this, there is an increasing interest to obtain alternative antimicrobial agents to use in food preservation systems. It is well established that some plants contain compounds able to inhibit microbial growth (Naqui et al., 1994; Matasyoh et al., 2007, 2009). These compounds can have different structures and different action when compared with conventional antimicrobials used to control microbes (Nascimento et al., 2000). The potential antimicrobial properties of plants had been related to their ability to synthesize several chemical compounds of relatively complex structures with antimicrobial activity (Nychas, 1996). Essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from plants. They are obtained from plant materials (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). They can be obtained by expression, fermentation or extraction but the method of steam Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

8 Inhibitory Action of some Essential Oils on Growth of Various Moulds 3 distillation is most commonly used for commercial production. Essential oils are volatile plant oils (steam distillable) which are composed chiefly of terpenoids with frequently occurring aromatic compounds arising from the phenylpropanoid pathway (e.g. eugenol and safrole). Use of essential oils with antifungal activities in food preservation would provide a technology to ensure that the foods and feeds are free of moulds and mycotoxins. Such essential oils of recognized antimicrobial spectrum could appear in food preservation systems as main antimicrobial compounds or as adjuvant to improve the action of other antimicrobial compounds (Kaur and Arora, 1999). This project aimed at identifying aromatic plants with essential oils that can control the growth of moulds in maize and, therefore inhibit the production of associated mycotoxins in the grains. These antifungal essential oils can be used by farmers, food processors and consumers as cheap, affordable and environmentally-friendly antimould products for food preservation systems. This would help alleviate great losses of grains encountered due to moulds and prevent mycotoxin poisoning outbreaks. Materials and Methods Extraction of Essential Oils The target aromatic plants that were used in the extraction of essential oils were selected from Lamiaceae, Asteraceae, Piperaceae and Apiaceae families which are known to contain antimicrobial essential oils (Matasyoh, et al., 2005; 2006; 2007). Fresh leaves of Artemisia vulgare (Mugwort), Foeniculum vulgare (Camphor bush), Piper capense (Wild pepper), Tarchonanthus camphoratus (Sweet fennel) and Rosmarinus officinalis (Rosemary) were collected from Kakamega forest and the nearby fields. The fresh leaves were packed into two litre flasks up to three-quarter full and weighed at 400 grams. About 500 ml of tap water was added and the leaves were subjected to hydro-distillation, using modified Clevenger- type apparatus for 4 hours as described by Matasyoh et al. (2008). The resultant mixture of steam and essential oil were passed through a Liebig condenser and cooled by a continuous flow of cold water. Essential oils are less dense than water and were separated as an upper layer, floating on the distillation water. The oil was then collected by decanting into sample bottles and dried using anhydrous sodium sulphate (Na 2 SO 4 ). The procedure was repeated until a sufficient amount of oil for antifungal tests was obtained. The oils were stored in a sealed glass vial (Bijoux bottle) at 4 o C for further analyses. Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

9 4 Nyukuri, et al., (2013) Isolation of Moulds from Maize Maize samples were collected from various households and market centres in Western Kenya. Moulds were isolated from these samples using the direct plating technique. The grains were surface sterilized in 2.5% sodium hypochlorite for 30 seconds and rinsed in three changes of sterile distilled water. The grains were blotted with sterile filter papers and plated on potato dextrose agar containing 7.5% sodium chloride and 133 mg streptomycin sulphate (for 1 litre of media). The plates were incubated at 25 o C and monitored daily for fungal growth for seven days. The resulting cultures were identified based on cultural and morphological characteristics using taxonomic keys (Nelson et al., 1983; Siefert, 1996; Klich, 2002). Target moulds were sub-cultured to obtain pure single-spore cultures. Screening Essential Oils for Antimould Activity Paper disc diffusion inhibition test was used to screen for antimould activity of the essential oils as described by Souza et al. (2005). One hundred microliters of mould suspension (approximately 10 6 spores/ml measured using a haemocytometer) prepared with sterile 0.85% physiological saline solution was uniformly spread on sterile potato dextrose agar in a petri dish. Sterile filter paper discs (Whatman No. 1, 6mm in diameter) were soaked with 10µl of the essential oil and placed at the centre of the inoculated culture plates. The plates were incubated at 25 o C for 7-14 days. At the end of the incubation period, diameters of the inhibition zones were measured to the nearest millimeter (mm) and recorded. Nystatin discs were used as the reference standard. Data on inhibition zones was analyzed to derive mean diameter. Separation of means for antifungal activity was done using LSD. Results and Discussions All the five essential oils extracted from Tarchonanthus camphoratus, Rosmarinus officinalis, Artemisia vulgare, Foeniculum vulgare and Piper capense were screened for their activity and they presented inhibitory action on 14 Aspergillus and 13 Fusarium species assayed. The moulds isolated and identified among the genus Aspergillus were 14 species which included A. parasiticus, A. flavus, A. wentii, A. ustus, A. nidulans, A. ochraceus, A. tamarii, A. niger, A. versicolor, A. flavipes, A. terreus, A. fumigatus, A. humicola and A. sparsus. Among the genus Fusarium, 13 mycotoxigenic species were identified and these included F. solani, F. proliferatum, F. sporotrichioides, F. moniliforme, F. scirpi, F. trincinctum, F. oxysporum, F. subglutinans, F. semitectum, F. nivale, F. avenaceum, F. graminearum and F. culmorum. Screening results for antimould activity of the oils on the Aspergillus strains are shown in Tables 1 and for Fusarium strains on 2. The Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

10 Inhibitory Action of some Essential Oils on Growth of Various Moulds 5 best antifungal activity for T. camphoratus was on A. nidulans at an inhibition zone of 14.3 mm followed by A. fumigatus (12.3 mm) and the least activity of the oil was observed against A. terreus with an inhibition zone of 6.7 mm. The highest activity of the oil on the genus Fusarium was observed against F. oxysporum with the largest inhibition zone of 24.3 mm as compared to 11 mm inhibition by Nystatin. This was followed by activity against F. solani (21.3 mm) and lastly F. nivale at (11.3 mm). The best activity for R. officinalis was exhibited on A. nidulans with an inhibition zone of 18.3 mm as compared to the standard Nystatin that had an inhibition of 20 mm. The oil showed least activity on A. niger with a diameter of 6.3 mm. The oil did not show any inhibition on A. tamarii. For Fusarium species, R. officinalis had good activity against F. trincinctum at 17.3 mm and the least activity was exhibited on F. graminearum at 6.3 mm. Though in some species there was minimal activity, the oil had activity on all the Fusarium species tested. Artemisia vulgare showed best results on A. nidulans with inhibition zone of 21.3 mm which was higher compared to Nystatin (20 mm). This was followed by an activity on A. fumigatus at 11.7 mm. The least inhibition was exhibited on A. parasiticus, A. flavus, A. humicola and A. ustus. The oil did not show any antifungal activity against A. ochraceus, A. tamarii, A. flavipes, A. wentii, A. terreus and A. versicolor. Table 1: Inhibition zones (mm) induced by essential oils of Tarchonanthus camphoratus, Rosmarinus officinalis, Artemisia vulgare, Foeniculum vulgare and Piper capense on Aspergillus species isolated from maize samples after 14 days of incubation Mould Essential oils a species T. R. A. F. vulgare P. STD b Control camphoratus officinalis vulgare capense A. flavus A. ochraceus A. parasiticus A. niger A. tamarii A. fumigatus A. flavipes A. ustus A. nidulans A. wentii A. sparsus A. versicolor A. terreus A. humicola a Absolute concentration; b Nystatin Control: Pure culture with no disc Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

11 6 Nyukuri, et al., (2013) Table 2: Inhibition zones (mm) induced by essential oils of Tarchonanthus camphoratus, Rosmarinus officinalis, Artemisia vulgare, Foeniculium vulgare and Piper capense on Fusarium species isolated from maize samples after 14 days of incubation Mould species Essential oils a T. R. A. F. P. STD b camphoratus officinalis vulgare vulgare capense F. avenaceum F. sporotrichiodes F. subglutinans F. culmorum F. trincinctum F. oxysporum F. proliferatum F. semitectum F. scirpi F. nivale F. graminearum F. moniliforme F. solani a Absolute concentration; b Nystatin Control: Pure culture with no disc The essential oil of Artemisia vulgare had activity on all the Fusarium species screened. The oil had good activity on F. semitectum at 15.7 mm and least activity was exhibited on F. graminearum and F. moniliforme. Foeniculum vulgare had good antifungal activity against A. nidulans at 12.3 mm. Although this activity was low compared to the standard (Nystatin=20 mm), it was the highest zone of inhibition for the oil. The least antifungal activity was shown on A. wentii at 6 mm. The oil had no activity on A. parasiticus. Piper capense had good activity on A. fumigatus with an inhibition zone of 18.3 mm, followed by A. niger. The least activity was exhibited on A. flavus at a diameter of 6.7 mm. The oil had no activity against A. parasiticus, A. tamari, A. nidulans and A. versicolor. The essential oil of F. vulgare had good activity on F. semitectum and least activity on F. solani and F. trincinctum. No antifungal activity was exhibited by the oil on F. avenaceum and F. moniliforme. Piper capense had a good activity on F. nivale at mm. This zone was higher than the standard which induced an inhibition zone of 10 mm. The least activity was on F. avenaceum and F. scirpi at 6.7 mm. However, the oil had activity against all the Fusarium species screened. Among the five oils tested, T. camphoratus had the highest antifungal activity against the moulds. The high antifungal activity of T. camphoratus oil is mainly due to the presence of the two isomers; geranial and neral, although other minor constituents like α-pinene have been reported to be the main cause of the antifungal activity of the oil from Pistacia lentiscus Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No Control

12 Inhibitory Action of some Essential Oils on Growth of Various Moulds 7 (Anacardiaceae) (Matasyoh et al., 2010). The antifungal activity of the oil varied with the type of mould species. According to Matasyoh et al. (2007), the essential oil of T. camphoratus was analyzed by gas chromatography mass spectrometry (GC MS) and was found to be dominated by monoterpenes, which accounted for 80.9% of the oil. The study indicated the presence of a high percentage of oxygenated monoterpenes (62.3%), of which the main constituents were fenchol (15.9%), 1, 8-cineole (14.3%) and α-terpineol (13.2%). Other monoterpenes present in fairly good amounts were α-pinene (6.87%), trans-pinene hydrate (6.51%), terpinen-4-ol (4.74%) and camphene (3.76%). These could explain the good antimould activity exhibited by the oil on different fungal species screened. Literature shows that R. officinalis has antimicrobial activities against a variety of bacteria, fungi, moulds and viruses (Salehi et al., 2007). The antimicrobial activity of the oil was tested on various microorganisms and it showed promising antimicrobial activity according to Antony, (2007). This explains why the oil had good antimould activity against the Aspergillus and Fusarium species screened in this study. Artemsia vulgare oil has been evaluated and results presented a great variety of sesquiterpenes that could be considered responsible for the antimicrobial activity. Although they usually occur as complex mixture, their activity may generally account for in terms of their major components (Glauciemar et al., 2009). The oil of F. vulgare has been shown to be effective against fungal pathogens causing diseases in plants and human beings according to Sunita and Mahendra (2008). According to their findings, the oils are important source of fungitoxic compounds and they may provide a renewable source of useful fungicides that can be utilized in antimycotic drugs against A. fumigatus and A. niger. The results support the notion that plant essential oils have a role as pharmaceuticals and preservatives. The good activity of P capense owes to the leaves having a high content of sesquiterpenes ( %) (Tchoumbougnang et al., 2009). The most prominent compound in the leaves was found to be α- pinene (12.8%) and β-pinene (50.1%). Consequently research shows that the oil is quantitatively rich in monoterpenes (80.8%). The activity of this oil against Aspergillus and Fusarium species can be attributed to the monoterpenes found in the oil. This plant has also been shown to possess high levels of insecticidal activity in a study done in Cameroon (Singh and Sumitra, 2005). In general, the essential oils evaluated in this study are reported to have a great variety of phytochemicals that could be considered as responsible for a higher or lower antimicrobial activity. Phytochemicals are chemical substances characterized as organic biomolecules found and isolated from different plant derivatives such as tea, extracts and essential oils. These compounds are responsible for Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

13 8 Nyukuri, et al., (2013) the biological activities exerted by several plants and their derivative products (Souza et al., 2005). In general, the inhibitory action of natural products on mould cells involves cytoplasm granulation, cytoplasmic membrane rupturing and inactivation and/or synthesis inhibition of intercellular and extracellular enzymes. These actions can occur in isolate or concomitant way and culminate with inhibition of the mycelium growth (Souza et al., 2005). The results of this study show that essential oils of T. camphoratus, R. officinalis, A. vulgare, F. vulgare and P. capense possess varying levels of antifungal activity against Aspergillus and Fusarium species and can possibly be used as viable alternatives to control microbial growth and mycotoxin production in foods. It would be important to establish the actual bioactive component/s and the mode of action of the essential oils against the Aspergillus and Fusarium species. Of interest also would be to determine the effect of the oils on mycotoxin production ability of the moulds. Acknowledgements The authors are grateful to the Lake Victoria Basin Research Initiative (VicRes) for the financial support. References Agrios, G. N. (1997). Plant Pathology, 4 th edition. Academic Press, New York. Pp 635. Akinpelu, D. A. (2001). Antimicrobial activity of Anacardium occidentale bark. Fitoterapia 72: Alan, H. and Cornie H. (2005). Reference guide for Essential oils. Abundant Health Press, U. S.A. Pp 2. Antony C. D. (2007). A Research Paper on Natural Preservatives. Research Director, Peter Black Medicare Ltd, White Horse Business Park. Aintera Avenue, Trowbridge, Wilt shire, U.K BA14OxB. Gibbons, A. (1992). Exploring new strategies to fight drug resistant microbes. Science 29: Glauciemar, D. V., Orlando V. S., Celia H. Y and Maria A. C. (2009). Chemical composition and Antimicrobial Activity of Essential oils of Ageratum fastigiatum (Asteraceae). Records of Natural Products Journal. 3(1): Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

14 Inhibitory Action of some Essential Oils on Growth of Various Moulds 9 Kaur, J. and Arora, D. (1999). Antimicrobial activities of species. International Journal of Antimicrobial Agents 12: Klich M. (2002). Identification of common Aspergillus species. ASM Press, Washington, DC, p Matasyoh, J. C., Kiplimo, J. J. and Karubiu, N. M. (2005). Chemical composition and antimicrobial activity of essential oils of Leonotis nepetifolia. International Journal of BiochemiPhysics 14: Matasyoh, J. C., Kiplimo, J. C., Karubiu, N. M. and Hailstorks, T. P. (2006). Chemical composition and antimicrobial activity of essential oil of Satureja biflora (Lamiaceae). Bulletin of the Chemical Society of Ethiopia 21: 1-6. Matasyoh, J. C., Kiplimo, J. J., Karubiu, N. M. and Hailstorks, T. P. (2007). Chemical composition and antimicrobial activity of essential oil of Tarchonanthus camphoratus. Food Chemistry 101: Matasyoh, L. G., Matasyoh, J. C., Wachira, F. N., Kinyua, M. C., Thairu, M.W. and Mukiama, T. K. (2008). Antimicrobial activity of essential oils of Ocimum gratissimum L. from different populations in Kenya. African Journal of Traditional Complementary and Alternative Medicine 5: Matasyoh, J. C., Maiyo, Z. C., Ngure, R. M. and Chepkorir, R. (2009). Chemical composition and antimicrobial activity of essential oil of Coriandrum sativum. Food Chemistry 113: Naqui, S. H. A., Khan, M. S. Y. and Vohora, S. B. (1994). Antibacterial, antifungal and antihelminthic investigation of Indian medicinal plants. Fitoterapia 62: Nascimento, G. G., Locatelli, J. and Freitas, P. C. (2000). Antibacterial activity of plant extracts on antibiotic resistant bacteria. Brazilian Journal of Microbiology 31: Nelson, P. E., Toussoun, T. A. and Marasas, W. F. O. (1983). Fusarium species. An illustrated manual for identification. The Pennsylvania State University Press, University Park, PA, USA. Nychas, G. J. E. (1996). Natural antimicrobial from plants. In: Gould, G. W. New methods of food preservation. Londres : CRC Press. Pp Ryan, K. J and Ray, C. G. (2004). Sherris Medical Microbiology (4 th edition). Mc Graw Hill. Pp Salehi, P., Fakhani, A. R., Samad, N. E. and Rouhollan, H. (2007). Rapid oil screening of Rosmarinus officinalis L. by hydrodistillation headspace Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

15 10 Nyukuri, et al., (2013) solvent microextraction. Flavour and Fragrance Journal 22(4): Singh G. and Sumitra M. (2005. Antimicrobial, antifungal and insecticidal investigation on Essentila oils. Natural Product Radiance 4(3): Smith, J. E., Solomons, G. L., Lewis, C. W. and Anderson, J. G. (1994). Mycotoxins in human nutrition and health. European Commision, Directorate-General XII, Science, Research and Development. EUY16048EN, Brussels, Belgium. Souza, E. L., Lima, E. O, Freire, K. R. and Sousa, C. P. (2005). Inhibitory action of some essential oils and phytochemicals on the growth of various moulds isolated from foods. Brazilian archives of Biology and Technology 48: Sunita B. and Mahendra R. (2008). Antifungal activity of essential oils from Indian medicinal plants against human pathogenic A. fumigatus and A. niger. World Journal of Medicinal Sciences 3(2): Tchoumbougnang F., Jazet P. M., Sameza M. L., Fombotich, N. A., Wouatsa N. and Menut C. (2009). Comparative Essential Oils Composition and Insecticidal effects of different tissues of Piper capense L., Piper giuneense Schum.et Thunn., Piper nigrum L. and Piper umbellatum L. grown Cameroon. African Journal of Biotechnology 8(3): Egerton J. Sci. & Technol. Volume 13: 1-10 ISSN No

16 Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding on Dumpsite Waste in Urban Slums of Industrial Towns in Western Kenya Muleke I. C.* 1, Maina, J.N 1., Osuga M. I. 2, Mutai, J. K 1. O. Karubiu N. 1, Bebe B. 1 1 Egerton University, Faculty of Veterinary Medicine and Surgery, Box , Njoro, Kenya 2 Kenyatta University, Department of Agricultural Resource Management, School of Agriculture and Enterprise, Development, Kenya. *Correspondence author: cimuleke@yahoo.com Submitted 5 th Feb. 2012; Reviewed 22 nd Jan. 2013; Accepted 7 th April 2013 Abstract Knowledge of toxic metal levels in livestock feeding on dumpsite waste is important for assessing the effects of pollutants in animals and contaminant intakes by human. A study was conducted in western Kenya to determine the lead and copper levels in the soil, water, serum and tissues of livestock feeding on refuse dumpsite waste in Webuye and Mumias urban towns suspected to be polluted with these metals. Indigenous free ranging cattle, sheep, goats and chicken were investigated. The samples: soil (5), water (5), dumpsite wastes (7), serum (76) and 17 kidney/liver were analyzed. Lead and copper levels were determined by the conventional aqua regia digestion protocol and read using the atomic absorption spectrophotometer. The mean lead and copper levels in the soil and water of mg/kg and 0.001mg/L were below acceptable risk limits of 5 25mg/kg and 0.015mg/L, respectively. The mean lead levels in serum of cattle, sheep, goats and chicken were 0.86, 0.82, 0.54mg/L and 0.76, 0.46, 0.21mg/L in Bungoma and Mumias towns respectively, and they exceeded acceptable risk limit levels of 0.01mg/L. The lead levels in kidney/livers of cattle, shoats and chicken were 1.2; 1.5; 1.0mg/kg respectively, and exceeded the acceptable risk limits of 0.5 mg/kg. Based on this study, the lead and copper levels in the soil, water and dumpsite wastes in Bungoma and Mumias towns are below the acceptable risk limits. However due to bio-accumulation process observed in animal tissues, there is need to continuously monitor the status of these heavy metals and create public awareness on good animal feeding practices in the urban slums to avoid mineral toxicity in animals and man through the food chain. Egerton J. Sci. & Technol. Volume 13: ISSN No

17 12 Muleke et al., (2013) Key words: Heavy metals, urban slums; dumpsite wastes, livestock feeding, human. Introduction Food safety is a growing concern in the urban areas dependent on food chains and where food scares are increasingly frequent (Bellaiche, 2007). The Food and Agricultural Organisation (F.A.O) emphasises that safe, good quality food is essential to food security, public health and economic development (FAO, 2007). The livestock population in urban slums in Kenya is expanding and the livelihood opportunities linked to livestock are increasingly in competition with domestic, industrial and other service uses for fresh water (FAO, 1995 and 1997; Waters-Bayer, 1995). Majority of livestock keepers in urban slums are the vulnerable groups of female headed households, children, retired people, widows and the less educated who lack access to inputs. It is estimated that drinking and service water needs vary from 30 to 148 litres for a lactating cow, 65 to 95 litres for a lactating sow and 25 to 65 litres for every 100 layer birds daily (Rosegrant et al, 2002; Chapagain et al, 2003). This additional demand is largely unaccounted for by the urban authorities whose water supply is unable to match the increasing demand hence livestock accessed contaminated water sources. Presently, urban authorities and policy makers lack relevant information to inform their decisions on regulating framework in the design of water-use efficiency and mitigation of pollution associated with livestock production in the urban slums. The wastewater and manure discharged locally are of major environmental concerns. The associated pollutants of public health concern include surplus nutrients (N, P and K), bacterial and parasitic pathogens, heavy metals and drug residues. Heavy metals are incorporated in animal feeds and water in low concentration for health and growth enhancement, but not all are metabolised, ending up in the environment via wastewater and manure. The commonly used heavy metals are Cu, Zn, Se, Co, Ar, Fe, Mn and Cd. Lead and copper are among the heavy metals implicated to cause toxicity in animals and man. They are found in paints, pipes, batteries, soldering rods, gunpowder, pesticides, fungicides, gasoline, engine oils, some anthelmintics, chemical fertilizers or soil, water, plants and other compounded animal feeds (Farr, 2004). While copper is a trace element required in small amounts in various metabolic functions, lead and other heavy metals can be highly toxic due to interference directly in the metabolic pathways or indirectly by causing deficiencies of other trace metals (Farr 2004). Excessively higher levels of these metals in blood and tissues of animals suggest an exposure Egerton J. Sci. & Technol. Volume 13: ISSN No

18 Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding 13 either from the air, soil, water or feeds or all of theses sources (Dupler 2001; Farr 2004). Man is exposed to the risk by eating food and drinking fluids contaminated with heavy metals, through air, or direct contact with the metals for people working in industries/factories dealing with heavy metals and their derivatives (Farr 2004). The industrial towns of Webuye and Mumias in western Kenya are densely populated urban slums with prominent livestock keeping and large volumes of waste discharges from industrial, domestic and livestock units. However the disposal practices of waste discharges are poor and indiscriminate. There is a paper mill factory in Webuye and sugar milling factory in Mumias. Closely linked to these industries are numerous small to large factories dealing with metal works, car maintenance and repair, construction works and backyard farming that use agro-chemicals (pesticides, herbicides, fungicides, anthelmintics), soap and leather factories that use a lot of chemicals. These activities pose a risk of contaminating the environment with hazardous substances including heavy metals. The broad objective of this study was to assess the food safety risks associated with livestock feeding on dump site waste feeds and drinking polluted waters in the urban industrial towns in Western Kenya. The specific objectives were to determine the levels of lead and copper in the soils, refuse dumpsite waste, drinking water, serum and kidney/liver tissues of livestock feeding on dumpsite wastes in urban slums of Webuye and Mumias towns. This was to create awareness on the safety of consuming drinking water and livestock products (blood, liver and kidney) sourced from Mumias and Webuye urban areas. Appropriate interventions to protect the consumers from the health risks from the heavy metals will be devised. Materials and Methods Study Area The study was carried out in Bungoma and Mumias urban towns of Western Kenya. The two towns were chosen since they suggested evidence of food safety concerns linked to livestock feeding practices in urban slums. Bungoma Town: The town lies between latitude and North and longitude and East. It covers an area of 2,068.5km2, which is about 25 per cent of the total area of Western Province. Major industries found here include sugarcane processing factory (Nzoia sugar Company, 10km from Bungoma town), Pan African Paper Mills at Webuye (processing paper from wood) and several coffee pulping factories located in the district. Other people are involved in off-farm businesses such as retail trading (own small shops, food kiosks, butcheries, small hotels). Others trade Egerton J. Sci. & Technol. Volume 13: ISSN No

19 14 Muleke et al., (2013) in food commodities (buying and selling), grains, pulses, vegetables, fruits, and even second hand clothes. Mumias Town: The town is 1258 m above sea level and lies between latitude 0 20'11.000" North and longitude '21.120" East. Mumias sugar factory is the major source of employment. Livestock Samples were taken from free ranging mature cattle, sheep, goats and chicken owned by smallholder urban town farmers who readily availed their animals in the study. Inventory of Unconventional Refuse Dumpsite Feeds A reconnaissance survey was undertaken to identify major dump sites within the urban slums. A careful examination of what scavenging livestock ingested at urban dumpsites was undertaken and inventory of unconventional refuse dumpsite materials recorded. The species of ruminant livestock were also recorded and numbers obtained from local government livestock records. Soil, refuse dump site feeds and water sampling Soil and refuse dumpsite feeds were collected in different dumpsites from urban slums where cattle, sheep, goats forage and where poultry scavenge. Collected dump site feed materials were put in a clean polythene bags and divided into two portions. One half was used for DM determination while the remaining was rinsed with de-ionized water several times and dried for lead and copper determination. At the centre of the 1.5x1.5m 2 area where refuse dump site samples were taken, the topsoil was dug to 12 cm depth in an area of 24 x 24cm square. The soil was carefully mixed and put in clean polythene bags, in the laboratory, it was ground in a mortar to obtain small particles of uniform particle size, and then DM, lead and copper levels were determined. About 50ml of water samples were collected in clean unused 50ml plastic bottles from each dump site areas where livestock drink. Each sample was filtered through Whatman filter paper grade 40 to remove debris before analysis for lead and copper using atomic absorption spectrophotometer. Serum Sampling A random sample of adult free ranging cattle, sheep and goats were bled from the jugular vein. At each bleeding, 5ml of whole blood was collected Egerton J. Sci. & Technol. Volume 13: ISSN No

20 Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding 15 from each animal into sterile plain universal bottles and kept at 4 0 C overnight to clot. The supernatant was centrifuged at 10,000rpm for 20min. Separated serum was stored at -20ºC until used for determination of lead and copper levels. Liver and Kidney Samples Liver and kidney samples were collected in the abattoirs from cattle, sheep and goats kept in urban slums from and preserved in ice packed cold boxes until used for heavy metal analysis. A few scavenging chicken raised around the urban dump sites were sacrificed to obtain liver, kidney and whole blood specimens for determination of lead and copper levels. Laboratory Analysis Proximate analysis of dump site feeds The proximate composition of refuse dump site feeds were determined using the AOAC (1990) procedure. Determination of lead and Copper Levels Conventional aqua regia digestion was performed in 250-mL glass beakers covered with watch glasses. A well-mixed sample of 0.5 g soil, ground dump site feeds, kidney and liver was digested in 12 ml of aqua regia on a hotplate for 3h at 110 C. After evaporation to near dryness, the sample was diluted with 20 ml of 2% (v/v with H 2 O) nitric acid and transferred into a 100-mL volumetric flask after filtering through Whatman filter paper grade 40 and diluted to 100 ml with dionized distilled water then analyzed for levels of lead and copper using the atomic absorption spectrophotometer. Statistical Analysis The data were analyzed using SPSS version 11.5 statistical package to obtain the mean, standard deviation, standard error of the mean, range and student s test on the measured parameters. Results Unconventional Feed Resources at Refuse Dumpsites The most common unconventional feed resources at refuse dumpsites were peels from potatoes, cassava, maize stalk residues, maize cob, sugarcane tops, baggase, residues from groundnuts and cowpea haulm. Other dumpsite materials were papers, rags, rope and jute bags; house hold waste polythene bags that are either degradable or non-biodegradable; remains of fruits and vegetables such as orange, pineapple melon; pumpkin, lettuce, cabbage and Egerton J. Sci. & Technol. Volume 13: ISSN No

21 16 Muleke et al., (2013) carrots. The species of ruminant livestock recorded and numbers obtained from local government livestock records are shown in Table 1. Table 1: The population of free ranging ruminant livestock in Mumias and Bungoma urban slums Mumias town Bungoma town Species Males Females Males Females Cattle Sheep Goats Table 2: Proximate composition of feed resources at refuse dumpsites Dumpsite location % DM %CP % Ash % CF % Fat Bungoma Bungoma Bungoma Mumias Mumias The nutrient composition of unconventional ruminant feeds sampled from five different dump sites in Bungoma and Mumias urban slums had a mean % DM of 22.5, % CP of 9.5, % Ash 11.0, % CF 53.5, % Fat 0.35 respectively. Dumpsite feeds obtained from Bungoma and Mumias had high % DM and % CF levels as indicated in Table 2. Lead and Copper Levels The mean lead and copper levels in ascendig order of the food chain from soil, dump site feeds, water, serum and kidney/liver in Bungoma and Mumias urban slums are shown in Figures 1 and 2, respectively. The highest lead and copper levels were recorded in soil and kidney/liver samples that ranged between 0.4mg/kg 3.5mg/kg. Of all the samples analyzed, water, serum and refuse dump feeds had negligible quantities of lead and copper that ranged between mg/L. Egerton J. Sci. & Technol. Volume 13: ISSN No

22 Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding 17 Conc. (mg/kg) Mumias [Pb] Bungoma [Pb] Sample Identity Figure 1: Lead concentration [Pb] in ascending order of the food chain from soil, water, dump site feeds, serum and kidney/liver in Bungoma and Mumias urban slums Conc. (mg/kg) Mumias [Cu] Bungoma [Cu] Sample Identity Figure 2: Copper concentration [Cu] in ascendig order of the food chain from soil, water, dump site feeds, serum and kidney/liver in Bungoma and Mumias urban slums Discussion This study shows that livestock in urban slums feeding on refuse dumpsite material and drinking waste water are likely to ingest heavy metals implicated to cause toxicity through the human food chain. The free ranging livestock (cattle, sheep and goats) in the urban slums were mainly indigenous to the environment in the study area, thus adapted to the vagaries of climatic Egerton J. Sci. & Technol. Volume 13: ISSN No

23 18 Muleke et al., (2013) and feed scarcity. This perhaps explains their ability to subsist on materials obtainable at the refuse dumpsites in these urban centres. Proximate composition of dumpsite wastes was 22.5 and 53.5% DM and CF, respectively. The degradable or non-biodegradable open refuse dumps not only make the environment filthy, but also provide unconventional feed resources for livestock roving in urban centres. The repercussion for consuming indigestible polythene materials is rumen impaction (Mohammed et al., 2007) and poor performance that is a drawback to scavenging livestock in the urban metropolis. The soil lead levels in the range ( mg/kg), were higher than those reported in uncontaminated urban soils of 0.50mg/kg (Mlay et al, 2008), suggesting there was heavy lead contamination in the study area. The soils in the proximity of Mumias sugar factory and Pan African Paper Mills at Webuye (processing paper from timber) had higher lead and copper levels that were almost three times the level from other sites. Elevated mineral levels in Mumias and Bungoma soils were expected due to pollution from the factories in the localities. The water lead and copper levels of 0.001mg/L were lower than minimum acceptable risk levels of 0.015mg/l (ILO 1983; Fitzgerald 1998). Drinking water from unknown sources predispose animals to ingestion of parasite eggs and heavy metals (Na, Cu, Pb, Cu, etc) that would accumulate leading to parasitosis and mineral toxicosis, consequently affecting humans through the food chain. Although the results here indicate refuse water could be safe for use by animals in as far as the levels of lead and copper are concerned, it is recommend that animals raised in urban centres be provided with clean drinking water and mineral supplements to enhance performance. The lead levels in serum of cattle, sheep and chicken were 0.86, 0.82, 0.54mg/L and 0.76, 0.46, 0.21mg/L in Bungoma and Mumias, respectively. The elevated lead levels in Bungoma urban slums were probably due pollution through numerous small factories dealing with metal works, garages within Webuye township, exhaust fumes from motor vehicles that use leaded gasoline. The possibility of cattle, sheep and even chicken to obtain extra lead through ingestion of dump site refuse feeds, soil and water cannot be ruled out since these metals tend to bio-accumulate in animal kidney and liver tissues as evidenced in this study pausing danger to the human food chain. The serum lead levels in this study, exceeded those reported by Lopez et al (2000) of (0.01mg/L) suggesting that animals in the urban slums were at risk of exposure to this metal contaminant. Egerton J. Sci. & Technol. Volume 13: ISSN No

24 Lead and Copper Levels in the Soil, Water, Serum and Tissues of Livestock Feeding 19 The mean serum copper concentration in the animals was mg/L. This was less than mg/l level reported by Mlay et al., 2008, and much less than the lower risk level of 0.6mg/L (range mg/L) recommended by the environmental Protection Agency. The unexpectedly low copper levels could have been due to the assay failing to detect the mineral below a certain threshold in the serum. Based on this study and inconsistent findings elsewhere (Fitzgerald,1998, Mlay et al., 2008), one should interpret the lead and copper levels in serum and water with caution and where applicable utilize soil, grass, kidney, liver and adipose tissue in assessing heavy metals risks associated with livestock feeding on refuse dumpsite material in urban slums instead of serum and water. Conclusions Despite presence of lead and copper in soil, water, dump site feeds, serum and kidney/liver of livestock in Bungoma and Mumias urban slums they have not reached the high risk level. However, there is need to continuously monitor the status of these heavy Metals in the urban slums to avoid toxicity in animals and man through the food chain. Acknowledgements The research work was funded by a grant from the Division of Research and Extension, Egerton University. The authors are grateful to Mr. Kamiti Ndegwa for his assistance in data analysis. References AOAC (1990). Association of official Agricultural Chemists. Official Methods of Analysis. 15thg Edition. AOIAC Washington D. Volume 1, 62pp. Bellaiche, M. (2007). Public health problems associated with animal feed. Chemical and microbiological hazards, sources and routes of contamination. Chapagain, A. K. and Hoekstra, A. Y., (2003). Virtual water flows between nations in relations to trade in livestock and livestock products. Value of water research report series number 13. UNESCO-IHE. Dupler, D., (2001). Heavy metal poisoning. Gale Encyclopedia of Alternative Medicine. Farmington Hills, MI: Gale Group. Egerton J. Sci. & Technol. Volume 13: ISSN No

25 20 Muleke et al., (2013) FAO. (1995). Global issues in the supply of livestock food products to urban populations. In R.T. Wilson, Ed. Supply of livestock products to rapidly expanding urban populations, pp Proceedings of the Joint FAO/WAAP/KSAS Symposium, May 1995, Hoam Faculty Club, Seoul National University, Seoul. Rome. 224 pp. FAO, (1997). Animal feeding and food safety. FAO Food and Nutrition Paper no. 69 / Rome (Italy), FAO, 48 p FAO (2007). Animal feed impact on food safety report of the FAO/WHO Expert Meeting. pre-publication version. Rome. 49 pp. Farr G, Why Heavy Metals are a Hazard to Your Health. accessed on 26th June Fitzgerald, D. J., (1998). Safety Guideline for copper in water. American Journal of Clinical Nutrition 67: reprint/67/5/ 1098S.pdf ILO (1983). International Labor Office Encyclopedia of Occupational Health and Safety. 3d edition. Geneva, Volume 1: Lopez, A. M., Benedicto, J. L., Miranda, M., Castillo, C., Hernandez, J. and Shore, R. F., (2000). Arsenic, cadmium, lead, copper and zinc in cattle from Galicia, Spain. The Science of the Total Environment 246 (2-2): Mlay,P. S. and Mgumia Y. O., (2008). Levels of lead and copper in plasma of dairy cows, pastures, soil and water from selected areas of Morogoro suburbs, Tanzania. Livestock Research for Rural Development 20 (4): Mohammed, A. K, and Mohammed, I. R., (2007). Fatal polythene bag rumen impaction in cattle at Shikaria, Nigeria. J. Animal Sci: 6-8. Rosegrant M.W., Cai, X. and Cline S. A., (2002). Global water outlook to Averting impending crisis. A 2020 vision for food, agriculture and environment initiatives. International food policy research institute (IFPRI) and International water management institute (IWMI). Waters-Bayer, A., (1995). Living with livestock in town: urban animal husbandry and human welfare. Keynote paper. Eighth International Conference of Institutions of Tropical Veterinary Medicine. Livestock Production and Diseases in the Tropics: Livestock Production and Human Welfare, September, Berlin, Germany, ETC Foundation, Leusden, the Netherlands Egerton J. Sci. & Technol. Volume 13: ISSN No

26 Ogendo, et al., (2013) 21 Management of stem borers using selected botanical pesticides in a maize-bean cropping system Ogendo, J. O. 1ψ, Deng, A. L. 2, Omolo, E.O. 1, Matasyoh J. C 3., Tuey, R.K. 4, Khan, Z. R. 5 Departments of 1 Crops, Horticulture and Soils; Biological Sciences and, 3 Chemistry, Egerton University, P.O. Box ,Egerton, Kenya., 4 Entomologist, KARI, P.O. Private Bag, Njoro. Kenya; 5 Insect Behavioural Science Department, icipe-mbita, P.O. Box 30, Mbita. Kenya ψ Corresponding Author: jogendo@egerton.ac.ke; ogendojoshua@gmail.com Cellphone: / Submitted 19 th April 2012; Reviewed 31 st Dec. 2012; Accepted 7 th April 2013 Abstract Sustainable maize production in sub-sahara Africa causing 20-80% grain yield losses. Although effective synthetic pesticides are available, the smallholder farmers are yet to fully integrate them in their crop production practices hence the need for scientific rationalization of indigenous pest management options. Aqueous extracts (0, 0.85 and 1.70% w/v) and crude powders (0, 15 and 30 kg/ha) of three botanicals, Lantana camara L., Tephrosia vogelii Hook, Tagetes minuta L. were evaluated for efficacy against stem borers and their effects on productivity of a maize-bean intercrop. Field experiments were laid out in a randomized complete block design (RCBD) with three replicates per site. Results from the field bioassay trials showed that application of crude powders of T. vogelii, T. minuta and general plant ash at kg/ha reduced (P<0.005) the stem borer population by 18-63% and increased (P<0.05) the maize grain yields by 30-70% ( ton/ha) compared to the untreated control (1.534 ton/ha). Irrespective of site, season or cropping system, the application of botanical crude powders and general ash, at 30 kg/ha, were as effective as the synthetic insecticide, Stalk borer 2% dust, applied at 10 kg/ha. The powder treatments, at kg/ha, increased the bean yield by % (av. 150%) in the intercrop plots compared to untreated control. Similar result trends on stem borer population and maize and bean yields were observed for aqueous botanical extracts applied as seed-soaking and sprays treatments. The choice of botanical plant to use against stem borers in maize-based cropping is largely governed by the availability and ease of agronomic manipulation by target users. The results of this study are discussed in the context of their relevance, acceptability, economic viability and suitability as good Egerton J. Sci. & Technol. Volume 13: ISSN No

27 22 Management of stem borers using selected botanical pesticides agricultural practices consistent with food security and wealth creation strategies in smallholder agriculture. Key words: Lantana camara, Tagetes minuta, Tephrosia vogelii, stem borer, botanical pesticide Introduction Smallholder agriculture in sub-saharan Africa (SSA) is characterized by perennial food deficits, cyclic famines and poverty. Grain pests pose the greatest threat to food grain production, storage and handling in smallholder agriculture. Literature survey in the farming communities in western Kenya shows that insect pests cause, on average, 16% field food grain losses (Ogendo et al., 2003a, 2004). Occurrence, diversity and severity of field insect pest damage vary with season, crops, stage of crop growth and agronomic practices. The cereal stem borers, cutworms, aphids, bean fly, armyworms and termites have emerged as major field insect pests in smallholder agriculture (Ogendo et al., 2003a). Although the use of synthetic pesticides have been recommended and promoted in Kenya for close to 5 decades, smallholder farmers are yet to fully integrate (only < 10% of farmers use) their use in the field grain crops (Saxena et al., 1990; Ogendo et al., 2003a, 2004). Information available shows that majority ( 70%) of smallholder farmers never apply any pest control measures (PCM) against field insect pests and where any PCMs are used the farmers perceived efficacy were either unknown or no rating available (Ogendo et al., 2003a). Various field studies in western Kenya, have identified several bio-intensive pest management options (ashes, crude powders and extracts) used for the control of field and storage insect pests (Saxena et al., 1990; Ogendo et al., 2003a) In his documented information, Pottorf (2004) reported that micro organisms, microbial products, mineral-bearing rocks and plant /animal derivatives (extracts, powders, ashes, manure, etc) were among the recommended pesticides in organic agriculture. With the knowledge of adverse effects of synthetic pesticides worldwide, attention is rapidly shifting to eco-friendly non-chemical options. The noble promise for the development of suitable simple natural anti-pest products has provided impetus for the scientific rationalization, improvement and packaging of the existing indigenous knowledge base and practices. Despite the enormous potential that has existed for generations, the non-chemical indigenous insect pest management practices have remained largely unexploited with little local research intervention and resources being committed. Field studies were, therefore, Egerton J. Sci. & Technol. Volume 13: ISSN No

28 Ogendo, et al., (2013) 23 conducted to evaluate the efficacy of aqueous extracts and powders of three indigenous pesticidal plants, Lantana camara L., Tephrosia vogelii Hook and Tagetes minuta L. against stem borers and their effects on productivity of a maize-bean intercrop system. Materials and Methods Three field trials were conducted in representative on-farm sites in Lambwe division, Suba District, during the short rains (September-January, 2003) and long rains (March- July, 2004; 2005) and at Egerton University, Njoro (October 2003-May 2004; May-October, 2005). The objective of the field studies was to evaluate the pesticidal potency of aqueous extracts and crude powders of L. camara, T. vogelii and T. minuta and general plant ash against stem borers in a maize-bean intercrop and monoculture of maize. The aqueous extracts and crude powders were evaluated at three rates (0, 0.85 and 1.70% w/w) and (0, 15 and 30 kg/ha), respectively. The effect of botanical treatments on the insect pest population dynamics, insect damage rating, and on the grain yield of the two cropping systems were studied in randomized complete block design (RCBD) arrangement replicated three times per site. Composite plant samples, of each botanical, were collected in Suba district, shade-dried for one week and further oven dried at 35 0 C to constant weight before grinding to fine powder using a laboratory electric mill. For the aqueous extract (spray and seed soaking) experiments, desired quantities mentioned above were dissolved in 5 litres of ordinary water for 24 hours (h), filtered using cloth mesh and the filtrate collected. The filtrate aqueous solutions were sprayed in maize-bean plots 50 days after planting (DAP) or used as an overnight (24 h) seed soaking treatment (on both maize and bean seeds) prior to planting. On the basis of modal farmer practice, exploratory rates of individual botanical pesticides were used to generate response curves. In particular, the efficacies of the test botanicals were evaluated against stem borers. Data on counts were first subjected to appropriate angular transformation before performing analysis of variance (ANOVA), Regressions and means separated using Tukey s HSD test (Gomez and Gomez, 1983; Scheiner and Guvevitch, 1993; SAS, 1995). Results and Discussion Results of field evaluations, from the two seasons each at Egerton University and Suba district, showed that crude powders and aqueous extracts (spray and seed-soaking) obtained from the three test botanical pesticides significantly (P<0.05) reduced stem borer incidences, increased grain yield and yield components of maize and beans in both maize monocrop and Egerton J. Sci. & Technol. Volume 13: ISSN No

29 24 Management of stem borers using selected botanical pesticides maize-bean intercrop systems (Fig.1-2; Tables 1-3). Irrespective of site, season or cropping system, the application of botanical powders of L. camara, T. vogelii, T. minuta and general plant ash, at kg/ha, reduced the stem borer load by 18-63% and increased the maize grain yields by 30-70% ( kg/ha) compared to the untreated control (1534 kg/ha). At 30 kg/ha, botanical powders and general ash were as effective as the synthetic insecticide, Stalk borer 2% dust, applied at 10 kg/ha. Although the botanical powders and general plant ash were not directly applied to bean plants in a maize-bean intercrop, the treatments (15-30 kg/ha) significantly increased the grain yield of beans by % (av. 150%) (Fig.2b). All botanicals at 30 kg/ha produced yields that were equally as high as the synthetic insecticide treated plots. The major implication of these results is that the choice of local plant to use as botanical pesticide, powder formulation, against stem borers in a maize based cropping system is largely governed by the availability and ease of agronomic manipulation by the target users. Irrespective of cropping system, locality and season, Lantana, Tephrosia and Tagetes aqueous botanical extracts sprayed at 1.70% w/v, reduced the stem borer loads by 53, 59 and 47%, respectively (Tables 1a-b) compared to the untreated control. The same treatments increased the grain yields by 48, 54 and 35% for Lantana (2343), Tephrosia (2425) and Tagetes (2128 kg/ha) compared to untreated control (1579 kg/ha). Lantana and Tephrosia treatments, sprayed at 1.70% w/v, were more effective in reducing stem borer loads and also produced higher maize grain yields compared to the synthetic insecticide, Karate (2164 kg/ha). Bean data generated over two seasons showed that spraying of aqueous botanical extracts, at % w/v, in a maize-bean intercrop significantly increased the grain yield of beans by 22-65% (av.46%) compared to the untreated control (171 kg/ha) (Fig.1b; 2a). Although the three botanical pesticides at the higher dose (1.70% w/v) increased the grain yield of beans by 53-65% ( kg/ha), the synthetic insecticide treatment (Karate ) produced significantly higher yields (348 kg/ha). Egerton J. Sci. & Technol. Volume 13: ISSN No

30 Ogendo, et al., (2013) 25 YIELD (KG/HA) (a) Seed-soaking with aqueous botanical extracts Botanical Extract (% w/v) YIELD (KG/HA) (b) Spray with aqueous botanical extracts Botanical Extract (% w/v) YIELD (KG/HA) (c) Crude botanical powders Botanical Crude Powder (kg/ha) Figure 1: Effect of (a) seed soaking with aqueous extracts, (b) spraying aqueous extracts and (c) crude powders of three botanical plants on bean yield at Egerton University, Njoro (2004); SR = short rains and LR = long rains Egerton J. Sci. & Technol. Volume 13: ISSN No

31 26 Management of stem borers using selected botanical pesticides YIELD (KG/HA) (a) Aqueous botanical sprays (SR 2004 and LR 2005) LR2005 SR2004 Botanical Extract (% w/v) YIELD (KG/HA) (b) Effect of botanical powders (SR2004 and LR2005) SR 2004 LR 2005 YIELD (KG/HA) Botanical Crude Powder (kg/ha) 2(c) Seed-soaking with botanical extracts (SR2004 and LR2005) SR 2004 LR 2005 Botanical Extract (% w/v) Figure 2: Effect of (a) spraying aqueous extracts, (b) crude powders and (c) seed-soaking with aqueous extracts of three botanical plants on bean yield in Suba district ( ); SR = short rains and LR = long rains Egerton J. Sci. & Technol. Volume 13: ISSN No

32 Ogendo, et al., (2013) 27 Table 1a: Effect of aqueous botanical extracts on stem borers and maize yield during short rains 2004 LAMBWE-SUBA, 2003/04 INTERCROP MONOCROP TREAT Yield Yield Sbor Cobindex (Kg/ha) Sbor Cobindex (Kg/ha) Control 2.667± ± ± ± ± ±47 Karate 1.333± ± ± ± ± ±46 Lan ± ± ± ± ± ±44 Lan ± ± ± ± ± ±55 Tep ± ± ± ± ± ±35 Tep ± ± ± ± ± ±56 Tag ± ± ± ± ± ±36 Tag ± ±0.07 ges but 1.333± ± ±39 TREAT EGERTON UNIVERSITY, NJORO, 2003/04 INTERCROP Yield MONOCROP Yield Cobindex BD% (Kg/ha) Cobindex BD% (Kg/ha) Control 0.613± ± ± ± ± ±50 Karate 0.634± ± ± ± ± ±51 Lan ± ± ± ± ± ±60 Lan ± ± ± ± ± ±63 Tep ± ± ± ± ± ±69 Tep ± ± ± ± ± ±79 Tag ± ± ± ± ± ±48 Tag ± ± ± ± ± ±76 Sbor = number of stem borer infested plants; Cobindex = ratio of cobs harvested to stand count at harvest; BD% = percent bird damaged cobs Means in a column whose SE values do not overlap are significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

33 Ogendo, et al., (2013) 28 Table 1b: Effect of aqueous botanical extracts on stem borers and maize yield during long rains, 2005 FARMER 1 (ADA OMULO) LAMBWE DIVISSION, SUBA DISTRICT 2005 (MARCH-JULY) FARMER 2 (PERPETUA OLANDO) Intercrop Monocrop Intercrop Monocrop Yield Yield Yield Stborco Yield TREAT Sbor1 Stborcob Kg/ha Sbor1 Stborcob Kg/ha Sbor1 Stborcob Kg/ha Sbor1 b Kg/ha Control 2.00 a 2.33 a 2731 b 1.00 ab 1.33 ab 2720 b 1.17 ab 4.17 a 2109 a 1.50 a 4.00 a 2227 a Karate 0.67 ab 1.00 ab 4138 ab 0.33 b 1.00 ab 2691 b 1.00 ab 4.00 a 2591 a 0.50 ab 2.83 a 2429 a Lan ab 1.67 ab 3798 ab 1.00 ab 1.33 ab 3566 ab 0.67 ab 3.33 a 2601 a 0.83 ab 2.83 a 2388 a Lan b 1.00 ab 4111 ab 0.00 b 0.67 ab 3684 ab 0.17 b 2.50 a 2564 a 0.50 ab 2.50 a 2545 a Tep ab 1.33 ab 3847 ab 0.33 b 1.00 ab 3496 ab 1.17 ab 4.17 a 2507 a 1.00 ab 3.00 a 2336 a Tep b 0.67 ab 3886 ab 0.33 b 0.33 b 4414 a 0.67 ab 2.67 a 2532 a 0.33 b 2.50 a 2721 a Tag ab 1.67 ab 3271 ab 1.00 ab 1.33 ab 3006 ab 0.83 ab 3.67 a 2205 a 0.83 ab 3.00 a 2359 a Tag b 1.33 ab 3287 ab 0.00 b 1.00 ab 3429 ab 0.83 ab 3.17 a 2208 a 0.17 b 2.33 a 2697 a Means in a column followed by same alphabetical letters are not significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

34 Ogendo, et al., (2013) 29 Table 2a: Efficacy of botanical powders against stem borers incidence and on maize yield, 2004 LAMBWE,SUBA 2003/04 EGERTON UNIVERSITY, NJORO, 2003/04 Intercrop Monocrop Intercrop Monocrop Yield Yield Yield Yield TREAT Sbor Kg/ha Sbor Kg/ha Cobindex BD% Kg/ha Cobindex BD% Kg/ha Control 3.8± ±27 7.4± ± ± ± ± ± ± ±37 Stdust10 1.7± ±60 1.6± ± ± ± ± ± ± ±53 Ash15 2.4± ±93 4.3± ± ± ± ± ± ± ±60 Ash30 1.9± ± ± ± ± ± ± ± ± ±57 Lan15 4.8± ±48 3.1± ± ± ± ± ± ± ±55 Lan30 1.6± ±55 1.7± ± ± ± ± ± ± ±60 Tep15 3.4± ±59 2.8± ± ± ± ± ± ± ±44 Tep30 0.7± ± ± ± ± ± ± ± ± ±56 Tag15 2.6± ±86 2.9± ± ± ± ± ± ± ±47 Tag30 2.3± ± ± ± ± ± ± ± ± ±43 Sbor = number of stem borer infested plants; Cobindex = ratio of cobs harvested to stand count at harvest; BD% = percent bird damaged cobs Any two means in a column whose SE values do not overlap are significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

35 30 Management of stem borers using selected botanical pesticides Table 2b: Efficacy of botanical powders against stem borers incidence and on maize yield in Lambwe during long rains, 2005 FARMER 1 (MARY RABILO) LAMBWE DIVISSION, SUBA DISTRICT 2005 (MARCH-JULY) FARMER 2 (HERINE AMAYO) Intercrop Monocrop Intercrop Monocrop Yield Yield Yield Yield TREAT Sbor1 Stborcob Kg/ha Sbor1 Stborcob Kg/ha Sbor1 Stborcob Kg/ha Sbor1 Stborcob Kg/ha Control 3.00 a 3.67 ab 3061 b 2.67 ab 4.00 ab 3137 b 2.44 a 4.00 a 2135 a 2.11 ab 2.89 a 2052 a Stdust ab 3.00 ab 3786 ab 2.00 ab 3.00 ab 4097 ab 1.89 ab 2.33 a 2009 a 1.22 ab 3.00 a 2097 a Ash ab 4.00 ab 3079 b 1.33 ab 3.67 ab 3222 b 2.00 ab 3.67 a 2116 a 2.00 ab 4.00 a 1727 a Ash ab 0.67 b 3337 ab 0.00 b 3.00 ab 3291 b 1.78 ab 1.89 a 2362 a 0.56 b 3.56 a 2177 a Lan ab 4.00 ab 3823 ab 1.67 ab 4.00 ab 3629 ab 1.33 alb 3.89 a 2298 a 2.33 ab 3.11 a 1848 a Lan b 1.67 ab 3949 ab 0.00 b 3.00 ab 3728 ab 0.67 ab 1.78 a 2543 a 0.56 b 2.22 a 2109 a Tep ab 3.00 ab 3521 ab 3.00 ab 4.67 a 4179 ab 1.67 ab 3.00 a 2455 a 2.11 ab 2.78 a 2323 a Tep ab 1.33 ab 4020 ab 0.33 b 3.33 ab 4211 ab 1.44 ab 2.33 a 2224 a 1.44 ab 2.78 a 2333 a Tag ab 2.67 ab 3741 ab 2.67 ab 3.33 ab 3815 ab 2.44 a 3.89 a 2410 a 2.11 ab 3.33 a 1919 a Tag ab 2.00 ab 5239 a 0.33 b 2.67 ab 4345 ab 1.67 ab 2.44 a 2422 a 1.78 ab 3.11 a 2155 a Means in a column followed by same alphabetical letters are not significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

36 Ogendo, et al., (2013) 31 In the seed soaking trials (Tables 3a-b), general ash, Lantana, Tephrosia and Tagetes extracts, at % w/v, significantly (P<0.05) reduced the stem borer loads by 29-79% (av. 58%) and increased the maize grain yields by 10-62% (av. 31%) compared to the untreated control. All the botanical pesticides and general plant ash treatments, at 1.70% w/v, significantly (P<0.05) reduced stem borer population and produced higher maize grain yields than synthetic insecticide, Furadan 5G applied at 0.567% w/v. These treatments reduced stem borer population to compared to 1.71 for Furadan 5G and improved maize grain yields to kg/ha against 2415 kg/ha for the synthetic insecticide treatment. Generally, planting maize in maize-beans intercrop significantly reduced the stem borer incidence and hence damage. Seed-soaking with botanical pesticides and general plant ash increased (P<0.05) the bean grain yields by % (av. 210%) compared to the untreated control. Aqueous extracts of general ash, Tephrosia and Tagetes, applied at 1.70% w/v, produced significantly higher bean yields than synthetic insecticide, Furadan 5G. These results suggest that the botanical treatments possibly have systemic action that lasts upto one month after application. Egerton J. Sci. & Technol. Volume 13: ISSN No

37 Ogendo, et al., (2013) 32 Table 3a: Effect of seed soaking in aqueous plant extracts on stem borer incidence, bird damage and yield of maize (SR 2004) LAMBWE-SUBA, 2003/04 EGERTON UNIVERSITY, NJORO, 2003/04 Intercrop Monocrop Intercrop Monocrop Yield Yield Yield Yield TREAT Sborfd Sborcob Kg/ha Sborfd Sborcob Kg/ha Cobindex BD% Kg/ha Cobindex BD% Kg/ha Control 4.4± ± ±49 6.5± ± ± ± ± ± ± ± ±102 Furad ± ± ± ± ± ± ± ± ± ± ± ±147 Ash ± ± ± ± ± ± ± ± ± ± ± ±101 Ash ± ± ± ± ± ± ± ± ± ± ± ±147 Lan ± ± ±55 3.9± ± ± ± ± ± ± ± ±100 Lan ± ± ±95 1.5± ± ± ± ± ± ± ± ±118 Tep ± ± ±52 3.2± ± ± ± ± ± ± ± ±135 Tep ± ± ± ± ± ± ± ± ± ± ± ±151 Tag ± ± ± ± ± ± ± ± ± ± ± ±109 Tag ± ± ± ± ± ± ± ± ± ± ± ±124 Sbor fd= number of stem borer infested plants; Sborcob = number stem borer infested cobs; Cobindex = ratio of cobs harvested to stand count at harvest; BD% = % bird damaged cobs Any two means in a column whose SE values do not overlap are significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

38 Ogendo, et al., (2013) 33 Table 3b: Effect of seed-soaking in aqueous plant extracts on stem borer incidence and yield of maize (LR 2005) LAMBWE DIVISSION, SUBA DISTRICT 2005 (MARCH-JULY) FARMER 1 (CECILIA AYOO) FARMER 2 (BOAZ NYATENG) Intercrop Monocrop Intercrop Monocrop TREAT Sbor1 Stborco b Yield Kg/ha Sbor1 Stborco b Yield Kg/ha Sbor1 Stborcob Yield Kg/ha Sbor1 Stborcob Yield Kg/ha Control 1.33 a 1.67 ab 3320 ab 1.33 a 1.33 ab 2693 b 1.67 a 8.33 a 3579 abc 0.67 b 6.00 ab 3977 abc Furadan 1.00 ab 1.00 ab 3514 ab 0.33 ab 0.67 ab 2556 bc 0.33 b 4.67 b 3554 abc 0.00 b 0.67 d 3310 bc Ash ab 0.33 b 3909 ab 0.67 ab 0.00 b 3455 ab 0.00 b 2.00 cd 3504 abc 0.67 b 1.00 d 3770 abc Ash b 0.00 b 4025 ab 0.33 ab 0.00 b 3310 ab 0.00 b 1.00 d 5100 a 0.67 b 2.00 cd 4140 abc Lan ab 0.67 ab 2955 ab 0.00 b 0.67 ab 3935 ab 0.00 b 5.00 ab 4647 ab 0.33 b 6.00 ab 3255 bc Lan ab 0.00 b 4078 ab 0.00 b 0.33 b 3883 ab 0.00 b 0.67 d 4722 ab 0.00 b 5.33 ab 3599 abc Tep ab 2.33 a 3329 ab 0.67 ab 1.33 ab 2601 bc 0.33 b 1.67 cd 2922 c 1.00 ab 2.67 c 3715 abc Tep b 0.00 b 4436 ab 0.00 b 0.33 b 4048 ab 0.00 b 0.00 d 3241 bc 0.33 b 2.33 c 4400 abc Tag b 0.33 b 4211 ab 1.33 a 0.67 ab 3486 ab 1.67 a 3.67 bc 3584 abc 0.67 b 8.33 a 4017 abc Tag b 0.00 b 3904 ab 0.33 ab 0.00 b 3412 ab 0.67 b 0.00 d 3741 abc 0.00 b 5.00 ab 4090 abc Means in a column followed by same alphabetical letters are not significantly different at α = 0.05 by Tukey s HSD test Egerton J. Sci. & Technol. Volume 13: ISSN No

39 Ogendo, et al., (2013) 34 The results obtained from the application of crude powders and aqueous extracts of the test botanicals, L. camara, T. vogelii, T. minuta and general ash in the control of field insect pests of maize and beans showed significant reductions in the insect pest populations particularly the stem borers (as reflected in the field and cob counts). These results are in agreement with the findings elsewhere in which use of botanicals such as neem-based preparations have proven effective in the management of insect pests such diamondback moth, onion thrips, voles, etc (Curtis et al., 2002; Koschier et al., 2002; Liang et al., 2003). The mechanisms of action for the various botanicals may be assumed to be on the basis of insecticidal activity (toxicity), repellence and anti-feedant. However, further studies are recommended to determine the mode(s) of action involved. It is important to identify with the farmers knowledge and perceptions of pests in the cereal-legume cropping systems. Nyeko et al. (2002) reported that farmers were generally more knowledgeable on insect and disease infestations but were weak in their ability to manage the pests. It emerges from this argument that farmers might have known the value of botanical pesticides over several generations but lack the innovative capacity to exploit such resources for effective and socio-economically viable improvement of their crop production and hence the food security and family incomes. That their use in agriculture promises to reduce the insect pest loads (by av. 55%) and increase the cereal-legume productivity by 50% is welcome scientific news which must be exploited for the benefit of the resource poor farmer if the perennial food deficits and high rural poverty is to be alleviated. On the basis of the preliminary scientific rationalization and validation results a major breakthrough is imminent especially for the crude powder recommendations which have so far demonstrated significant efficacy at rates (15-30 kg Powder/ha) comparable to the synthetic insecticides such as Stalk borer 2% dust at 10 kg/ha. However, there is need for further verification trials, in an up-scaling programme, to test these botanicals against recommended synthetic pesticides before a recommendation package may be proclaimed and made available as new technology. Additionally, domestication of these botanical plants must be pursued alongside the development of appropriate accompanying agronomic / cultural practices and home-based processing / preparation procedures for the aqueous extracts and crude powders in the the current and potential user domains in subsistence agriculture. Egerton J. Sci. & Technol. Volume 13: ISSN No

40 Ogendo, et al., (2013) 35 Conclusions and Recommendations Conclusions It can be concluded from these findings that aqueous extracts (seed-soaking and sprays) and crude powders obtained from three botanical plants, Lantana camara, Tephrosia vogelii and Tagetes minuta and general plant ash, have demonstrated significant reductions in stem borer incidences and pest load in both mono- and inter-cropped maize crops. The efficacy levels for the test botanical pesticides, especially the crude powder applications, were comparable to the synthetic insecticides (Karate, Furadan 5G or Stalk borer 2% dust). At the present farmer s crop production practices, the adoption of the aqueous extracts (sprays), powders and extracts (seed soaking) promises to reduce the stemborer insect pest load, compared to the untreated control, by 47-60, and 30-79% respectively with corresponding grain yield increases (and food reserves) of 30-70% based on current study. Recommendations Since the field evaluations in this study engaged a participatory bottoms up approach in which the smallholder farmers indigenous knowledge and practices were incorporated, the results obtained are considered compatible with and highly acceptable in the existing farmer circumstances. On the basis of research results presented above and other research information available, it is strongly recommended that further in-depth studies on the mode /mechanism of action and efficacy duration (residual effect) be conducted using the botanical powders and extracts against stem borers in maize-based cropping systems and validated/up-scaled in the target wider user domains. Such research-extension pursuits should incorporate findings from on-going biological and IPM studies in the region (Imbuga et al., 2002; Nabirye et al., 2003; Khan et al., 2008). It is further recommended that, given their insecticidal potential, the three botanical plants be subjected to in-depth laboratory evaluations on bioactivity (toxicity, repellence, anti-feedant and related studies) against the major insect pests, including (the bostrichid beetles (Prostephanus truncatus and Rhyzopertha dominica) and effects on quality parameters of stored cereal and legume grains in tropical agriculture. Such studies should be backed by bio-safety studies to establish any harmful effects of powder and extract formulations on man and other non-target organisms. It is further recommended that suitable multiplication,harvesting and botanical Egerton J. Sci. & Technol. Volume 13: ISSN No

41 36 Management of stem borers using selected botanical pesticides preparation (formulation) protocols for these promising botanical plants (Tephrosia vogelii, Lantana camara and Tagetes minuta) be developed and local farmers empowered through targeted training of farmers and being encouraged to grow the plants in designated points in their farms. There is need to integrate the use of these botanicals with the other eco-friendly stemborer control strategies such as Push-Pull System, developed by ICIPE, which uses a maize-desmodium intercrop for soil fertility (BNF) improvement, stem borer repellence and suppression of Striga hermonthica. ICIPE should provide lead on the appropriate powder formulation methods. Acknowledgements The research grant provided by the African Institute for Capacity Development (AICAD), Grant No. AICAD/RD-07/SED/02-001, to the first author is herein acknowledged. The authors wish to thank Kenya Agricultural Research Institute (KARI), Njoro, icipe-african Insect Science for Food and Health, Mbita Point and Egerton University for laboratory space, supply of test insects and other forms of institutional support during conduct of the study. References Curtis, P.D., Rowland, E.D., Good, G.L. (2002) Developing a plant-based vole repellent: screening of ten candidate species. Crop Protection 21(4): Finney, D.J. (1964) Statistical Methods in Biological Assay. 2 nd edition. Charles Griffin and Co. Ltd, London. 668p.Gomez, K. A. and Gomez, A. A. (1983) Statistical Procedures for Agricultural Research. 2 nd Edition. An International Rice Research Institute Book. A Wiley-Interscience Publication. Haines, C.P. (ed.) (1991) Insects and Arachnids of tropical stored products: their biology and identification. A Training Manual. 2 nd edition. NRI: UK. Imbuga, M., Namungu, P., Wanga, B. (2002) An overview: Isolation & characterization of bioactive compounds from Tephrosia vogelii Hook F. its use as a biorational for the control of pigeon peas (Cajanus cajan) pests. Forum 5: Programme and Extended Abstracts. 5 th Regional Meeting of the Forum for Agricultural Resource Husbandry, August 2002, Entebbe, Uganda. Egerton J. Sci. & Technol. Volume 13: ISSN No

42 Ogendo, et al., (2013) 37 Kéïta, S.M., Vincent, C., Schmit, J.P., Ramaswamy, S. and Bélanger, A. (2000) Effect of various essential oils on Callosobruchus maculatus F. (Coleoptera: Bruchidae). J. Stored Products Research 36: Khan, Z.R., Pickett, J.A., Wadhams, L.J., Hassanali, A., Midega, C.O. (2006). Combined control of Striga hermonthica and stemborers by maize Desmodium spp. Intercrops. Crop Protection 25(9): Khan, Z.R., Midega, C.A.O., Njuguna, E.M., Amudavi, D.M., Wanyama, J.M., Pickett, J.A. (2008). Economic performance of the push pull technology for stemborer and Striga control in smallholder farming systems in western Kenya. Crop Protection 27(7): Koschier, E.H., Sedy, K.A., Novak, J. (2002) Influence of plant volatiles on feeding damage caused by the onion thrips, Thrips tabaci. Crop Protection 21(5): Liang, G.M., Chen, W., Liu, T.X. (2003) Effects of three neem-based insecticides on diamondback moth (Lepidoptera: plutellidae). Crop Protection 22(1): Nabirye, J., Nampala, P., Ogenga-Latigo, M.W., Kyamaywa, S., Wilson, H., Odeke, V., Iceduna, C., Adipala, E. (2003) Farmer-participatory evaluation of cowpea integrated pest management (IPM) technologies in Eastern Uganda.Crop Prot. 22: Ngatia, C.M. (2000). The Pest Complex of stored pulses as a consequence of the grain market liberalization. 7 th Biennial KARI Scientific Conference, November Nairobi. Kenya. Ogendo, J.O., Deng, A.L., Belmain, S.R., Walker, D.J., Musandu, A.O.and Obura, R.K. (2004) Pest status of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), control methods and constraints to safe maize grain storage in western Kenya. Egerton J. Science and Technology Series 5(1): Ogendo, J.O., Deng, A.L., Omolo, E.O., Matasyoh, J.C. and Tabu, I.M. (2003a) Grain Pests Management Practices in Subsistence Agriculture: A Case of farmers in Suba District, Nyanza Province, Kenya. Farm Survey Report. AICAD Grant No. AICAD/RD-07/SED/ Ogendo, J.O., Belmain, S.R., Deng, A.L. and Walker, D.J. (2003b) Comparison of toxic and repellent effects of Lantana camara L. with Tephrosia vogelii Hook and a synthetic pesticide against Sitophilus zeamais Motsch. (Coleoptera: Curculionidae) in stored maize grain. Insect Science and Its Application, Vol.23(2): Egerton J. Sci. & Technol. Volume 13: ISSN No

43 38 Management of stem borers using selected botanical pesticides Ogendo, J.O. (2000). Evaluation of insecticidal and repellent properties of Lantana camara L. and Tephrosia vogelii Hook against the maize grain weevil, Sitophilus zeamais Motschulsky, in maize grain storage in Kenya. M.Sc. Thesis. Univ. of Greenwich, UK. Papachristos, D.P. and Stamopoulos, D.C. (2002) Toxicity of vapours of three essential oils to the immature stages of Acanthoscelides obtectus Say (Coleptera: Bruchidae). J. Stored Products Research 38: Pottorf, L.P. (2004) Friendly pesticides for Home Gardening. Colorado State University Cooperative Extension ( Talukder, F.A. and Howse, P.E. (1994) Laboratory evaluation of toxic and repellent properties of the Pithraj tree, Aphanamixis polystachya Wall & Parker, against Sitophilus oryzae L. International Journal of Pest Management, 40(3): Zibokere, D.S. (1994) Insecticidal potency of red pepper (Capsicum annum) on pulse beetle (Callosobruchus maculatus) infesting cowpea (Vigna unguiculata) seeds during storage. Indian Journal of Agricultural Sciences 64(10): SAS Institute Inc. (1995) SAS/STAT User s Guide. Release 6.03 Edition. SAS Institute Inc., SAS Campus drive, Cary, NC Saxena, K. N.; Pala-Okeyo, A.; Seshu-Reddy, K. V.; Omolo, E. O. and Ngode, L. (1990) Insect Pest Management and Socio-economic circumstances of small-scale farmers for food crop production in western Kenya: A Case Study. Insect Science & Its Application 10(4): Shaaya, E., Kostjukovski, M., Eilberg, J., and Suprakarn, C. (1997) Plant oils as fumigants and contact insecticides for the control of stored-product insects. J. Stored Products Research 33(1): Scheiner, S.M. and Guvevitch, J. (ed.) (1993) Design and Analysis of Ecological Experiments. Chapman and Hall, New York. Egerton J. Sci. & Technol. Volume 13: ISSN No

44 39 Screening for host plant resistance to Helicoverpa armigera (Lepidoptera: Noctuidae) in selected chickpea (Cicer arientinum L.) genotypes in Kenya Ruttoh E. K 2., Mulwa R.M.S 1, Ngode L. 2, Gohole L. 2, Towett, B. 1, Njogu N. 1, Silim, S 3., Rao G. V. R 3, and Kimurto P. K. 1 * 1 Department of Crops, Horticulture and Soil Sciences, Egerton University 2 Department of Crop and Seed Science, University of Eldoret, P.O. Box, 3900 Eldoret, Kenya 3 ICRISAT, Regional Office Nairobi, Kenya *Corresponding author: kimurtopk@gmail.com, Pkimurto@egerton.ac.ke Submitted 16 th June 2012; Reviewed 22 nd July 2012; Accepted 24 th April 2013 Abstract Helicoverpa armigera is a major pest on the chickpea (Cicer arientinum) world. In Kenya, it causes up to 80% yield losses of the crop. Control measures include application of pesticides and cultural methods which have become less feasible due to their associated costs. Host plant resistance can offer long-term benefits in managing this pest. The objective of this study was to screen and identify chickpea genotypes that are tolerant to Helicoverpa armigera infestation under field conditions. Thirty chickpea genotypes were screened at Agricultural Training Centre, Koibatek under field conditions for two seasons in RCBD design in 3 replicates. Data on larval densities, percent pod damage, and amount of leaf consumed were transformed using angular transformation and then subjected to ANOVA, while treatment means separated using Duncan s Multiple Range Test at P Correlation analysis between larval densities, yield and yield components was done using Genstat 12. The genotypes were classified into various categories of resistance and susceptibility on a visual leaf damage rating scale of 1-9. The results showed that there was significant variation in larval densities among the genotypes. At vegetative stage, mean larval densities ranged from 0.15 to 1.2 Genotypes EC and EC had lowest larval densities while ICC4973 and ICC3137 had highest densities. Larval densities increased from 0.3 to 1.97 during flowering stage. The larvae population increased drastically to a mean of 3.58 at podding. Genotype EC had the least larval density of 2.2. The genotypes, EC583318, EC583250, EC and EC were least infested by the larvae with percent pod damage ranged from 3% on EC with high yields while ICC3137 had highest pod damage of 20.2%. EC was found to be low yielding (1051kg ha -1 ) while ICC4958 was the high yielding Egerton J. Sci. & Technol. Volume 13: ISSN No

45 Ruttoh, et al., (2013) 40 (2205kg ha -1 ). Genotypes EC58318, ICCV10, ICC14831, EC583260, EC583264, and EC had high resistance and could be exploited to chickpea breeding programmes as source to resitance. Keywords: Chickpea, Helicoverpa armigera, larval density, resistance, germplasm. Introduction Chickpea is the third most important legume crop in the world after beans and peas. In Asia, chickpea is second in importance to rice (ICRISAT, 2005). The global annual production of chickpea is Metric tonnes grown on million hectares with average yields of Kg/ha (FAOSTAT, 2009). About 90% of the global area and 88% of production is concentrated in Asia. India is the leading chickpea growing country with over 60% share in acreage and production. In Africa, the annual production is approximately 320,000 tons, which accounts for about 5% of global production. The leading producers are Ethiopia (268,000 tons), Malawi (37,000), Tanzania (31,000 tons) Sudan (12,000), and Kenya (<10,000 tons) (ICRISAT, 2012). In Kenya, recent reports indicate that chickpea was introduced in Kenya in 1980s in Eastern province and in Rift valley (Bomet and Njoro area) in early 1990s (ICRISAT, 1989; Metto, 2002), but a recent survey (Kaloki, 2009) indicates that local accessions have been in existence under cultivation in coastal and Eastern parts of Kenya for the last 40 years. Since then, the crop has since spread in Kenya and is currently adapted to varied agro-ecological zones such as dry highlands (Naivasha, Njoro, Nakuru, Uasin Gishu and Timau), medium altitudes (Bomet, Kabete, Mbeere) and also in dry lowlands (Baringo, Kerio valley, Machakos and Koibatek) with annual rainfall range of mm per annum (Jaetzold and Schmidt, 1983, Kibe and Onyari, 2007, Onyari et al., 2010). The crop that is currently expanding to new areas from the original semi arid areas to the Rift Valley highlands and mid altitudes as a relay crop during the short rains (Kimurto et al., 2010; Mulwa et al., 2010). Currently chickpea has been introduced in dry highlands like Bomet, Koibatek, Naivasha and Nakuru as relay crop after harvesting cereals and in dry low lands like Baringo and Kerio valley during the short rains (Kimurto et al., 2010). To date 4 chickpea varieties (LTD068-ICCV00108, LTD064-ICCV00305, EU- Chania Desi 1-ICCV97105 and SAINA-K1-ICCV95423) have been released for commercial production by 3 research institutions (KARI, Leldet Seed Company and Egerton University) (KEPHIS, 2010, 2012). They are grown extensively specifically in Mbeere, Koibatek and Bomet districts (Kaloki, Egerton J. Sci. & Technol. Volume 13: ISSN No

46 41 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes 2010). Previous work at KARI Naivasha (dry lowland) also reported mean yields of 1604 and 1894 kg ha -1 during the short and long rains (late sowing) respectively from Kabuli (Onyari et al. 2010). Growing chickpea in relay with wheat has the potential to contain the current threat of ug99 strain of wheat stem rust since could break the lifecycle of the pathogen. Work done recently in dryland areas has shown that several varieties (ICCV92944, ICCV 92318, ICCV 96329, ICCV 97037, ICCV10 and ICCV 97126) are well adapted with yields ranging between tons ha -1 (Kimurto et al, 2008; ICRISAT, 2005; Kimurto et al., 2009; Thagana et al., 2009). This indicates that Kenya, like many African countries has a high potential for chickpea production and export to deficit countries such as India, China, and Pakistan. In spite of this, Helicorverpa armigera remains the single most serious insect pest that causes significant yield losses of up to 80% due to its mobility, high polyphagy, short generation duration, and high reproductive rate (Suma et al., 2009). Currently, the application of chemical spray insecticides is the most common method of controlling this pest on crops including chickpea (Sharma et al., 2007). However, H. armigera is known to have developed resistance to almost all insecticides used for its control (Kranthi et al., 2002). The chemical sprays are also of environmental concern and are responsible for human health problems. Limited success has been attained in the development of cultivars with tolerance to pod borers and its control has relied heavily on the use of chemicals. Chickpea pod borer causes yield losses of over US $2 billion in the semi-arid tropics despite application of insecticides costing $ 500 million annually (ICRISAT, 1992; Sharma et al., 2005). H. armigera has developed resistance to several pesticides, especially synthetic organophosphates, carbamates, and organ chlorines insecticides (Harender, 2003), leading to excessive use of more chemicals, which also leads to environmental pollution. Use of insecticides also increases cost of production for the small-scale farmers, since they are not affordable and are increasingly becoming less feasible. Biological control methods such as Bacillus thuringiensis (Bt) toxin, H. armigera polyhedrosis virus and Entomopathogenic fungi have been developed but they are not stable (Lewis, 1997; Ranga-Rao and Shanower, 1999). Weeding as a cultural control where is effective for avoiding oviposition of H. armigera eggs, and biological control with egg parasitoids from Trichogramma is used for inoculative and inundative releases against the pest. Genetic transformation with the Bt genes has been developed in India, however the deployment of transgenic crops for pest management is raising concerns and may take time to be fully integrated Egerton J. Sci. & Technol. Volume 13: ISSN No

47 Ruttoh, et al., (2013) 42 in cultivation. Thus, host plant resistance (HPR) along with natural enemies and cultural practices remain the backbone of pest management systems favourable to most agro-ecosystems (Sharma, 2007). Despite the importance of HPR in integrated pest management, breeding for plant resistance to insect pests has not been in rapid development as the case may be in disease resistance. With the development of H. armigera resistance to insecticides, there is an urgent need to develop chickpea cultivars with native resistance to the pest. Thus this work was aimed at investigating and identifying chickpea germplasm with host plant resistance to H. armigera. Materials and Methods This study was conducted at the Agricultural Training Centre (ATC) Koibatek, in the Rift Valley of Kenya. The site lies in Agro-ecological zone between UM2 and UM3 with an average annual rainfall of 767 mm and mean annual minimum and maximum temperatures of C and C respectively. The soils are vitric, well drained, deep to very deep, brown to dark loams, sandy to clay loam andosol (Jaetzold et al., 2007). Thirty chickpea genotypes comprising one known tolerant check (ICC506) and a test susceptible commercial check (ICC4973) were evaluated. The experiment was conducted for two seasons; short rains (Nov 2008-March 2009) and long rains (April -August 2009) to ensure that material with good levels of resistance was identified under the different environmental conditions. During planting, no fertilizer was applied to the experimental plots. The plots were kept weed free throughout the season by manual weeding. The crop was protected against fungal diseases such as Aschochyta blight and Fusarium wilt with occasional sprays of Ridomil at rates of 2.5g/litre of water. Data collected included number of larvae per plant (larvae density) at the vegetative, flowering and podding stages, total number of pods and the damaged pods per plant at podding stage. Leaf feeding damage, rated visually on a 1 to 9 scale, where 1 = <10% while 9 = >80% leaf area damage was determined. The data on leaf damage scores was used as a resistance rating for the genotypes where a score of 1 stood for very high resistance with leaves free from any damage and 9 representing very high susceptibility with >90% of leaves damaged (Singh and Weigand, 2004). Additionally, the number of days to 50% flowering, number of days to maturity, plant height (cm), biomass (kg/ha), 100-kernel weight (g), grain yield (kg/ha) and harvest index (HI) were measured. Egerton J. Sci. & Technol. Volume 13: ISSN No

48 43 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Data were subjected to analysiss of variance (ANOVA) using the GenStat Release 12.1 software (2009) and means separated using the Duncan s multiple range test (DMRT) at P Data on larval densities and percent pod damage were transformed using angular transformation, before analysis. Correlation analysis was done to determine the relationship between the H. armigeraa damage at various crop phenological stages, yield components and overall crop yield. Results Herlicoverpa Infestation Vegetative Stages The 30 genotypes screened showed significant variability (P<0.05) to H. armigeraa infestationn at this stage in the two seasons. Genotype EC was least infested by H. armigera with larval density of in season I compared to a larval density of recorded on the resistant check (ICC506). Similarly, low infestation densities of 0.23 and 0.27 weree observed in genotypes EC and EC in season I, respectively. These genotypes were not significantly different from the resistant check (Figure 1). Key: Resistant check Susceptible check Test genotypes Figure 1: Larval densities at vegetative stage in season I. Bars show standardd errors The highest larval density of 1.13 was recorded on genotype ICCVX while the susceptible check; ICC4973 was heavily infested, recording a density of Genotypes ICC4058, ICCV07105, ICCVX , ICCVX , ICCVX , and ICC3137 were also not significantly different from the susceptible check with larval densities ranging from 0.13 on EC to 1.13 on ICCVX in season I (Figure 1). Egerton J. Sci. & Technol. Volume 13: ISSN No

49 Ruttoh, et al., (2013) 44 In season II, the levels of infestation were comparatively higher at vegetative stage than in season I. Infestations ranged from on EC to on ICCC37 (Figure 2). Genotypes ICC4533, EC583250, EC583260, EC and EC posted an average larval density of 0.2 against a density of in the resistantt check (Figure 2). Larval densitiess of more than 1.0 were observed in genotypes ICCC37, ICC3137 and ICC637. Key: Resistant check Susceptible check Test genotypes Figure 2: Larval densities at vegetative stage in season II. Bars show standard errors Infestation at Flowering Stage The second instar larvae were common and fed on the flowers, flower buds and foliage. There was no much variation on the mean larvall density recorded between the vegetativee and flowering stages in season I. A mean larval density of was recorded at this stage as compared to at vegetative stage. The larval density ranged from 0.2 on EC to on ICC3137. The density of larvae decreased drastically on the resistant check from 0.2 at vegetative stage to 0.1 at flowering stage. Larvae densities increased from vegetative on genotypes EC583250, EC583318, EC583264, ICC14402, ICC4958, ICCVX and ICCV The genotypes ICCC37, ICC3137, ICC07105, 1CCVX maintained an average of 1.0 larval density between vegetative and flowering stages. The genotypes ICC10393, EC had the least larval density and were not significantly different from the resistant check at this stage (Figure 3). Egerton J. Sci. & Technol. Volume 13: ISSN No

50 45 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Key: Resistant check genotypes Susceptible check Test Figure 3: Larval densities at flowering stage in season I. Bars show standardd errors The insect infestation levels increased especially in season II from a mean density of at vegetative stage to 1.02 at flowering stage. There was a high population build up of larvae between the two phenological stages as compared to season I. In season II, the larval density varied from 0.4 on genotypes EC and EC to on ICC3137. The genotype ICC506 (check) had the least larval density of The genotypes EC583250, EC583260, EC583264, EC583318, ICCV07106 and ICCCVX did not differ significantly on supporting the average larval density of 0.42, which was the lowest at this stage (Figure 4). Key: Resistant check genotypes Susceptible check Test Figure 4: Larval densities at flowering stage in season II. Bars show standardd errors Egerton J. Sci. & Technol. Volume 13: ISSN No

51 Ruttoh, et al., (2013) 46 Infestation at Podding Stage In both seasons, the density of the larvae increased drastically at this stage. A grand mean larval density of and was obtained in season I and II respectively. There was significant variation among the 30 genotypes in response to insect larvae infestation levels in the two seasons at P In season I, the larval density increased from a mean of at flowering to at podding whereas in season II it increased from 1.02 at flowering to at podding stages. The fourth and fifth instars weree common and found chewing veraciously on buds, flowers and pods, leaving characteristic round holes on the chickpea pods and feed on developing grain. In season I, the lowest larval density of was recorded on genotype EC The same density of larvae was recorded on the resistant check (ICC506). The larval density ranged from on the genotype EC to on ICC The genotypesec583250, EC583260, EC583264, EC583318, and ICC14402 were not significantly different in supporting the larvae at this stage of plant growth (Figure 5). The genotypes ICCV07108, ICCV07104, and ICCV07105 were not significantly different from the susceptible check (ICC4973) and an average larval density of 8.0 and were the heavily infested genotypes at this phenological stage of plant growth (Figure 5). Key: genotypes Resistant check Susceptible check Test Figure 5: Larval densities at podding stage in season I. Bars show standardd error In season II, the genotype ICCV07105 had the lowest larval density of 0.30 while the resistant check, ICC506 had The genotypes EC583250, EC583260, EC583264, and ICCV10, were not significantly different in Egerton J. Sci. & Technol. Volume 13: ISSN No

52 47 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes supporting the larval densities and had an average density of 1.67 larvae. The highest larval density of was recordedd on genotype ICC637, while the susceptible check had larval density of 3.5. The genotypes, ICC3137 and ICC637 were not significantly different from the susceptible check (Figure 6). Key: genotypes Resistant check Susceptible check Test Figure 6: Larval densities at podding stage in season II. Bars show standardd errors Pod Damage The Helicoverpa damaged pods turned whitish due to drying and can easily be distinguished from undamaged pods. In season I, mean pod damage of percent was obtained as compared to 9.78 percent recorded in season II. The pod damage ranged from 2.53 percent on genotype EC to 25.6 percent on ICC10393 (Figure 7). The resistant check recorded 6.57 % pod damage while the susceptible check had percent pod damage. The genotypes ICC and ICCV07104 recorded the highest pod damage of 25.6 and percent respectively. The genotypes EC583250, ICCVX , ICCVX were not significantly different from resistant check. ICC16903, ICC14402, ICCV07113, ICCV07106, ICC3137 and the susceptible check weree not significantly different in pod damage percent obtained (Figure7). Egerton J. Sci. & Technol. Volume 13: ISSN No

53 Ruttoh, et al., (2013) 48 Key: genotypes Resistant check Susceptible check Test Figure 7: Percentage larval pod damage in season I. Bars show standard errors In season II, the mean damage percent decreased by 3.17 as compared to season I. The pod damage percentage ranged from 0.57 on the genotype EC to 29.7 on ICC3137, EC and ICCVX had the lowest pod damage percent recorded. The genotypes EC583264, EC and ICCVX were not significantly different in terms of pod damage percent obtained and recorded low pod damage percent as compared to resistant check, ICC506 (Figure 8). Key: genotypes Resistant check Susceptible check Test Figure 8: Percentage larval pod damage percent in season II. Bars show standard errors Egerton J. Sci. & Technol. Volume 13: ISSN No

54 49 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Leaf Damage The 30 genotypes showed high variability to leaf damage by the pod borer larvae. General observations indicated that genotypes with light coloured foliage and more spread were preferred by the pest than those with deep green colour and having a compact canopy structure. These genotypes also tended to be more hairy. Genotypes displaying these traits included EC583318, ICC506, EC583260, ICCV10, and EC To the touch, genotype EC583260, had tough, hard leaflets and was found to be the least preferred by the pest. The pest preferred genotypes (ICC4973, ICCC37, ICC1356, and ICC3137) also had large leaflet area and soft leaves. The lowest leaf damage score of 1.3 was recorded on the resistant check, ICC506. Genotypes ICCV07113, ICC14831, EC583250, and EC were not significantly variable in terms of leaf damage, recording the same leaf damage score (Figure 9). Genotypes ICC4058, ICC1356, ICC07106, ICCVX , ICCC37, and ICC3137 averaged 5.0 on leaf damage score while the susceptible check ICC4973 registered the highest leaf damage score of 7.7 (Table 1). Table 1: Classification of the 30 genotypes by their resistance/ susceptibility to the pod borer Genotypes Category reactions ICC506 Very highly resistant EC583250, EC583318, ICC14831, ICC5383 Highly resistant EC583260, EC583264, EC583311, ICC14402, Resistant ICCV10, ICCV07105, ICC10393 ICC16903, ICC4533, ICC867, ICCVX Moderately resistant 72, ICCVX , ICC867 ICCV07104, ICCV07108, ICCV07106, Intermediate ICC4058, ICCVX , ICCV07113 ICC1356, ICCVX Moderately susceptible ICC3137, ICCC37, ICC637 Susceptible ICC4973 Highly susceptible Egerton J. Sci. & Technol. Volume 13: ISSN No

55 Ruttoh, et al., (2013) 50 Key: Resistant check Susceptible check genotypes Figure 9: Leaf damage in the field. Bars show standard errors Test Correlation Analysis of Larval Density, Yield Components and Yields Results of the correlation analysis indicated a significant positive correlation of larvall densities at vegetative, flowering, and podding stages of the chickpea crop in both seasonss I and II. In season I, the pod damage percentage was positively correlated to larval densities at vegetative, podding stages but negatively correlated at flowering stage (Table 2). Larval densities at vegetative, flowering, and podding stagess were positively correlated with plant height. The larval densities at podding stage were positive and significantly correlated to harvest index and plant height. The number of days to 50% flowering was significant and positively correlated to yield while the number of days to maturity was negatively correlated to the realized yield. Plant vigour was significant and positively correlated to the yield obtained and the number of larvae at vegetative and podding stages, but negatively correlated to 50% flowering. Plant biomass and kernel weight were positively correlated to plant vigour (Table 2). Egerton J. Sci. & Technol. Volume 13: ISSN No

56 51 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Table 2: Correlation coefficients of H. armigera larvae infestation at vegetative, flowering, podding, pod damage percent, yield and yield components. (Season 1) LCV LCF LCP PDP BIO HI KWT DF DM PHGT PV LCV 1 LCF 0.76** 1 LCP 0.65** 0.54** 1 PDP BIO HI * KWT * DF ** DM * * 1.00 PHGT 0.22* 0.33* 0.28* PV * * Y/HA ** 0.62** ** ** Key: LCV-larval counts at vegetative stage, LCF-larval counts at flowering stage, LCP -larval counts at podding stage, PDP-pod damage percent, BIO-plant biomass, HI- harvest index, KWT-kernel weight, DF-days to 50 % flowering, DM- days to 75 % physiological maturity, PHGT-plant height, PV-plant vigour, Y/HA-yield per hectare, *-significant at P 0.05 and **-significant at P In season II, the pod damage percentage had a positive and significant correlation to larval density at vegetative, flowering and podding stages of plant growth (Table 3). Biomass was significantly correlated to larvae density at podding stage and negatively correlated to the density of larvae at vegetative and flowering stages. Harvest index had a positive significant correlation with larvae density at vegetative and flowering stages; however, the correlation was not significant with larval density at podding stage and pod damage percentage. Plant height was positively correlated with larval density at flowering, but was negatively correlated with the number of days to flowering. The yield obtained per hectare in season II correlated positively with biomass, harvest index, plant vigour, kernel weight and days to maturity. The yield also positively correlated with larval density at the vegetative and flowering stages but was negatively correlated at the podding stage. Though not significant the pod damage percentage was negatively correlated to the yield realized in the two seasons; in season I it was significantly correlated to harvest index (Table 3). Egerton J. Sci. & Technol. Volume 13: ISSN No

57 Ruttoh, et al., (2013) 52 Table 3: Correlation coefficients of H. armigera larvae infestation at vegetative, flowering, podding, pod damage percent, yield and yield components. (Season II) LCV 1 LCV LCF LCP PDP BIO HI PV KWT DF DM PHGT Y/HA LCF ** 1 LCP ** ** 1 PDP ** ** * 1 BIO * HI * * PV KWT DF ** DM ** PHGT * * * Y/HA **.6518**.6518* * ** Key: LCV-larval counts at vegetative stage, LCF-larval counts at flowering stage, LCP -larval counts at podding stage, PDP-pod damage percent, BIO-plant biomass, HI- harvest index, PV-plant vigour, KWT-kernel weight, DF-days to 50 % flowering, DM- days to 75 % physiological maturity, PHGT-plant height, Y/HA-yield per hectare, *-significant at P 0.05 and **-significant at P Egerton J. Sci. & Technol. Volume 13: ISSN No

58 53 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Discussion Field screening of Helicoverpa armigera The field studies have shown that the chickpea selections have variable response to levels of infestation in the field and damage by Helicoverpa armigera larvae. The variability in the infestation levels per genotype can be attributed to varying amounts of chickpea foliar secretions containing high concentrations of malic acid. These volatiles from the plants play an important role for host location. This explains the observed patterns of variability in the larval counts per genotype. The genotypes, ICCV10, EC583318, EC583260, EC and EC attracted fewer larvae as compared to ICCC37, ICC1356, ICC4973 and the difference could be explained by the varying amounts of foliar volatiles. The pest can therefore be controlled by selection of these genotypes which are believed to release low foliar concentrations of malic acid. The preference or non-preference for a given genotype is because of difference in canopy structure of the plant (Muhammed et al., 2009). The genotypes, EC583318, EC583260, ICCV10, ICC14831, EC and EC with a dense kind of canopy, which influences the movement and feeding of the borer s larvae and as result low larval density was recorded on these genotypes. These genotypes were also associated with smaller leaflets and high density of trichomes per unit leaf area. The higher trichome density has a role in imparting resistance /tolerance against chickpea pod borer (Girija et al., 2008). Other resistance mechanism as exhibited by EC could be hardiness of the leaves due to high contents of lignin, hemicellulose and cellulose. The low infestation of larvae in this genotype can be attributed to pod borers inability to chew this genotype with ease and non-preference for this genotype. The genotypes such as ICC3137, ICC1356, ICCC37, ICC637, ICCVX and ICC4973 had spread pattern of canopy structure with large soft leaflets and were more preferred by the pest. These genotypes tended to be susceptible by supporting high a population of the pod borers in the two seasons. The larval counts increased with crop growth in the field by 346 and 175 per cent in both Seasons II and I respectively from vegetative to podding stage. The amount of foliar exudate and the concentration of malic acid depend on temperature, growth stage and has been shown to increase at the reproductive stages of the plant (Muhammed et al., 2009). This gives a reason why there were high larval counts during podding stage as compared to vegetative and flowering stages. The population fluctuations of H. armigera on the chickpea can also attributed to the weather patterns during the cropping season. The important factors indicating the probability of population build up were high temperatures and low rainfall. In season II, occasional heavy torrents of rain Egerton J. Sci. & Technol. Volume 13: ISSN No

59 Ruttoh, et al., (2013) 54 over the growing period, tended to wash and destroy the noctuid eggs on the plant and led to breakdown of pupation chambers in the soil preventing the adult emergency resulting to low population over the cropping season. The genotypes having light green colour foliage (ICC4973, ICC1356, ICC37 and ICC637) were preferred by the pest and had high larval counts. The difference in foliage colour is a good criterion for the determination of resistance in chickpea against gram pod borer (Susanne, 1990). The colour changes or difference unveils the role of the plant constituents in its formation, which could act as anti-feedants to the insect larvae. The genotypes EC583250, EC583260, ICCV10, ICC14402 and EC with deep green colour were non-preferred and attracted low larval counts. The possible mechanisms of resistance could be physical factors/morphology of the leaflets (hardiness, hairiness and size), composition and amount of leaf exudates. Conclusion The chickpea genotypes EC583250, EC583318, EC583260, ICCV10, ICC14831 and EC were identified to have resistance to H. armigera with comparable yields. We therefore recommend them for use in chickpea breeding programmes to confer resistance to the susceptible and high yielding varieties. Specifically, genotype EC showed high resistance to Helicoverpa larvae and possesses other outstanding desirable traits such as high yields, biomass and harvest index and can be advanced as a variety for release to farmers. References ICRISAT. (2005). International Crop Research Institute for the Semi-Arid Tropics. Annual Report, Nairobi, Kenya. pg Jaetzold, R., Schmidt, H., Shisanya, C and Honetz, B. (2007). Farm Management Handbook of Kenya-Natural Conditions and Farm Management Information, 2 nd Edition. Vol.II/B: Central Kenya (Rift Valley and Central Provinces). Ministry of Agriculture. pg , Kaloki, P. (2010). Sustainable Climate Change Adaptation Options in Agriculture: The Case of Chickpea in the Semi-Arid Tropics of Kenya. Final Technical report. Pg Egerton J. Sci. & Technol. Volume 13: ISSN No

60 55 Screening for host plant resistance to Helicoverpa armigera in chickpea genotypes Kimurto, P.K., Towett, B.K., Gangarao, V.R., Rajeev, V.R and Gaur, P. (2010). Annual Report on Generation Challenge Program Chickpea annual meeting, Presentation paper in Lilongwe Malawi. Pg 5. Kranthi, K.R., Jandhay, D.R., Kranthi, S., Wanjari, R.R., Ali, S.S. and Russel, D.A. (2002). Insecticide resistance in five pod borer pests of cotton in India. Crop Protection Journal 21: Kranthi, K.R., Jandhay, D.R., Kranthi, S., Wanjari, R.R., Ali, S.S. and Russel, D.A. (2002). Insecticide resistance in five pod borer pests of cotton in India. Crop Protection Journal 21: Muhammed., Nazir, A. and Muhammed, T. (2009). Host plant resistance Relationship in chickpea (Cicer arietinum L.) against gram pod borer (Helicoverpa armigera Hubner) Pakistan Journal of Botany. 41(6) Mulwa, R.M.S., Kimurto, P.K. and Towett, B.K. (2010). Evaluation and selection of drought and pod borer (Helicoverpa armigera) tolerant chickpea genotypes for introduction in Semi-arid areas of Kenya. Research Application Summary. Second RUFORUM Biennial Meeting September 2010, Entebbe, Uganda. pp Sharma, H.C. (2007). Host plant resistance to insects: Modern Approaches and Limitations. Indian Journal of plant protection 35(2) Suma, S., Biradar, O. and Salimath, P.M. (2009). Genetic enhancement of chickpea for pod borer resistance through expression of CryIAc protein. Karnataka J. Agric. Sci., 22(3-Spl. Issue): ( ). Egerton J. Sci. & Technol. Volume 13: ISSN No

61 The prevalence and effects of Plasmodium falciparum, Schistosoma mansoni and soil-transmitted helminth co-infections on haemoglobin concentration amongst school-aged children in Kisumu Municipality, Kenya Ngetich C. E. * 1, Odhiambo R. 1, Mwandawiro C. 2, Kihara J. 2 1 Biological Sciences Department, Egerton University, P.O. Box 536, Egerton, Kenya 2 Kenya Medical Research Institute, Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC), P.O. Box Nairobi *Corresponding author: emyngetich@gmail.com Tel: Submitted 16 th Nov., 2012; Reviewed 21 st March 2013; Accepted 12 th May 2013 Abstract Schistosomosis and soil-transmitted helminths regarded as neglected tropical diseases affect the poor in rural and urban areas of developing countries. In this study the prevalence and effects of Plasmodium falciparum, Schistosoma mansoni and soil-transmitted helminth infections on haemoglobin concentration in school-aged children was examined. Up to three hundred and fifty six (356) children were randomly selected in four different primary schools in Kisumu municipality, Kenya. It was found that, school age children had high malaria parasites levels with 62% harbouring more than one parasite. Only 8.6% of the total children examined had no parasites under the study. The prevalence of anaemia was 12%, where P. falciparum and S. mansoni had a positive association with low haemoglobin concentration with increasing parasite intensity. However, soil-transmitted infections were not significantly found to be associated with low haemoglobin levels among the study population. The high prevalence of parasite parasitaemia among the children (91.2%) is important for instituting regular de-worming and treatment of the children and possible integration of intestinal worm control into the National Malaria Control programmes. Measures that reduce P. falciparum and helminths infections particularly S. mansoni will consequently reduce anaemia among school children. Key Words: Co-Infections, Malaria Parasites, Helminths, Anaemia, School Children Egerton J. Sci. & Technol. Volume 13: ISSN No

62 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 57 Introduction Malaria is a public health problem in more than 90 countries, inhabited by 2.4 billion people representing about 40% of the world s population (Murphy and Breman, 2001). Annually, an estimated million clinical cases of malaria are reported and million deaths occur worldwide mostly among children (Snow, et al, 1999). Approximately 90% of the clinical cases have been shown to occur in Africa (Murphy and Breman, 2001). In Kenya, malaria is still a major cause of morbidity and mortality and it is the leading cause of outpatient and inpatient mortality (MOH, 2000). About 30% of the estimated 8.2 million outpatients in Kenya attending health facilities are diagnosed with malaria annually and of these, 22,000 develop severe complications necessitating inpatient admission (MOH, 2001). During blood stage of its life cycle, the parasites cause destruction of infected red blood cells (RBCs) resulting in anaemia. Non-parasitized RBCs too are removed from the circulation by complement-mediated lysis and phagocytosis resulting from immune complex deposition and complement activation (Menendez, et al. 2000). Anaemia can also occur because of reduced young RBCs production due to suppression of the normal response of erythropoietin in the spleen and bone marrow (Mackintosh, et al. 2004). Previous studies on anaemia among the population in malaria endemic areas have recognized P. falciparum as the main cause of anaemia (Brooker, et al., 1999 and, Akhwale et al., 2004). Cases of hookworm infection worldwide are estimated to be 740 million, with the highest prevalence of hookworm infection occurring in sub-saharan Africa and Eastern Asia (De Silva, et al., 2003). Schistosomosis too, categorized as one of the neglected diseases, affects exclusively the poor in rural and impoverished urban areas in developing countries (Engels, et al. 2002). It is estimated that at least 200 million people are currently infected with schistosomosis and its mortality is estimated at 11,000 deaths per year while the burden is estimated at 1.7 million disability-adjusted life years (DALYs) lost per year (TDR/WHO, 2002). Schistosomosis and soil-transmitted helminths (Hookworms, Ascaris lumbricoides and Trichuris trichiura) are among the most neglected infections that affect mostly the poor in the rural and impoverished urban areas (Hotez, et al. 2005). Heavy infections with schistosomosis and hookworm are known to lower haemoglobin concentration leading to anaemia. For instance, hookworm infections account for up to 35% and 73% Egerton J. Sci. & Technol. Volume 13: ISSN No

63 58 Ngetich et al., (2013) of the iron-deficiency anaemia and severe iron-deficiency anaemia in Africa, respectively (Hotez, et al. 2005). Blood loss may also occur in heavy infections of T. trichiura while contribution of ascariasis to low haemoglobin has been found to in some populations (Stephenson et al., 1993 and Curtale et al., 1993). Anaemic condition often occurs due to multiple causes such as nutritional deficiency, genetic malfunctions, as well as from parasitic infections (Phillips and Pasvol, 1992, Menendez, et al., 2000). It affects all population groups but the most susceptible are pregnant women and young children (WHO, 2002). Helminthic infections, particularly schistosomes and STHs, which are also prevalent in malaria endemic areas, require further investigation on their influence on anaemia in malaria prone areas. Studies have shown that these parasitic infections contribute to the occurrence of low haemoglobin concentration especially in areas with poor sanitation, unavailability of safe water and lack of good water management practices in a community (Handzel, et al. 2003). Previous studies on anaemia in Kenya shows the prevalence of malaria as the main factor in contributing to anaemia in endemic areas (Akhwale et al. 2004). The present study reports a baseline surveys on prevalence and intensity of P. falciparum, Schistosoma mansoni and soil-transmitted helminth (STHs) infections among school-age children. The study also sought to investigate the effect of P. falciparum, S. mansoni and STHs infections in either single or co-infections on haemoglobin concentration among school going children in Kisumu Municipality. The study revealed high prevalence of multi-parasitism among schoolchildren, the most common parasites being P. falciparum, S. mansoni, A. lumbricoides and T. trichiura. However, hookworms were least prevalent and were not significantly associated but negatively correlated with low haemoglobin levels in the study population. Plasmodium falciparum and S. mansoni were significantly associated with low haemoglobin concentration among the schoolchildren. Egerton J. Sci. & Technol. Volume 13: ISSN No

64 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 59 Materials and Methods Study Population Four schools with total of 356 children participated in the study i.e. Kasagam (55), Nanga (90), Ogal (166) and Gongo (45). Schoolchildren aged between 9 and 16 years with a mean age of 11.8 participated in the study. About fiftyfive percent (54.8%; N= 356) of the children were girls. The children were randomly selected to meet selection criteria, which included the 9 16 years age bracket and willing by a written consent through their guardians and teachers to participate in the study. Kisumu municipality lies along the shores of Lake Victoria in Nyanza Province, Kenya with an altitude of 1200 meters above sea level. The main occupations of the residents are fishing and small-scale farming near the lake while others are employed or running various businesses. The main crops grown in the area include finger millet, maize, groundnuts and cassava, sweet potatoes, beans, and various horticultural crops. Zebu cattle, chickens and goats are abundant especially in the outskirts of the city. Children from the urban areas in this region have access to the lake and small ponds where snail breeding occur exposing them to schistosomosis infection. With availability of fresh water, breeding of mosquitoes also takes place throughout the year, increasing the chances of intense malaria transmission. The moist conditions of the soil provide suitable habitat for hookworms and other geohelminths to thrive in areas where sanitation is poor. Participants names were coded for confidentiality. Children were stratified into four age groups namely:. 9-10, 11-12, 13-14, yrs. The Plasmodium falciparum density was classified into low and high, while helminth infection was classified into light, moderate and heavy infection (WHO, 1994). Measurement of Plasmodium Parasitaemia and Haemoglobin Levels Thin and thick smears were prepared from a drop of blood (approximately 10µl) on the microscope slides. Using a swab moistened with 70% alcohol, the lobe of the left hand third finger was cleansed and allowed to air-dry. The ball of the finger was punctured with a sterile lancet and the first drop wiped off using cotton wool. Using another slide at an angle of 45 o, a fast spread was made on the blood drop on one slide to obtain a thin smear. The blood smear was air-dried thoroughly then fixed using absolute ethanol. Each slide smear was then individually stained using 5% Giemsa stain for 45 minutes. The slides were rinsed using tap water and air-dried in a vertical position. A Egerton J. Sci. & Technol. Volume 13: ISSN No

65 60 Ngetich et al., (2013) thin smear was used for species identification while thick blood smear was used for enumeration of the parasites density per microlitre of blood. Asexual stages (trophozoites, schizonts and gametocytes) of malaria parasites were counted against 200 white blood cells where the density of asexual parasites was calculated, assuming that there are 8000-leucocytes/μl of blood (Warhurst, and Williams, 1996). The Plasmodium parasites were expressed as number of parasites/μl of blood. For negative blood slides, the counting of white blood cells was increased to 500, before the slide was reported negative. Haemoglobin concentration estimation was carried out in the field using HemoCue haemoglobinometer (HemoCue AB, Angelhom, Sweden) according to manufacturer s instructions. The blood from a finger prick was collected in micro-cuvette (HemoCue strip), and inserted into HemoCue heamoglobinometer where the haemoglobin concentration was read from the screen and expressed as the amount of Hb in gms/dl. Anaemia was defined as haemoglobin concentration <11g/dl and cut-off 7.5g/dl indicated as severe anaemia. Malaria-related anaemia is defined as Hb 11g/dl and severe anaemia Hb 7.5g/dl in the presence of malaria parasitaemia of any density in endemic areas (Menendez et al., 2000). Quantification of Schistosoma species and STHs Kato Katz technique method was used to determine helminth egg count in the stool (Katz, et al., 1972). The technique allows easy identification of eggs in stools and indirect estimation of the intensity of infection, which directly relates to morbidity (WHO, 1994). Each child provided a stool sample in a labelled container with unique identification number. A fixed quantity of sieved 41.7 mg (WHO Kit) of stool was obtained by filling a punched template. It was then deposited on a glass slide, covered with malachite green/glycerine impregnated cellophane, and left to clear for about 45 minutes. The slides were examined for S. mansoni, A. lumbricoides and T. trichiura eggs. However, hookworm eggs were counted within an hour to avoid clearing. Egg-counts were expressed as number of eggs/gm of stool by multiplying the total eggs with 24 (WHO factor). Haradi-Mori culture technique was utilized to verify hookworm species in which pear-size stool from positive samples was smeared on to a thin Whiteman s filter paper I. The filter papers were immersed into clean distilled water, filled to 1 / 2 of a test tube. The water was allowed to percolate, and kept out in the sunlight for 10 days to allow hatching of the eggs. Within 7 days, the eggs started hatching into rhabditiform larvae. The larvae were allowed to transform into filariform stages by leaving them in a moist filter Egerton J. Sci. & Technol. Volume 13: ISSN No

66 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 61 paper in a Petri dish for a few hours for easy identification of hookworm species. Parasitological examination of S. haematobium eggs in urine was undertaken by Nuclear Pore Filtration technique as described by Kahama et al., (1998). Three urine samples per study subject were collected between 10 a.m. and 2 p.m. on three consecutive days. A duplicate 10ml aliquot of urine was filtered through 15-mm polycarbonate filters (Nuclear pore R; Costar Europe Ltd., Badhoevedorp, the Netherlands). It was then placed on a labelled slide and examined under a microscope within 6 hours and measured as mean egg counts of the two filters expressed per 10 ml of urine. Treatment and Ethical Consideration The permission to carry out the study was obtained from ethical review committee of the Ministry of Science and Technology and Kisumu Division of Vector Borne Diseases (DVBD) authorities. The purpose of the study was carefully explained to the teachers, guardians and children before the start of the sample collection. After the examination, all children were treated with a single dose of Praziquantel at 40mg/Kg of body weight to treat schistosomosis, and Albendazole 400mg single dose, against geo-helminths. Any malaria positive cases with definite clinical symptoms were treated with Coartem drugs under supervision of the area clinical officer. Data Analysis The data was entered into excel computer programme and analysed using SPSS (version 11.5) software for windows. Descriptive statistical analysis was used to compute malariometric indices and the difference in mean haemoglobin concentration values in single and multiple parasite infections were tested for statistical significance using Chi-square test. ANOVA statistical package was used to analyse comparisons of continuous variables among individuals in the different schools and health centres. Kruskall- Wallis analysis was used to assess the variation of egg output by age and sex of the children. The significant level throughout the analysis was regarded as P-value < Results Prevalence of Anaemia and Severe Anaemia The prevalence of anaemia among schoolchildren was high with those having heavy intensity of S. mansoni, 29.4% (Table I). High intensity of P. falciparum infection had high prevalence (16.7%) of anaemia cases. Children Egerton J. Sci. & Technol. Volume 13: ISSN No

67 62 Ngetich et al., (2013) with both light and moderate infections of A. lumbricoides and T. trichiura had anaemia while it occurred in children with light infections with hookworm. There were no cases of severe anaemia diagnosed but 11.2% of the children had anaemia (Hb 11g/dl). The average haemoglobin concentration was 12.6g/dl ± Table 1: Severe Anaemia and Anaemia Percentage Prevalence Light/Low Moderate High/Heavy P. falciparum Anaemia 11.7% % S. mansoni Anaemia 21.3% 9% 29.4% Hookworm Anaemia A lumbricoides Anaemia T. trichiura Anaemia 12.2% 4.6% 7% 0% 6.4% 12.7% 0% 0% 0% NB: The worm loads were classified as low, moderate or heavy intensity while intensity of Plasmodium species were classified as light or heavy intensity (WHO, 1994). There was significant difference in the occurrence of anaemia with different age groups with higher number of children in between ages years more likely to be anaemic (χ 2.= , P =000). There was no significant difference in occurrence of anaemia between different sexes (Table 2). Table 2: Prevalence of Anaemia by age group and sex Haemoglobin concentration Anaemia Normal χ 2 p-value Age Groups (21.4%) 76 (24.2% (38.1%) 143 (45.5%) (33.3%) 82 (45.5%) >15 3 (7.1%) 13 (4.1%) Sex of children Male 19 (45.2%) 142 (45.2%) Female 23 (54.8%) 172 (54.8%) Parasite infection and haemoglobin concentration The findings showed high significance negative correlation between haemoglobin concentration and P. falciparum intensity implying that; as the parasite densities increased, the haemoglobin concentration was reduced Egerton J. Sci. & Technol. Volume 13: ISSN No

68 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 63 (correlation coefficient = , P = 0.003). There was significant negative correlation between haemoglobin concentration and S. mansoni, (correlation coefficient = , P = 0.031) but there was no significant difference in haemoglobin concentration between sexes and among different age groups; df = 1, P = 0.643, and df = 3, P = respectively. On the effect of coinfection with P. falciparum, S. mansoni and N. americanus on the haemoglobin concentration, the study revealed that 64% (N=42) of the anaemic children (Hb 11g/dL) had parasite co-infection with two or more of the parasites (Table 3). Although, A. lumbricoides and T. trichiura did not show significant association with low haemoglobin concentration, 0.5% coinfected with the two parasites were anaemic (Hb<11g/dl). Table 3: Prevalence of Specific Parasite Co-Infection in Relation to Low Hb Levels Parasite and co-infection Number % prevalence Plasmodium S. mansoni Hookworms Plasmodium +S. mansoni All 5 parasites Plasmodum, S. mansoni + 1 or more STH Plasmodium and 1 or more STH S. mansoni + 1 or more STH STH alone None Total Parasite Rate/Prevalence Plasmodium falciparum and S. mansoni infections ranked the highest in occurrence among the parasites diagnosed (Table 4) Table 4: Overall Parasite Prevalence Gongo Kasagam Nanga Ogal Total %Prevalence P. falciparum S. mansoni Hookworm A.lumbricoides T. trichiuris No. of children Egerton J. Sci. & Technol. Volume 13: ISSN No

69 64 Ngetich et al., (2013) Malaria Endemic malaria, mainly caused by P. falciparum, which accounts for 99% prevalence in this study, appeared to be the main cause of malariaa cases in the population. One percent (1%) of Plasmodium positive children had P. malariae whereas 5.6% were diagnosed with other P. falciparum stages (schizonts or gametocytes). Plasmodium falciparum was more prevalent among children in Ogal and Gongo with prevalence rate of 77.7% and 48.9% respectively (Fig. 1). Infection rate in the other schools was below 20% in all the samples examined. It was noted that many school age children seeking health care had high rates of malaria parasites. Twenty-two percent (N = 50) of anaemic children (Hb 11g/dl) had mono-infection with P. falciparum Plasmodium S. mansoni Hookworms A. lumbricoides T. trichiura Gongo Kasagam Nanga Ogal Figure 1: The Percentagee Prevalence of Parasites per Study Site Schistosomosis Schistosomosis due to S. mansoni was predominant helminth studied in all the schools with Nanga having higher prevalence when compared to the other sites. There was no transmission of Schistosoma haematobiumm among the study population. Soil Transmitted Helminths The highest prevalence (56.4%) was reported in Kasagam followed by Gongo primary school with 33.3%. Using morphological features examined under dissecting microscope, filariform larvae that were obtained after culturing positive stools for ten days revealed that Necator americanus was responsible for the hookworm infection among the study population in Kisumu municipality. Mono-infection with hookworm (N. Americanus) accounted for only 4% of the total parasitess prevalent in the schools. About 5% of children with Hb<11g/ had hookworm mono infection. The occurrence of Ascaris lumbricoides and T. trichiura were evenly distributed Egerton J. Sci. & Technol. Volume 13: ISSN No

70 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 65 among schoolchildren under the study and the prevalence rates were almost similar in all the study sites. Parasite Density Plasmodium falciparum intensity was classified as high and low while worm intensities were classified into three categories; light, moderate and heavy infections. Most of the children had light intensity of the parasitic infections diagnosed. Generally, high or heavy parasite densities were only observed in P. falciparum and S. mansoni infections. Overall, there were higher incidences of light intensity in all the parasite infections as compared to moderate or heavy/high infections. The results showed no relationship between the sex of the children and parasite intensity. There was no statistical difference among different age groups except for strong significance in P. falciparum intensity (X 2 = , df = 3, P = 0.007) occurring in different age groups with less parasite intensities among the older children. Parasite intensities in different schools showed significant variation in infection intensities (df 3 P < 0.001). Parasite Interactions There was significant negative correlation between P. falciparum intensity and hookworm intensity (Corr. Coeff. = , P 0.041). Using linear regression model, no significant interaction was observed among different parasitic infections. Discussion This study has demonstrated that malaria and schistosomosis significantly contribute to anaemia among primary school children in Kisumu municipality, and that both girls and boys are equally infected. The children with P. falciparum and S. mansoni had a risk of developing anaemia with increasing densities for both parasites. Co-infection with P. falciparum and S. mansoni are high predictor to anaemia (<11g/dl) as also reported in by Koukounari et al (2008). Despite high prevalence of single and multiple coinfections among the children, anaemic cases were few and this was perhaps due to many children having light or moderate intensities to either of the parasite infections. The results concur with previous findings on the epidemiology of Plasmodium and helminth co-infections (Brooker, et al. 2007). A quarter of the anaemic cases had P. falciparum and S. mansoni coinfection although even in single infection, both P. falciparum and S. mansoni there were higher percentages of anaemia cases as opposed to STH infection. Furthermore, the contribution of malaria and schistosomosis to Egerton J. Sci. & Technol. Volume 13: ISSN No

71 66 Ngetich et al., (2013) anaemia, could not be ruled out in anaemic cases where the two parasitic infections either one or both, in the presence of STHs infections occur in the same individual. A similar finding was reported in a study on intensity and prevalence of anaemia among adolescent girls in Western province, in which schistosomosis and falciparum malaria were found to be responsible for the anaemia (Leenstra, et al., 2004). In the later study, soil-transmitted helminths were not found to contribute to anaemia levels in the study population, not even hookworm, a parasite that is usually associated with high anaemia levels (Guyatt et al., 2000, and Brooker et al., 2007). The little or no effect in some children with multiple parasites infections on haemoglobin concentration could be attributed to low parasite intensities, good iron nutritional status and possibly attributes related to genetic factors and this is in consistent with other similar studies in western Kenya (Koukounari et al., 2008). The contribution of hookworm infections to anaemia is well established, even light hookworm infections have been implicated with low haemoglobin levels among individuals with poor iron nutritional status (Brooker, et al., 1999, Crompton, 2000). Although their effect on haemoglobin was not significant in the population under this study, their effect even in low worm burdens as reported in earlier studies would still worsen the health status of an individual (Crompton, 2000). Therefore, it is not surprising that about 5% (N = 42) of anaemic cases were children positive with only N. americanus. Although hookworm infections did not seem to significantly contribute to anaemia levels, it is possible that their presence may increase the risk of children presenting clinical malaria attack, as it has been demonstrated by Spiegel and others, thereby indirectly contributing for anaemia levels in malaria patients (Spiegel, et al., 2007). The results from the study has shown that malaria and helminth parasites are acquired at an early age with over 30% of 9 10 age group being infected with majority of the parasite types and peak at the age of for all parasite infections diagnosed, thereafter there was a decline with increase in age. The high incidence rates of STHs particularly A. lumbricoides and T. trichiura among the younger age group reveal their vulnerability probably due to their young age, and fact that due to their young age, may not be able to observe proper hygiene thus get more exposed to parasite infection than older children. The decline of parasite incidence rate with increasing age may be attributed to acquisition of immunity to parasites particularly P. falciparum with continuous exposure, (Hommel & Gilles, 1998). Egerton J. Sci. & Technol. Volume 13: ISSN No

72 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 67 This study demonstrated high prevalence rates of P. falciparum (49.7%) and S. mansoni (62.4%) among school-aged children in Kisumu Municipality, which may indirectly indicate these parasite infections in the general population as reported in another study in Busia district, Kenya (Brooker, et al., 2000). Many infections among the children were remarkably light, with over 30% of the children having light infections of S. mansoni and P. falciparum. The proximity to the Lake played a role in transmission and sustenance of these parasitic diseases (malaria and schistosomosis) in this region by providing habitat and breeding sites of Anopheles gambiae and fresh water snails (Biomphalaria species) respectively. Children in this region easily get access to the lake and river water as they go about their daily chores. Conclusion (i) Schistosomosis was the most prevalent parasite diagnosed in the study. (ii) The study revealed that P. falciparum, S. mansoni and STHs peak in school age children probably due to predisposing factors in the surrounding areas where the children reside including absolute poverty, lack of proper drainage and poor sanitation. (iii) The occurrence of high rates of helminthic infections (schistosomosis, N. americanus, A. lumbricoides and T. trichiura), could be indicators of faecal contamination of the water sources which play a big role in completion of the parasites life cycle and a reflection of low standards of hygiene in the study area. (iv) (v) (vi) The study provides quantitative evidence on the degree of parasite infection among schoolchildren that could inform the policy makers on the actual benefits of incorporating anthelmintic treatment in malaria control programmes Over 12% of the children with low haemoglobin concentration, could potentially be prevented from developing anaemia if the parasites diagnosed in the study were treated and controlled, in time. There is therefore a need to examine the nutritional status of the people living in the study area, in order to appreciate the contribution of local available foods like fish and green vegetables on haemoglobin concentration despite high intensities of parasitic infections and high occurrences of co-infections. Egerton J. Sci. & Technol. Volume 13: ISSN No

73 68 Ngetich et al., (2013) (vii) Future research is required to evaluate evidence for antagonistic or synergistic interactions among the parasites when they occur together in the same individual since so far there are still conflicting results (Brutus, et al., 2006). Acknowledgements We are thankful to the schools, and children who agreed to participate in the study. We show gratitude to Egerton University for the Research grant, Ministry of Health for the drugs used to treat the children, and East and Southern Africa Centre of International Parasite Control (ESACIPAC) for providing portable digital haemoglobinator used for haemoglobin level estimation. We also wish to thank Mr. E. Oloo, A. Juma, J. Nyatigi, J. Nyagool assisting in the data collection and examination of samples. We are indebted to P. Shumanant of Duke University (US) and B. Kinyua of KEMRI for assisting in the data entry and analysis. References Akhwale, W. S., Lum J. K., Kaneko, A., Eto, H., Obonyo, C., Bjorkman A. and Kobayakawa (2004). Anaemia and malaria at different altitudes in the western highlands of Kenya. Acta Tropica. 91: Brooker, S., Akhwale, W., Pullan, R., Estambale, B., Clarke, S. E. and Hotez, P. J. (2007): Epidemiology of Plasmodium-Helminth Co- Infection in Africa: Populations at Risk, Potential Impact on Anemia, and Prospects for Combining Control. American Journal of Tropical Medicine and Hygiene, 77 (Suppl 6), Brooker, S., Miguel, E. A., Moulin, S., Luoba, A. I., Bundy, D. A. P. and Kremer, M. (2000). Epidemiology of Single and Multiple Species of Helminth infections among School Children in Busia District, Kenya. East Afr. Med. J. 77(3): Brooker, S., Peshu, N., Warn, P.A., Mosobo, M., Guyatt, H.L., Marsh, K., Snow, R.W. (1999): The epidemiology of hookworm infection and its contribution to anaemia among pre-school children on the Kenyan coast. Trans. R. Soc. Trop. Med. Hyg. 93: Brutus, l., Watier, l., Briand, V., Hanitrasoamampionona, V., Razanatsoarilala, H. and Cot, M. (2006). Parasitic Co-Infections: Does Ascaris lumbricoides protect against Plasmodium falciparum infection? Am. J. Trop. Med. Hyg. 75 (2): Egerton J. Sci. & Technol. Volume 13: ISSN No

74 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 69 Curtale, F., Tilden, R., Muhilal, Vaidya Y., Pokhrel, R.P., Guerra, R. (1993): Intestinal helminths and risk of anaemia among Nepalese children. Panminerva Medicine 35: Crompton, D. W. (2000). Public Health importance of hookworm disease. Parasitology; 121 S39 - S50 De Silva, N. R., Brooker, S., Hotez, P. J., Montresor, A., Engels, D. and Savioli L. (2003). Soil-transmitted helminth infections: Updating the global picture. Trends in Parasitology.19: Engels, D., Chitsulo, L., Montresor, A. and Savioli, L. (2002). The global epidemiological situation of schistosomosis and new approaches to control and research. Act Trop. 82: Guyatt, H.L., Brooker S., Peshu, N. and Shulman, C.E. (2000). Hookworm and anaemia prevalence. The Lancet; 356: 2101 Handzel, T., Karanja, D.M.S., Addiss, D.G., Hightower, A. W., Rosen, D. H., Colley, D.G., Andove, J., Slutsker, L. and Secor, W.E. (2003). Geographic distribution of schistosomiasis and soil-transmitted helminths in Western Kenya: Implications for antihelminthic mass treatment. Am. J. Trop. Med. Hyg. 69(3): Hommel, M., Gilles, H.M. (1998). Malaria, in Collier L., Ballows A., Sussman M. Topley & Wilson s Microbiology and Microbial Infections, (9 th ed) Cox F.E.G., Hotez, P. J., Bethony, J., Bottazzi, M. E., Brooker, S. and Buss, P. (2005). Hookworm: The Great Infection of Mankind. PLoS Med. 2(3): 67 Kahama, A.I., Kremsuer, P.G., Van Dan, G.J. and Deelder, A.M. (1998). The dynamic of soluble egg antigen of S. haematobium in relation to egg counts, circulating anodic and cathodic antigen and pathology markers before and after chemotherapy. Trans. R. Soc. Trop.Med. Hyg. 92: Katz, N., Chaves, A., and Pellegrino, J. (1972). A simple device for quantitative stool thick-smear technique in Schistosoma mansoni. Revista do Instituto de Medicina Tropical de Sao Paulo. 14: Koukounari, A., Estambale, B.B.A., Njagi K.J., Cundill, B., Ajanga, A., Crudder, C., Otido, J., Jukes, M.C.H., Clarke, S.E., and Brooker S. (2008). Releationship between anaemia and parasitic infections in Kenya schoolchildren; A Bayesian hierarchial modelling approach. Int. J. Parasitol. 38 (14-4): Leenstra, T., Kariuki, S. K., Kurtis, J.D., Oloo, A. J., Kager P.A. and Ter Egerton J. Sci. & Technol. Volume 13: ISSN No

75 70 Ngetich et al., (2013) Kuile, F. O. (2004). Prevalence and severity of anemia and iron deficiency: cross-sectional studies in adolescent schoolgirls in western Kenya. Eur. J. Clin Nutr. 2004; 58: Mackintosh, C. L., Beeson, J. G., and Marsh, K. (2004). Clinical Features & Pathogenesis of Severe malaria: Trends in Parasitology. 20 (12): Menendez, C., Fleming, A. F., Alonso, P. L. (2000). Malaria-related Anaemia. Parasitology Today. 16 (11): Ministry of Health (MOH, 2000). Health information reports. Ministry of Health, Kenya Annual Report. MOH (2001). National Malaria Strategy. Ministry of Health, Kenya Annual Report. Murphy, S. C. and Breman, J. G. (2001). Gaps in the childhood malaria burden in Africa: Cerebral Malaria, Neurological Sequelae, Anaemia, Respiratory Distress, Hypoglycaemia and Complications of Pregnancy. Am. J. Trop. Med. Hyg., Suppl. 1-2, Phillips, R. E. and Pasvol, G. (1992). Anaemia of Plasmodium falciparum malaria. Baillieres Clin Haematol. 5: Snow, R.W., Graig M., Deichmann, U. and Marsh, K. (1999). Estimating mortality, morbidity and disability due to malaria among Africa's nonpregnant population. Bulletin of the WHO. 77(8): Spiegel, A., Tall, A., Raphenon, G., Trape, J. F. and Druilhe, P. (2003). Increased frequency of malaria attacks in subjects co-infected by intestinal worms and Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg. 97: Stephenson, L.S., Latham, M.C., Kinoti, S.N., Kurz, K.M., Brigham, H. (1998). Improvement in physical fitness of Kenya schoolboys infected with hookworm, Trichuris trichiura and Ascaris lumbricoides following a single dose of Albendezole. Trans. R. Soc. Trop. Med. Hyg. 84: TDR/WHO (2002). Special Programme for Research and Training in Tropical Diseases. Schistosomiasis. TDR strategic direction. Warhurst, D.C. and Williams, J. E. (1996). Laboratory Diagnosis of Malaria. J. Clin Pathol. 49: World Health Organization (WHO, 1994). Bench Aids for the Diagnosis of Intestinal Parasites. (Eds. LR Ash et al,) World Health Organization, Egerton J. Sci. & Technol. Volume 13: ISSN No

76 The prevalence and effects of P. falciparum, S. mansoni and soil-transmitted 71 Geneva. WHO (2002). The World Health Organization report; Reducing risks, promoting healthy life. Geneva: World Health Organization Egerton J. Sci. & Technol. Volume 13: ISSN No

77 Creating a grading scheme for fresh ice-stored White snapper (Lutjanidae spp.) and Rabbit fish (Siganus sutor) using quality indices Lokuruka, M. N. I. 1 *, Muyela, B. 2, Okeyo, G. O. 3 and Anakalo, A. 4 Karatina University, P.O Box , Karatina, Kenya. Tel: ; mlokuruka@gmail.com 2 Department of Biochemistry and Molecular Biology, Egerton University, P.O Box , Egerton 3 Micro-Enterprise Support Programme Trust, P.O. Box , Nairobi 4 Division of Research, Extension and Linkages, Kisii University, P.O. Box Kisii, Kenya 1 *Corresponding Author, Department of Food Science and Nutrition, Submitted 21 st Dec. 2012; Reviewed 8 th March 2013; Accepted 8 th April 2013 Abstract Currently, the Kenya fisheries industry does not have an official grading scheme for fresh fish. Due to lack of an appropriate and locally developed grading scheme, this study used the Quality Index Method to develop a grading scheme for iced Rabbitfish and White snapper through a shelf life study. The two fish were bought from fishermen at the landing beach after delayed icing of 4-6 hr. The two fish were placed into gutted and ungutted lots. Both lots were layered with ice and stored in insulated boxes. Three iced fish from each batch were sampled on days 4, 8, 12, 16, 20, 24 and 30 for ungutted lots but up to 34 days for the gutted fish. Sensory attributes of the fish including appearance of skin, color of slime, color and odor of gills were selected for development of a quality index scheme by a trained sensory panel. Total viable counts (TVC) were determined at 22 and 37 o C as well as hydrogen sulphide-producing bacterial counts at 22 o C. Free fatty acid level, ph and total volatile basic nitrogen were also determined on experimental fish over the storage period. The shelf life of gutted Rabbitfish and White snapper was 32 and 28 days, respectively; ungutted Rabbitfish and White snapper, had shelf lives of 28 and 24 days, respectively. The TVC and hydrogen sulphide-producing bacterial log was 6.1±0.21 and 5.8±0.55 CFU/g at the point of rejection of ungutted Rabbitfish and log 6.3±0.24 and 5.8±0.28 CFU/g for ungutted White snapper. For gutted Rabbitfish and White snapper, TVC and hydrogen sulphide-producing bacterial log were 5.9±0.19 and 5.3±0.11 CFU/g and log 5.9±0.22 and 5.4±0.21 CFU/g, respectively at point of rejection by the taste panel. Gutting the two fish Egerton J. Sci. & Technol. Volume 13: ISSN No

78 Lokuruka, et al., (2013) 73 species increased shelf life by about 4 days. The quality index scheme developed in this work was appropriate for creating commercial grades of the two marine fish. The select sensory parameters and QI are suitable, while TVBN, FFA, ph, TVC and hydrogen sulphide-producing bacterial count are not appropriate for rapid commercial grading of fresh, ice-stored White snapper and Rabbitfish. Key words: Grading, White snapper, Rabbitfish, ice, quality index Introduction The fisheries sector plays a significant role in the socio-economic development of Kenya through the sector s positive contribution to employment creation, revenue generation and food security all of which are crucial to the attainment of the Millennium Development Goals and Vision 2030 targets for the country. During the year 2008, the sector supported a total of 62,324 people directly as fishers deriving their livelihood from the various fishery resources in the country (MoFD, 2009). During the same period the sector supported about a million people directly and indirectly, working as fishers, traders, processors, suppliers and merchants of fishing accessories and employees and their dependants. The Kenyan fishery is mainly artisanal with very few commercial/industrial vessels. The vessels target mainly shrimps using purse seines and long liners which are owned by Distant Water Fishing Nations operating under Kenyan licenses in the Economic Exclusive Zone (EEZ) targeting tuna and tuna-like fish species. The artisanal fishery accounts for almost all marine fish catch even though Kenya s EEZ which is predominantly for commercial fishing is under-exploited with an estimated annual potential of between 150,000 to 300,000 metric tons (MOFD 2003, 2005, 2009). In 2008, total national fish production was 133,600 metric tons with an ex-vessel value of Kshs 13,001,017,000 (approximately US $ 174 million). During the same period, the country earned over Kshs. 3.7 billion (approximately US $ 49 million) in foreign exchange from exports of fish and fishery products. Fish production in the country has been declining since 1999 when the highest quantity (214,709 metric tons) of fish was landed but the ex-vessel value has been increasing year after year (MOFD, 2009). The coastal fishery off the Indian Ocean contributes 3-5% to the annual output of fish, but 100% of crustaceans and molluscs. Fish production from the marine artisanal fishery has remained fairly constant at between 5,000 and 8,000 metric tons annually over the years and showing only marginal Egerton J. Sci. & Technol. Volume 13: ISSN No

79 74 Grading marine fish using Q1, biochemical and microbiological indices fluctuations. The marine fish species are diverse with over 200 species recorded. Between 2007 and 2009, the pelagic fishery contributed 3,500-4,100 metric tons of fish annually therefore providing close to or slightly more than 50% of the marine contribution to the national fishery and fish products output. In declining order by quantity, the five major pelagic fish species of the marine fishery are rabbbitfish, scavenger, parrotfish, the snappers (white and red snapper), and the sturgeon (MOFD 2005, 2009). Between 2007 and 2009, rabbit fish and the snappers were 640 and 750 metric tons (MOFD, 2009), which was about 10% of the total marine fishery output or 20% of the pelagic fish output. The quantity of both fish equals or slightly exceeds the total crustacean and molluscan annual output, indicating their important economic and food value to the marine fishery and the country. Fish quality assurance is a composite of measures carried out to ensure that fish and fishery products maintain prime quality and are safe for human consumption. The quality index method (QIM) is a reliable, fast and speciesspecific method of fish freshness assessment that can be applied in quality assurance (Martinsdottir et al., 2001). It is a tool that can be used to measure the freshness of a fish species after a quality index (QI) scheme has been developed for the specific fish species. It is based on scoring selected attributes of a fish species and determining the sum of scores of attributes in the course of cold storage. The sum of scores of all selected attributes is the QI of the fish species and is an indicator of its freshness at any stage of storage. The QI can therefore be used to determine the maximum shelf life of a fish species, with cooked fish from the same day s experimental batch being used for comparison. Since microbiological and biochemical indices are conventionally used to establish the end of shelf life and the edibility of fish, the QI can be correlated to the commonly used microbiological and biochemical indices to prove its usefulness for shelf life estimation and for following the rate of loss of freshness. The species, time of storage, temperature and other handling conditions determine the rate of loss of quality and therefore the freshness of fish (Olafsdottir et al., 1997). However, the low temperature of ice slows the rate of loss of freshness and as was shown for Nile perch, shelf life is considerably long when the commodity is iced immediately after harvest (Okeyo and Lokuruka, 2010a). Currently, the few land-based fish processing industries mainly based in Mombasa use an EU-developed fish grading scheme, which is based on European fish species and which are not found in Kenya s coastal fishery. Although it is based on the QIM, it may not therefore be appropriate as it is developed using temperate fish species. Although it is used (importers Egerton J. Sci. & Technol. Volume 13: ISSN No

80 Lokuruka, et al., (2013) 75 recommend it), it is not recognized as official due to the lack of an appropriate, locally developed grading scheme. This current experiment was done in order to establish quality grades of two important fish species of Kenya s marine fishery through a shelf life study in ice, the use of the QI and selected biochemical and microbiological indices. Grading normally depends on the use of sensory attributes to gauge the economic value of a food commodity. For fresh fish and fish products, the same sensory attributes that are used to create the QI scheme by the QIM can also be used to grade fish, since economic value is related to food freshness if consumers desire that quality food is sold in the fresh state. The microbiological indices selected for the study were the total viable counts (TVC) at 22 and 37 o C, and hydrogen sulphide-producing bacteria counts at 22 o C; total volatile basic nitrogen (TVBN), ph and free fatty acids (FFA) amount were the biochemical indices selected for study. The sensory attributes selected for the QI scheme development were: appearance of the skin; slime color; clarity and shape of the eyes; skin texture; the belly elasticity; gill colour, odour and gill slime appearance. These sensory attributes were assessed based on scores of 0-3 (Table 1 and 2). Cooked fish sensory assessment was done concurrently with the raw fish sensory evaluation using the experimental fish for the day. The microbiological and biochemical indices were correlated to the sum of sensory scores referred to the QI. The microbiological, biochemical indices and the QI were used to create grades for the two fish. Materials and Methods Raw Material and Sampling Fresh White snapper (Lutjanidae spp.) and Rabbitfish (Siganus sutor) were purchased at random from fishermen at Kenyatta beach and Likoni fish landing sites, respectively. Both landing sites are on Mombasa Island, Kenya, and are about 20 km apart. The fish samples were collected between September 2010 and February The time interval between harvesting and arrival of the fish at the landing sites was estimated to be about 4-6 hours. After taking the weights and lengths (controlled) of the beachpurchased fish, the samples were divided into two groups-ungutted and gutted. The gutting process was hygienically done at the landing beaches, by removing the offals and washing the fish well with running potable tap water to remove any dirt and slime before layering with flaked ice; the iced fish were then packed in insulated containers. The iced fish were stored in a walk-in cold room maintained at 0-2 C for 2 nights before being transported the 3 rd day within 7-8 hr to cold rooms at Egerton University, 690 km away, where they were stored at 0-2 o C throughout the experimental period. Four fish (the experimental unit) each of both gutted and ungutted fish lots were Egerton J. Sci. & Technol. Volume 13: ISSN No

81 76 Grading marine fish using Q1, biochemical and microbiological indices randomly sampled on days 4, 8, 12, 16, 20, 24, 28, and 30 for the ungutted experimental White snapper and Rabbit fish. The gutted fish of both fish species were additionally sampled on day 32 and 34. All experimental fish samples were analyzed according to the sensory, proximate, biochemical and microbiological procedures described in the sections that follow. Sensory Evaluation The sensory characteristics of the two fish species were assessed over the storage period of 30 days for ungutted and up to 34 days for the gutted fish by a panel of ten previously trained assessors belonging to the Department of Dairy and Food Science and Technology of Egerton University, Kenya. The appearance of the skin; skin slime color; clarity and shape of the eyes; skin elasticity; belly integrity, gill colour, odour and gill slime appearance were assessed based on scores of 0-3 (Table 1). The attributes (odour, taste and texture) of cooked fish were evaluated by the same laboratory expert panel of ten people on each sampling day, simultaneously. The odour, taste and texture were evaluated using a structured acceptability scale of 0-10 with the rejection threshold set at 4. The skin appearance and skin slime color were eventually not included in the sensory assessment of Rabbitfish due to the dark color of its skin. Proximate Analysis Moisture Content Determination Moisture content was determined by oven drying of 5 g of fish flesh in triplicate at 105 C until a constant weight was reached (AOAC, 2000). Crude Protein Determination This was done to determine if there was any variation in the protein content over the storage period of the fish in ice. An AOAC (2000) procedure was used. Approximately 0.2 g of fish flesh was accurately weighed in triplicate into six Kjeldahl digestion tubes. Ten ml of concentrated sulphuric acid was added into each of the tubes and one selenium tablet added to act as a catalyst. The samples were then digested at a temperature of 445 C for 3 hours. The samples were cooled at room temperature and then distilled using a Kjeldahl distillation unit (UDK 127, VelpScientifica, Spain). The distillate was collected in 15 ml of 0.1 N HCL in which a mixed indicator of methyl red and methylene blue had been added. The HCL-distillate mixture was then titrated against 0.1 N NaOH. The % crude protein was determined by the formula: Egerton J. Sci. & Technol. Volume 13: ISSN No

82 Lokuruka, et al., (2013) 77 % Crude protein = {(V1-V2) x N x 14 x 6.25/W}, where V2 was the volume of the titre, V1 was the volume of the HCL used for the blank test, N was the normality of the acid and W being the weight of the test portion. Crude lipids Determination This was done to establish if there was any variation in lipid content during the storage period of fish in ice. The AOAC (2000) procedure was used. Five grammes of fish flesh were accurately weighed into a Soxhlet thimble in triplicate. The sample was covered with cotton wool and the thimble placed in the soxhlet extractor (CX7/06, Quickfit, England). The extractor was connected to a flat bottomed flask in which 200 ml of hexane had been added and the mixture heated on a heating mantle, with refluxing for 6-8 hours; the solvent was eventually evaporated in a rotary evaporator (RE52CS-2, Shangai, China). The sample left in the flat-bottomed flask was oven dried at 80 C for 30 minutes (to constant weight) and cooled in a desiccator and then weighed. The lipid content was determined by the formula: % crude lipid content= (weight of residue/original weight of sample) x 100 Biochemical Analyses Total Volatile Basic Nitrogen (TVBN) Approximately 10 g of skinless fish flesh from the tail region was homogenized with 50 ml of doubled distilled water. The mixture was centrifuged in a Sorval RC-285 centrifuge (Dupont, Wilmington, Delaware, U.S.A) at 400 rpm for 5 minutes and the supernatant filtered through a Büchner funnel using Whatman No.1 filter paper. Two grammes of MgO was added followed by one drop of antifoaming agent were added into a 250- ml Erlenmeyer flask containing 25 ml of 3% aqueous solution of boric acid; 0.04 ml of a mixture of methyl red and methylene blue indicators was used as the indicator. Distillation was continued until a final distillate volume of 125 ml was obtained in the Erlenmeyer flask. The distilled TVBN was titrated with an aqueous 0.1 N HCL solution. TVBN content was expressed as mg N/100 g of fish flesh (Goulas and Kontominas, 2005). TVBN = (V C ) /10, where V was the volume of hydrochloric acid added and its concentration (C), 10 represented the weight of the sample, while 14 is the molecular weight of nitrogen. Free Fatty Acids (FFA) Determination Percent FFA was determined by a standard AACC method (AACC, 2004) and was expressed as percent oleic acid equivalent. Egerton J. Sci. & Technol. Volume 13: ISSN No

83 78 Grading marine fish using Q1, biochemical and microbiological indices Microbiological Analyses A 25-g fish sample from the dorsal region with skin on, was mixed with 225 ml of peptone water diluent, and homogenized. An estimation of the total viable counts that could be responsible partly for deterioration in quality both as a result of contamination due to poor handling and autolysis was made. The TVC and H 2 S-producing bacteria counts were done on Iron Agar (Oxoid, UK) by pour plate technique with an overlay as described by Gram et al. (1987). The plates were incubated at 22 C and 37 o C for 3 days. TVC was obtained by registering all bacterial colonies on the incubated plates from each dilution of the sample showing growth. The number of colonies showing a clear dark colour represented the bacterial population able to produce H 2 S, while the white colonies represented the non- hydrogen sulphide producing viable count at 22 o C. Data Analysis The data from the two sampling trips were pooled and the mean values for the sensory, microbiological and biochemical parameters were determined for each of the treatments. The means of the selected sensory, microbiological and biochemical parameter determinations analyzed for each of the treatments (at each sampling period) were subjected to analysis of variance (ANOVA). The least significance difference (LSD) procedure of Fisher was used to test for the differences between treatments means (significance was defined as p 0.05). Results Sensory Evaluation Analysis The quality index scheme developed for this study is shown in Table 1. Egerton J. Sci. & Technol. Volume 13: ISSN No

84 Lokuruka, et al., (2013) 79 Table 1: The QIM scheme developed for White snapper (Lutjanidae spp.) and Rabbitfish (Siganus sutor) stored in ice Parameter Attribute Description Demerit Points Appearance *Skin Very bright, pearl shiny 0 Bright 1 Dull 2 Slime Clear-transparent 0 Slightly cloudy/milky 1 Cloudy 2 Eyes Clarity Clear-translucent 0 Slightly opaque 1 Opaque 2 Shape Convex 0 Flat 1 Concave/Sunken 2 Texture Elasticity Elastic 0 Slightly marked by pressure 1 Clearly marked by pressure 2 Belly Intact 0 Slightly intact 1 Soft 2 Very soft 3 Gills Colour Bright/dark red 0 Brownish red 1 Discoloured/brown 2 Gill Odour Fresh/seaweedy 0 Neutral 1 Fishy/sour 2 Off-odour/rotten 3 Gill slime Clear-translucent 0 Slightly cloudy 1 Cloudy 2 Total demerit points (QI scores) 20 *Attribute not considered for rabbitfish due to the dark color of its skin There was an increase in the QI scores with storage time in ice for both gutted and ungutted experimental Rabbitfish (Table 2), and for the gutted and ungutted experimental White snapper (Table 3). The relationship between QI and time in ice was linear for both fish species (Figures 1 and 2). Egerton J. Sci. & Technol. Volume 13: ISSN No

85 80 Grading marine fish using Q1, biochemical and microbiological indices Table 2: QI scores for select sensory parameters of iced gutted and ungutted Rabbitfish Days of sampling Parameter Skin slime G 0.125±0.24 a 0.5±0.11 a 0.5±0.2 a 1.125±0.05 b 1.625±0.04 b 1.375±0.05 b 1.75±0.03 b 1.875±0.15 c U 0.25±0.31 a 0.375±0.2 a 0.5±0.17 a 1.625±0.11 b 1.625±0.1 b 1.625±0.08 b 1.875±0.16 c N/a Eye clarity G 0.25±0.05 a 0.375±0.15 a 0.5±0.02 a 1.25±0.16 b 1.25±0.22 b 1.375±0.01 b 1.75±0.05 b 1.875±0.17 b U 0.25±0.07 a 0.375±0.22 a 0.625±0.0 a 1.375±0.33 b 1.375±0.18 b 1.625±0.06 b 1.75±0.01 b N/a Eye shape G 0±0.21 a 0.375±0.01 a 0.625±0.05 a 1±0.2 b 1±0.15 b 1.625±0.02 c 1.375±0.3 b 2±0.10 c U 0.25±0.0 a 0.625±0.11 a 0.625±0.13 a 0.875±0.16 a 1.625±0.06 b 1.625±0.14 b 1.875±0.15 b N/a Elasticity G 0.125±0.12 a 0.25±0.18 a 0.375±0.08 a 1.125±0.21 b 1±0.14 b 1.25±0.1 b 1.75±0.04 c 1.875±0.5 c U 0.25±0.14 a 0.375±0.05 a 0.5±0.04 a 1.375±0.03 b 1.625±0.01 b 1.875±0.02 c 1.75±0.02 c N/a Belly G 0.25±0.05 a 0.25±0.12 a 0.625±0.24 b 1.125±0.13 b 1.375±0.2 b 1.25±0.18 b 1.875±0.17 c 2.25±0.12 c U 0.375±0.01 a 0.375±0.01 a 0.75±0.06 b 1.625±0.0 b 1.875±0.17 c 1.625±0.11 b 2.125±0.17 c N/a Gill color G 0.125±0.17 a 0.375±0.06 b 0.625±0.05 b 1.625±0.05 c 1.375±0.11 c 1.75±0.05 c 1.875±0.11 d 2±0.05 d U 0.125±0.04 a 0.375±0.0 b 0.625±0.1 b 1.375±0.07 c 1.375±0.01 c 1.75±0.06 c 2±0.01 d N/a Gill odor G 0.125±0.02 a 0.5±0.16 b 0.875±0.14 b 1.375±0.12 c 1.625±0.04 c 1.625±0.12 c 2±0.01 c 2.5±0.16 d U 0.125±0.02 a 0.375±0.01 b 0.75±0.01 b 1.875±0.34 c 1.75±0.21 c 1.875±0.1 c 2.125±0.41 c N/a Gill slime G 0.375±0.33 a 0.5±0.1 a 0.625±0.07 a 1.375±0.14 b 1.25±0.07 b 1.75±0.08 b 1.75±0.08 b 2±0.18 c U 0.375±0.01 a 0.625±0.13 a 0.75±0.05 a 1.625±0.18 b 1.75±0.01 b 1.875±0.05 b 1.875±0.04 b N/a Total QI G U n=9: Mean values in the same row with the same superscript are not significantly different (p>0.05) Legend: G-gutted, U-ungutted N.B: No data was taken for the ungutted fish on day 30 as the fish were unfit for human consumption on the day. Egerton J. Sci. & Technol. Volume 13: ISSN No

86 Lokuruka, et al., (2013) 81 Table 3: QI scores for select sensory parameters in iced gutted and ungutted White snapper Days of Sampling Parameter Skin appearance G 0.5±0.35 a 0.625±0.17 a 0.75±0.25 a 1.375±0.15 b 1.625±0.01 b 1.625±0.11 b 2±0.01 c U 0.5±0.36 a 1.125±0.37 b 1.125±0.1 b 1.375±0.06 b 1.625±0.03 c 1.875±0.01 c N/a Slime G 0.375±0.2 a 0.875±0.11 b 0.625±0.2 b 1±0.01 b 1.625±0.17 c 1.625±0.00 c 1.875±0.05 c U 0.5±0.36 a 0.875±0.34 a 0.75±0.6 a 1.25±0.31 b 1.625±0.41 b 1.625±0.10 b N/a Eyes clarity G 0.375±0.06 a 0.375±0.01 a 0.5±0.4 a 1±0.2 b 1.25±0.12 b 1.625±0.02 b 1.875±0.16 c U 0±0.04 a 0.75±0.14 b 0.75±0.5 b 1.25±0.02 c 1.25±0.1 c 1.75±0.17 c N/a Eye shape G 0±0.05 a 0.625±0.05 b 0.75±0.0 b 1±0.02 b 1.125±0.02 b 1.25±0.15 c 1.75±0.21 c U 0±0.05 a 1.125±0.01 b 1.00±0.14 b 1.125±0.12 b 1±0.05 b 1.75±0.06 b N/a Elasticity G 0.25±0.01 a 0.375±0.11 b 0.625±0.08 b 0.875±0.22 b 1.375±0.16 c 1.625±0.04 c 1.875±0.11 c U 0.375±0.01 a 0.5±0.21 a 0.75±0.12 b 1.25±0.11 b 1.75±0.07 c 1.75±0.14 c N/a Belly G 0.25±0.02 a 0.375±0.30 a 0.875±0.16 b 1.125±0.01 b 1.625±0.10 c 1.75±0.05 c 2.25±0.10 d U 0.875±0.05 a 0.75±0.02 a 1.375±0.02 a 1.625±0.05 b 2±0.2 b 2.00±0.22 b N/a Gill color G 0.375±0.11 a 0.5±0.05 a 1.25±0.16 b 1.125±0.17 b 1.625±0.01 c 1.625±0.12 c 2±0.05 c U 1±1.10 a 0.875±0.49 a 1.25±0.02 a 1.25±0.07 a 1.625±0.15 b 1.50±0.05 b N/a Gill odor G 0.25±0.15 a 0.375±0.07 a 1±0.05 b 1±0.01 b 2±0.02 c 2.25±0.10 c 2.25±0.01 c U 0.625±0.25 a 0.75±0.33 a 1.25±0.21 b 1.375±0.06 b 2.375±0.06 c 2.75±0.02 c N/a Gill slime G 0.375±0.02 a 0.75±0.04 b 0.75±0.2 b 1.125±0.21 b 1.25±0.30 b 2.5±0.01 c 2±0.12 c U 0.625±0.12 a 0.625±0.10 a 0.75±0.04 a 1.25±0.02 b 1.625±0.10 b 2.75±0.3 c N/a Total QI G U n=9: Mean values in the same row with the same superscript are not significantly different (p>0.05) Legend: G-gutted, U-ungutted N.B: No data was taken for the ungutted fish on day 26 as the fish were considered unfit for human consumption by the day. Egerton J. Sci. & Technol. Volume 13: ISSN No

87 82 Grading marine fish using Q1, biochemical and microbiological indices QI scores Rabbit Fish y = 0.614x R² = Sampling Time (Days) Figure 1: Storage period vs. QI scores for ungutted Rabbitfish White snapper y = 0.647x R² = QI scores Sampling Time (Days) Figure 2: Storage period vs. QI scores for ungutted White snapper The highest QI scores for all the parameters for the ungutted and gutted rabbitfish samples were reached on day 28 (QI score out of a maximum score of 18) and day 32 (QI score out of a maximum score of 18) of storage in ice, respectively, whereas for the ungutted and gutted White snapper, the highest QI scores for all the sensory parameters were Egerton J. Sci. & Technol. Volume 13: ISSN No

88 Lokuruka, et al., (2013) 83 reached on day 24 (QI score, out of a maximum score of 20) and day 28 (QI score, out of a maximum score of 20) of storage in ice, respectively. At this point both fish were considered unfit for human consumption. Hygienic gutting increased the shelf life of both Rabbitfish and White snapper bought from the beaches by approximately 4 days, irrespective of whether gutted or not. Proximate Analysis There was no significant change (p>0.5) in lipid, protein and moisture contents in all the fish samples of the same fish and between the two fish species, over the storage period (Table 4). Table 4: Proximate composition of iced gutted and ungutted White snapper and Rabbitfish over the storage period Days of sampling Parameters Moisture (%) GW 72.12±0.11 a 72.45±0.30 a 74.39±0.23 a 72.22±0.16 a 75.02±0.33 a 73.57±0.61 a 75.61±0.41 a N/a UW 73.07±0.24 a 72.52±0.59 a 75.14±0.54 a 75.11±0.15 a 76.09±0.18 a 74.48±0.22 a N/a N/a GR 74.31±0.42 a 74.24±0.81 a 72.89±0.15 a 75.66±0.66 a 76.52±0.12 a 76.14±0.38 a 77.13±0.18 a 75.79±0.31 a UR 74.32±0.21 a 72.55±0.28 a 75.38±0.13 a 75.41±0.35 a 74.18±0.28 a 77.22±0.25 a 74.91±0.56 a N/a Crude Lipids (%) GW 0.57±0.03 a 0.56±0.13 a 0.61±0.18 a 0.60±22 a 0.62±05 a 0.59±0.14 a 0.61±0.35 a N/a UW 0.61±0.18 a 0.60±0.05 a 0.62±0.34 a 0.62±0.55 a 0.61±0.21 a 0.60±0.10 a N/a N/a GR 0.62±0.05 a 0.61±0.15 a 0.63±0.12 a 0.63±0.08 a 0.65±0.03 a 0.63±0.05 a 0.64±0.05 a 0.64±0.12 a UR 0.60±0.41 a 0.63±0.53 a 0.63±0.61 a 0.65±0.26 a 0.65±0.44 a 0.66±0.31 a 0.64±0.00 a N/a Crude Protein (%) GW 17.2±33 a 18.0±14 a 17.10±12 a 18.59±11 a 18.0±05 a 16.98±21 b 18.23±44 a N/a UW 17.55±10 a 17.69±04 a 18.41±15 b 17.14±66 a 16.20±20 a 16.58±14 a N/a N/a GR 19.0±36 a 19.45±12 a 20.23±44 a 22.59±05 b 21.67±09 a 21.18±35 a 18.56±43 a 20.01±40 a UR 20.0±58 a 19.32±21 a 19.09±41 a 18.72±02 a 18.38±14 a 17.01±61 a 18.22±02 b N/a n=9: Mean values in the same row with the same superscript are not significantly different (p>0.05) Legend: GW-gutted white snapper, UW-ungutted white snapper, GR-gutted rabbitfish, URungutted rabbitfish Microbiological and Biochemical Indices Table Viable Counts (TVC) TVC increased significantly from the 12 th day onwards, irrespective of the temperature of incubation (22 and 37 o C) (Table 5 and 6). Also, the significant change in TVC (p<0.05) occurred on the 12 th day at both incubation temperatures, whether the fish were gutted or not. At 22 o C, the inadmissible microbial load of log 6.0 was reached on 24 day irrespective of whether the fish were gutted or not. But at 37 o C incubation temperature, the microbial load of log 6.0 was reached on the 32 nd and 24 th day for the gutted and ungutted fish, respectively. The correlation between TVC and QI scores Egerton J. Sci. & Technol. Volume 13: ISSN No

89 84 Grading marine fish using Q1, biochemical and microbiological indices was positive, and was 0.89 and 0.98 in Rabbitfish and White snapper, respectively (Table 7 and 8). Table 5: Values of Microbiological and biochemical parameters in gutted and ungutted Rabbitfish over the storage period Days of Sampling Parameter Coliforms cfu/g (37 C) G 1.2±0.28 a 1.4±0.33 a 1.5±0.24 a 1.4±0.25 a 1.6±0.21 a 1.4±0.14 a 1.6±0.41 a 1.8±0.22 b U 1.6±0.02 a 1.6±0.71 a 2.4±0.68 a 3.0±0.39 b 3.8±0.14 b 4.8±0.23 c 4.8±0.11 c N/a H 2 S log cfu/g 22 C G 1.6±0.15 a 1.6±0.69 a 1.8±0.05 a 3.1±0.12 b 3.7±0.68 b 3.2±0.24 b 4.2±0.59 c 5.3±0.11 c U 2.4±0.11 a 2.2±0.78 a 3.6±0.56 b 4.9±0.33 c 5.2±0.62 c 5.2±0.35 c 5.8±0.55 c N/a ph G 8.00±0.14 a 8.12±0.29 a 8.34±0.35 a 8.11±0.38 a 8.41±0.41 b 8.40±0.01 b 8.28±0.51 a 8.34±0.08 a U 8.18±0.09 a 8.22±0.05 a 8.20±0.64 a 8.35±0.13 a 8.43±0.25 a 8.39±0.71 a 8.44±0.68 b N/a FFA (%) G 1.90±0.13 a 1.88±0.09 a 2.15±0.03 a 2.18±0.21 b 2.12±0.61 a 2.08±0.39 a 2.23±0.05 b 2.19±0.12 b U 1.91±0.34 a 1.94±0.41 a 2.06±0.78 a 2.10±0.23 a 2.14±0.18 a 2.25±0.29 b 2.19±0.34 b N/a TVBN mg N/100g G 9.80±0.09 a 10.62±0.19 a 11.29±0.68 a 15.14±0.33 b 20.06±0.04 b 24.18±0.72 c 27.12±0.21 c 28.01±0.36 c U 11.05±0.66 a 11.98±0.52 a 13.41±0.81 a 18.18±0.52 b 18.34±0.10 b 26.41±0.11 c 28.63±0.23 c N/a TVC log cfu/g (22 C) G 2.5±0.23 a 2.7±0.11 a 4.5±0.14 a 4.9±0.32 a 5.3±0.15 b 5.5±0.25 b 5.8±0.37 c 6.0±0.23 c U 2.5±0.32 a 3.3±0.22 a 4.7±0.50 a 5.1±0.23 b 5.4±0.24 b 5.8±0.22 c 6.4±0.14 d N/a TVC log cfu/g (37 C) G 2.0±0.14 a 2.1±0.23 a 3.6±0.15 a 4.3±0.35 a 4.8±0.14 b 5.1±0.28 b 5.3±0.33 c 5.9±0.22 c U 2.0±0.21 a 2.5±0.13 a 3.7±0.17 a 4.7±0.14 b 5.0±0.35 b 5.5±0.33 c 6.1±0.21 d N/a n=9: Mean values in the same row with the same superscript are not significantly different (p>0.05) Legend: G-gutted, U-ungutted N.B: No data was taken for the ungutted fish on day 32 as it was unfit for human consumption by the day. Egerton J. Sci. & Technol. Volume 13: ISSN No

90 Lokuruka, et al., (2013) 85 Table 6: Values of Microbiological and biochemical parameters in gutted and ungutted White snapper over the storage period Days of sampling Parameter Coliforms cfu/g (37 C) G 1.4±0.12 a 1.6±0.71 a 1.6±0.32 a 1.8±0.05 a 2.1±0.44 b 1.8±0.61 a 1.8±0.54 a U 1.60±0.08 a 2.0±0.10 a 2.4±0.14 b 3.1±0.48 b 3.8±0.66 b 5.2±0.29 c N/a H 2 S log cfu/g (22 C) G 1.80±0.19 a 1.62±0.26 a 2.01±0.43 a 2.28±0.53 b 3.89±0.55 c 3.43±0.14 c 5.4±0.21 d U 1.70±0.20 a 2.2±0.51 a 4.0±0.42 b 4.8±0.69 b 5.2±0.27 b 5.8±0.28 c N/a ph G 8.14±0.25 a 8.21±0.41 a 8.33±0.65 a 8.31±0.18 a 8.28±0.22 a 8.38±0.32 a 8.38±0.61 a U 8.21±0.12 a 8.28±0.48 a 8.32±0.05 a 8.31±0.11 a 8.41±0.66 b 8.40±0.18 b N/a FFA (%) G 1.82±0.14 a 1.84±0.64 a 1.96±0.39 a 2.0±0.24 b 1.98±0.19 a 2.08±0.54 b 2.18±0.11 b U 1.90±0.01 a 1.89±0.50 a 2.10±0.31 b 2.12±0.55 b 2.05±0.61 a 2.18±0.35 b N/a TVBN mg N/100g G 11.14±0.22 a 10.72±0.31 a 12.19±0.68 a 15.38±0.55 b 16.20±0.51 b 22.75±0.13 c 24.68±0.35 c U 10.91±0.21 a 11.66±0.11 a 14.12±0.23 a 16.48±0.06 b 21.39±0.41 b 26.20±0.05 c N/a TVC log cfu/g (22 C) G 2.7±0.23 a 3.3±0.21 a 3.9±0.42 a 4.4±0.35 b 5.0±0.32 b 5.3±0.11 c 5.9±0.19 d U 4.0±0.14 a 4.9±0.32 a 5.3±0.25 b 5.9±0.24 b 6.3±0.24 c 7.0±0.11 e N/a TVC log cfu/g (37 C) G 2.1±0.11 a 2.9±0.45 a 3.2±0.44 a 4.0±0.27 b 4.4±0.17 b 5.0±0.28 c 5.4±0.21 d U 3.1±0.17 a 4.3±0.27 a 5.0±0.22 b 5.5±0.37 b 5.9±0.40 c 6.7±0.33 d N/a n=9: Mean values in the same row with the same superscript are not significantly different (p>0.05) Legend: G-gutted, U-ungutted; N.B: No data was taken for the ungutted fish on day 28 as it was unfit for human consumption by the day. H 2 S-producing Bacteria In the both fish, a significant change in the microbial count occurred in the gutted and ungutted fish on the 16 th and 12 th day, respectively. The microbial load of log 5.8 was reached on the 28 th day in the ungutted fish but beyond 30 days in the gutted fish. The correlation between H 2 S- producing bacterial load and QI was 0.96 and 0.90 for Rabbitfish and White snapper, respectively. Coliforms No significant change in coliform counts occurred over the storage period in the gutted fish. However, the significant change in the coliform count occurred on the 12 th day in the ungutted fish of both species. The coliform counts were high and would be of concern as far as food safety is concerned. Egerton J. Sci. & Technol. Volume 13: ISSN No

91 86 Grading marine fish using Q1, biochemical and microbiological indices Table 7: Correlations between the different indices studied in Rabbitfish Indices ph TVBN FFA QI scores TVC H 2 S- producing bacterial count ph TVBN FFA QI Scores TVC H 2 S- producing bacterial count Table 8: Correlations between the different indices studied in White snapper Index ph TVBN FFA QI scores TVC H 2 S-producing bacterial count ph TVBN FFA QI Scores TVC H 2 S-producing bacterial count Table 9: Physical descriptors developed for grading Rabbitfish and White snapper Grade A (Prime grade) Grade B (Select grade) Grade C (Acceptable) Grade D (Reject grade) *Pearl shiny skin *Bright skin *Slightly dull skin *Dull skin Clear transparent Slightly cloudy Cloudy slime Cloudy slime slime slime Clear translucent Slightly Slightly opaque eyes Opaque eyes eyes translucent eyes Convex eye shape Flat eye shape Slightly concave eye Concave eye shape shape Elastic texture Slightly elastic texture Slightly marked by pressure Clearly marked by pressure Intact belly Slightly intact Soft belly Very soft belly belly Bright red gill Brownish red gill colour Slightly discoloured gill Discoloured/brown gills Seaweedy odour Neutral odour Slightly fishy odour Off odour/rotten odour Clear transparent gill slime Slightly cloudy slime Cloudy gill slime Cloudy gill slime *Attribute not considered for Rabbitfish Egerton J. Sci. & Technol. Volume 13: ISSN No

92 Lokuruka, et al., (2013) 87 Table 10: QI score, microbiological and biochemical values developed for grading Rabbitfish and White snapper Fish type Grade TVBN (mgn/ 100g) H2Sproducing bacteria 22 o C (log cfu/g) TVC at 22 C (log cfu/g) TVC at 37 C (log cfu/g) FFA (%) ph QI score Shelf life in ice (days) Rabbitfish A (Prime) 11.45± ± ± ± ± ± /18 10 White snapper B (Select) 15.18± ± ± ± ± ± /18 16 C (Acceptable) 24.04± ± ± ± ± ± /18 22 A (Prime) 11.11± ± ± ± ± ± /20 6 B (Select) 16.15± ± ± ± ± ± /20 14 C (Acceptable) 26.84± ± ± ± ± ± /20 22 Discussion Sensory Evaluation The quality index scheme developed for this study is shown in Table 1. There was an increase in the QI scores with storage time in ice for both gutted and ungutted experimental Rabbitfish (Table 2), and the gutted and ungutted experimental White snapper (Table 3). The increases in QI showed the progressive deterioration of the fish as shown in the changes in sensory parameters as noted by the sensory panel. The highest QI scores for all the parameters for the ungutted experimental and gutted Rabbitfish were reached on day 28 (QI score of out of a maximum score of 20) and day 32 (QI score of out of a maximum score of 20) of storage in ice, respectively. For ungutted and gutted experimental White snapper, the highest QI scores were reached on day 24 (QI was out of 20) and 28 (QI was out of maximum of 20 points) of storage in ice, respectively; at this point the fish was considered unfit for human consumption. The beach-purchased and gutted Rabbitfish had a longer shelf life of 32 days as compared to the ungutted fish which had a shelf life of 28 days. Hygienic gutting increased the shelf life of both Rabbitfish and White snapper bought from the beaches by approximately 4 days. Guts removal slowed the rate of the observed sensory changes in both fish species, consequently resulting in extended prime quality and shelf life. Guts are reservoirs of bacteria, whose rate of multiplication influences the rate of fish deterioration as was evident in changes in the sensory parameters and the value of biochemical and microbiological indices. Table 9 gives the gradual and progressive changes in the sensory descriptors which were used to follow the changes in the sensory parameters selected for this study. The sensory descriptors of the 4 grades as described in Table 9 are generally Egerton J. Sci. & Technol. Volume 13: ISSN No

93 88 Grading marine fish using Q1, biochemical and microbiological indices similar for both Rabbitfish and White snapper. When the sensory parameters are turned into a semi-quantitative tool through the use of the QI score, both the sensory descriptors and the QI score become useful tools for grading the two fish studied in this work. Therefore the fish grader can use either tool for fish grading as long as the relationship between the two parameters is established. However, when using the sensory descriptors, it is recommended that more than one parameter be used to establish the fish grade for the particular fish. Table 10 gives the QI score for Grade 1-4 of both fish (e.g., for Rabbbitfish, the QI score was 4.9, 10.8 and 14.1 for grade A, B and C, respectively, while the QI score was 5.3, 10.0 and 17.8, for grades A, B and C of White snapper). Where the fish grader has a large number of fish to deal with, a fairly accurate range of QI score can be established for each grade of fish. However, despite the small difference in the QI score at the same grade for the two fish studied here, it is apparent that the QI score range is speciesspecific. Therefore, a QI score range and the corresponding sensory descriptors as given in Table 9 should be established for each fish species for the purpose of grading. The QI score and the sensory parameters selected in this study are therefore suitable for commercial fish grading. Biochemical Parameters TVBN The TVBN of the ungutted and gutted experimental Rabbitfish increased with storage time in ice (Table 5). There was a significant (p<0.05) increase in the values of this index from the 16 th day of storage in ice for both the ungutted and gutted experimental Rabbitfish. However, these values were significantly lower (p<0.05) for the gutted experimental Rabbitfish as compared to the ungutted experimental fish. The TVBN value at the end of shelf life (day 28) for the ungutted Rabbitfish was mg N/100 g flesh, while for the gutted (day 32) was mg N/100 g flesh. A significant increase (p<0.05) in TVBN values was also observed in ice-stored ungutted and gutted White snapper with values of and mg N/100 g, respectively, at the end of shelf life. A significant increase (p<0.05) in TVBN was observed with storage in ice for both the ungutted and gutted White snapper with values of and mg N/100 g, respectively, at the end of shelf life in ice. There was no significant difference (p>0.05) in TVBN values between the two species of fish at the end of shelf life. These results showed that there may be similarity in the proteolytic activity that results in TVBN in the two fish species, whose rate was slower in the gutted fish. Further, the levels of TVBN in hygienically gutted fish samples (compared to the ungutted fish) were lower due to the reduced gut microbial load and hence reduced bacterial decomposition of nitrogenous compounds Egerton J. Sci. & Technol. Volume 13: ISSN No

94 Lokuruka, et al., (2013) 89 in the fish flesh. Although different TVBN values distinguished the different grades of each fish, the values were not significant between fish species for the same grade. Therefore, TVBN is a significant parameter for fish grading. However, due to the long time required for its analytical determination, it may not be appropriate for rapid grading of fresh fish. ph In this study, ph was considered a factor that determines the survival and growth of micro-organisms during storage, processing and distribution of fish. For both the ungutted and gutted experimental Rabbitfish, there was no significant increase (p>0.05) throughout the period of storage (Table 5); the trend was the same in the White snapper (Table 6). For Rabbitfish, the initial ph was 8.14 and 8.00 in the ungutted and gutted samples, respectively. At rejection, the ph was 8.44 and 8.34 on day 28 and 32 for the ungutted and gutted fish, respectively. For the White snapper, the initial ph was 8.21 and 8.14 but 8.38 and 8.40 in the gutted and ungutted White snapper, on day 24 and 28 (rejection point), respectively. The initial high ph in both fish species indicated some proteolysis already having taken place before icing. The small variation after icing and during subsequent storage indicates the arrest of the proteolytic activity by icing. The ph at rejection point by the taste panel was almost similar for both species, with treatment. The increase in ph indicates the accumulation of volatile amines such as ammonia mainly derived from microbial actions through bacterial degradation or deamination of proteins, peptides and amino acids in the fish muscle. There was a significant difference (p<0.05) in ph variation between the gutted and ungutted fish samples of each fish species, showing that gutting influenced ph variation. ph is not appropriate for fish grading at it changes very little during iced storage, and was not influenced by the species in this experiment although it seemed to be influenced by treatment (gutting). FFA There was no significant increase (p>0.05) in FFA content in the ungutted and gutted samples of both Rabbitfish and White snapper over the storage period. Although the White snapper had an initial FFA value of oleic acid equivalent, the values at rejection point on day 24 and 28 were 2.18 and 2.18 mg oleic acid equivalents for the gutted and ungutted fish. For Rabbitfish the initial values were mg oleic acid equivalents and 2.19 mg oleic acid equivalents on day 24 and 28 of storage for both gutted and ungutted fish. At the point of rejection for both fish species, %FFA values were mg oleic acid equivalents. The progressive increase in the %FFA values over the storage time showed the progressive breakdown of lipids in fish muscle with time in iced storage. Gutting, however, reduced the Egerton J. Sci. & Technol. Volume 13: ISSN No

95 90 Grading marine fish using Q1, biochemical and microbiological indices rate of lipid breakdown and the subsequent formation of FFA in both fish species. However, the rate of FFA formation was greater in the White snapper than in Rabbitfish for some unclear reason, despite both having a comparable total lipid content of % for Rabbitish and % for White snapper. Postmortem hydrolysis of fish muscle lipids leads to the release of free fatty acids. During storage, a considerable amount of FFA appears. This phenomenon is more profound in ungutted than gutted fish, and is catalyzed by lipases and phospholipases (Pacheco-Aguilar et al., 2000). From the results of this study, the rate of breakdown of fish lipids to form FFA varied with the marine fish species, although the content at the end of shelf life was of similar magnitude for each species (2.18% for gutted and gutted White snapper and 2.19% FFA for ungutted and gutted Rabbitfish). Percent FFA may not be a useful parameter in fish grading, and the long time required for its analytical determination currently makes its adoption unsuitable for rapid commercial grading. Microbiological Parameters TVC In both fish species, the significant increase in TVC (p<0.05) was observed on the 12 th day of iced storage irrespective of the storage temperature and treatment (gutting or ungutting). As TVC did not give an indication of the arrival of the point of consumer rejection, it may not be a good guide for fish grading. H 2 S-producing Bacteria A significant increase (p<0.05) in H 2 S-producing bacteria was observed in the ungutted Rabbitfish from the 16 th day while in the gutted fish this was apparent from the 20 th day of storage in ice onwards. There was a significant difference (p<0.05) in H 2 S-producing bacterial count between the gutted and ungutted Rabbitfish with higher counts being observed in the ungutted fish samples. The gutted and ungutted iced experimental White snapper showed H 2 S-producing bacteria counts that also increased significantly (p<0.05). However, higher counts were observed at any one stage of storage in the ungutted White snapper as compared to the gutted fish (Table 6). At the point signifying the end of shelf life, the level of these bacteria was log 5.3 and log 5.8 in gutted and ungutted Rabbitfish, respectively, but log 5.4 and 5.8 in gutted and ungutted White snapper, respectively. The increase in the bacterial load in the gutted fish of both species was slower as a result of gut material removal. Guts are therefore sources of hydrogen sulphide-producing bacteria in two fish species. Despite different initial values of bacterial loads, the values at the end of shelf life were of similar magnitude (CFU log 5.4 Egerton J. Sci. & Technol. Volume 13: ISSN No

96 Lokuruka, et al., (2013) 91 and 5.8 for ungutted and gutted white snapper, respectively, and log 5.8 and 5.3 for ungutted and gutted Rabbitfish, respectively). The magnitude of the counts in both fish species at the point of rejection by the taste panel was similar irrespective of whether they were gutted or not. Coliforms A significant increase (p<0.05) in coliform count was observed in both gutted and ungutted fish samples as storage progressed in ice. The difference in coliform count between the gutted and the ungutted was significantly different (p<0.05) in both fish species with higher values observed in the ungutted batch (Tables 5 and 6). This signifies that guts are sources of coliforms. At the point signifying the end of consumer acceptability, the coliform count was log 5.3 (up from CFU log 1.2) in gutted Rabbitfish as compared to log 5.4 (up from CFU log 1.4) in the gutted White snapper. The values in the ungutted Rabbitfish and White snapper were CFU log 5.8 (up from CFU log 1.6 in both fish species) (Table 5 and 6). The removal of gut material considerably reduced the rate of increase of coliforms in both fish species. However, the level of coliforms at the end of shelf life was of similar magnitude for the ungutted and gutted fish of each fish species. Similar to the hydrogen sulphide-producing bacteria, guts are sources of coliforms. The high level of coliforms in both gutted and ungutted fish of both species is a cause for concern as far as food safety is concerned. The high levels may be attributed to the poor personal hygiene of fishers and poor hygiene standards on the artisanal, outrigger boats, high ambient seawater temperatures (23-24 o C) and the poor handling before icing. For Rabbitfish, the initial CFU was log 1.6 and 1.2 but at the end of shelf life, the CFU was log 5.8 and 5.3 in ungutted and gutted fish, respectively. But for White snapper, the initial values were CFU log 1.4 and 1.6 for gutted and ungutted, which were CFU log 5.4 and 5.8 on day 28 and 24, respectively. Therefore, the magnitude of the final level of coliforms at rejection point by the taste panel for both fish species was similar in the gutted and ungutted lots. There was no E. coli bacteria detected in both fish species over the entire storage period whether the fish were gutted or left ungutted. Proximate Composition The moisture content and the chemical composition of fish normally influence the rate of microbial multiplication in fish, and therefore would be expected to influence the rate of change of the microbiological and biochemical indices studied in this study. However, from Table 4, this was not apparent. Therefore, the grading of the two fish species cannot be based at the current level of knowledge on changes in the moisture, protein and lipid content during iced storage. As no instrument or sensory parameter can Egerton J. Sci. & Technol. Volume 13: ISSN No

97 92 Grading marine fish using Q1, biochemical and microbiological indices be used currently to rapidly gauge the proximate composition when grading fish, none of the 3 proximate parameters is suitable for rapid fish grading. However, if some instrument can be used to approximate composition and the composition can be related to quality as judged by sensory parameters, then it may be used. X-rays have been used since the 1930s for measuring the fat content in animals (Hogreve, 1938) but the technique was not widely adopted as it was not promising and was therefore replaced by ultrasound (Hazel and Kline, 1959). Ultrasound is therefore the most commonly used method for subcutaneous fat determination in all domestic animals and has been validated extensively. But this method has not been applied to small animals such as fish. It is currently being used only in large livestock. Conclusions The QI score and the sensory parameters studied in this study are suitable for commercial grading of iced, fresh fish. Their rapid determination makes their adoption for iced fresh fish grading attractive. ph is not appropriate for fresh fish grading at it changes very little during iced storage, and was not influenced by fish species in this experiment, although it seemed to be influenced by treatment (gutting). Percent FFA and TVBN are potentially useful parameters in iced fresh fish grading, but the long time required for their analytical determination makes their adoption inappropriate for rapid grading of iced, raw fish in commerce currently. Therefore, based on the results of this experiment, we recommend the adoption of select sensory parameters (Table 1) and the QI for rapid commercial grading of iced, fresh fish, but where high quality or prime freshness is a critical requirement for fish processing, and more time is available for other considerations, TVBN and %FFA are recommended as additional indices for potential use during grading of iced fresh fish. The microbiological indices studied in this work are not appropriate for iced, fresh fish grading, but can be used as guides of the extent of loss of freshness or prime quality, generally after 2 weeks of iced-storage of fresh fish. The shelf life of ice-stored Rabbitfish was 24 and 32 days when ungutted and gutted, respectively; but for White snapper, it was 24 and 28 days when ungutted and gutted, respectively. Recommendations The authors recommend the following: 1. The QI score and select sensory parameters can potentially be used for commercial grading of fresh and iced Rabbitfish and White snapper Egerton J. Sci. & Technol. Volume 13: ISSN No

98 Lokuruka, et al., (2013) The TVBN and FFA cannot be used for rapid commercial grading of fresh and iced Rabbitifsh and White snapper but would complement the QI score and select sensory parameters when speed is not of essence during fish grading 3. The microbiological indices studied in this experiment would not be useful for rapid grading of fresh and iced Rabbitfish and White snapper under 2 weeks of storage in ice. References AACC. (2004). Official Methods of Analysis, 10 th edn. American Association of Cereal Chemists, Washington, D.C. pp AOAC. (2000). Official Methods of Analysis, 17 th edition, Association of Official Analytical Chemists, Washington, D.C., pp Goulas, A.E. and Kontominas, M.G. (2005). Effect of salting and smoking method on the keeping quality of chub mackerel (Scomber japonicus): biochemical and sensory attributes. Food Chemistry 93: Gram, L., Trolle, G. and Huss, L.L. (1987). Detection of specific spoilage bacteria on fish stored at high (20ºC) and low (0ºC) temperatures. Int. J. Food Microbiol. 4: Hazel L.N., and Kline, E. A. (1959). Ultrasonic measurement of fatness in swine. J. Animal Sci. 1959; 18: Found at Accessed on 8th November, Hogreve, F. (1938). A new experimental method for determining fat deposition and fat production at different stages of fattening in live pigs of various breeds. Z. Zucht B. 1938; 40: Found at Accessed on 8th November, Ministry of Livestock and Fisheries Development. (2005). Annual Report. Fisheries Development Division, Nairobi. Ministry of Fisheries Development (MOFD). (2009). Annual Statistical Fisheries Bulletin. MOFD, Nairobi. Okeyo G.O. and Lokuruka, M.N.I. (2010a). Deterioration profile of iced Lake Victoria Nile Egerton J. Sci. & Technol. Volume 13: ISSN No

99 94 Grading marine fish using Q1, biochemical and microbiological indices perch (Lates niloticus) in relation to some microbiological and chemical indices. Egerton J. Sci. Tech. 10: Olafsdottir, G., Martinsdottir, E., Oehlenschlager, J., Dalgaard, P., Jensen, B., Undeland, I., Mackie, I. M., Henehan, G. and Nielsen, J. (1997). Methods to evaluate fish freshness in research and industry. Trends Food Sci. Technol. 8: Egerton J. Sci. & Technol. Volume 13: ISSN No

100 Histopathological effects of Trypanosoma brucei brucei infection on the master circadian rhythm pacemaker in rats Maina C. I. 1, Ng wena G. M. 2, Mwaniki D. M. 3, Oucho A. O. 3 1 Department of Biological Sciences, Egerton University, P.O. Box , Egerton, Kenya. Tel cimaina@yahoo.com 2 Department of Medical Physiology, School of Medicine, Moi University, P.O. Box , Eldoret, Kenya. 3 Department of Biological Sciences, Chepkoilel University College, P.O. Box , Eldoret, Kenya. Submitted 6 th Nov. 2012; Reviewed 24 th May 2013; Accepted 5 th June 2013 Abstract Trypanosomiasis is an infectious disease of humans and animals caused by protozoan parasites of the genus Trypanosoma and transmitted by the bite of infected tsetse flies of the Glossina species. The disease is hallmarked by sleep/wake disturbances and disruptions in other circadian rhythm activities. Although trypanosomiasis has a well known aetiology, histopathological studies on brain regions involved in the control of circadian rhythms are scanty. This study investigated the histopathological effects of Trypanosoma brucei brucei infection on the suprachiasmatic nucleus, the master circadian rhythm pacemaker, in the hypothalamus of rats. It also investigated the effects of the infection on the rat s body weight and temperature. Twelve control and twelve experimental male albino rats, weighing g, were used in this study. Body weight and temperature of each rat was measured four days in a week. The experimental rats were inoculated intraperitoneally with 0.2ml of infected blood containing 1 x 10 4 T.b.brucei parasites. The infected animals were allowed to go through the full course of infection and sacrificed when they were in extremis. Each rat was decapitated and the brain immediately extracted from the skull. The brain was fixed in buffered neutral formalin (10%) for at least 48 hours. The brain was later removed from the formalin solution and a coronal section made. The coronal section was processed histologically and stained using the haematoxylin and eosin method. The stained slides were examined under a microscope and photomicrographs taken. Significant differences in body weight and temperature were recorded between the control and experimental rats. Histopathological changes, including tissue degeneration and infiltration and proliferation of cells, were observed in the suprachiasmatic nucleus of infected rats. The findings of this study provide a basis for understanding the Egerton J. Sci. & Technol. Volume 13: ISSN No

101 96 Maina, et al., (2013) mechanisms underlying functional alterations in circadian rhythms that characterize animal trypanosomiasis. Keywords: Trypanosomiasis, Circadian rhythms, Pacemaker, Histopathology Introduction African trypanosomiasis is an infectious disease of both humans and animals of similar aetiology and epidemiology. The causative agents of the disease are protozoan parasites of the genus Trypanosoma that live and multiply extracellularly in the blood and tissue fluids of their mammalian hosts and are transmitted by the bite of infected tsetse flies of the Glossina species (Sterverding, 2008). Throughout history, African trypanosomiasis has severely threatened the lives and livelihood of entire communities and constitutes a major constraint to livestock and crop production in Africa (PATTEC, 2001) The World Health Organization (WHO) estimates that over 60 million people in Africa are at risk of contracting human trypanosomiasis. Besides, at least 300,000 to 500,000 people are presently infected and 100,000 deaths can be attributed to human trypanosomiasis each year (WHO, 2012). The situation could be worse than this because of the many unreported cases from inaccessible rural and war-ravaged areas. The disease is endemic in southeast Uganda and western Kenya, and is reported more frequently among tourists, especially those traveling to wild park areas near Lake Victoria, to the Tarangire, Serengeti and Lake Manyara National Parks, all of which are located in East Africa (Visser, 2010). Tsetse flies infest about 10 million km 2 of fertile land spread across 37 countries on the African continent, from Senegal in the North to South Africa in the south (Oluwafemi, 2009). Countries particularly affected include Sudan, the Democratic Republic of Congo, Angola, Kenya, Uganda and Tanzania. Many areas that are infested with tsetse flies are the most suitable areas for livestock and crop production. Tsetse transmitted trypanosomiasis is accorded little attention and priority because it is essentially a rural problem, which occurs only in Africa. However, its negative impact on the history and socio-economic development of most of the continent has been, and continues to be, very devastating (PATTEC, 2001). Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major public health problem in many countries in Africa. The disease is Egerton J. Sci. & Technol. Volume 13: ISSN No

102 Histopathological effects of Trypanosoma brucei brucei infection 97 caused by two subspecies of Trypanosoma brucei; T. b. gambiense and T. b. rhodesiense. The former is responsible for the chronic form of sleeping sickness in West and Central Africa, and it accounts for more than 95% of reported cases of sleeping sickness (WHO, 2012). On the other hand, T. b. rhodesiense gives rise to the acute form of the disease in East and Southern Africa, and it accounts for less than 5% of reported cases of sleeping sickness (WHO, 2012). There are two distinct stages during the course of sleeping sickness. The first or early stage of the disease, also known as the haemolymphatic phase, is defined by the restriction of the trypanosomes to the blood and lymph system (WHO, 2012). The symptoms of this stage include fever, headache, joint pains and itching. The second or late stage of the disease, also known as the neurological phase, is characterized by the presence of the parasites in the cerebrospinal fluid. In general, this is when the typical signs of the disease occur: confusion, sensory disturbances, extreme lethargy, poor condition and coma. Disturbance of the sleep/wake cycle, which gives the disease its name, is an important feature of the second stage of the disease. If left untreated, sleeping sickness patients die within months when infected with T. b. rhodesiense or within years when infected with T. b. gambiense (Steverding, 2008; WHO, 2012). African animal trypanosomiasis (AAT), on the other hand, is caused by other tsetse-fly-transmitted trypanosomes, namely T. congolense, T. vivax, and T. brucei brucei (Mare, 2008). The disease is most important in cattle but can also cause serious losses in goats, sheep, pigs, horses, camels, dogs, and cats. The disease in domestic animals and particularly cattle is a major obstacle to the economic development of the rural areas affected (WHO, 2012). In South Africa the disease is widely known as Nagana, which is derived from a Zulu word meaning "to be in low or depressed spirits" a very apt description of the disease (Mare, 2008). In domestic animals, the trypanosome parasites cause a severe, often fatal disease while in wild animals, they cause relatively mild infections. All domestic animals can be affected by trypanosomiasis and the symptoms include fever, listlessness, emaciation, hair loss and discharge from the eyes, oedema, anaemia, and paralysis (Mare, 2008). As the illness progresses the animals weaken more and more and eventually die. Animals can host the human pathogen parasites, especially T. b. rhodesiense; thus both domestic and wild animals are an important parasite reservoir (Simo et al., 2006; WHO, 2012). Egerton J. Sci. & Technol. Volume 13: ISSN No

103 98 Maina, et al., (2013) African Animal trypanosomiasis causes death of well over 3 million cattle annually with an estimated lost potential of US$ 6 12 billion (Adeiza et al., 2008). The disease has rendered approximately a quarter of African arable land mass unsuitable for profitable livestock farming, and has profoundly affected settlement and economic development in much of the African continent, especially south of the Sahara desert (Nga yo et al., 2005; Adeiza et al., 2008). Trypanosomiasis is hallmarked by sleep/wake disturbances, and disruptions in other circadian rhythm activities like temperature and hormone secretion. These disruptions are an indication of some changes in the suprachiasmatic nucleus, the master circadian rhythm pacemaker, and, hence, the biological clock in mammals. This study was, therefore, undertaken to investigate the effects of T,b,brucei infection on the histology of the suprachiasmatic nucleus in rats. The study also investigated the effects of the infection on body weight and temperature of the rats. Materials and Methods Experimental Setup Twenty four male albino rats, aged 3-3½ months and weighing g, were used in this study. The rats were randomly divided into two groups of twelve rats each. The first group of twelve rats constituted the uninfected controls while the other twelve were inoculated with a T.b. brucei isolate and constituted the infected experimental group. The rats were housed at room temperature ( C) in the mini-laboratory animal house of the Department of Biological Sciences, Chepkoilel University College, Kenya. They were housed three per cage and were exposed to 12/12hours of light/dark cycle throughout the study period. The rats were provided with food (Mice Pencil, Unifeed Millers Ltd, Kisumu, Kenya) and water ad libitum. Two weeks prior to actual pre-infection baseline data collection, the rats were observed and accustomed to routine handling. They were also screened for ectoparasites besides being injected subcutaneously with 0.01ml of Ivermectin (Ivermin, Sinochem Ningbo Ltd., China), a broad- spectrum parasiticide that effectively controls both ecto- and endo-parasites. The research committee of the Department of Biological Sciences, Chepkoilel University College, Kenya, approved this study and the animals were handled in accordance with internationally accepted principles for laboratory animal care and use. Egerton J. Sci. & Technol. Volume 13: ISSN No

104 Histopathological effects of Trypanosoma brucei brucei infection 99 Determination of Body Weight and Temperature The body weight of each rat was determined by weighing each rat on an electronic weighing balance (Deliver Instrument Co., USA). The weighing was done four days in a week between Hrs. The body temperature of each rat was measured at the same time through rectal insertion of a digital clinical thermometer (Model ECT-1, China). The tip of the thermometer was sterilized by wiping it with a cotton swab dipped in methylated spirit before and after use. Inoculation of Experimental Rats An isolate of the parasite Trypanosoma brucei brucei (ILTat1.4) was obtained from the International Livestock Research Institute (ILRI), Nairobi, Kenya. The parasite was originally obtained from the blood of a naturally infected cow in Uhombo, Kenya. The isolate was inoculated intraperitoneally to a donor rat for the purpose of harvesting enough parasites for subsequent inoculation into experimental rats. The donor rat was transported to Chepkoilel University College, Eldoret, Kenya, where the study was carried out. The donor rat was monitored for the presence of parasites daily by direct microscope observation of trypanosomes in wet smears of blood samples obtained from tail bleeds. When parasitaemia was established, the donor rat was anaesthetised with ether and 1ml of blood obtained from it through a cardiac puncture. Then, 0.2ml of this blood, diluted with phosphate buffered saline solution (ph 7.4), and containing approximately 1.0 x 10 4 T.b.brucei parasites was injected intraperitoneally to each of the twelve rats in the experimental group. The number of parasites was determined using the Neubauer haemocytometer method (Herbert and Lumsden, 1976). Tail blood samples were collected daily from the infected rats and screened for trypanosomes using the standard trypanosome detection methods, namely wet and thin blood smear films (OIE, 2008). The rats were also observed for any abnormal behaviour and/or any clinical signs of infection. The rats were infected after four weeks of pre-infection baseline data collection. Control rats were, concurrently, injected intraperitoneally with 0.2ml normal saline. Histopathological Evaluations The infected animals were allowed to go through the full course of infection without treatment and sacrificed when they were in extremis. Uninfected control rats were concurrently sacrificed too. Each rat was anaesthetised with ether, decapitated and the brain immediately extracted from the skull. The brain was immediately put in buffered neutral formalin (10%) and fixed for at least 48 hours. Thereafter, the brain was removed from the formalin Egerton J. Sci. & Technol. Volume 13: ISSN No

105 100 Maina, et al., (2013) solution and a coronal section made. The coronal section of the brain was processed histologically using an automated tissue processor (Tissue-TEK E150, Miles Co., USA). The well processed tissue was embedded in paraffin wax and sectioned at 5µm using a microtome (Leica Co., USA). The thin sections were stained using the haematoxylin and eosin method (Lowe, 1998), examined under a microscope, and photomicrographs taken. Histological differences in these tissues between control and experimental rats were determined. Statistical Analysis The data obtained from this study were summarized as means ± standard deviation and the differences between the means analyzed using Student s t- test (SPSS v19 for Windows) at 5% significance level. Results Parasite Detection Parasites became detectable in the blood of experimental rats 5 to 8 days after infection (Figure 1). Figure 1: Blood smear of an infected rat showing T.b.brucei (arrows) parasites. (Mg x1000) The experimental rats showed no obvious signs of disease for the first fifteen days after infection. Thereafter, they showed apparent fatigue, decreased activity, reduced appetite, discharge from the eyes and nose, and paralysis of limbs and tail. The infected rats became emaciated and weighed about 15% less than the uninfected control rats at the time of sacrifice. On the other Egerton J. Sci. & Technol. Volume 13: ISSN No

106 Histopathological effects of Trypanosoma brucei brucei infection 101 hand, the uninfected control rats showed no signs of trypanosomiasis or other infections throughout the period of experimentation. Effect of T.b.brucei Infection on Body Weight The uninfected control rats recorded a steady increase in body weight throughout the study period, from a mean of ± g at the start of the study to ± g at the end of the study (Figure 2). Likewise, the experimental rats recorded an increase in body weight during the preinfection period, from ± g to ± 20.73g. The body weight continued rising one week after infection (to ±19.94 g) and then decreased to ± g at the end of the study. The difference in body weight between the uninfected control rats and the infected experimental rats was significant (P = ). Figure 2: Body weight of control and experimental rats Effect of T.b.brucei Infection on Body Temperature The body temperature of uninfected control rats remained normal, ranging between 34.8 ± C and 36.0 ± C, throughout the study period. Similarly, the body temperature of experimental rats remained normal during the pre-infection period. However, the temperature rose to 38.4 ± C two weeks after infection (Figure 3), and remained significantly higher (P = ) for the rest of the study period. Egerton J. Sci. & Technol. Volume 13: ISSN No

107 102 Maina, et al., (2013) Figure 3: Body temperature of control and experimental rats Effects of T.b.brucei Infection on Suprachiasmatic Nucleus Histology Light microscopic examination of the suprachiasmatic nucleus of uninfected control rats revealed normal neurons with clearly visible round nuclei and fewer glial cells (Figure 4a). On the other hand, the suprachiasmatic nucleus of infected experimental rats showed neurons with smaller and shrunken (pyknotic) nuclei, and degenerative patches were evident. There was also a marked increase in cellularity of glial and other cells (Figure 4b). Figure 4: Photomicrographs of the suprachiasmatic nucleus in (a) uninfected control, and (b) T.b.brucei-infected rats. Note the increased cellularity in the infected animals Egerton J. Sci. & Technol. Volume 13: ISSN No

108 Histopathological effects of Trypanosoma brucei brucei infection 103 Discussion Detection of Parasites The present study presents an experimental model of African trypanosomiasis in rats infected with T.b.brucei. In this study, T.b.brucei parasites became detectable in the tail blood of experimental rats 5 8 days after infection. This finding concurs with those of other investigators (Lundkvist, 2001; Darsaud et al., 2003; Abenga et al., 2005) who reported similar results on rats inoculated with T.b.brucei. Once inside the body, the parasite is completely exposed to the host s immune system but, in many instances, it survives and proliferates, resulting in characteristic waves of parasitaemia every three to five days (Pays, 1999; Lundkvist, 2001). The immune system kills subpopulations of the parasite, but because of the variable surface glycoprotein (VSG) coat on the trypanosome in some subpopulations, a proportion of the trypanosome population escapes the immune system actions, proliferates and another relapse of parasites is observed in the blood (Pays, 1999). The successful switching of the antigenic coat of the trypanosome plays a key role in the avoidance of the host immune system. At first, the immune system was, thus, able to control the parasite population. This could explain the lack of any obvious clinical signs of infection in the experimental rats for the first two weeks after infection. Thereafter, the immune system became overwhelmed, setting the stage for the emergence of signs of infection which ranged from apparent fatigue and reduced activity, to paralysis of limbs and tail at the terminal stages of the disease. Body Weight and Temperature Experimental rats, contrary to control animals, experienced weight loss after a little less than two weeks of infection. A notable lack of appetite, and drop in food intake, always preceded the drop in body weight. Similar findings have been reported in rats infected with T. b. brucei (Kristensson et al., 1998; Nyakundi et al., 2002) as well as in rats infected with T. b. gambiense (Nishimura et al., 2001) and in rabbits infected with T. b. brucei (Toth et al., 1994). Loss of body weight among domestic animals infected with trypanosomes has also been reported. Trypanosoma congolense-infected West African dwarf goats (Faye et al., 2005) and sheep (Katunguka- Rwakishaya et al., 1993), and T. brucei-infected pigs (Fagbemi et al., 1990) recorded lower body weights than their uninfected control counterparts. Likewise, Mutayoba et al., (1989) reported loss of body weight in small East African goats infected with T. congolense. Egerton J. Sci. & Technol. Volume 13: ISSN No

109 104 Maina, et al., (2013) Loss of body weight, and food intake disturbances, are also observed in humans suffering from African trypanosomiasis. Patients infected by T. b. gambiense recorded loss of body weight during the neurological phase of the disease, with many of the patients dying in severe cachexia (Dumas and Bisser, 1999). Even though some rare cases of bulimia were observed, most often the patients were anorectic (Dumas and Boa, 1988; Dumas and Bisser, 1999). The drop in body weight during trypanosomiasis could be attributed to the lack of appetite and depletion of food reserve in the host by the parasite (Adamu et al., 2008). Although not investigated in this study, pro-inflammatory cytokines have been implicated as the major cause of reduced appetite and body weight during trypanosomiasis. It has been shown that the cytokines interleukine- 1(IL-1), interferon-γ (IFN-γ), and tumor necrosis factor -α (TNF-α) decrease food intake by a direct effect on glucose-sensitive neurons of the hypothalamus (Plata-Salaman et al,. 1988) or indirectly through a stimulation of the hypothalamic prostaglandin E 2 synthesis (Dinarello et al., 1986), which in turn stimulates the release of hypothalamic corticotropinreleasing factor, an endogenous anorexigenic neuropeptide (Uehara et al., 1989). A study by Quan et al., (1999) found out that the body weight loss of T.b. brucei-infected rats correlated with mrna expression of pro-inflammatory cytokines IL-1and TNF-α, and with the progression of terminal and axonal degeneration in the brain. These authors proposed that the cytokines, especially TNF-α, may have caused the pronounced weight loss they observed in these rats. In another study, Rhind et al., (1997) reported an increase in concentration of TNF-α in the blood of T. b. gambiense-infected patients at the neurological phase. In addition, this increase was concomitant to changes in neuroendocrine circadian rhythms (Radomski et al., 1994; Reincke et al., 1998) and was also related to the disease severity (Okomo- Assoumou et al., 1995). Other factors that may contribute to reduced food intake, and hence, reduced body weight, include fever and stress caused by the presence of trypanosomes inside the animal s body (Pathak, 2009). In the present study, the experimental rats showed a sharp increase in body temperature two weeks after infection. This finding concurs with those of Grassi-Zucconi et al., (1995) who also reported an increase in body temperature of rats infected with T.b. brucei. Other investigators (Mutayoba et al., 1989; Adeiza et al., 2008) working with trypanosome-infected goats have reported similar results. Fever is one of the typical clinical features of trypanosomiasis and even humans suffering from African trypanosomiasis Egerton J. Sci. & Technol. Volume 13: ISSN No

110 Histopathological effects of Trypanosoma brucei brucei infection 105 are not spared of it (Meunier, 2009; Odero et al., 2012). These findings suggest that trypanosome infection disrupts the temperature set point in the hypothalamus of infected animals, adjusting it to a higher value. Parasitic infections are associated with inflammatory processes and release of pyrogens that lead to body temperature changes (Baracos et al., 1987). The mammalian body always attempts to maintain its temperature around a fixed set-point. When the set-point is raised the result is fever, a condition in which the body produces more heat without changing heat loss or by reducing heat loss through vasoconstriction and high tissue insulation (Pathak, 2009). When the heat regulating mechanism of the body becomes overwhelmed, the temperature becomes elevated uncontrollably. In this study, all the infected experimental rats became hyperthermic in the postinfection period with body temperature rising up to C, from C. The presence of parasites in the rats probably caused a dysfunction of the thermoregulatory system through modulation of metabolic processes or release of pyrogens that changed the thermal set-point in the hypothalamus. The results of this study indicate that T.b.brucei infection is accompanied by a decrease in body weight, and an elevation of body temperature in rats. The loss in body weight was preceded by a notable lack of appetite and reduced food intake in the rats. The body temperature set point in the hypothalamus could have been shifted upwards under the influence of pyrogenic stimuli released during the infection. Suprachiasmatic Nucleus Histopathological Changes This study revealed that trypanosomiasis affects the hypothalamic suprachiasmatic nucleus in rats. The neuronal nuclei in the infected rats appeared smaller and shrunken, and there was marked infiltration and proliferation of glial cells. These observations mirror those in human patients (Adams et al., 1986) and, as such, provide a good model system for pathogenesis studies. Histopathological studies from human brain specimens of sleeping sickness victims have reported the occurrence of glial cell proliferation and hypertrophy (Poltera, 1985; Hunter and Kennedy, 1992; Manuelidis et al., 1995). In experimental T.b.brucei infection in mice, infiltration and proliferation of cells was reported to appear throughout the brain and this was proposed to be associated with a transient increase of proinflammatory cytokines (Hunter et al., 1991). Similar findings were reported on experimental T.b.brucei infections in different animal species (Stevens and Moulton, 1977; Kristensson et al., 1995; Chianella et al., 1999). The present observation provides evidence that experimental African trypanosomiasis in rats results in a marked infiltration and proliferation of cells in the suprachiasmatic nucleus. Egerton J. Sci. & Technol. Volume 13: ISSN No

111 106 Maina, et al., (2013) The suprachiasmatic nucleus is the master circadian rhythm pacemaker in mammals (Abe et al., 2002; Welsh et al., 2010). It is the site of an endogenous clock mechanism, exhibiting circadian rhythms of metabolism, electrophysiological activity, and gene expression (Schwartz, 2008). Individual suprachiasmatic nucleus neurons in dispersed culture have, in fact, been shown to generate independent circadian oscillations of clock gene expression and neuronal firing (Welsh et al., 2010). Suprachiasmatic nucleus is, thus, a circadian oscillator that functions as a biological clock. Retinal efferent fibers originating predominately from intrinsically photosensitive retinal ganglion cells (iprgcs) and travelling via the retinohypothalamic tract (RHT) provide irradiance signals to the suprachiasmatic nucleus that entrain the circadian oscillator to the 24 hr environmental day/night cycle. Entrainment provides a predictable and appropriate phase relationship between the endogenous and the day/night cycles, in effect enabling recognition of local time (Pickard and Sollars, 2007). Since individual cells of the suprachiasmatic nucleus are capable of functioning independently from one another, they must, therefore, form a cohesive circadian network through intercellular coupling. The network properties of the suprachiasmatic nucleus lead to coordination of circadian rhythms among its neurons and neuronal subpopulations (Mohawk and Takahashi, 2011). From the suprachiasmatic nucleus, electrical signals carry timing information, via complex multineuronal pathways, to other control centers in the brain, especially the paraventricular nucleus, and the pineal gland. Because of the disturbance in the suprachiasmatic nucleus histology, this information may be altered in trypanosome-infected rats. The histopathological changes observed in the suprachiasmatic nucleus of T.b. brucei-infected rats in the present study strongly suggest a disturbance of the functional rhythmic properties of suprachiasmatic nucleus neurons. This may result in an alteration of the suprachiasmatic nucleus output to other brain regions and, consequently, a dysregulation in the circadian control of physiological, hormonal and behavioural rhythms. Experimental studies in rodents have shown that at early stages of infection the T.b.brucei parasite is confined to regions of the nervous system located outside the blood-brain barrier (Schultzberg et al., 1988), whereas at late stages the blood-brain barrier is disrupted and a number of trypanosomes can be found scattered in the brain and spinal cord, without any specific distribution (Philip et al., 1994). Invasion by trypanosomes, furthermore, causes release of a vast number of immune response molecules (Lundkvist, 2001) which may affect the suprachiasmatic nucleus function and output. Egerton J. Sci. & Technol. Volume 13: ISSN No

112 Histopathological effects of Trypanosoma brucei brucei infection 107 Indeed, during trypanosome infection, there is a marked release of proinflammatory cytokines such as IL -1, TNF-α and IFN-γ, as well as nitric oxide (Quan et al., 1999; Rhind and Shek, 1999) and prostaglandins (Kubata et al., 2000). Radomski et al. (1994) found a positive correlation between plasma levels of cytokines, in particular IFN-γ, and the severity of the disease, in patients with trypanosomiasis. Other studies have reported increased levels of TNF-α, IFN-γ, and IL-l during the course of trypanosome infection (Bancroft et al., 1983; Askonas and Bancroft, 1984; Le and Vilcek, 1987) and, Bocci (1990) suggested that these cytokines are involved in nervous system histopathology. Nitric oxide (NO) has been demonstrated to alter the circadian firing pattern in the suprachiasmatic nucleus in vitro (Ding et al., 1994). Cytokines may therefore alter the diurnal increase in spontaneous firing in the biological clock via a nitric oxide-mediated pathway. Nitric oxide may also be involved in the pathogenesis of the sleep-wake disturbances in experimental trypanosomiasis. In primates infected with T.b. brucei, increased nitric oxide concentrations were found in plasma and cerebrospinal fluid (Sternberg et al., 1998). Nitric oxide is also released by macrophages in trypanosomeinfected mice (Millar et al., 1999) and may cause immunosuppression (Sternberg and Mcguigan, 1992). Besides, mrna for inducible nitric oxide synthase was found in the choroid plexus, meninges and brain parenchyma in rats infected with T.b. brucei (Quan et al., 1999). Pentreath (1989) and Hunter et al. (1991) have suggested that production of cytokines within the brains of infected individuals may give rise to the symptoms of late-stage trypanosomiasis and be important in generating the changes seen in the central nervous system. The results of this study show that experimental trypanosome infection causes histopathological changes in the suprachiasmatic nucleus of infected rats. These observations point to a suprachiasmatic nucleus dysfunction during trypanosome infection and suggest that the synchronizing action of the suprachiasmatic nucleus could be altered during the disease. Factors released by trypanosomes or the host animal defense during the infection could account for such impairment. Since the suprachiasmatic nucleus plays an important role as a pacemaker for circadian rhythms, the findings of this study provide a basis for understanding the physiological, hormonal, and behavioural changes that characterize African trypanosomiasis. In conclusion, this study has revealed that rats experimentally infected with T. b. brucei develop trypanosomiasis which is associated with decreased body weight and elevated body temperature. The study has also Egerton J. Sci. & Technol. Volume 13: ISSN No

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118 Histopathological effects of Trypanosoma brucei brucei infection 113 Randomski, M.W., Buguet, A., Bogui, P., Doua, F., Lonsdorfer, A.T., Tapie, P. and Dumas, M. (1994). Disruptions in the secretion of cortisol, prolactin, and certain cytokines in human African trypanosomiasis patients. Bull Soc Pathol Exot. 87: Reincke, M., Heppner, C., Petzke, F., Allolio, B., Arlt, W., Mbulamberi, D., Siekmann, L., Vollmer, D., Winkelmann, W. and Chrousos, G.P. (1994). Impairment of adrenocortical function associated with increased plasma tumor necrosis factor-alpha and interleukin-6 concentrations in African trypanosomiasis. Neuroimmunomodulation 1: Rhind, S.G. and Shek, P.N. (1999). Cytokines in the pathogenesis of human African trypanosomiasis; antagonistic roles of TNF-α and IL-10. In: M. Dumas, B. Bouteille, and A. Buguet. eds. Progress in human African trypanosomiasis, sleeping sickness. Springer Verlag, Paris. pp Rhind, S.G., Sabiston, B.H., Shek, P.N., Buguet, A., Muanga, G., Stanghellini, A., Dumas, M. and Radomski, M.W. (1997). Effect of melarsoprol treatment on circulating IL-10 and TNF-_ levels in human African trypanosomiasis. Clin Immunol Immunopathol 83: Schultzberg, M., Ambatsis, M., Samuelsson, E.B., Kristensson, K. and van Meirvenne, N. (1988). Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia. J. Neurosci. Res. 21: Schwartz, W.J. (2008). Circadian Oscillations in the Suprachiasmatic Nucleus. Encyc Neurosc: Simo, G., Asonganyi, T., Nkinin, S.W., Njiokou, F. and Herder, S. (2006). High prevalence of Trypanosoma brucei gambiense group 1 in pigs from the Fontem sleeping sickness focus in Cameroon. Vet Parasitol.139: Sternberg, J. and Mcguigan, F. (1992). Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection. Eur J Immunol 22: Sternberg, J.M., Njogu, M.N., Gichuki, C.W. and Ndung, U.J. (1998). Nitric oxide production in vervet monkeys (Cercopithecus aethiops) infected with Trypanosoma brucei. Parasite Immunol 20: Stevens, D.R. and Moulton, J.E. (1977). Experimental meningoencephalitis in Trypanosoma brucei infection of deer mice Peromyscus maniculatus., Acta Neuropathol. 38: Egerton J. Sci. & Technol. Volume 13: ISSN No

119 114 Maina, et al., (2013) Steverding, D. (2008). The history of African trypanosomiasis. Parasites and Vectors 1:3-8. Toth, L.A., Tolley, E.A., Broady, R., Blakely, B. and Krueger, J.M. (1994). Sleep during experimental trypanosomiasis in rabbits. Proc Soc Exp Biol Med 205: Uehara, A., Sekiya, C., Takasugi, Y., Namiki, M. and Arimura, A. (1989). Anorexia induced by interleukin-1: involvement of corticotropinreleasing factor. Am J Physiol 257:R613 R617. Visser, B.J. (2010). Human African Trypanosomiasis; A neglected disease. Accessed 20/07/2010 Welsh, D.K., Takahashi, J.S. and Kay, S.A. (2010). Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol 72: World Health Organization. (2012). Human African Trypanosomiasis (Sleeping Sickness). Fact sheet No Accessed 15/07/2012. Egerton J. Sci. & Technol. Volume 13: ISSN No

120 Growth Performance, Haematological Indices and Carcass Characteristics of Broilers Fed Diet Containing Different levels of Chromolaena odorata Leaf Meal Bonsu F. R. K. 1, Kagya-Agyemang J. K. 1, Kwenin W. K. J. 1, Hope K. N. 2 and Sekyere F. O. 1 1 Department of Animal Science Education. University of Education, Winneba, Mampong-Ashanti, Ghana, E- mail:fritzobonsu@yahoo.com 2 Department of Crop and Soil Science Education, University of Education, Winneba, Mampong-Ashanti, Ghana Submitted 12 th Jan. 2013; Reviewed; 24 th April 2013; Accepted 21 st May 2013 Abstract A 5-week study was carried out to determine the effect of Chromolaena odorata leaf meal (COLM) on growth performance, haematological parameters and carcass characteristics of commercial COBB-500 broiler chickens. The study was set up as a completely randomized design with four (4) treatments replicated three (3) times. The four dietary treatments contained 0, 10, 20 or 30 g kg -1 of COLM. There were no significant differences (P>0.05) among treatments in feed intake, water consumption, final body weight, weight gain and carcass characteristics. Feed conversion ratio was significantly influenced (P<0.05) in favour of birds fed the control and 2% COLM diet. Haematological indices including haematocrit (HCT), haemoglobin (Hb), red blood cells (RBC) and white blood cells (WBC) were significantly influenced (P<0.05) by the inclusion of COLM. However, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), and Platelet (PLT) were not significantly different (P>0.05) among treatments. Inclusion of COLM significantly reduced serum cholesterol concentration from mg dl -1 of the control to 81.8 mg dl -1 in 3% COLM. It was concluded that COLM could be included in broiler finisher diets up to a level of 30 g kg -1 without any deleterious effects. Key words: Chromolaena odorata, broilers, haematology, leaf meal, performance Introduction Poultry industry continues to play a major role in the socio-economic development of people in developing countries. This is evident by the Egerton J. Sci. & Technol. Volume 13: ISSN No

121 116 Bonsu, et al., (2013) employment opportunity offered and the provision of protein from chicken meat and eggs in solving malnutrition problems. Food security concerns have been raised in recent times as a result of relatively slower growth rate of the poultry industry which has not matched with the rate of population growth in developing countries. The major factor affecting the growth of the industry is the high cost of feed stuff (Amerah et al., 2007). The overreliance of the poultry industry in Ghana on conventional feed ingredients such as maize, fishmeal, soybean meal and wheat bran, inter alia, which are inadequate in supply to satisfy the nutritional requirements of humans as well as the animal industry especially in the lean season is another contributing factor. Imported feed stuffs have become scarce and more expensive due to the widening gap between the local currency and the US Dollar. In this regard, it has become expedient to explore alternate and cheap sources of feed stuff to reduce cost of production. Chromolaena odorata leaf meal popularly called Acheampong in Ghana is considered a serious weed pest on farmlands. Chromolaena odorata, previously known as Eupatorium odorata is a perennial shrub which belongs to the family Asteraceae. Its common name is Siam weed (Abbiw, 1990). Chromolaena odorata has been reported to have multipurpose medicinal properties (Iwu, 1993; Akinmoladun et al., 2007). The fresh leaves when applied to cuts or wounds drastically reduce bleeding (Bedi et al., 2001; Phan et al., 2001). Mashed green leaves are also used for traditional preservation of corpse. Dried leaves of Chromolaena odorata are also used as mosquito repellant, as antimicrobial agent against Bacillus cereus and antifungal agent against Aspergillus niger (Moses et al., 2010). Another important use of the leaves is for improving soil fertility. Farmers have commended its high class manure properties for maize, plantain and other field crops when the leaves are left to decompose on the field or used for compost preparation. Although nutritive research on Chromolaena odorata leaves remains scanty probably because it has been reported to be poisonous to livestock as a result of high nitrate content in the leaves and young shoots (Sajise et al., 1974), they are high in crude protein, dry matter, vitamins and minerals (Apori et al., 2000). The present study was conducted to investigate the effect of Chromolaena odorata on the growth performance, haematological parameters and carcass characteristics of broiler chickens. Egerton J. Sci. & Technol. Volume 13: ISSN No

122 Growth Performance, Haematological Indices and Carcass Characteristics 117 Materials and Methods Experimental Location The experiment was conducted at the Poultry Section of the Department of Animal Science Education, University of Education, Winneba, Mampong- Ashanti, Ghana. Mampong-Ashanti is located at N, W and at an altitude of 457 m above sea level. The mean annual rainfall was about 1500 mm. The mean monthly temperature was 32 o C while the mean relative humidity was 90% in the morning (06.00h) and 55% in the afternoon (15.00h). Experimental Design and Procedure A total of 300 day-old unsexed COBB-500 hybrid broiler chicks were procured from Darko Farms and Company Limited, Kumasi, Ghana and reared for an initial period of three (3) weeks on a 230 g kg -1 crude protein broiler starter mash in a deep litter house. Thereafter, a sample of 240 birds of similar weights were selected for the 35-day experiment and randomly allocated to the four dietary treatments. Each treatment was replicated 3 times and each replicate had 20 birds made up of male and female ratio 2:3. Each bird had a space of 0.2 m 2. Finisher diet (Table 1) for the 4 treatments and water were offered ad libitum. Processing of COLM The fresh leaves of green unflowered Chromolaena odorata were harvested and subjected to thermal drying in the solar drier at an optimum temperature between 37 0 C-41 0 C for a period ranging from hours depending on the amount of solar radiation received and the quantity of leaves fed into the drying unit. The solar dried leaves totaling 50 kg was milled through a 2 mm sieve and kept in air tight sacks for utilization in ration formulation. A sample of milled Chromolaena odorata leaves was analyzed at the Animal Nutrition laboratory for proximate composition using the standard method of AOAC (2000). Data Collection Data were collected on feed intake, water consumption, body weight and weight gain, feed conversion ratio for five (5) weeks (starting from week 4 and ending at week 8). Haematological parameters and carcass characteristics were taken at the end of the experiment. Egerton J. Sci. & Technol. 13(2013):

123 118 Bonsu, et al., (2013) Haematological Parameters After feed withdrawal for 12 h, blood samples were collected randomly from the jugular vein of two birds from each replicate into anticoagulant (EDTA) bottles and analyzed for total red blood cells (RBC), white blood cells (WBC), platelets, haemaglobin (Hb), haematocrit (HCT), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) using a Haematological Auto Analyzer. Serum cholesterol was analyzed according to the procedure outlined by Cheesbrough (1987). Carcass Analysis Two (2) birds (one male and one female) were randomly picked from each replicate at the end of the experiment. Birds picked were starved overnight to empty their crop after which live weight were taken. Birds were slaughtered and bled. After bleeding, carcasses were then manually de-feathered after scalding in hot water (85 0 C) for 20 seconds. The birds were decapitated and eviscerated and weighed to obtain the dressed weight. Internal organs and isolated abdominal fat were weighed separately. Sensory Analysis Fresh dressed carcasses and steamed chicken were used for the sensory analysis. Meats from different treatments were salted and steamed separately (100 o C for 20 minutes). Cooked meat were cut into cubes (2 cm x 2 cm x 2 cm), wrapped in foil and labeled with random 3-digits codes and presented randomly to eight trained panelists in different orders for evaluation. The sensory attributes of chicken were evaluated for skin and fat colour, beak and shank colour, aroma, texture, taste and over-all acceptability with a 5-point category scale as follows: Skin colour, fat colour, beak and shank colour were assessed using RAL Color of Maryland Metrics (2011). Aroma: 1-very weak; 2- weak; 3 moderate; 4 strong; 5- very strong Texture: 1 very tender; 2 tender; 3 moderate; 4 tough; 5 very tough Taste: 1 highly unacceptable; 2- unacceptable; 3- moderate; 4- acceptable; 5- highly acceptable Egerton J. Sci. & Technol. Volume 13: ISSN No

124 Growth Performance, Haematological Indices and Carcass Characteristics 119 Table 1: Composition of experimental diets Feed Ingredients (Kg) 0% COLM 1.0% COLM 2.0% COLM 3.0% COLM Maize Fish meal Soybean meal Wheat bran Oyster shells Dicalcium phosphate Premix Sodium Chloride COLM Calculated Analysis Crude protein (%) Crude fibre (%) Ether extract (%) Calcium (%) Phosphorus (%) Metabolisable energy (MJ kg -1 ) Feed Cost kg -1 (GH ) *Vitamin mineral premix provided the following per kg of diet: Fe 100 mg, Mn 110 mg, Cu 20 mg, Zn 100 mg, I 2 mg, Se 0 2 mg, Co 0 6mg, sanoquin 0 6mg, retinal 2000mg, cholecalciferol 25 mg, α-tocopherol mg, menadione 1 33mg, cobalamin 0 03 mg, thiamin 0 83 mg, riboflavin 2 mg, folic acid 0 33 mg, biotin 0 03 mg, pantothenic acid 3 75 mg, niacin 23 3 mg, pyridoxine 1 33 mg. Statistical Analysis Data collected was subjected to Analysis of Variance using GENSTAT Release 7 (2007) software. The least significant difference (LSD) at 5% was used to separate treatment means that were significantly different. Results and Discussion Proximate Composition The result of the proximate composition of the Chromolaena odorata is presented in Table 2. Egerton J. Sci. & Technol. 13(2013):

125 120 Bonsu, et al., (2013) Table 2: Proximate Composition of Chromolaena odorata leaf meal NUTRIENT PERCENTAGE (%) Crude Protein (CP) Crude Fibre (CF) Ether Extract (EE) Ash Content Dry matter Nitrogen-free extract The crude protein content of 22.80% obtained in this study is lower than the 25.80% and 26.20% reported by Apori et al. (2000) and Ngozi et al. (2009) respectively but higher than the 18.67% reported by Aro (1990). The crude fibre content of 11.49% obtained is also lower than the 31.1% and 26.57% reported by Apori et al. (2000) and Ngozi et al. (2009) respectively but similar to the 11.86% reported by Aro (1990). The ash composition (11.50%) was similar to the 10.9% reported by Apori et al. (2000) but higher than the 6.17% and 3.63% reported by Ngozi et al. (2009) and Aro (1990) respectively. Differences in nutrient composition are attributed to differences in age of leaves and edaphic factors which probably differed at the geographic location (Wood, 1998). Growth Performance The result of COLM inclusion on growth performance of finisher broilers is presented in Table 3. Mean feed intake, water consumption, final body weight and body weight gained were not significantly influenced (P>0.05) by the levels of Chromolaena odorata leaf meal in the finisher diet of broilers. However, birds fed the controlled diet had numerically higher values when compared with that obtained by birds fed the diets that contained the COLM. Feed conversion ratio was significantly influenced in favour of birds fed the control diet and the diet that had 2% COLM as compared with birds fed the diet that contained the Chromolaena odorata leaf meal at 3% level but not COLM at 1%. Results of most leaf meal studies have had depressed body weight gain when included in the diet at higher levels. COLM has been reported to contain antinutritional factors including cyanogenic glycosides, phytate, saponins and tannins (Ngozi et al., 2009) which are known to cause growth depression in broilers. The present study did not result in body weight gain depression probably due to the relatively lower inclusion rate of COLM. It is worth noting that feed intake was decreased as the level of COLM increased and resulted in a similar pattern in the body weight gain. The cost for body weight gain reduced as the level of COLM increased and this improved on the overall profit accrued for birds fed the COLM diets. Egerton J. Sci. & Technol. Volume 13: ISSN No

126 Growth Performance, Haematological Indices and Carcass Characteristics 121 Table 3: Performance indicators of broilers fed finisher diets containing varying levels of COLM Parameter Experimental Treatments SEM 0% COLM 1% COLM 2% COLM 3% COLM Initial body weight (g) Final body weight (g) Feed intake (g) Body weight gain (g) Water intake (l) Feed conversion ratio 2.36 b 2.47 ab 2.38 b 2.51 a 0.57 *Cost of gain GH *$1.00=GH 1.80 as at the time of experiment a-d Means in the same row with different superscript are significantly (P<0.05) different SEM- Standard error of the means Haematological Parameters The effect of Chromolaena odorata leaf meal on haematological parameters is presented in Table 4. Blood Haemoglobin (Hb), White Blood Cells (WBC), red blood cells (RBC) and haematocrit (HCT) were significantly (P<0.05) influenced by COLM inclusion in the diet. However, blood platelet, Mean Corpuscular Volume (MCV), and Mean Corpuscular Haemaglobin Concentration (MCHC) were not significantly (P>0.05) different among dietary treatments. Birds fed 1% COLM diet had the highest Hb and RBC concentration, and recorded the blood with greater proportion of red blood cells to whole blood (HCT). The WBC was highest in blood of birds fed the control diet as compared to that of the COLM diets. It could be possibly due to a disease challenge which caused birds on the control diet to produce more cells in an attempt to fight it. Conversely, COLM has some medicinal properties which could fight the potential pathogenic threat and therefore there was no need for the body system to produce extra WBC. However, there is the need to carry out further research to establish the specific poultry disease(s) that COLM can effectively control. Blood platelet was not significantly (P>0.05) influenced by COLM. Chromolaena leaves are known to stop bleeding when there is a cut (Bedi et al., 2001) - an attribute of the blood platelet. This hemostatic activity of the chromoleana leaves was therefore expected to cause a significant influence on the platelet content of the blood but that was not the case in the present study. It is possible that the biochemical properties responsible for hemostasis can not readily get absorbed into the blood. Serum cholesterol was reduced significantly (P<0.05) from mg dl -1 in the control to 81.8 mg dl -1 in those birds fed 3% COLM. Saponins reduce uptake of certain nutrients including cholesterol at the gut through intra-luminal physio-chemical interaction and have been Egerton J. Sci. & Technol. 13(2013):

127 122 Bonsu, et al., (2013) reported to have hypocholesterolemic effect (Moses et al., 2010). This might account for the serum cholesterol reduction observed. Table 4: Effect of COLM on haematological indices Parameter Experimental Treatments SEM 0% COLM 1% COLM 2% COLM 3% COLM Hb(g/dl) ab a b b 0.50 RBC (M/µL) 2.61 ab 2.87 a 2.56 b 2.41 b 0.12 WBC (K/µL) a bc ab c 2.10 HCT (%) b a b b 1.10 MCHC (g/dl) Blood Platelet (K/µL) MCV (fl) Serum cholesterol (mg/dl) a 98.2 a 99.7 a 81.8 b a-d Means in the same row with different superscript are significantly (P<0.05) different SEM- Standard error of the means Hb-Haemaglobin, RBC- Red Blood Cells, WBC-White Blood Cells, HCT- Haematocrit, MCHC- Mean Corpuscular Haemaglobin Concentration, MCV- Mean Corpuscular Volume. Carcass Characteristics The overall carcass characteristics were not influenced by the inclusion of Chromolaena odorata leaf meal in the diets (Table 5). Organ weight and dressing percentage were not significantly (P>0.05) influenced by the levels of COLM in the diet. This indicated that the present levels of COLM used may not have adverse effect on organs. Fat deposition in the lower abdomen reduced as the level of COLM in the diet increased. However, the abdominal fat deposited was not significantly (P>0.05) influenced possibly due to lower levels of COLM used in this study. Yellow pigmentation was observed on the skin, intermuscular fat, shank and beak of birds fed the COLM diet with high intensity in birds fed the 3% COLM. Consumer acceptability and patronage was higher for birds with the yellow pigmentation as a result of additional xanthophylls, and carotene (vitamin A) which improves vitamin status of the meat. Egerton J. Sci. & Technol. Volume 13: ISSN No

128 Growth Performance, Haematological Indices and Carcass Characteristics 123 Table 5: Carcass-organ characteristics of broilers fed different levels of COLM Parameter Experimental Treatments SEM 0% COLM 1% COLM 2% COLM 3% COLM Dressing percent (%) Abdominal Fat (g) Gizzard Weight (g) Heart Weight (g) Intestine Weight(g) Liver Weight (g) Means in the same row with different superscript are significantly (P<0.05) different SEM- Standard error of the means Table 6: Sensory Evaluation of Chicken Meat ATTRIBUTE 0% COLM 10% COLM 20% COLM 30% COLM SEM Aroma Texture Taste Acceptability b 4.0 ab 4.2 a 4.2 a 0.05 Means in the same row with different superscript are significantly (P<0.05) different SEM- Standard error of the means Sensory analysis (Table 6) indicated that the COLM inclusion in broiler diet at this present level did not influence aroma, taste and texture. However, most of the birds fed the COLM diet had better acceptability (P<0.05) as compared with the control as a result of the yellow pigmentation of fresh and cooked carcasses which was more appealing. Conclusion The results of the present study show that COLM has nutritional potential that can be utilized to reduce the overall production cost of broilers without adversely affecting the haematological indices, carcass characteristics and meat quality. The present study suggests potential medicinal properties of the COLM and future research will seek to explore this potential. Acknowledgement The Department of Animal Science Education of the University of Education, Winneba, is acknowledged for allowing their facilities to be used for this work. Egerton J. Sci. & Technol. 13(2013):

129 124 Bonsu, et al., (2013) References Abbiw, D.K. (1990). Useful plants of Ghana. 2 nd Edition, Intermediate Technology Publication. Akinmoladun, A.C., Ibukun, E.O. and Dan-Ologe, I.A. (2007). Phytochemical constituents and antioxidant properties of extracts from the leaves of Chromolaena odorata. Sci. Res. Essays, 2: Amerah, A.M., Ravindra,V., Lentle, R.G. and Thomas, D.G. (2007). Feed particles size: implication on the digestion and performance of poultry. World s Poultry Science Journal 63: A. O. A. C. (2000). Official methods of analysis of the Association of Official Analytical Chemists, 17 th edition Gaithersburg, MD. Apori, S.O., Long, R.J., Castro, F.B. and Ørskov, E.R. ( 2000). Chemical composition and nutritive value of leaves and stems of tropical weed Chromolaena odorata. Grass Forage Sci., 55: Aro, S.O. (1990) The effect of Chromoleana odorata leaf meal on the performance, egg quality characteristics, nutrient utilization, haematological and biochemical indices of layers. Department of Animal Science, University of Ibadan, Ibadan, Nigeria.Pp Bedi, G.,Tonzibo, Z.F.,Oussou, K.R., Choppard, C., Mahy, J.P. and N Guessen, T.Y. (2001). Effect of essential oil of Chromolaena odorata (asteraceae) from Ivory Coast on cyclooxygenase function of prostaglandin-h synthase activity. African Journal of Pharmacy and Pharmacology, 4(8): Cheesbrough, M. (1984). Medical Laboratory Manual for Tropical Countries- Microbiology. Volume 2, 2 nd edition, Butterworth- Heinemann Ltd. Halley Court, Jordan Hill, Oxford OX2 8EJ. Genstat Release 7.2 DE. (PC/Windows )( 2007). Lawes Agricultural Trust (Rothamsted Experimental Station). Iwu, M.M., (1993). Handbook of African medicinal plants. CRC Press Inc., Boca Raton. ISBN: X. Maryland Metrics. (2011). RAL Color Chart. Available at: Moses, S.O., Akintayo, O., Kamil, O.Y., Labunmi, L., Heather, E.V., Jessika A.T. and William, N. (2010). Chemical composition and bioactivity of the essential oil of Chromolaena odorata from Nigeria. Records of Nat. Prod., 4(1): Egerton J. Sci. & Technol. Volume 13: ISSN No

130 Growth Performance, Haematological Indices and Carcass Characteristics 125 Phan, T.T., Wang, L., See, P., Grayer, R.J., Chan, S.Y. and Lee, S.T. (2001). Phenolic compounds of Chromolaena odorata protect cultured skin cells from oxidative damage: Implication for cutaneous wound healing. Biol. Pharm. Bull., 24: Ngozi, M.I., Ikewuchi, C.J. and Ikewuchi, C. (2009). Chemical profile of Chromolaena odorata L (King and Robinson) leaves. Pakistan Journal of Nutrition, 8(5): Sajise, P.E., Palis, R.K., Norcio, N.V. and Lales, J.S. (1974). The biology of C. odorata L. King and Robinson. 1. Flowering behaviour, pattern of growth and nitrate metabolism. Phil. Weed Sci. Bull., 1:17-24 Wood, C.D. (1998) Genetic variation in the nutritive value of Gliricidia. Animal Feed Science and Technology 75: Egerton J. Sci. & Technol. 13(2013):

131 Free surface Flow in Open Channels over Complex Shapes Gathia P.W. 1* and Lonyangapuo, J. K. 1 1 Mathematics Dept., Egerton University, P.O. Box 536, Egerton *Correspondence Author: patricia_wanjiru@yahoo.com & amtjklonya@yahoo.com Submitted 23 rd March 2012; Reviewed 18 th Feb 2013; Accepted 22 nd May 2013 Abstract In this study, the problem of free surface fluid flowis investigated in an arbitrary shaped channel in which the form of the free surface profile and the fluid velocity on the free surface is given explicitly, whereas the exact shape and location of the solid bottom boundary and the fluid velocity on this boundary are unknown. Two problems are considered in this paper, the first is when a step occurs on the bottom surface of a channel. The second is when a single depression occurs on the bottom surface of a channel. This approach was based upon the minimization of the sum of the squares of the distances between the simulated free surface position from the direct problem and the computed value determined by the direct formulation from the estimated solid bottom surface position. The extremum total pressure technique to the case when the geometrical configuration of the bottom surface is a depression. An iterative technique is used for certain configurations of the lower surface, the inverse procedure could accurately recover this shape using only the known position of the free surface. However, the failure of the minimization procedure to incorporate any physical features meant that only the simplest of geometries could be successively recovered. To conclude, for free surfaces corresponding to flow over monotonically increasing steps or over single depression, the maximization of the pressure functional is capable of retrieving the geometrical shape of the lower boundary. Introduction The present problem involves the potential flow between fixed boundaries when the extremum of the function containing the kinetic energy is appropriate in the formulation which is a minimum. However, the continual changing of the lower boundary position in any iterative procedure together with the constraints of the free surface position means that in the present situation any such function is unknown yet. In addition to these difficulties, it is noted that in certain situations the domain occupied by the fluid over which the integration is occurring may itself be expected to be a minimum Egerton J. Sci. & Technol. Volume 13: ISSN No

132 Gathia and Lonyangapuo 127 or a maximum depending on the actual problem under consideration. This aspect of the domain itself being a maximum or a minimum according to the problem can be implied by fluid flow over a single trough (depression) and over a single step in which case one would expect within the constraits of the problem, the deepest possible water depth and the shallowest water depth to occur respectively. Further, the fact that the functional is physically conjectured means that the nature of the extremum required is somewhat unclear and in any case highly dependent on the chosen functional itself. Therefore the occurrence of both types of extremum is investigated in this study. In this paper, the extremum of the total pressure is developed and used to identify the bottom surface which may not necessarily be represented by a monotonically increasing function as was the situation investigated by Lonyangapuo et al (1998). This formulation is derived from Bernoulli s equation by use of the variational principle. In order to generate this numerical procedure, first, an initial estimate for the height of the bottom surface and hence the angle that the solid bottom boundary makes with the horizontal is given. Based upon this initial estimate of the position of the solid bottom boundary, the resulting nonlinear algebraic equations are computed and the corresponding height of the surface by using the numerical procedure developed for the direct problemtezduyar at al (1992). The pressure over the total fluid domain which is termed the objective function is then minimized subject to simple bounds and nonlinear constraits on the independent variable and the new height of the bottom surface is then determined. For this problem the nonlinear constraits are taken to be the free surface position where it is required that the computed and the known values agree to some specified degree of accuracy. This minimization uses a Sequential Quadratic Programming (SQP) algorithm in which the search direction is the solution of a Quadratic Programming problem (QP). The complete mathematical formulation of this problem is derived by use of the boundary integral method, the Riemann-Hilbert method and Mushkhelishlivilli s singular integral equation theory (1953). First problem to be considered in this paper is when a step occurs on the bottom surface of a channel. Far upstream of the step, the fluid is of depth h and flow horizontally in the positive X direction with a uniform speed of magnitude U. The upstream and downstream infinity positions are denoted by X E and X F respectively. Egerton J. Sci. & Technol. Volume 13: ISSN No

133 128 Free surface Flow in Open Channels over Complex Shapes The step is assumed to be located in the region X1 X X 2. The shape of the bottom surface is initially assumed to be governed by the function Y b = f(x) and the angle the solid bottom surface makes with the horizontal is 1 1 given by β ( X ) = tan f ( X ). The maximum height reached by the step is when X = X F and is denoted by h s, figures 1(a) and 1(b). On the bottom of the channel the stream function ψ is chosen such that ψ = 0, therefore on the free surface, let ψ = U h. Also investigated is when a single depression occurs on the bottom surface of a channel. The depression is assumed to be located in the region X1 X X 2 and the maximum depression height also occurs in this region. At X X F, the fluid flow is assumed to be uniform and the fluid depth again reaches h, figure 1(b). For the two problems, the equation governing the fluid motion is the Laplace equation, and may be expressed as 2 2 φ φ + = 0, - < X <, Yb < Y < Y 2 2 f (1) X Y where Y b and Y f are the bottom surface and the free surface heights respectively. This equation is solved subject to the boundary condition φ = 0, where n is the outward normal to the free surface and the bottom n φ surface boundaries. In addition, is specified at large distances both X upstream and downstream of the step namely, at X E and X F. Egerton J. Sci. & Technol. Volume 13: ISSN No

134 Gathia and Lonyangapuo 129 y 1 A N M D 1 Y f 1 U U h Y b F h s D A E x y (a) A step U 1 A N M D 1 h Y f 1 U x A E Y b h s D F (b) A depression Egerton J. Sci. & Technol. Volume 13: ISSN No

135 130 Free surface Flow in Open Channels over Complex Shapes y 1 A N U τ M D 1 h ψ = 0 α = β D A E F φ (c) w-plane η α = β τ A 1 A E D D 1 F φ (d) t-plane Figure 1: (a) and (b) show a schematic diagram of the physical problems and the notation used, while ( c ) and (d) show the infinite plane and the upper plane respectively. A rectangular coordinate system Z = X +iy is introduced such that the X axis is horizontal and the Y-axis is vertically upwards. A velocity potential φ and a stream function ψ is introduced such that the complex potential W = φ +iψ is an analytic function in the domain occupied by the fluid. Bernoulli s equation at any point in the fluid flow in the channel is given by 2 2 P U p U + + h = + + Y g 2g g 2.(2) ρ ρ g Egerton J. Sci. & Technol. Volume 13: ISSN No

136 Gathia and Lonyangapuo 131 where U is the velocity far upstream, P is the atmospheric pressure, ρ is the fluid density, g is the gravitational acceleration, U and P are the velocity and pressure respectively at any general point in the fluid flow. At the free surface on which P = P, equation (2) gives the free surface non- dimensional fluid velocity, that is, u f U = U f = 2 [ 1+ (1 y 2 f Fr )] (3) where Y f y f = is the non-dimensional free surface height and Fr = h U gh is the upstream Froude number. Next, the complex velocity which is expressed as dw dz iα = Ue (4) where α is the angle the fluid velocity vector makes with the positive X- axis. On the bottom and the free surface, α becomes β and θ respectively. Then the logarithm of the complex fluid velocity is given by 1 dw Ω = ln( ) = τ iα... (5) U dz U dw where τ = ln( ) and τ,α, and Ω are the analytical functions in an U dz infinite strip of the flow field, figure 1(c ). This infinite strip is transformed onto the upper half plane of the auxiliary plane by applying a conformal mapping function derived by use of the Schwartz- Christoffel transformation, figure 1(d). Equation (5) is in the form of a Riemann-Hilbert problem whose solution is obtained by using the Riemann-Hilbert method U b with, τ = ln( ) and α = θ being unknown quantities on the bottom and U the free surface respectively. The Extremum Pressure Method and Numerical Procedure The required pressure functional developed in this thesis is obtained from Bernoulli s equation (2) in the form: Egerton J. Sci. & Technol. Volume 13: ISSN No

137 132 Free surface Flow in Open Channels over Complex Shapes 2 2 Fr 2 PFr = H y 2 u..(6) X Y U P where P = 0 and the transformations x =, y =, u =, p = h h U 2 ρu 2 Fr and H = + 1 are applied. Other non-dimensional quantities obtained 2 following this transformation are: X 1 X 2 X E X F X b U b x1 =, x2 =, xe =, xf =, xb =, ub =, ψ = h h h h h U U Ψh and φ =. U Φh On using the irrotational conditionu = φ, the total fluid pressure is obtained by integrating equation (6), to get 2 Fr 2 F = H y ( φ) dv 2 V (7) xf xf Fr φ = Hy f y f dx + Hyb + yb dx φ ds n where xe F = V PFr 2 dv enclosing the volume of the fluid V between and lower surfaces. xe is the total pressure functional, S denotes the surface x = xe, xf x = and the upper The last term in equation (7) has been obtained by use of the Divergence 2 theorem and φ = 0. Then the integral over the surface S may be expanded as follows: φ φ φ φ φ φ ds = φ dy + φ ds + φ ds + φ dy n n n n n s in bot free out.(8) φ φ = φ dy + φ dy n n in out where the notation in, free, bot and out are used to show the integration across the upstream inlet flow, the free surface, the bottom surface and across the outlet respectively. The last expression in equation (8) is arrived Egerton J. Sci. & Technol. Volume 13: ISSN No

138 Gathia and Lonyangapuo 133 at since on both the free and the bottom surfaces the fluid does not penetrate φ the boundary, that is, = 0. Using the fact that the constant integral sum n involving y in expression (7) does not affect the position of the extremum f φ of the functional, φ and are known constants at the inlet, then equation n * (7) reduces to finding the extremum of the modified functional F which is given by F * = xf xe Hy b y 2 b Fr dx 2 2 φ φ dy (9) n out A minimization method which is based on the Quadratic Programming (QP) algorithm is used which minimizes any smooth nonlinear functional subject to a set of pre-assigned constraits on the independent variables, y b and a set of nonlinear constraits. The parameters y min max b, y b, Fr, 1 and 2 play a significant role in the min max solution technique for this problem where y b and yb are the lower and upper bounds on the independent variable y b respectively and 1 and 2 are accuracy parameters. Using the mean value theorem, the numerical integration of equation (9) becomes: n * H min F = - { yb ( i) yb ( i 1) } { yb ( i) yb ( i 1) } n y R b i= (10) 2 Fr k [ xb ( i) xb ( i 1) ] φb ( xf ) ub ( xf )[ y f ( xf ) yb ( xf )] 2 * where F is a nonlinear functional, n is the total number of grid points used, ( x b, yb ) is the position of the bottom surface, φ b ( x F ) and u b ( x F ) are the values of the velocity potential and the fluid velocity respectively on the bottom surface at the far downstream infinity position. This equation is solved subject to the simple bounds min max y y y, x x x..(11) b b b E and the nonlinear constraits [ y k ( i) ] y c ( i) [ y k ( i) + ],i = 1,2, ,n.. (12) f 1 f b 2 b F Egerton J. Sci. & Technol. Volume 13: ISSN No

139 134 Free surface Flow in Open Channels over Complex Shapes k c where n is the total number of nonlinear constraits used, y f and y f are the known and the computed free surface height, respectively. If in solving c k equations (10) (12) the condition that y f be approximately equal to y f is not satisfied, then there is no feasible solution to the nonlinear constraits and therefore the accuracy parameters 1 and 2 may have to be relaxed. In all the problems investigated in this paper while the value of 2 is fixed at zero, the value of 1 is allowed to vary until a particular small value is identified that = 2 0 corresponds to when there in no feasible solution to the nonlinear constraits. Letting means the computed free surface height not allowed to be greater than the known free surface height c y f is k y f. For the present problem, we found that the best values of 1 lie in the range The constrained minimization problem (10) is solved 1 opt numerically using MATLAB until an optimum solution denoted by y b is obtained or until a local minimum is attained and no further improvement can be made. est The choice of the initial estimate for the height of the bottom surface, y b is not crucial here. Whether or not a solution may be obtained which is in this feasible region. This is in contrast with the case considered in Wen et al (1987), where the choice of the independent variable β was in a feasible region and the regularization parameter β max was well identified. In this investigation we start with a rough guess of the initial estimate of the bottom surface and then find by the direct approach the corresponding initial height of the free surface y. Then by choosing the first predicted int f solution as the next initial estimate, with 1 taking on a smaller value than that used for the first approximation can progressively improve on the predicted solution until an optimal solution opt y b occurs when the computed free surface height the known height y is attained. This solution opt b c y f is in agreement with k y f to the desired accuracy controlled by the value of 1. Fluid Flow over a Step The minimum or maximum pressure method is now applied to the first example, namely, the case when the bottom solid obstacle is a step. The analytical geometry for such a bottom surface is given as Egerton J. Sci. & Technol. Volume 13: ISSN No

140 Gathia and Lonyangapuo 135 y b = b 0, x E x < 0 (13) hs h yb = / x 1- cos( bπ ) xb L L 0.(14) 2 hs yb =, L < x b xf (15) h By using the direct approach, with h s = 0. 4, L = 2.0, Fr = 2.0 and m = 40, h k k the numerical free surface position, ( x f, y f ), the fluid speed on the free k surface u f and the potential function along the free surface φ k f are obtained. Identical results are obtained when infinity positions are taken to lie in the ranges 7 x E 3 and 5 x F 15. Therefore for all the results presented in this paper, x = 3 and x = 5. E By observing the profile of the given free surface, we conclude the possible position of the obstacle to lie in the range 2 x b 4. Hence we prescribe the domain of the step to be constrained between x 1 and x 2 with x 1=-2, x2 int =4.0 and therefore L = 6. 0 where the superscript int is used inorder to discriminate between L int and L where L is the true step length. Similar results are obtained when the possible position of the step is not dependent on observing the free surface profile, that is, the range is the whole domain xe xb xf so that x 1 = xe, x 2 = xf and L int = xf xe. Then the simple constraints given by equations (13) (15) now take the form y b = 0, - 3 xb < 2.(16) min max y b yb y b, -2 x b 4...(17) hs yb =, 4< xb 5...(18) h where y min max hs b = 0 and yb = although bounds of y min b = and have also h been successively used. It is appropriate to investigate the functional for * both minima and maxima. First we solve F prescribed in equation (10) with the initial estimate for the height of the bottom surface given by est y = 0, -3 x 5... (19) b b F Egerton J. Sci. & Technol. Volume 13: ISSN No

141 136 Free surface Flow in Open Channels over Complex Shapes An iterative procedure is used to solve equations (3), (5), (10) (11) with 1 = 10 3 and = for the first and second computed solutions respectively. The optimal value of F min for the two sets of solutions are and respectively which are both very close to the numerical value obtained using the analytical expression for yb, namely, It is 4 seen that these values of F min differ from the later by and respectively. This indicates a gradual improvement towards the numerical value found using the analytical yb as the value of 1 decreases. The sigh of the functional is now changed so that the minimization process produces the required maximum solution following a simple sigh change to the final result. The predicted value of Fmax obtained for both solutions is and they differ from the numerical value by The profiles of these results are compared with the analytical one, and it is seen that for both Fmin and Fmax the predicted profiles for both the free and bottom surfaces are graphically indistinguishable from their known profiles. The results for this problem show that both minima and maxima extrema can be used to accurately determine both the shape and the position of the unknown step. Fluid Flow over a Single Depression The extremum total pressure technique is applied to the case when the geometrical configuration of the bottom surface is a depression, that is, y b = 0, -3 x b < 0..(20) hsh 2xbπ yb = [1 cos( )], 0 x b L.(21) 2 L y = 0, L<x 5 (22) b b where h s / h is taken to be 0.4 and as in the previous example, we take xe = 3, L = 2.0, Fr = 2.0, m = 40 and x F = 5 to find the analytical known k position for the free surface y f. Again by observation of the profile of the given free surface,then the possible position of the obstacle is found to lie in the range 2 x b 4, so that x1 and x2 now assume the values 2 and 4 int respectively and hence L = 6.0. The extremum functional F is defined by equation (20), the simple and the nonlinear costraints are given by equations (21) and (22), respectively, whilst the initial estimate of height of the lower min max surface, and especially around the region occupied by the y b y b Egerton J. Sci. & Technol. Volume 13: ISSN No

142 Gathia and Lonyangapuo 137 min max depression are not known a priori and therefore assign both y b and y b to large negative and positive values respectively. For the free surface fluid flows where the free surface is explicitly defined but the bottom surface min max k height is unknown, it is appropriate to take y b = and y b = max( y f ). As in the preceding investigation, the computed solutions are obtained by taking 1= 10 3 and 1= 10 4 respectively. The minimization problem (20) int min (22) is solved when 2 = 0, L = 6. 0, Fr = 2.0, y b =, max k y = max( ), m = 40 and n = 20, Behr,M (2001). b y f The results obtained are optimal but the predicted values of are not in agreement with the analytical solution. The values of F min obtained are and and those values do not compare well with the numerical value obtained using the analytical expression for y b, namely, These values differ from the numerical value obtained using the analytical 3 4 expression for by and respectively. y b F max It is observed that is able to identify the exact shape and position of the analytical bottom surface while F min cannot. This shows that inorder to retrieve the shape of a single depression, maximize the pressure functional F has to be maximized and not minimized. Numerical Results and Discussion In order to illustrate the computational procedure developed in this paper, the numerical calculations for the direct approach were performed on an h obstacle of one-dimensional fixed height s such that 0. 6 and for h h a given free surface profile with the upstream Froude number greater than one. These conditions were considered so that the fluid flow over the obstacle always remained supercritical. In the direct approach, the angle that the solid bottom surface makes with the horizontal, β, is found analytically. The angle β plays a crucial role in the determination of and therefore it is necessary to calculate its value accurately. In this study, the values of β is generated numerically, that is, by using central differences so that the numerical free surface profile is slightly different from that obtained using the analytical expression for y b but the agreement improves as the number of grid points increases. For the two particular Egerton J. Sci. & Technol. Volume 13: ISSN No h s y b c y f

143 138 Free surface Flow in Open Channels over Complex Shapes examples considered in this paper, all the information about the free surface height y is found from the direct approach and hence the analytical form k f of the solid bottom surface is known. This information is useful when discussing the accuracy of the predicted solution. 1.0 y 0.8 (1) Numerical profiles for both surfaces 0.6 (2) Profiles for the initial estimate 0.4 (3) First predicted profiles, = (4) Second predicted profiles, = x Figure 2: The free and bottom surface profiles for the fluid flow over a depression when F defined by equation (21) is minimized with 1= 10 3 and 2 = The parameter values are 0, L int 2 = = 6.0, m = 40, n =20, Fr = max k 2.0, y = max( ) and infinity conditions at x = -3 and x = 5 respectively. b y f Figure 2 also shows comparison of the analytical bottom surface profile, the free surface profile and the initial estimate profiles for both surfaces. It is observed that when using the profile value of = 10 3, the solution is considered as being converged and it is this value that has been taken in most of the calculations presented in this paper although numerous checks have been made to check whether this is appropriate. Egerton J. Sci. & Technol. Volume 13: ISSN No

144 Gathia and Lonyangapuo 139 y (1) Numerical profiles for both surfaces 0.6 (2) Profiles for the initial estimate 0.4 (3) First predicted profiles, = (4) Second predicted profiles, = x Figure 3: The free and bottom surface profiles for the fluid flow over a depression when F defined by equation (3) is maximized with 1= 10 3 and 2 = The parameter values are 0, L int 2 = = 6.0, m = 40, n =20, Fr = min max k 2.0, y b = y b = max( y f ) and infinity conditions at x = -3 and x = 5 respectively. Figure 3 shows comparison of the analytical bottom surface profile, the numerical free surface profile and the initial estimate profiles for both surfaces. The convergence criterion applied in all the results presented in this paper is that the maximum difference between the values of the velocity potential at all the points on the free surface corresponding to two consecutive iterations is less than some small prescribed quantity called the tolerance. Figure 2 represents the free surface profiles which are generated by fluid flows over the step given by equation (21) for 0 x L. It is observed that the height of the free surface increases and there is a symmetrical rise and fall in the free surface elevation as the fluid passes over the step. This result is in good agreement with similar results obtained by King and Bloor (1987) for the free surface fluid flow over a hump. It is observed that an increase in the step length is accompanied by an increase in b Egerton J. Sci. & Technol. Volume 13: ISSN No

145 140 Free surface Flow in Open Channels over Complex Shapes the length of the perturbed surface and the maximum surface height. It is also observed that each curve follows a similar path until it reaches the vicinity of the step. Beyond the step, the numerical calculations broke down when the value of the step height exceeds the value of and therefore conclude that the critical non-dimensional step height is h h s = When the function of the step is given by equation (21), then the maximum non-dimensional step height for which convergent solutions are found was about and for L =1.5 π and 2π respectively and that is the reason why in this paper these two situations have been considered in detail. It is seen that just as in the situation when L = π, there is a region over the hump where the local Froude number is less than one. Conclusion The position and shape of obstacles on the bottom surface of a channel are investigated by establishing the extrema of the pressure functions in a twodimensional, steady, inviscid, incompressible and irrotational fluid flow under the effects of gravity has been investigated. It is evident from results obtained that the numerical model introduced is good enough in identifying the shape and position of an obstacle that has a monotonically increasing file regardless of the nature of the extremum. However, in the case of the free surface values are simulated from a depression of the lower surface, then only an extremum possessing the nature of a maximum can succeed as the minimization fails to retrieve the required configuration. To conclude, for free surfaces corresponding to flow over monotonically increasing steps or over single depression, the maximization of the pressure functional is capable of retriving the geometrical shape of the lower boundary. References Behr, M.( 2001). Stabilized space-time finite element formulations for freesurface flows. Communications in Numerical Methods in Engineering. 17: Chang, Y. C., Hou, T.Y, Merriman,B, and S. Osher,S(1996). A level set formulation of Eulerian interface capturing methods for incompressible fluid flows. Journal of Computational Physics. 124: Farhat, C., M. Lesoinne,M. and Maman, N.( 1995). Mixed explicit/implicit time integration of coupled aeroelastic problems: Three-field Egerton J. Sci. & Technol. Volume 13: ISSN No

146 Gathia and Lonyangapuo 141 formulation, geometric conservation and distributed solution. International Journal of Numerical Methods in Fluids. 21: Hirt, W., and Nichols, B.D. (1981). Volume of fluid (VoF) method for the dynamics of free boundaries. Journal of Computational Physics. 39: King, A.C and Bloor, M.(1987). Free surface flow for a stream obstruction by an arbitrary bed topography, Quart. J. Mech. Applied. Method. 43: Lonyangapuo, J.k, Elliot, L and Ingham(1998). Identification of the shape of a channel from a given free surface profile. Computational Mechanics Publ. UK, Muskhelishvilli, N.I., (1953). Singular integral equations, Holland. Tezduyar, T. E., Behr. M, and Liou, J., (1992). A new strategy for finite element computations involving moving boundaries and interfaces the deforming-spatial-domain/space-time procedure: I. The concept and the preliminary tests. Computer Methods in Applied Mechanics and Engineering. 94: Wen. X, Ingham, D. B. and Widodo, B., (1997). The free surface fluid flow over an open channel. Engineering Analysis with Boundary Elements. 9: Egerton J. Sci. & Technol. Volume 13: ISSN No

147 Influence of Feeding Fat During Lactation on Growth Performance of Pups and Maternal Organ Morphology in Laboratory Mice Kagya-Agyemang, J. K. 1, 2, Król E. 1 and Speakman J. R. 1 1 School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK 2 Department of Animal Science Education, University of Education, Winneba, P. O. Box 40, Mampong-Ashanti, Ghana *Author for correspondence ( jkagyaagyemang@yahoo.com) Submitted 16 th Aug. 2012; Revised 21 st March 2013; Accepted 15 th May 2013 Abstract Laboratory mice (strain MF1) were used as a model to investigate the influence of dietary fat intake on lactation performance and maternal organ morphology. High fat (60%), medium fat (45%) and low fat (10%) diets were fed ad libitum to mice between days 4 and 18 of lactation. Low fat (LF) was used as the control diet and each diet contained 20% protein. Mice fed on high fat (HF), medium fat (MF) and low fat (LF) diets reached asymptote in their daily food intakes at 14.95±1.14 g day -1, 16.30±0.61 g day -1 and 16.57±0.26 g day -1 respectively between days of lactation. At weaning, litters from HF and MF-fed mice were significantly heavier (109.33±27.31 g and ±20.02 g) than pups on LF diet (80.85±20 g). That was because the HF and MF-fed mice did not only consumed more energy (306.52±25.03 kj day -1 and ±13.49 kj day -1 ) at peak lactation but also delivered more milk energy (203.20±49.92 kj day -1 and ±42.21 kj day -1 ) to their pups than the LF-fed mice (164.60±30.59 kj day -1 ). Evidence suggests that the positive effects of feeding fat to mice were in part due to the low specific dynamic action (SDA) and probably low heat production for milk synthesis. Probably, the ability of the HF and MFfed mice to directly transfer absorbed fat into the milk might have reduced the heat production of lactogenesis. The mice made morphological changes to cope with the high fat intake by way of increased mass of the stomach and liver and fat deposition around the liver and gonads. HF and MF diets had beneficial effects on lactation performance because they increased the capacity of mice to generate milk more efficiently and weaned heavier offspring than mice fed LF diet. Key words: Dietary fat, Apparent digestibility, Milk energy output, Lactation performance, Laboratory mice, Maternal organ morphology. Egerton J. Sci. & Technol. Volume 13: ISSN No

148 Influence of Feeding Fat During Lactation on Growth Performance of Pups 143 Introduction Energy is a key resource and limits on its availability and expenditure have a potentially profound effect on the evolution of many morphological and behavioural traits (Stenseth et al., 1980; Speakman and McQueenie, 1996). The sustained maximal rate of energy intake (SusEI) is an important factor that imposes an upper limit on animal performance that depends critically on energy, such as reproductive output. This is because reproduction is known to be energetically expensive (Thompson, 1992; Hammond and Diamond, 1997; Bryan and Bryant, 1999; Johnson et al., 2001a; Bacigalupe and Bozinvic, 2002; Król and Speakman, 2003a,b; Król et al., 2007) and thermoregulatory capabilities, which may define the global distribution limits of endotherms (Root, 1988; Bozinovic and Rosenmann,1989; Bacigalupe and Bozinovic, 2002). SusEI is the maximum rate of energy intake that animals can sustain over protracted period of days to weeks so that energy demands are fuelled only by food intake (Speakman and Król, 2005). Limits on SusEI are likely to be particularly important during peak lactation which is the time of greatest energy demand on female mammals (Hammond and Diamond, 1994; Speakman and McQueenie, 1996; Rogowitz, 1998; Johnson and Speakman, 2001; Johnson et al., 2001a, b, c; Król and Speakman, 2003a; Król et al., 2007). Limitations on SusEI at this time may determine the total investment that mammals can make in their offspring and may therefore define maximum litter and offspring sizes, which are important life history traits (Johnson et al., 2001a, b, c, Król et al., 2007). The specific dynamic action (SDA) of diets with high carbohydrate content is higher than those with high fat content (Kriss et al., 1934, Gawecki and Jeszka, 1978; Kagya-Agyemang et al., 2010). Based on the difference in SDA, it could be predicted that mice fed a high fat/medium fat diet should be able to consume more food at peak lactation with positive effects on milk production and growth of their offspring. The present study was designed to feed diets with constant protein but variable contents of fat and carbohydrate to MF1 mice between days 4 and 18 of lactation at 21 C. Previous studies have focused on energy requirements without considering that the limits on growth of offspring might be due to fat content. Hence, the maximal intake of a high/medium fat diet could be set by lower heat production which translates to faster growth of offspring. In view of this, the present study examined two hypotheses. First, if SDA of high/medium fat diet was important, it should lead to a higher total energy intake when lactating mice were fed a high/medium fat diet. Second, if the fat content of a diet affects growth, the energy intake by lactating mice may be affected by changing fat contents and higher fat content will translate into greater offspring growth. Egerton J. Sci. & Technol. Volume 13: ISSN No

149 144 Kagya-Agyemang, et al., (2013) Materials and Methods Animals and Experimental Protocol Sixty virgin female mice (Mus musculus L.: out bred MF1) aged 9-10 weeks old (Harlan UK Limited, England) were used in this study. The animals were individually housed in cages (44 cm x 12 cm x 13 cm) with sawdust. Rat and mouse breeder and grower diet, (15.60 kj/g gross energy, 18.80% crude protein, 60.30% carbohydrate, 3.40% crude oil, 3.7% crude fibre, 90% dry matter and 3.80% ash, Special Diets Services, BP Nutrition, Witham, UK) and water were supplied ad libitum. The environment was regulated at 21 ºC (range 20 to 22 ºC) on a 12 L: 12 D photoperiod with lights on at 07:00 h. Baseline measurement of body mass was used to allocate mice into three experimental groups with 20 animals per group. Completely randomized design was used for the study. Each female was paired with a male for 11 days after which the males were removed. Thereafter, female body mass and food remaining in each hopper were weighed each morning between 08:00 h and 10:00 h, using a (Mettler Toledo, Switzerland) top-pan balance (± 0.01 g). Food intake was calculated from the difference between the amount of food provided and that left in the hopper. On the day of parturition (day 0 of lactation), no measurements were made on the lactating mothers and their pups. From days 1-18 of lactation, maternal body mass, litter size, litter mass and pup mortality were monitored daily. On days 2-3 of lactation, mothers from each experimental group were presented with either high fat (HF), medium fat (MF) or low fat (LF) diet while still supplied with standard rodent chow ad libitum. The diets used were HF diet (D12492) with 60% kcal from fat, MF diet (D12451) with 45% kcal from fat and LF diet (D12450B) with 10% kcal from fat (Research Diets, New Brunswick, NJ, U.S.A). The composition of experimental diets is shown in Table 1. From day 4 of lactation onwards, the animals were switched from the chow diet to HF, MF or LF diet exclusively. Low fat (LF) was used as the control diet. Maternal food intake was measured between days 5-18 of lactation. Food intake was not monitored when mixed diets were presented on days 1-4. All animals were maintained in accordance with the United Kingdom Home Office animals (Scientific procedures) Act Egerton J. Sci. & Technol. Volume 13: ISSN No

150 Influence of Feeding Fat During Lactation on Growth Performance of Pups 145 Table 1: Composition of macronutrient diets Diet code D12492 D12451 D12450B Diet High Fat Medium Fat Low Fat (Control) % kcal kcal kcal Fat Carbohydrate Protein Total Ingredients (g/kg diet) Casein L-cystine Corn starch Maltodextrin Sucrose Cellulose Soya oil Lard Mineral mix Dicalcium phosphate Calcium carbonate Potassium citrate Vitamin mix Choline bitartrate Gross energy (kj g -1 ) Source: Research Diets, New Brunswick, NJ, U.S.A Body Composition and Organ Morphology Body composition (fat and fat free mass) was derived from organ morphology data. On day 18 of lactation, 60 mothers (HF, N=20, MF, N=20, LF, N=20) were weighed and killed by CO 2 inhalation and immediately dissected. The brain, intrascapular brown adipose tissue, thyroid gland, liver, kidneys, lungs, heart, spleen, gonadal fat, gonads, abdominal fat, mesenteric fat, subcutaneous fat, pancreas, stomach, small intestine, and large intestine were removed. The remaining parts were divided into tail, pelage and carcass. The wet mass of tissues was recorded (± g; Ohaus Analytical Plus) and tissues dried (Gallenkamp oven at 60 ºC) for 14 days (Król and Speakman, 1999) and re-weighed to determine dry mass. Faecal Production and Gross Energy Analysis Total faeces produced by mice between days of lactation were collected. The faeces were separated from the sawdust. Samples of each macronutrient diet and faeces were weighed and dried (Gallenkamp oven at 60 ºC) for 14 days to obtain dry mass. These were subsequently analysed for gross energy content using bomb calorimetry (Gallenkamp Autobomb Adiabatic Bomb Calorimeter; Rowett Research Institute Analytical Services, Egerton J. Sci. & Technol. Volume 13: ISSN No

151 146 Kagya-Agyemang, et al., (2013) Bucksburn, Aberdeen, UK) using benzoic acid as the standard. The female dry mass food intake for each macronutrient diet (FI DM ; g) was calculated as: FI DM = (mass food given x dry mass content) (1) The apparent digestibility of dry mass food (d m ; %) and energy (d e ; %) were calculated following Król and Speakman, (2003a) as: FI DM DM f d = m 100 x, (2) FI DM (FI DM x GE food ) (DM f x GE faeces ) d e =100 x, (3) FI DM x GE food where DM f is dry mass of faeces (g) and GE is gross energy. Digestible energy intake was obtained by subtracting the energy measured in faeces from the gross energy intake as measured from daily food consumption. Doubly Labelled Water Measurements of Daily Energy Expenditure and Total Water Turnover The doubly labelled water (DLW) method was used to measure daily energy expenditure (DEE) from the elimination rates of 2 H (deuterium) and 18 O in females during peak lactation. The total water turnover (rh 2 O) from the elimination rates of 2 H was also calculated. Measures of DEE were made to determine the milk energy output (MEO) from the difference between metabolizable energy intake (MEI) and DEE (Król and Speakman, 2003b).The DLW measurements were conducted on 60 lactating females (HF, N=20 and MF, N=20 and LF, 20). On day 16 of lactation (between 8:00 h and 11:00 h) individual mice were weighed to ±0.01 g using a balance (Mettler Toledo, Switzerland) and labelled with an intra-peritoneal injection of approximately 0.2 g of water containing enriched 2 H (36.3 atoms%) and 18 O ( 59.9 atoms%). The syringe used to inject the DLW was weighed (± g; Ohaus Analytical Plus, Brookylin, USA) immediately before and after the injection to provide an accurate measurement of the amount of the isotope injected. Mice were placed back in their cages during the 1 h equilibration period (Speakman, 1997). An initial µl blood sample was collected by tail tipping 1 h after the injection, which was the time generally assumed to be required for the isotope to reach equilibrium (Król and Speakman, 1999). Blood samples were immediately flame-sealed into pre- Egerton J. Sci. & Technol. Volume 13: ISSN No

152 Influence of Feeding Fat During Lactation on Growth Performance of Pups 147 calibrated 50 µl pipettes (Vitrex, Camlab Limited, Cambridge, UK) and stored at 4 C until analysis. A final blood sample was collected approximately 48 h after the initial blood sample was collected to estimate isotope elimination rates. Samples of blood in capillaries were vacuumdistilled (Nagy, 1983) and water from the resulting distillate was used to produce CO 2 and H 2 (Speakman and Król, 2005). Gas source isotope ratio mass spectrometry was used to analyse the isotope ratios of 18 O: 16 O and 2 H: 1 H. The samples were run alongside high enrichment standards that were used to correct the raw data to these standards. For each lactating mouse, initial 2 H and 18 O dilution spaces were calculated by the intercept method (Coward and Prentice, 1985; Speakman and Król, 2005) and then converted to mass assuming a molecular mass of body water of and expressed as a percentage of body mass before injection. The intercept method was used since the actual body water pool estimated by desiccation using the intercept method was more accurate than the plateau method (Speakman and Król, 2005). The final 2 H and 18 O dilution spaces were inferred from the final body mass, assuming the same percentage of body mass as measured for the initial dilution spaces. The isotope elimination rate (k) was calculated following Nagy (1975). For calculation of DEE based on CO 2 production, single pool model equation 7.17 (Speakman, 1997) was used. Energy equivalents of rates of CO 2 production were calculated using a conversion factor of J ml -1 CO 2 derived from the Weir equation (Weir, 1949). Female total water turnover (rh 2 O) was calculated by multiplying the fractional turnover rate by the total body water (TBW). It was assumed that 25% of the water leaving the body was fractionated (Speakman, 1997). Therefore, a fractionation factor of was applied for deuterium (Speakman, 1997). This approach assumes that rates of water influx and efflux are constant, so rh 2 O = total water influx = total water efflux (Nagy and Costa, 1980). Milk Energy Output Milk energy output (MEO) was evaluated as the difference between metabolizable energy intake (MEI) and daily energy expenditure (Król and Speakman, 2003b). Statistical Analyses The differences in baseline and pregnancy data between the groups for body mass and food intake were analysed using repeated measures analysis of variance (ANOVA). The significance of changes in body mass and food intake over time was assessed by general linear modeling (GLM). Significant differences between days and diets were detected using the post-hoc Tukey test. Asymptotic food intake across the three dietary treatment groups in late lactation was compared using ANOVA. The asymptotic food intake in late Egerton J. Sci. & Technol. Volume 13: ISSN No

153 148 Kagya-Agyemang, et al., (2013) lactation was defined as the period during which no significant differences in food intake between days were detected. The difference in pup mortality for the HP, MF and HC diets was assessed using GLM. Least squares regression analysis was used to examine the relationship between milk energy output (MEO) and litter size, and litter growth and MEO. Organ morphology data was analysed using GLM and ANOVA. All statistical analyses were performed using Minitab for Windows (version 14; Minitab Inc., State College, PA, USA; Ryan et al., 1985). All statistical significance were determined at P<0.05. Results Maternal Body Mass The body mass of HF (29.51±1.29 g), MF (29.52±1.33 g) and LF mice (29.52±1.42 g) was not significantly different (P>0.05) before mating. Body mass increased significantly during days of pregnancy in HF (P<0.001), MF (P<0.001) and LF mice (P<0.001) reaching a peak of 53.96±4.18 g, 55.83±5.06 g and 56.28±5.24 g, for HF, MF and LF mice, respectively just before parturition (Figure 1). Mean body mass of mice in the three dietary groups was not significantly different during pregnancy (P>0.05) and averaged 44.74±7.04 g, 45.41±7.30 g, and 44.75±7.99 g for HF, MF and LF mice, respectively. Between days 1-4 of lactation when mice had a choice of standard rodent chow or their selected macronutrient diets, maternal body mass of mice in each group increased. Maternal body mass of HF, MF and LF mice did not show any significant difference (P>0.05) and averaged 41.39±3.82 g, 41.15±2.04 g, and 41.79±3.05 g on the day after parturition and increased to 42.67±3.18 g, 42.36±2.55 g, and 42.39±3.25 g on day 4 of lactation, respectively for HF, MF and LF mice. Female mice in all the three groups had a stable body mass between days 5-18 of lactation when the diet manipulation took place. The trend of change in body mass of HF, MF and HC mothers followed a similar pattern (Figure 1). There was a highly significant effect of day of lactation (P<0.001) on maternal body mass and also there was a highly significant effect of diet (P<0.05). However, there was no significant diet by day interaction (P>0.05). All Tukey pairwise comparisons among levels of diet showed that the LF-fed mice were significantly heavier (P<0.05) than HF and MF-fed mice between days 5-18 of lactation. The body mass of HF and MF-fed mice showed no significant difference (P>0.05). Egerton J. Sci. & Technol. Volume 13: ISSN No

154 Influence of Feeding Fat During Lactation on Growth Performance of Pups Baseline Pregnancy Lactation Body m ass (g) Males with females No measurements. Diet manipulation HF MF LF 0 Parturition Day of reproduction Figure 1: Mean body mass of mice throughout baseline, pregnancy and lactation. Day 0 was parturition. Diet manipulation started on day 4 of lactation. Mice were fed high fat (HF), medium fat (MF), and low fat (LF) diets. Maternal body mass was measured between days 1-18 of lactation. Error bars represent 1 sd of the mean Food Intake The food intake of mice in the three dietary groups was not significantly different before mating (P>0.05). The animals consumed a mean of 5.28±0.51 g day -1, 5.28±0.27 g day -1 and 5.27±0.36 g day -1 for HF, MF and LF mice, respectively. Food intake increased significantly during days of pregnancy in HF (P<0.001), MF (P<0.001) and LF mice (P<0.001) reaching a maximum of 8.16±1.15 g, 8.04±1.13 g and 8.49±1.09 g before decreasing to 6.94±0.40 g, 6.92± 0.59g and 7.20± 0.57 g for HF, MF and LF mice, respectively on the day before parturition (Figure 2). The mean food intake of mice in the three groups (6.54±1.05 g day -1, 6.29±0.94 g day -1, and 6.57±1.24 g day -1 ) was not significantly different throughout pregnancy (P>0.05). Diet manipulation started on day 4 of lactation, so there was no food intake data for days 1-4 of lactation when animals were fed on a mixture of rodent chow and the target diets. There was a significant increase in food intake between days 5-11 of lactation in mice fed HF (P<0.001), MF (P<0.001) and LF diets (P<0.001) from 10.45±1.86 g, 12.27±2.26 g and Egerton J. Sci. & Technol. Volume 13: ISSN No

155 150 Kagya-Agyemang, et al., (2013) 12.43±2.68 g on the day after the animals were fed macronutrient diets exclusively to 13.20±2.60 g, 14.89±2.39 g and 16.33±1.88 g on day 11, respectively. Over the next 6 days (days 12-17), daily food intake remained constant (P>0.05) at an average of 14.95±1.14 g day -1, 16.30±0.61 g day -1 and 16.57±0.26 g day -1 for mice fed HF, MF and LF diets, respectively. The food intake averaged over these 6 days was termed the asymptotic daily food intake (Figure 2). The corresponding asymptotic gross energy intakes were ±26.41 kj day -1, ±13.92 kj day -1 and ±4.64 kj day -1 (equivalent to digestible energy intakes of ±25.03 kj, ±13.49 kj and ±4.45 kj) for mice fed HF, MF and LF diets, respectively. On day 18 of lactation, the daily food intake increased above the asymptotic level (Figure 2). This was because the pups started feeding directly on the food in the hoppers on this day. Over all, there was a highly significant effect of day of lactation (P<0.001) on maternal food intake and a highly significant effect of diet (P<0.001) on maternal food intake between days 5-18 of lactation. All Tukey pairwise comparisons among levels of diet showed that the mass of food intake of LF mice was significantly higher (P<0.05) than that of HF and MF mice between days 5-18 of lactation. The food intake of HF and MF-fed mice was not significantly different (P>0.05). There was no significant interaction effect (P>0.05) on maternal food intake during lactation. Egerton J. Sci. & Technol. Volume 13: ISSN No

156 Influence of Feeding Fat During Lactation on Growth Performance of Pups Baseline Pregnancy Lactation 25 Food intake (g/day) Males with females No measurements Day of reproduction Diet manipulation Parturition HF MF LF Figure 2: Mean daily food intake of mice throughout baseline, pregnancy and lactation. Day 0 was parturition. Diet manipulation started on day 4 of lactation. Maternal food intake was measured between days 5-18 of lactation. Lactating mice were fed high fat (HF), medium fat (MF), and low fat (LF) diets. Error bars represent 1 sd of the mean Apparent Assimilation Efficiency and Digestibility of Energy The gross energy content of faeces was significantly different between HF, MF and LF lactating dams (P<0.001) and averaged 20.91±1.26, 20.96±0.59 and 15.93±0.70 kj g -1 dry mass for HF, MF and LF lactating dams, respectively. The gross energy content of faeces of HF and MF mice did not differ significantly (P>0.05) but this was significantly different (P<0.001) from that of LF mice. The energy content of the faeces was however lower than that of the food (19.90 kj g -1, kj g -1 and kj g -1 ) for HF, MF and LF mice, respectively. The mean apparent assimilation efficiency was 87.34±1.79%, 88.73±1.51% and 92.17±1.33%, between days of lactation for mice fed HF, MF and LF diets, respectively (P<0.001). The mean apparent assimilation efficiency of mice fed LF diet was significantly higher (P<0.05) than that of mice fed HF and MF diets, respectively. The mean apparent digestibility of energy was significantly different (P<0.001) Egerton J. Sci. & Technol. Volume 13: ISSN No

157 152 Kagya-Agyemang, et al., (2013) between the three dietary treatment groups during lactation (days 11-15) and averaged 88.54±1.62%, 89.68±1.38% and 92.99±1.19%, respectively for mothers fed on HF, MF and LF diets. The mean apparent digestibility of energy of LF mice was significantly higher (P<0.05) than that of mice fed HF and MF diets, respectively. Litter Size and Mass The HF, MF and LF dams gave birth to an average of 10.5±2.86 (range 3-15), 10.7±2.31 (range 7-14) and 12.2±2.39 pups (range 8-17), respectively and weaned an average of 10.3±2.77 (range 3-14), 10.6±2.38 (range 7-14) and 11.2±2.78 pups (range 4-16), respectively. The litter masses of HF, MF and LF pups increased from 19.18±4.87 g, 19.69±4.25 g and 21.48±3.22 g at day 1 of lactation to ±27.31 g, ±20.02 g and 80.85±20.0 g, respectively at weaning. The mean mass of individual pups of HF, MF and LF mothers increased from 1.85±0.18 g, 1.84±0.13 g and 1.78±0.17 g at day 1 of lactation to 10.98±2.59 g, 10.26±2.10 g, and 7.68±2.56 g, respectively over the same period of time. The mean number of pups of HF, MF and LFfed dams at day 1 of lactation were not significantly different (P>0.05) and they were also not significantly different (P>0.05) at weaning (day 18 of lactation). However, pups from dams fed HF and MF diets were significantly heavier than pup from dams fed LF diet (P<0.05) (Figure 3) at weaning. Pups from dams fed HF and MF diets were not significantly (P>0.05) different from each other in terms of mass (Figure 3) Litter mass (g) HF MF LF Age (days) Figure 3: Growth of pups from dams fed high fat (HF), medium fat (MF) and low fat (LF) diets between days 4-18 of lactation. Error bars represent 1 sd of the mean Pup Mortality Litter size at day 1 of lactation did not differ significantly between the three groups of mice (P>0.05). The HF, MF and LF dams lost 2, 1 and 17 offspring prior to weaning from 199, 204 and 220, respectively which were Egerton J. Sci. & Technol. Volume 13: ISSN No

158 Influence of Feeding Fat During Lactation on Growth Performance of Pups 153 born. Therefore, the mean number of offspring lost during the 18 days of lactation was 0.11, 0.06 and 0.94, respectively for HF, MF and LF dams, respectively. The number of pups lost by LF-fed dams was significantly higher (P<0.001) than that of HF and MF-fed dams. Doubly Labelled Water (DLW) Measurements of Daily Energy Expenditure (DEE) and Total Water Turnover of Lactating Mice The DEE measured on day 16 of lactation was not significantly different (P>0.05) between lactating mice fed on HF, MF and LF diets and averaged ±12.64 kj day (range kjday -1 ), ±9.71 kj day -1 (range kjday -1 ) and ±14.29 kj day (range kjday -1 ), respectively (Table 2). The total water turnover (rh 2 O) of lactating mice fed on MF, HF and LF diets averaged 23.59±6.26 g day -1 (range g day -1 ), 24.12±588 g day -1 (range g day -1 ) and 26.76±6.58 g day -1 (range g day -1 ), respectively (Table 2). Comparison of the DEE of lactating mice in the three dietary groups using body mass as a covariate showed significant (P<0.05) diet effect. Table 2: Results of the doubly labelled water measurements of energy expenditure and water turnover (rh 2 O) in lactating HF and MF fed mice and lactating controls fed on LF diet Trait Lactating mice ANOVA HF mice MF mice LF mice F 2, 49 P BM (g) a 45.85± ± ± k d (h -1 ) b 0.031± ± ± k o (h -1 ) c 0.041± ± ± k o /k d 1.330± ± ± N d (% of BM) d ± ± ± N o (% of BM) d ± ± ± N d /N o 1.049± ± ± DEE (kj day -1 ) e ± ± ± rh 2 O (g day -1 ) f ± ± ± Values are means ± S.D. a Body mass before injection; b deuterium elimination rate; c18 O elimination rate; d deuterium (N d ) and 18 O (N o ) dilution spaces (moles) were converted to g assuming a molecular mass of body water and were expressed as percentage of body mass before injection; e daily energy expenditure; f total water turnover. HF, high fat, MF, medium fat and LF, low fat Milk Energy Output The milk energy output (MEO) calculated on day 16 of lactation was significantly different (P<0.001) between lactating mice fed HF and MF diets and and those fed LF diet and averaged ±49.92 kj day -1 (range kjday -1 ), ±42.21 kj day -1 (range kjday -1 ) and ±30.59 kj day (range kjday -1 ), respectively. Tukey Egerton J. Sci. & Technol. Volume 13: ISSN No

159 154 Kagya-Agyemang, et al., (2013) pairwise comparisons among levels of diet indicate that MEO of both MF and HF mice were significantly higher (P<0.05) than that of LF mice. The MEO of MF mice was not significantly different (P>0.05) from that of HF mice. The relationship between growth of litters from HF, MF and LF mice and MEO showed a highly significant effect of MEO on litter growth (P<0.001) as well as a highly significant effect of the group (P<0.001). 350 Milk energy output (kj/day) Litter size Figure 4: Milk energy output as a function of litter size for lactating dams fed on HF diet (filled circles, y=-0.52x , r 2 =0.001), MF diet (open circles, y=2.98x , r 2 =0.033), and control dams fed on LF diet (square symbols, y=-2.48x , r 2 =0.051) Litter growth (days 6-18) Milk energy output (kj/day) Figure 5: Litter growth as a function of milk energy output for lactating dams fed on HF diet (filled circles, y=0.29x + 765, r 2 =0.737), MF diet (open circles, y=0.20x , r 2 =0.437), and control dams fed on LF diet (square symbols, y=0.19x +8.14, r 2 =0.362) Effect of Macronutrient Diets on Maternal Organ Morphology To evaluate the effect of the HF, MF and LF dietary treatments on maternal organ morphology, the dry masses of 20 organs were compared. The wet masses and standard deviations of all the tissues removed from the mice in each group are as shown in Table 4. Analysis of variance (ANOVA) showed that there were significant differences in the masses of only the gonadal fat, Egerton J. Sci. & Technol. Volume 13: ISSN No

160 Influence of Feeding Fat During Lactation on Growth Performance of Pups 155 stomach and liver (Table 4). The mean total wet mass of the HF, MF and LF mice were not significantly different (P>0.05) (Table 3). The dry masses and standard deviations of all the tissues are shown in Table 5. There were significant differences between groups in the masses of gonadal fat, stomach and liver. When compared with the mice fed on LF diet, the HF mice deposited fat mass of g day -1 (equivalent to 0.57 kj day -1 ) into the gonadal fat. The MF mice also deposited fat mass of g day -1 (equivalent to 0.53 kj day -1 ) into the gonadal fat. Comparison of the dry tissue masses of mice in the three dietary groups using body mass a covariate also showed that only the masses of gonadal fat, stomach and liver were significantly different (Table 5). ANOVA showed that the mean total dry mass of HF and MF mice was not significantly different from that of the mice fed LF diet (Table 3). The body water mass and the percent body water content of HF, MF and LF mice were not significantly different (Table 3). Table 3: Overall body composition and organ morphology in lactating mice fed on macronutrient diets at 21 C Macronutrient diets ANOVA Trait HF MF LF F 2, 53 P Body mass (g) ± ± ± Total wet mass (g) ± ± ± Total dry mass (g) ± ± ± Body water mass (g) ± ± ± Body water content (%) ± ± ± HF, high fat; MF, medium fat; LF, low fat Values are means ±SD. Egerton J. Sci. & Technol. Volume 13: ISSN No

161 156 Kagya-Agyemang, et al., (2013) Table 4: Wet organ masses in lactating mice fed on macronutrient diets at 21 C Macronutrient diets ANOVA GLM Organ HF MF LF F 2, 53 P F 2, 52 P Su fat 5.359± ± ± Mefat 0.369± ± ± Ab fat 0.505± ± ± Go fat 0.566±0.265 a 0.520±0.182 a 0.378±0.230 b BAT 0.272± ± ± Sto 0.265±0.051 b 0.295±0.079 b 0.317±0.060 a SI 1.176± ± ± LI ± ± ± Gon 0.263± ± ± Thy 0.205± ± ± Spl 0.184± ± ± Pan 0.380± ± ± Liv 3.289±0.544 a 3.077±0.702 b 3.086±0.562 b Kid 0.614± ± ± Hea 0.286± ± ± Lun 0.426± ± ± Bra 0.423± ± ± Tai 1.001± ± ± Pel 4.095± ± ± Car ± ± ± Su fat, subcutaneous fat; Me fat, mesenteric fat; Ab fat, abdominal fat; Go fat, gonadal fat; BAT, brown adipose tissue; Sto, stomach; SI, small intestine; LI, large intestine; Gon, gonads; Thy, thyroid; Spl, spleen; Pan, pancreas; Liv, liver; Kid, kidneys; Hea, heart; Lun, lungs; Bra, brain; Tai, Tail; Pel, pelage; Car, carcass. HF, high fat; MF, medium fat; LF, low fat. Values are means ±SD. For organs with significant P values (bold type), different letters indicate significant differences between the groups, as assessed by the Tukey pairwise comparisons. The P values in bold type are significant after Bonferroni correction (P=0.05 divided by 20 comparisons becomes P=0.003). Egerton J. Sci. & Technol. Volume 13: ISSN No

162 Influence of Feeding Fat During Lactation on Growth Performance of Pups 157 Table 5: Dry organ masses in lactating mice fed on macronutrient diets at 21 C Macronutrient diets ANOVA GLM Organ HF MF LF F 2, 53 P F 2, 53 P Su fat 2.059± ± ± Mefat 0.203± ± ± Ab fat 0.284± ± ± Go fat 0.430±0.210 a 0.415±0.14 a 0.224±0.151 b BAT 0.151± ± ± Sto 0.075±0.012 b 0.101±0.03 a 0.097±0.018 a SI 0.304± ± ± LI 0.098± ± ± Gon 0.098± ± ± Thy 0.060± ± ± Spl 0.040± ± ± Pan 0.139± ± ± Liv 0.968± b 0.844±0.159 c 1.139±0.216 a Kid 0.149± ± ± Hea 0.067± ± ± Lun 0.094± ± ± Bra 0.092± ± ± Tai 0.420± ± ± Pel 1.748± ± ± Carc 5.207± ± ± Su fat, subcutaneous fat; Me fat, mesenteric fat; Ab fat, abdominal fat; Go fat, gonadal fat; BAT, brown adipose tissue; Sto, stomach; SI, small intestine; LI, large intestine; Gon, gonads; Thy, thyroid; Spl, spleen; Pan, pancreas; Liv, liver; Kid, kidneys; Hea, heart; Lun, lungs; Bra, brain; Tai, Tail; Pel, pelage; Car, carcass. HF, high fat; MF, medium fat; LF, low fat. Values are means ±SD. For organs with significant P values (bold type), different letters indicate significant differences between the groups, as assessed by the Tukey pairwise comparisons. The P values in bold type are significant after Bonferroni correction (P=0.05 divided by 20 comparisons becomes P=0.003). Discussion The body masses of mice increased significantly during pregnancy (Figure 1). During lactation the HF and MF-fed mice also increased in body mass (Figure 1). The increase in body mass of the mice was associated with their high energy intake. Contrary to previous studies (Hammond and Diamond, 1994; Speakman and McQueenie, 1996), the mice studied by Johnson et al. (2001a) and the mice in the present study did not continue to increase their food intake until the end of lactation, but instead reached an asymptote from days 12-17, respectively (Figure 2). In the study by Johnson et al. (2001a) when the MF1 mice were fed rodent chow only, the asymptotic daily food intake was 23.1 g day -1 and the corresponding net energy intake was 301 kj Egerton J. Sci. & Technol. Volume 13: ISSN No

163 158 Kagya-Agyemang, et al., (2013) day -1. In the present study, the asymptotic daily food intake s (14.95±1.14 g day -1 and 16.30±0.61 g day -1 ) corresponding gross energy (345.40±26.41 kj day -1 and ±13.92 kj day -1 ) and net energy intakes (306.52±25.03 kj day -1 and ±13.49 kj day -1 ), litter mass at day 18 ( g and g) and milk production (203.20±49.92 kj day -1 and ±42.21 kj day -1 ) of HF and MF-fed mice were significantly higher than that of the LF-fed mice (164.60±30.59 kj day -1 ). The feeding of fat (Table 1) to lactating mice impacted positively on reproductive performance of the HF and MF-fed mice because the specific dynamic action (SDA) for HF (4.5%) and MF (3.9%) diets were significantly lower than that of the LF (6.1%) diet (Kagya- Agyemang et al., 2010). As a result, the HF and MF-fed mice consumed more energy (306.52±25.03 kj day -1 and ±13.49 kj day -1 ) at peak lactation so the energy available for milk production was greatly increased. Therefore, pups from dams fed HF and MF diets were heavier at weaning than those from LF-fed dams (Figure 3). Dietary fat elevates milk fat concentration in sows and promotes faster growth in piglets throughout lactation (Averette et al., 1999). Increasing the fat content in the milk increases the energy supply to offspring thereby improving survival (Pettigrew, 1981) and pup growth rate. This suggests that the fat intake of lactating mice is correlated with the growth of their pups (Rolls et al., 1984; Loh et al., 2002). In the present study, the faster growth rate of the pups from HF and MF-fed mice was associated with the high energy exported as milk to the pups (Figures 4 and 5). This corroborates the findings of Grigor and Warren (1980), Pettigrew (1981), Del et al. (1997), Averette et al. (1999), and Loh et al. (2002) that increasing the fat content in milk increases the energy supply to offspring, thereby improving their survival and growth rate. This is consistent with the idea that energy is the most important limiting factor during lactation, in that all of the energy provided to the young must first be consumed by the dam in addition to her own energy requirements to ensure reproductive success (Hanwell and Peaker, 1977; Oftedal, 1984; Kenagy et al., 1990; Speakman et al., 2001). In this study, positive effects occurred because the HF and MF-fed mice consumed much more food (14.95 g day -1 and16.30 g day -1 ) and energy ( kj day -1 and kj day -1 ) at peak lactation but their mean daily energy expenditure (DEE) (103.3 kj day -1 and kj day -1 ) was the same as that of the control mice fed LF diet ( kj day -1 ) (Table 2) so the energy available for milk production was greatly increased ( kj day -1 and kj day -1 ). Egerton J. Sci. & Technol. Volume 13: ISSN No

164 Influence of Feeding Fat During Lactation on Growth Performance of Pups 159 Fatty acids are the major form in which fat is made available as fuel for energy generation. The main ways by which lactating mice can increase the fat content of their milk to support the faster growth of their pups/offspring is through increased dietary fat intake and increased de novo lipogenesis. At weaning, litters from HF and MF-fed mice were significantly heavier (109.33±27.31 g and ±20.02 g) than pups on LF diet (80.85±20 g) (Figure 3). This was evidenced by the fact that the HF and MF-fed mice not only consumed more energy (306.52±25.03 kj day -1 and ±13.49 kj day -1 ) at peak lactation but also delivered more milk energy (203.20±49.92 kj day -1 and ±42.21 kj day -1 ) to their pups than the LF-fed mice (164.60±30.59 kj day -1 ). Probably, the HF and MF-fed mice had the ability to directly transfer absorbed fat into the milk to support the growth of their pups. In the present study, the LF diet contained 70% energy as carbohydrate. Conversion of carbohydrate to fat prior to oxidation is thermogenically costly and about 28% of the energy content of carbohydrate is lost as heat (Flatt, 1978; Hellerstein et al., 1996; Hellerstein, 1999). This indicates that the LF-fed mice had to expend part of energy consumed for de novo lipogenesis to produce enough milk to support the growth of their offspring. De novo lipogenesis is thermogenically costly and it is accompanied by energy expenditure of about 25% (Flatt, 1978). Overall, the growth of offspring from HF and MF-fed mice was better when compared with that of LF-fed mice. This shows that offspring growth improved on the HF and MF diets (Figures 3, 4 and 5). The mice in the different dietary treatments did not show significant differences in body mass and the overall daily energy expenditure did not differ between the three diets (Table 2). This shows that the positive effects of HF and MF diets on lactation performance were due to the ability of the HF and MF-fed mice to reduce the metabolic heat generated as a by-product of milk production (Kagya-Agyemang et al., 2010). It has been reported that feeding rats on a high fat diet depresses the rate of mammary gland lipogenesis (Agius and Williams, 1980; Grigor and Warren, 1980). Therefore, at peak lactation the HF and MF-fed mice assimilated more energy than the LF-fed mice and used the extra energy to generate more milk to support the faster growth of their offspring. The overall body composition and organ morphology in lactating mice fed on macronutrient diets at 21 o C did not differ from each other (Table 3). The lactating mice made morphological changes to cope with the high fat intake by way of increased mass of the stomach and liver and fat deposition around Egerton J. Sci. & Technol. Volume 13: ISSN No

165 160 Kagya-Agyemang, et al., (2013) the gonads (Tables 4 and 5). The MF-fed mice responded to changes in their reproductive states by increase in mass of the stomach to account for the large increase in food demands. Mass changes and lengthening of the gut and organs connected with food processing have been reported in several mammals during lactation (Myrcha, 1962, 1965; Campbell and Fell, 1964; Speakman and McQueenie, 1996; Król et al., 2003c). The HF-fed mice responded to changes in their reproductive state by increase in the wet liver mass (Table 4). Conclusion The MF1 mice fed on HF, MF, and LF diets reached an asymptote in their daily food intake at 14.95± 1.14 g day -1, 16.30± 0.61 g day -1 and 16.57± 0.26 g day -1 respectively, at peak lactation. At weaning, the pups from HF and MF-fed mice were significantly heavier than pups from LF-fed mice. This was because the peak lactation energy intake of HF and MF-fed mice was significantly higher than that of the LF-fed mice. Acknowledgement The financial support from the government of Ghana was very much appreciated. References Agius, L. Y. and Williams, D. H. (1980). Rapid inhibition of lipogenesis in vivo in lactating rat mammary gland by medium chain triacylglycerols and partial reversal by insulin. Biochem. J. 192: Averette, L.A., Odle, J., Monaco, M.H. and Donovan, S.M. (1999). Dietary fat during pregnancy and lactation increases milk fat and insulin-like growth factor I concentrations and improves neonatal growth rates in swine. J. Nutr. 129: Bacigalupe, L. D. and Bozinovic, F. (2002). Design, limitations and sustained metabolic rate: lessons from small mammals. J. Exp. Biol. 205: Bozinovic, F. and Rosenmann, M. (1989). Maximum metabolic rate of rodents- physiological and ecological consequences on distributional limits. Funct. Ecol. 3: Egerton J. Sci. & Technol. Volume 13: ISSN No

166 Influence of Feeding Fat During Lactation on Growth Performance of Pups 161 Bryan, S. M. and Bryant, D. M. (1999). Heating nest-boxes reveals an energetic constraint on incubation behaviour in great tits, Parus major. Proc. R. Soc. Lond. B 266: Campell, R. M. and Fell, B. F. (1964). Gastrointestinal hypertrophy in the lactating rat and its relation to food intake. J. Physiol. 171: Coward, W. A. and Prentice, A. m. (1985). Isotope method for the measurement of carbon dioxide production rate in man. Am. J. Clin. Nutr. 41: Del, P. M., Delgado, G. and Villalpando, S. (1997). Maternal lipid intake during pregnancy and lactation alters milk composition, production and litter growth in rats. J. Nutr. 127: Flat, J. P. (1978). The biochemistry of energy expenditure. Rec. Adv. Obesity Res. 2: Gawecki, J. and Jeszka, J. (1978). The effect of the extent of hydrolysis on casein on its specific dynamic action in the rat. Br. J. Nutr. 40: Grigor, M. R. and Warren, S. M. (1980). Dietary regulation of mammary lipogenesis in lactating rats. Biochem. J. 188: Hammond, K. A, and Diamond J. (1994). Limits to dietary nutrient intake and intestinal nutrient uptake in lactating mice. Physiol. Zool. 67 (1): Hammond, K. A, and Diamond, J. (1997). Maximal sustained energy budgets in humans and animals. Nutr. 386: Hanwell, A. and Peaker, M. (1977). Physiological effects of lactation on the mother. Symp. Zool. Soc. Lond. 41: Hellerstein, M. K., Schwarz, J-M. and Neese, R.A. (1996). Regulation of hepatic de novo lipogenesis in humans. Ann. Rev. Nutr. 16: Hellerstein, M. K. (1999). De novo lipogenesis in humans: metabolic and regulatory aspects. Eur. J. Clin. Nutr. 53: S53-S65. Johnson, M. S, Thomson S.C, and Speakman J. R. (2001a). Limits to sustained energy intake I. Lactation in the laboratory mouse, Mus musculus. J.Exp. Biol. 204: Johnson, M. S, Thomson S. C, and Speakman J. R. (2001b). Limits to sustained energy intake III. Effect of concurrent pregnancy and lactation in Mus musculus. J. Exp. Biol. 204: Egerton J. Sci. & Technol. Volume 13: ISSN No

167 162 Kagya-Agyemang, et al., (2013) Johnson, M. S, and Speakman J. R.(2001c). Limits to sustained energy intake V. Effect of cold exposure during lactation in Mus musculus. J. Exp. Biol. 204: Kagya-Agyemang, J. K., Król, E. and Speakman, J. R. (2010). Effects of macronutrient composition on specific dynamic action in the laboratory mouse, Mus musculus. Global J. Pure Appl.Sci. 16: Kenegy, G. J., Masman, D., Sharbaugh, S. M. and Nagy, K. A. (1990). Energy expenditure during lactation in relation to litter size in freeliving golden-mantled ground squirrels. J. Anim. Eco. 59: Kriss, M., Forbes, E. B. and Miller, R. C. (1934). The specific dynamic effects of protein, fat, and carbohydrate as determined with the albino rat at different planes of nutrition. J. Nutr. 8: Król, E, and Speakman J. R. (1999). Isotope dilution spaces of mice injected simultaneously with deuterium, tritium and oxygen-18. J. Exp. Biol. 202: Król, E, and Speakman J. R. (2003 a). Limits to sustained energy intake. VI. Energetics of lactation in laboratory mice at thermoneutrality. J. Exp. Biol. 206: Król, E, and Speakman J. R. (2003 b). Limits to sustained energy intake. VII. Milk energy output in laboratory mice at thermoneutrality. J. Exp.Biol. 206: Król, E, Johnson M. S, and Speakman J. R. (2003c). Limits to sustained energy intake. VIII. Resting metabolic rate and organ morphology of laboratory mice lactating at thermoneutrality. J. Exp. Biol. 206: Król, E, Murphy, M. and Speakman, J. R. (2007). Limits to sustained energy intake. X. Effects of fur removal on reproductive performance in laboratory mice. J. Exp. Biol. 210: Loh, T. C., Foo, H. L., Zurina, A. W. and Tan, B. K. (2002). Effect of feeding fat during pregnancy and lactation on growth performance, milk composition and very low density lipoprotein composition in rats. Mal. J. Nutr. 8(2): Myrcha, A. (1962). Variation in length and weight of the alimentary tract of Clethrionomys glareolus. Acta Theriol. 9: Myrcha, A. (1965). Length and alimentary tract of Apodemus flavicollis. Acta Theriol. 10: Egerton J. Sci. & Technol. Volume 13: ISSN No

168 Influence of Feeding Fat During Lactation on Growth Performance of Pups 163 Nagy, K. A. and Costa, D. P. (1980). Water flux in animals: analysis of potential errors in the tritiated water method: Am. J. Physiol. 238: R454-R465. Nagy, K. A. (1983). The Doubly Labelled Water ( 3 HH 18 O) Method: A Guide to its Use. UCLA Publication no Los Angeles, CA: University of California. Oftedal, O. T. (1984). Milk composition, milk yield and energy output at peak lactation: a comparative review. Symp.Zool.Soc. Lond. 51: Pettigrew, J. E. (1981). Supplemental dietary fat for peripartal sows: a review. J. Anim. Sci. 53: Rogowitz, G. L. (1998). Limits to milk flow and energy allocation during lactation in the hispid cotton rat, Sigmodon hispidus. Physiol. Zool. 71: Rolls, J. B., Van Duijvenvoorde, P. M. and Rowe, E. A. (1984). Effects of diet and obesity on body weight regulation during pregnancy and lactation in the rat. Physiol. Behav. 32: Root, T. (1988). Environmental factors associated with avian distributional boundaries. J. Biogeog. 15: Ryan, B. F., Joiner, B. L. and Ryan T. A., Jr. (1985). Minitab Handbook. 2 nd edition. Boston, MA: PWs-Kent. Speakman J. R, and McQueenie J. (1996). Limits to sustained metabolic rate: The link between food intake, basal metabolic rate and morphology in reproducing mice, Mus musculus. Physiol. Zool. 69: Speakman, J. R. (1997).Doubly Labelled Water: Theory and Practice. London: Chapman and Hall. Speakman J. R, Gidney A, Bett J, Mitchell I. P, and Johnson M. S. (2001). Limits to sustained energy intake IV. Effect of variation in food quality on lacatating mice, Mus musculus. J. Exp. Biol. 204: Speakman J. R, and Krol E. (2005). Limits to sustained energy intake IX: A review of hypotheses. J. Comp. Physiol. B 175: Stenseth, N. C., Framstead, E., Migula, P., Trojan, P. and Wojciechowska- Trojan, B. (1980). Energy models for the common vole (Microtus arvalis): energy as a limiting resource for reproductive output. Oikos 34: Egerton J. Sci. & Technol. Volume 13: ISSN No

169 164 Kagya-Agyemang, et al., (2013) Thompson S. D. (1992). Gestation and lactation in small mammals: basal metabolic rate and the limits of energy use. In: Tomasi T.E, and Horton T.H. (eds) Mammalian energetics. Interdisciplinary views of metabolism and reproduction. Comstock, Ithaca, pp Weir, J. B. de V. (1949). New methods for calculating metabolic rates with special reference to protein metabolism. J. Physiol. Lond. 109: 1-9. Egerton J. Sci. & Technol. Volume 13: ISSN No

170 Faecal bacterial contamination of borehole water between points-of-access and points-of-use in Naivasha, Kenya; Public health implication Donde O. O.* 1,2, Wairimu A. M. 2, Shivoga A. W. 3, Trick G. C. 4,5 and Creed F. I. 6 1 Kenya Marine and Fisheries Research Institute, Lake Turkana Research Station, P. O. Box , Lodwar, Kenya 2 Department of Biological Sciences, Egerton University, P.O. Box , Egerton, Kenya 3 Department of Biological Sciences, Masinde Muliro University of Sci. & Tech. P.O. Box , Kakamega, Kenya 4 Department of Biology, Western University, 1151 Richmond Street N. London, Ontario, Canada. 5 Schulich School of Medicine and Dentistry, Western University, 1151 Richmond Street N. London, Ontario, Canada. *Corresponding Author s Details: ; oscinho@yahoo.co.uk, Phone: +254 (0) Fax: +254 (020) Submitted 11 th Dec. 2012; Reviewed 18 th Feb. 2013; Accepted 15 th May 2013 Abstract Microbiological assessment of drinking water at Point of Access (POA), vendors and household levels is a major issue due to realization of the impact of poor microbial water quality on the general public health. The impact is even greater in densely populated areas as in the case of Naivasha. Assessment on the effects of poor handling of drinking water at different domains aims at protecting consumers from waterborne diseases. The study investigated the bacterial water quality levels at different handling domains; borehole POA, vendors and household within three villages (Karagita, Mirera and Kamere). Membrane Filtration Technique (MFT) and heterotrophic Plate Count (HPC) procedure was used in estimating the densities of E. coli, total coliforms, intestinal enterococci, Clostridium perfringens and hetetrophic bacteria in water samples from all the above domains. In addition, selected physico-chemical parameters were measured in situ using appropriate measuring probes. Data was analyzed using Sigmaplot analysis software version 12, with α = All the water samples from borehole POA, vendors and households had bacterial quality of above the recommended standards for drinking water. Median values for E. coli, intestinal enterococci and C. perfringens were not significantly difference between the sites (P>0.05). Total coliforms and HPC were Egerton J. Sci. & Technol. Volume 13: ISSN No

171 166 Faecal bacterial contamination of borehole water between points-of-access significantly different between the site (P<0.05). In conclusion, the study indicates that poor water handling has negative effect on the bacterial quality of drinking water, hence a public health concern. Hygienic water handling practices at both the supply sources and within households storage containers, proper sewerage systems and efficient pre-consumption water purification techniques are recommended. Key words: Bacterial, Boreholes contamination, handling, household, vendors. Introduction African population has not shown any sign of meeting the 2015 Millennium Development Goal (MDG) on drinking water target. The population without access to improved drinking water sources has even increased by 61 million, from 280 million in 1990 to 341 million in 2006 because the increase in coverage is not keeping pace with population growth (UNICEF & WHO 2008). Water-related diseases continue to be one of the major health problems globally. An estimated 4 billion annual diarrhoeal cases, represented by 5.7% of the global disease burden was recorded in the year 2000 (WHO 2002). This has made it necessary to put in place water quality security measures. Household water security comprise of water availability, good quality, easier accessibility and availability for consumption at household level (Asare, 2004). Water security and sanitation has become a major issue due to realization of the impact of poor microbial water quality on public health. Assessment of drinking water quality at public and private domains aims at ensuring that consumers use water that is free from pathogens of water related diseases (Jensen et al., 2002). Pollution of water resources in most parts of Kenya is in the rise (Kithiia and Ongwenyi, 1997). Vendors selling water to households or at collection points are common in many parts of Kenya where water scarcity limits access to suitable quantities of drinking-water as the case in Naivasha area. Within this area, vendors use a range of modes of transport to carry drinking-water for sale directly to the consumer (WHO 2009). Other studies have revealed that degradation trends in water quality within Naivasha is due to increased human population density (Okoth and Otieno, 2000; Mavuti, 2003 and Kithiia, 2006). The major source of water for the rapidly-expanding human population within Naivasha area is boreholes. However, increase in anthropogenic activities coupled with rise in human population growth has rendered the quality of water for domestic use unwholesome (Otiang a and Egerton J. Sci. & Technol. Volume 13: ISSN No

172 Donde, et al., (2013) 167 Oswe 2007). Therefore, there is need for regular water quality analyses at sources and within households. Regular sampling and analysis provide data on the quality of water, the efficiency of water treatment, the integrity of distribution systems as well as the sanitation status within the households. The range of pathogenic microorganisms is extensive and therefore water is examined for microbiological indicators of contamination (Frahm et al., 2003). The use of indicator organisms is based on the assumption that if they are present then the pathogen may also be present, and if absent then the water is suitable for consumption or pose a lower risk of transmitting WBDs. The principal bacteria indicators are the coliforms bacteria; including total coliforms, faecal coliforms and E coli, the faecal streptococci like intestinal enterocci and C. perfringens (Noble et al., 2003). Their presence in drinking water is undesirable, but does not necessarily indicate any health hazard. The faecal coliforms are a group of thermotolerant species which may indicate faecal contamination. The key species of the faecal coliform group is Escherichia coli commonly found in the faeces of human and is thus a definitive indicator of faecal contamination (Frahm et al., 2003). Concern about faecal coliforms densities in water for domestic use is of paramount importance when considering its safety (Japan International Cooperation Agency (JICA), 2003). The densities of faecal contamination indicators may change during the water handling process, for instance from the source to and within the households. The percentage of samples testing positive for indicator bacteria may decrease after collection from highly contaminated sources because of die-off as bacteria compete for limited oxygen and nutrients in the water (Momba & Notshe 2003). Conversely, the percentage of positive samples may increase after water is collected and stored from safe sources because of contamination through hands, unwashed containers and dippers. Where basic sanitation is lacking, there is more likelihood of indicator bacteria from faeces being introduced into stored water (Ologe 1989). Using uncovered water containers is likely to increase water contamination between source and point-of-use as hands are dipped into vessels to scoop a cupful of water (Chidavaenzi et. al., 1998). Various agencies such as World health Organization (WHO), National Environmental Management Authority of Kenya (NEMA-Kenya), United States Environmental protection Agency (US-EPA) and European Framework have outlaid standards for drinking water provision (Table 1). Therefore, concern about faecal coliforms densities in water is of paramount importance when considering the safety of drinking water in Naivasha region Egerton J. Sci. & Technol. Volume 13: ISSN No

173 168 Faecal bacterial contamination of borehole water between points-of-access (JICA, 2003). Ways of finding solutions to the problems of faecal contamination into water sources is very necessary. The objective of this study was to compare the quality of water that is at the borehole points of use against what is at the vendors and household domains as well as confirming whether these qualities meet the drinking water quality guideline by different agencies. It involved determining the densities of total coliforms, E. coli, intestinal enterococci, C. perfringens and live heterotrophic bacteria using Membrane Filtration Techniques (MFT) and Heterotrophic Plate Count (HPC) Procedure. Some selected physical and chemical parameters were also measured in situ from all the water sources sampled. For the household three villages (Karagita, Mirera and Kamere) were studied. Table 1: Drinking water quality guidelines by different Agencies Parameters Units Authority WHO NEMA- KENYA US-EPA EU- Framework Physico-Chemical ph ph units Conductivity μs/cm at 20 0 C 2500 Microbial parameters Total viable counts at 37 o C/ml CFU /ml Total coliforms CFU not detected/100ml shall be absent <1/100ml 0/100ml E. coli CFU not detected/100ml shall be absent <1/100ml 0/100ml Enterococci CFU not detected/100ml shall be absent 0/100ml Sulphite reducing anaerobes CFU Materials and Methods not detected/100ml shall be absent 0/100ml Study Area Lake Naivasha basin is a freshwater lake in the Rift Valley Province of Kenya (Fig 1). Its watershed covers parts of both the Rift Valley and Central Provinces and it lacks a well-established outlet. It is located in the rain shadow of the Aberdare Range with a mean annual rainfall of about 650 mm, with a heavy rainy season from March to May and a short rainy season in September and October. The mean annual temperature around Lake Naivasha is approximately 25 C, with the highest temperatures from December to March and the lowest in July. The Lake Naivasha watershed is drained by two perennial rivers that enter the lake from the north, the Malewa and Gilgil Rivers, with catchment areas of 1,700km 2 and 400km 2, Egerton J. Sci. & Technol. Volume 13: ISSN No

174 Donde, et al., (2013) 169 respectively. The lake is also drained by other seasonal rivers and streams, including the Kerati River to the north (Mireri, 2005). Karagita is a low income peri-urban settlement situated 6km from the centre of Naivasha Town, next to this internationally renowned Lake Naivasha, a designated Ramsar Site in the Rift Valley Province of Kenya. The community is large and growing fast with a current estimated total population of 54,000 people. This population is distributed in two major blocks; Karagita and neighbouring block Mirera. Karagita is a densely populated slum with a population of 27,000 people. Mirera has 27,000 people that surrounds Karagita and is more sparsely populated. The area is continuing to be built up and using conservative growth rates the population is expected to grow to almost 100,000 people by 2017 and to 173,000 people by People in 80 percent of the households in the area live on less than US $ 1 dollar each per day and boreholes, rivers and lake are the major sources of water for domestic consumption. ( Figure 1: Map of Lake Naivasha; Study sites (adopted from Martin et al., 2001) Egerton J. Sci. & Technol. Volume 13: ISSN No