III. MATERIALS AND METHODS

Transcription

1 Materials and Methods

2 III. MATERIALS AND METHODS The materials and methods adopted for the present study are described under the following headings: 3.1 Cultivation of Azolla 3.2 Chemical Evaluation of Azolla 3.3 Metabolism Assay in Broilers Experimental Diets, Birds and Management Collection of Samples and Analytical Procedure Determination of Metabolizability of Energy and other Nutrients of Azolla Ileal Nitrogen Absorbability Metabolism Assay in Layers Experimental Diets, Birds and Management Sample Collection and Analytical Procedure Determination of Metabolizability of Energy and other Nutrients of Azolla Growth Performance in Broilers Experimental Diets, Birds and Management Growth Performance Parameters Carcass Characteristics and Organometry Intestinal Tract Measurements Production Performance Indices Cost effectiveness of diets Production Performance in Layers Experimental Diets, Birds and Management

3 Egg Production Parameters Egg Characteristics Efficiency of Utilization of Protein and Energy Cost effectiveness of diets 3.7 Statistical Analysis 3.1 Cultivation of Azolla For initial screening, Azolla was cultivated, multiplied and harvested in cement tanks near KVAFSU Library Bangalore (Plate-1 and 2). The cultivation was done in 28x3x6ft cement tank covered with plastic sheets. The culture Azolla pinnata for multiplication was received from State Semen Collection Center, Hesseragatta and about 0.5kg/m2 of fresh Azolla seeds were inoculated into each tank. Each tank was dosed with 50 g of superphosphate at 15 days interval. First crop was harvested on 15 th day of postinoculum and the subsequent harvest was at two days interval. The azolla immediately after harvesting was washed with fresh water and dried in sun for 3 to 5 days and was stored in plastic containers for further chemical evaluation (Plate-3). However, the cultivated sample was not sufficient enough for carrying out biological evaluation and hence the dried Azolla meal (AZM) (Plate-4) was procured from Tamil Nadu Rice Research Institute, Aduthurai, Tanjore Dist. in bulk quantum. The procured Azolla pinnata material was dried in sunlight for one day and dried material was incorporated in the experimental rations.

4 32 Plate 1 : Azolla pinnata grown in a Plate 2 : Fresh Azolla pinnata cement tank Plate 3 : Sun dried Azolla pinnata Plate 4 : Azolla meal (AZM)

5 Chemical Evaluation of Azolla The samples of fresh and dried Azolla pinnata which were cultivated at Department of Animal Nutrition and the representative cultivated sample from farmers of Raichur district and also sample obtained from Central Seed Procuring Institute, Hessaraghatta and the sample from Tamil Nadu Rice Research Institute, Aduthurai, Tanjore Dist (TNRRI), were ground through 1mm sieve and then subjected for analysis of various nutrients. The dried Azolla meal (AZM) samples were analyzed in triplicate for proximate principles viz., dry matter (DM), crude protein (CP), ether extract (EE), total ash (TA), crude fibre (CF) and nitrogen free extractives (NFE) as per the methods described by AOAC (2005). The estimation of calcium (Ca) and phosphorus (P) was done by titration method as per the procedures of Talapatra et al. (1975). The gross energy content of dried AZM samples was determined using adiabatic bomb calorimeter (AOAC, 2005) using benzoic acid as standard at the Dept. of Animal Nutrition, Veterinary Research and Training Institute, TANUVAS, Namakkal, Tamil Nadu. 3.3 Metabolism Assay in Broilers The relative usefulness of a feed ingredient is measured by its metabolizable energy (ME) content. Since no data is currently available as regards the energetic worth of dried Azolla in poultry, an earnest effort was made to determine the ME value of dried Azolla for broilers and layers separately. The procedures adopted to determine metabolizabilty of energy in dried Azolla are explained hereunder:

6 Experimental Diets, Birds and Management The experimental diets were prepared based on conventional system of replacing varying level of basal diet by test ingredient as described by Sibbald and Slinger (1963). A practical starter type of diet comprising of maize, soybean meal and vegetable oil was prepared to serve as a basal mixture. The test diets were prepared by replacing 10 per cent, 20 per cent and 30 per cent of the basal ration by the sun-dried AZM i.e., basal mixture and test ingredient (dried Azolla) in the ratio of 90:10, 80:20 and 70:30, respectively. Further, the basal and test diets were supplemented with Fibre degrading enzyme 0.4g /kg to result in a set of 8 diets. The tailor made enzyme preparations employed were received from M/s Kay Pee Yes Biotech Pvt. Ltd., Hebbal Industrial Area, Mysore. The supplier reported that the fibre degrading enzyme preparation was derived from Asperigillus species and claimed to contain fibre degrading enzymes viz., xylanase-2500, beta-glucanase 0 and cellulase 500. The dietary description is: Enzyme supplementation Basal Mix 10% AZM 20% AZM 30% AZM - T1 T3 T5 T7 T2 T4 T6 T8 The mineral sources and other additives were added over and above per cent to all the diets, so that a uniform supply of these was ensured after substitution of AZM in the basal mixture. The chromic oxide (Cr 2O3) as an inert indicator was added to all the

7 35 diets at 0.5 per cent level to observe the ileal nitrogen absorbability. The detailed ingredient composition of each diet is given in Table 3.1. One hundred and sixty, one-day-old straight-run commercial broiler chicks (selected crossbred of Hubbard strain) were distributed in to sixteen groups of 10 chicks each (Plate-5). The birds were reared on raised wire floor battery brooders (plate-5) kept in a well-ventilated and hygienic house. Ground maize was offered on first day while a corn-soy type starter diet was prepared and offered to all the chicks uniformly for the next 6 days. From 8th day onwards, the experimental diets were assigned randomly to two duplicate of such groups. The diets were offered ad libitum to the corresponding birds in colony feeders and potable water in PVC fountains for initial two weeks and in a continuous channel from next week (3rd week) was provided. During the course of the experiment, standard vaccination and managemental practices including brooding were uniformly followed as per the commercial practice Collection of Samples and Analytical Procedure On 19th, 20th and 21st day of the experiment, feed consumed by each group was accurately measured and the excreta voided by such group was collected before the beginning of the subsequent day at a.m. for three consecutive days (Plate-6). Utmost care was exercised to avoid contaminations from feathers, scales and debris. The collected excreta samples were immediately weighed and dried for 24 h at 0C in hot air oven. The three-day dried excreta samples from each duplicate group were weighed and pooled duplicate wise. Then the samples were allowed to equilibrate to atmospheric conditions, ground to pass through 1mm sieve and well-homogenized samples were stored separately in airtight polythene bags for further analysis.

8 36 Table 3.1 : Ingredient and calculated nutrient composition of experimental diets used in broilers' metabolism trial Ingredients, Kg T1 T2 T3 T4 T5 T6 T7 T8 Basal Mix AZM Total Di-calcium phosphate Calcite powder Salt Trace Minerals premix Vitamin &additive mix Chromic oxide Fibredegradingenzyme4 Calculated composition ME. Kcal/kg CP, % EE, % CF, % Ca, % TP, % Pav, % Basal mix consisted of 61% maize, 38% soybean meal and 1% sunflower oil 2. Trace mineral premix contained Fe-90000, I-2000, Cu-15000, Mn-90000, Zn and Se300ppm 3. Vitamin and additive mix contained vitamin premix 0.05kg (each 0g contained vit A25MIU, vit D3 5.6MIU, vit E-60g, vit K -4g, vit B1-4g, vit B2-10g, vit B6-6g, vit B g, Niacin-80g, Cal-d-pantothenate-30g, Folic acid-2g, Biotin-0.16g, Organic nutritive carrierqs), Hepatocare 0.05kg, Tylosin phosphate 0.05kg, Curotox 0.05kg, Choline chloride 0.05kg, DL-methionine kg and L-lysine 0.094kg. 4. Fibre degrading enzymes contained Xylanase 2500, β-glucanase0 and cellulase 500 units per gram

9 37 Plate 5 : Metabolism trial in broiler birds Plate 6 : Collection of excreta in broiler trial

10 38 The dietary feed samples were also collected at the beginning and at the end of the metabolism trial and dried in hot air oven to arrive at the dry matter per cent of such samples. Further, the samples were ground to pass through 1mm sieve and stored in plastic containers for further analysis. The proximate analysis of experimental diets and excreta samples were performed according to standard procedures (AOAC, 2005). Gross energy content of such samples was determined by adiabatic bomb calorimeter (AOAC, 2005). Appropriate corrections were made for differences in moisture content. On completion of AME bioassay (end of 21st day), 5 birds from each duplicate group were slaughtered by cervical dislocation. The body cavity was cut opened and the entire intestinal tract was removed. The digesta content in the terminal 1/5 th segment of the ileum was gently squeezed into a 120 ml reagent bottle containing 20 ml of 5% H2SO4. The ileal digesta samples of birds from each duplicate group were pooled and stored at -200C for further analysis. At the time of analysis, the contents of ileal digesta were thawed, homogenized and were made up to ml volume. Duplicate samples of the homogenate were run each for nitrogen and chromium oxide as per the methods described by Hill and Anderson (1958). The nitrogen content of ileal digesta was obtained as per the standard procedure (AOAC, 2005) while the chromic oxide content was spectrophotometrically following the procedure of Fenton and Fenton (1979). analyzed

11 Determination of Metabolizability of Energy and other Nutrients of Azolla The metabolizability of various nutrients of assay diets including gross energy were calculated and then the metabolizability of energy and other components of AZM were arrived at by using simultaneous equations. a) Dry matter and other nutrients: The group wise dry matter metabolisability (DMM) of diets was determined using the following formula (Han et al., 1976). DMM (%) = } { Weight of the dry feed Weight of the dried consumed (g) excreta voided (g) X Weight of dry feed consumed (g) Similarly, the metabolisability of organic matter (OM) and other nutrients, crude protein (CP), ether extract (EE), crude fibre (CF) and nitrogen free extractives (NFE) were determined using the formula given below. Metabolisability coefficient of nutrient (%) = (Unit nutrient intake) (Unit nutrient out go) Unit nutrient intake X b) Metabolizable energy: Based on the gross energy (GE) values of assay diets and the excreta samples determined by bomb calorimetry, the apparent ME (AME) values of the diets were calculated by subtracting the GE of the excreta from GE intake divided by total dry matter intake using the following formula: AMEdiet (kcal/kg) = (Feed intake x GEdiet) - (Excreta output x GEexcreta) Feed intake The nitrogen corrected AME (AMEn) values were calculated using a factor of 8.22 kcal per g of nitrogen retained in the body (Hill and Anderson, 1958).

12 40 c) Metabolizabile Energy Content of AZM: The ME content of the test AZM were arrived by using simultaneous equation methods i.e., the difference between measured digestibility or the calorific value for basal and test diets. Similarly, metabolizability / digestibility coefficient of other nutrients of AZM were also arrived at. ME of test feed ingredient (kcal/g) ME of test diet (kcal/g) - ME of control diet (kcal/g) x 0.7 = Ileal Nitrogen Absorbability The ileal nitrogen digestibility (IND) of assay diets was calculated using Chromic oxide as a marker and given as follows: IND (%) = [(N/Cr)d (N/Cr)i] (N/Cr)d X Where (N/Cr) d = the ratio of nitrogen to Cr in diet and (N/Cr) i = the ratio of nitrogen to Cr in ileal digesta 3.4 Metabolism Assay in Layers Since there are differences within type of birds with respect to ability to metabolize energy or digest other nutrients, a metabolism trial was also conducted with egg type chicken to arrive at ME worth of AZM in layers as well Experimental Diets, Birds and Management The dietary design employed here is very much similar to that of broilers metabolism trial and described under Sec A conventional practical type layer diet comprising maize, soybean meal, sun flower extraction and DORB was prepared to serve

13 41 as basal mixture (T1). The test diets were prepared by incorporating sun dried AZM at 10 (T3), 20 (T5) and 30 % (T7) respectively replacing the corresponding proportion of basal mixture. Further, a parallel set of another 4 diets were prepared by supplementing each diet with fibre degrading enzyme 0.5g /kg to result in T 2, T4, T6 and T8 correspondingly. The enzyme preparations added in such diets were same as those employed in broiler metabolism trial. The rest of the components in the diets namely shell grit, mineral mixture, common salt and vitamin premix were maintained in all diets at uniform level. The chromic oxide (Cr2O3) as an external indicator was added to all diets at 0.5 per cent level. The detailed ingredient composition of each diet is given in Table 3.2. A total of one hundred twenty-eight BV-300 commercial layers of about 32 weeks age and uniform body weight with proper history were selected and randomly divided into 32 groups of 4 birds each in a commercial poultry farm (Sri Rama Poultry Farm, Channapatna) (Plate-7). The four groups were housed alternatively in colony cages with each cage unit measuring 15 x15 x18 size. Each of the 8 diets described above was offered to four replicates of four hens each. Partitioning was made in feed trough to avoid mixing up of test diets. The rest of the managemental practices were kept uniform Sample Collection and Analytical Procedure After conditioning the laying hens with their corresponding experimental diets for 11 days, a conventional 3-day metabolism trial was carried out, during which the feed intake and excreta output (Plate-8) from each group of birds was measured quantitatively to obtain the ME data.

14 42 Table 3.2 : Ingredient and calculated nutrient composition of experimental diets used in layer metabolism trial Ingredients, Kg T1 T2 T3 T4 T5 T6 T7 T8 Basal mix Azolla Total Mineral mixture Salt Vitamin and additive mix Chromic oxide Shell grit Fibre degrading enzymes4 Calculated composition ME. Kcal/kg CP, % EE, % CF, % Basal mix consisted of 55% maize, 14% soybean meal, 18% DORB and 13% sunflower extractions 2. Mineral mixture contained : Calcium-32%, Phosphorus-6% and other trace minerals 3. Vitamin and additive mix contained AB2D3K- 0.1kg (each 0g contained vit A-50MIU, vit D3 5.6MIU, vit E-60g, vit K -4g, B-complex-0.2kg, (vit B1-4g, vit B2-10g, vit B6-6g, vit B120.03g, niacin-80g, Cal-d-pantothenate-30g, folic acid-2g, biotin-0.16g, organic nutritive carrier-qs), Hepatocare 0.05kg,., Lysine kg, DL-methionine 0.07kg 4. Fibre degrading enzymes contained : Xylanase 2500, β-glucanase0 and cellulase 500 units per gram

15 43 Plate 7 : Metabolism trail in layer birds. Plate 8 : Collection of excreta in layers

16 44 The procedure adopted for collection and processing and chemical analysis of excreta and diet samples were similar to that of broilers metabolism trial, described under section Determination of Metabolizability of Energy and other Nutrients of Azolla The metabolizable energy and other components of AZM in layers were arrived by methods which were very much similar to those of broilers metabolism study (Section 3.3.3). 3.5 Growth Performance in Broilers A feeding trial was conducted to asses the effect of dietary inclusion of AZM at different levels on performance of broilers such as body weight gain, feed consumption and feed efficiency, carcass characteristics and organometry, nutrient utilization and cost effectiveness of AZM inclusion. A brief account about the performance trial in broilers is presented hereunder: Experimental Diets, Birds and Management Five iso-nitrogenous and iso-caloric diets were prepared by incorporating sun dried AZM at 0, 2.5, 5.0, 7.5 and 10 per cent levels to meet the nutrient requirements specified by Bureau of Indian Standards (BIS, 1992). Further, another set of five diets was prepared by supplementing each diet with fibre degrading enzyme 0.4g /kg. Such diets were prepared separately for each phase i.e., starter (0-14 days), grower (15-28 days) and finisher (29 to 42 days) phases. The ingredients calculated nutrient composition of different diets compounded for different phases are detailed in Tables 3.3.

17 Table 3.3 : Ingredient composition of experimental diets compounded for different phases during performance trial of broilers 1 Broiler finisher diet 10% 0% 2.5% 5% 7.5% 10% AZM AZM AZM AZM AZM AZM Additives contained Brovit plus- 0.5kg (each 500g contained vit A-12.5 MIU, vit D3-2.8 MIU, vit. E-30g, vit K-2g, vit B1-2g, vit B2-5g, vit B63g, vit B g, niacin-40g, Cal-d-panthothenate-15g, folic acid-1g, biotin-0.08g, organic nutritive carrier.-q.s), Tylosine phosphate-0.05kg; Hepatocare-0.1kg; Choline chloride-0.05kg; Curodox-0.05kg., DL-Methionine-200g, L-Lysine-g. Note: Parallely, another set of 5 diets with AZM at 0, 2.5, 5, 7.5 and 10% were also prepared using fibre degrading enzyme@ 0.05kg per quintal for all stages. 45 Broiler starter diet Broiler grower diet 0% 2.5% 5% 7.5% 10% 0% 2.5% 5% 7.5% AZM AZM AZM AZM AZM AZM AZM AZM AZM Maize Soybean meal Lecithin oil Di-calcium phosphate Calcite powder Salt Azolla meal Additives1 Total Chemical composition, % CP EE CF Ca TP Pav ME, kcal/kg Cost, Rs/kg Ingredients, Kg

18 46 The design was aimed to study the influence of inclusion of AZM at different levels on broiler performance as well as for increasing the efficiency of AZM utilization through biotechnological approaches and is described as follows: Enzyme supplementation 0% AZM 2.5% AZM 5% AZM 7.5% AZM 10% AZM - T1 T3 T5 T7 T9 T2 T4 T6 T8 T10 A total of 200 one-day-old straight-run commercial chicks (Hubbard) were randomly divided into 20 groups of 10 chicks each. The birds were housed in raised wire floor battery brooders of single tier system, which were kept in a well-ventilated hygienic house. The brooding was done up to two weeks of age as per the standard practice and all the birds were vaccinated against New Castle disease and Infectious Bursal disease on 8 th and 14th day of age, respectively. Each of the 10 diets were offered randomly to duplicate groups of 10 chicks each. The diets in colony feeders were provided to the corresponding birds ad libitum and potable water was provided in PVC fountains (initial 2 weeks) or in a continuous channel along with PVC fountains (3 rd week onwards) was made available round the clock. The rest of the managemental practices were accomplished uniformly for all the birds during the 42-day experimental period Growth Performance Parameters Several parameters were studied to observe the effect of inclusion of AZM in broiler diets with biotechnological approaches and are described hereunder:

19 47 a) Body Weight Gain: The weekly individual body weights of birds were recorded and the body weight gains were arrived at for each week. The body weight gain was also calculated for starter, grower and finisher phases and on a cumulative basis. Accordingly, the body weight gains in different dietary groups were compared as per treatment wise and main factors (AZM levels and Enzyme supplementation) wise. b) Feed Consumption: The daily feed offered to all individual groups was accurately recorded and at the end of each week, the residual feed, as well as spilled over feed from that group was accounted. Care was also taken to subtract the amount of feed consumed by the birds that died during a particular week by calculating the daily feed consumption during that week. Thus the average feed consumption in different groups was calculated during each week as well as during starter phase (0 to 14 days), grower phase (15 to 28 days) and finisher phase (29 to 42 days) and cumulatively (0 to 42 days). c) Feed Conversion Ratio: The feed conversion ratio (FCR) expressed as the ratio of amount of feed consumed to the body weight gained under each group of birds arrived at each week and each phase wise and also cumulatively. d) Mortality: Mortality in respective groups was recorded as and when the birds died. The dead birds were necropsied to identify the dietary cause, if any. The physical consistency and the colour of fecal droppings of various groups were under observation from time to time.

20 Carcass Characteristics and Organometry At the end of the experiment (42nd day), two birds (1 male and 1 female) from each replicate, were separated and starved for 12 hrs however with a provision of plenty of water. Then immediately after recording their live body weights (pre-slaughter bird weight), the birds were sacrificed by cervical dislocation and the carcasses were subjected for the study of following parameters: a) Dressing Percentage: The slaughtered birds were defeathered, denecked and eviscerated along with two legs beneath the hock joint to observe the effect of various experimental diets on the dressing percentage. The dressing percentage was calculated as the percent of the carcass weight obtained after removing the feathers, neck, legs and internal viscera, to its live body weight. b) Organometry: From the sacrificed birds, different organs viz., heart, liver, gizzard, proventriculus, spleen and bursa and the breast meat portion as well as abdominal fat were carefully separated and weighed to observe the effect of different dietary treatments on growth and development of certain organs. The procedures followed are briefed here under: 1. Giblets: The weight of the giblet organs viz., heart without pericardium, liver with out gall bladder, gizzard with out the food contents and internal lining membrane and proventriculus from each sacrificed bird were recorded (Plate-9) and expressed as the per cent of pre-slaughter bird weight as well as dressed weight as follows:

21 49 Relative heart percentage = Relative liver percentage = Relative gizzard percentage = Relative proventriculus percentage = Heart weigth (g) Live weight (g) Liver weight (g) Live weight (g) Gizzard weight (g) Live weight (g) Proventriculus weight (g) Live weight (g) x x x x 2. Lymphoid organs: The weights of the lymphoid organs viz., bursa and spleen from each sacrificed bird was recorded and expressed as the percent of pre-slaughter bird weight (g/g). 3. Abdominal fat weight: The weight of the fat in abdomen including the fat surrounding gizzard, bursa, cloaca and adjacent muscles of each bird was recorded and expressed as percent of concerned pre-slaughter bird weight. 4. Breast meat yield: The weight of the breast meat from each sacrificed bird was recorded (Plate-10) and expressed as the percent of pre-slaughter bird weight (g/g).

22 50 Plate 9 : Organometry of broiler performance trial. Plate 10 : Breast meat of broiler performance trial

23 Intestinal Tract Measurements Along with other parameters of organometry study, the intact digestive tract of all sacrificed birds were carefully removed and the length of different segments of intestine viz., duodenum, jejunum and ileum were measured as follows: Length of duodenum = the length of pancreatic loop Length of jejunum = from pancreatic loop to Meckel s diverticulum Length of ileum = from Meckel s diverticulum to ileocaecal junction The length of each segment was expressed in terms of percentage of pre-slaughter body weight (cm/g) Production Performance Indices The cost of the starter, grower and finisher types of all the experimental diets was arrived at by considering the prevailing prices of the constituent feed ingredients and feed additives including that of enzymes. The relative cost effectiveness of each diet was thus assessed. Further, by considering the prevailing sale price of the broiler bird, the Performance Index Score (PIS) and the Economic Index Score (EIS) were arrived at i.e., 1) PIS = 2) EIS = Average final body weight (g) x % livability FCR x No, of days reared x 10 PIS Cost of diet (Rs/kg) Cost effectiveness of diets The cost of each diet prepared during broilers feeding trial was arrived at by considering the prevailing prices of the constituent feed ingredients, mineral salts and

24 52 other additives including that of enzymes. Further, by considering the sale price of broiler chicken including the expenditure on chicks, labour, medicine etc., the net profit for each treatment was calculated separately for broilers under different treatments. The relative cost effectiveness of each diet was thus assessed. 3.6 Production Performance in Layers By making use of the ME value of AZM obtained from layers metabolism trial, AZM was included at 0, 2.5, 5.0, 7.5 and 10 per cent levels in layers diets and evaluated in terms of egg production, feed consumption, feed conversion efficiency, egg characteristics (egg weight, Haugh unit, shell thickness, yolk colour etc.), nutrient utilization and efficiency of utilization of energy and protein. At the end, the cost effectiveness of AZM was also being assessed. The 84-day layer production performance trial was conveniently divided into 3 periods of 28 days duration each. A brief description about the materials and methods adopted for feeding cum production performance trial in layers are presented hereunder: Experimental Diets, Birds and Management a) Experimental dietary design : The practical type layer diet (control) involving maize and DORB as the main energy sources and soybean meal and sunflower extraction as the protein sources with shell grits, calcite powder, di-calcium phosphate and salts of pertinent trace minerals was prepared to conformed to the nutrient specification of Bureau of Indian Standards (BIS, 1992). In the test diets, the sun dried AZM was included at four different levels (2.5, 5.0, 7.5 and 10 per cent, respectively). Further, these five iso-nitrogenous and iso-caloric diets were

25 53 supplemented with fibre degrading enzyme 0.5g/kg diet to result in a set of five test diets, a design similar to that described under section , was aimed to study the effect of inclusion of AZM at different levels as well as with the hope of increasing the efficiency of AZM utilization through biotechnological approach. The detailed ingredient and calculated nutrient composition of each diet is given in Table 3.4. b) Experimental birds: After a gap of 15 days following a metabolism trial, the same birds were used for production trial. One hundred sixty BV-300 commercial layers of about 35 weeks age were redistributed randomly into 10x4x4 birds. The birds were housed in three tier colony cages. Four birds were housed in each cage measuring 15 x15 x18 size to serve as a replication. Each of the 10 diets (Sec ) were offered to four such replications of 4 birds each in colony cage units. The birds were placed in cages in such a way that there was one empty cage between the two groups to limit the availability of particular diet to another group of birds. Added to it, the continuous feed trough was partitioned with a partition material to avoid mixing up of test diets, if any. By and large, the distribution of replications of treatments was essentially uniform. The birds were maintained under standard managemental conditions including periodical deworming, preventive or therapeutic disease control, lighting programme, feeding frequency, watering methods and other routine bio-security aspects. The experiment lasted for 84 days which was conveniently divided into three 28-day interval periods.

26 54 Table 3.4 : Ingredient and nutrient composition of experimental diets compounded during performance trial of layers Ingredients, kg 0 % AZM 2.5% AZM 5% AZM 7.5% AZM 10% AZM Maize De-oiled rice bran Soybean meal Sunflower extraction Di-calcium phosphate Calcite powder Shell grit Salt Azolla meal Additives1 Total Chemical composition, % CP EE CF Ca TP Pav ME, Kcal/kg Cost, Rs/kg Additives contained AB2D3K- g (each 500g contained vit A-12.5 MIU, vit D3-2.8 MIU, vit. E-30g, vit K-2g), B-complex-200g (vit B1-2g, vit B2-5g, vit B6-3g, vit B g, niacin-40g, Cal-d-panthothenate-15g, folic acid-1g, biotin-0.08g, organic nutritive carrier.-q.s), Tylosine phosphate-0.05kg; Hepatocare-0.1kg; Choline chloride-0.05kg and toxin binder-0.075kg., DLMethionine-0.05kg, L-Lysine-0.02kg Note: Parallely, another set of 5 diets with AZM at 0, 2.5, 5, 7.5 and 10% were also prepared using fibre degrading 0.05kg per quintal.

27 Egg Production Parameters Various egg production performance parameters were considered to critically evaluate the possibility of inclusion of AZM in layer diets in combination with biotechnological approach for enhancing its nutritive value and are described hereunder: a) Body weights: Individual body weights of birds were recorded at beginning of the experiment as well as at every 28-day interval to monitor the pattern of body weight changes, if any, due to dietary regimen. Group wise average weights under different treatments were arrived and the period wise changes (loss / gain) in body weight were arrived at. The weighing of the birds was done in the early hours of the day before feeding and oviposition. b) Egg production: Every day in late hours, the number of eggs produced in a particular replicate group was recorded and the hen day egg production was arrived at on the basis of three 28-day periods as per various treatments for further statistical analysis. The rate of egg production was calculated both for the individual periods and on cumulative basis as well. c) Daily feed consumption: An amount of 120 g of feed per bird per day was collectively offered to the four birds of replicate group during the experiment period. Subsequently, the daily required amount of concerned diet was adjusted based on the pattern of feed being consumed by the particular group of birds. The diets were offered in divided doses of about 50 per cent in morning and the remaining 50 per cent in the late afternoon hours. The feed residue, if any, was recorded in each group at the end of 28-day period.

28 56 d) Feed efficiency: The amount of feed consumed to produce one kg egg mass and/or dozen eggs in each dietary replication was calculated. Such feed efficiency values (kg feed/kg egg mass or kg feed/dozen eggs) were calculated both period-wise and cumulatively. e) Mortality: The health status of the birds of various groups was under constant observation from time to time. The dead birds were subjected for post mortem examination to identify the dietary causes, if any. The physical consistency of fecal droppings of various groups was under periodical observation Egg Characteristics On the terminal day of every 28-day interval, all the eggs produced from different replicate groups were collected and were weighed individually during the experimental period of 84 days. Further, on the immediate next day, each egg was broken and the entire contents were carefully placed on a glass slab for measurement of albumen and yolk indices. The values were then arranged according to treatments and main factors wise under each 28-day period as well as cumulatively. a) Egg weight: On every Tuesday and Friday of each week, egg weights were recorded and in addition, egg weights were also taken at the end of 28-day periods. The weights so obtained on eight occasions during a particular 28-day (4-week) period were averaged and the data were arranged as per treatments and main factors. b) Egg shape index: The breadth and length of each egg (unbroken) was measured by using Vernier Calipers as described by Reddy (1979) and egg shape index (ESI) was calculated as:

29 57 ESI (%) = Horizontal diameter (breadth) in cm Vertical diameter (length) in cm X c) Albumen index : The height and diameter of egg albumen were obtained using Ames Haugh Unit Spherometer and Vernier Caliper (Plate-11), respectively and the albumen index (AI) was calculated as: AI= Albumen height in mm Albumen diameter* in mm X *Average of albumen length (mm) and albumen width (mm) d) Haugh unit score: After recording the height of albumen at two places (one near to yolk and the other at the end of dense albumen) by using Ames Haugh unit meter and the relationship between egg weight and albumen height for each egg was calculated as Haugh unit score (Haugh, 1937) which was calculated as follows: Haugh unit = x log (H x W7) Where H= albumen height (mm) and W= egg weight (g) e) Egg yolk colour: The colour of yolk of every broken open egg was scored by matching (contrast) technique using Roche yolk colour fan (Roche Company, 1969). Its colour value denotes the colour intensity from 1 to 14 according to the degree of yolk colour (Plate-12, Plate-13 and Plate-14). f) Yolk index: The height (at centre place) and diameter of the yolk was measured using Ames Haugh Unit Spherometer and Vernier Calipers (Plate-15) and the yolk index (YI) was calculated as:

30 58 Plate 11 : Albumen index score Plate 12 : 28th day yolk colour Plate 13 : 84th day yolk colour

31 Plate 14 : The yolk colour of eggs collected during the 84-days layer production trial as influenced by AZM at different levels 7.5%AZM 2.5%AZM 10%AZM 5%AZM Magnified yolk colour at 10%AZM 59 0% AZM

32 60 Plate 15 : Yolk index score.

33 61 YI (%) = Yolk height in mm Yolk diameter in mm X g) Shell thickness: Following the breaking of egg, the shell pieces devoid of shell membranes at broad end and narrow end and at middle were carefully selected and their thicknesses were measured using a digital calipers as described by Ogunmodde and Oguntella (1971). The average of these two pieces was represented as shell thickness Efficiency of Utilization of Protein and Energy The gross efficiency of converting dietary protein and metabolisable energy to those of eggs under all treatments and during different periods was calculated based on intake and transfer of the said nutrients as described below: a) Protein utilization: Gross efficiency of protein utilization (EPU) was calculated as follows: Total egg mass produced X 0.12 EPU (%) = Total dietary protein intake X Where, 0.12 represents the unit of protein present in one unit of egg. b) Energy utilization: Gross efficiency of energy utilization (EEU) was calculated as follows: EEU (%) = Total egg mass produced X 1.6 Total dietary ME intake X Where, 1.6 represents the amount of kcal per every gram of egg mass as described by Reddy (1979).

34 Cost effectiveness of diets The cost of each diet prepared during layers feeding trial was arrived at by considering the prevailing prices of the constituent feed ingredients, mineral salts and other additives including that of enzymes.. Further, by considering the sale price of eggs including the expenditure on labor, medicine etc., the net profit for each treatment was calculated separately for layers under different treatment. The relative cost effectiveness of each diet was thus assessed 3.7 Statistical Analysis The data generated during the biological trials in broiler and layers involving AZM at different levels and enzyme supplementation were analyzed manually using Microsoft excel based one way ANOVA (CRD) for the treatments and two way ANOVA (CRD) for the main effect of dietary regimen (AZM level and Enzyme supplementation) according to the procedures described by Snedecor and Cochran (1989). All the percentage values were transformed to arc sin values before analysis. The differences in means were tested by using Duncan s Multiple Range Test (Duncan, 1955) when the test was significant.