LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY. Vaida Jokubauskienė

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY Vaida Jokubauskienė ALTERNATION OF SELECTED MICROELEMENTS IN THE BLOOD SERUM OF IN-CALF COWS AND MINERAL ADDITIVES, USED TO STABILIZE THE LEVEL OF MINERALS Summary of Doctoral Dissertation Agricultural Sciences, Veterinary (02A) Kaunas 2014

2 The research was done in Lithuanian University of Health Sciences, Veterinary Academy in Research supervisor Prof. Habil. Dr. Vytautas Špakauskas (Veterinary Academy of Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary 02A). Veterinary Science Cauncil: Chairman Prof. Habil. Dr. Saulius Petkevičius (Veterinary Academy of Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary 02A). Members: Dr. Raimundas Mockeliūnas (Institute of Microbiology and Virology of Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary 02A); Prof. Dr. Algimantas Paulauskas (Vytautas Magnus University, Science of Biomedicine, Biology 01B); Assoc. Prof. Dr. Antanas Šarkinas (Food Institute of Kaunas University of Technology, Technological Sciences, Chemical Engineering 05T); Prof. Dr. Rasa Želvytė (Veterinary Academy of Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary 02A). Opponents: Prof. Dr. Bronius Bakutis (Veterinary Academy of Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary 02A); Prof. Habil. Dr. Aniolas Sruoga (Vytautas Magnus University, Science of Biomedicine, Biology 01B). Public doctoral dissertation defence in Veterinary Science Council will take place at the Veterinary Academy of Lithuanian University of Health Sciences auditorium of Dr. S. Jankauskas 2 p. m. on 28 th of November, Address: Tilžės str. 18, LT Kaunas, Lithuania. The summary of doctoral dissertation was sent on 28 th of October, 2014, according to the confirmed list. The doctoral dissertation is available in the Library of Veterinary Academy of Lithuanian University of Health Sciences.

3 LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA Vaida Jokubauskienė PASIRINKTŲ MIKROELEMENTŲ KAITA VERŠINGŲ KARVIŲ KRAUJO SERUME IR MINERALINIAI PRIEDAI MINERALŲ KIEKIUI STABILIZUOTI Daktaro disertacijos santrauka Žemės ūkio mokslai, veterinarija (02 A) Kaunas 2014

4 Disertacija rengta metais LSMU Veterinarijos akademijoje Neužkrečiamųjų ligų katedros Eksperimentinės ir klinikinės farmakologijos laboratorijoje. Mokslinis vadovas prof. habil. dr. Vytautas Špakauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija 02 A). Veterinarijos mokslo krypties taryba: Pirmininkas prof. habil. dr. Saulius Petkevičius (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija 02A). Nariai: dr. Raimundas Mockeliūnas (Lietuvos sveikatos mokslų universiteto Mikrobiologijos ir virusologijos institutas, žemės ūkio mokslai, veterinarija 02A); prof. dr. Algimantas Paulauskas (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija 01B); doc. dr. Antanas Šarkinas (Kauno technologijos universitetas, Maisto institutas, technologijos mokslai, chemijos inžinerija 05T); prof. dr. Rasa Želvytė (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija 02A). Oponentai: prof. dr. Bronius Bakutis (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija 02A); prof. habil. dr. Aniolas Sruoga (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija 01B). Disertacija bus ginama viešame Veterinarinės medicinos mokslo krypties tarybos posėdyje 2014 m. lapkričio 28 d. 14 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos dr. S. Jankausko auditorijoje. Adresas: Tilžės g. 18, LT Kaunas, Lietuva. Disertacijos santrauka išsiuntinėta 2014 m. spalio 28 d. pagal patvirtintą adresų sąrašą. Su disertacija galima susipažinti LSMU Veterinarijos akademijos bibliotekoje.

5 LIST OF ABBREVIATIONS µg/kg a microgram per kilogram µg/l a microgram per liter µmol/l micromoles per liter Co cobalt Cu copper DM dry matter Fe iron g gramme GF AAS Graphite furnace atomic absorption spectrometry I iodine ICP MS inductively coupled plasma mass spectrometry kg kilogram LUHS Lithuanian University of Health Sciences M arithmetic medium Mn manganese Mo molybdenum n samples size NRC national research council p statistical significance r Pearson correlation coefficient SD Standard deviation VA Veterinary Academy Zn zinc 5

6 INTRODUCTION Scientific and technological progress in agriculture is of great importance in features of cattle exploitation. Higher milk yields, better quality of milk, larger augment and increased fertility rates are related to the nourishment of cattle. Adequate nutrition is one of the main factors, affecting the health of the cattle. It is therefore very important to ensure and balance the quantity of fodder s nourishment required to meet the needs of the body. Microelements are such elements, which are necessary for the body in quantities of milligrams and micrograms (NRC, 2001). Cows supply with all microelements in the fodder, with the exception of iron, is only percent out of the standard (Bartkevičiūtė, Černauskienė, 2004; Suttle, 2010; Lopez Alonso, 2012). Factors, stimulating quantitative changes of microelements in the body, are very diverse. Cattle needs for these minerals vary, depending on the body s condition, age, breed, feeding characteristics. Also climatic conditions, geographical location, characteristics of the soil, fodder and quantity of antagonistic agents in the fodder (phosphate, oxalate, antagonistic microelements) have a great effect on the change of microelements level in the body (Spears, 2003; Yokus, Cakir, 2006; Van De Weyer et al., 2010; Noaman, 2013(b)). Physiological role of microelements in animal health is well-defined and scientifically proven; however it receives little attention, since many cattle breeders believe, that relevant amounts of these minerals in the fodder fulfil animals feeding requirements (Suttle, 2010). Unfortunately, the lack of microelements or toxicosis symptoms in the body is not always obvious (Lopez Alonso, 2012). Many researchers have proven, that minor imbalance of level of microelements in the body causes disorders of metabolism, immune system, reproductive system, also productivity reduces and milk quality deteriorates (Akar, Yildiz, 2005; Akhtar et al., 2009; Badiei et al., 2011; Heidarpour et al., 2012). In order to strengthen the organism and improve features of animal exploitation, mineral additives of various chemical compositions, were created. Researchers (Griffiths et al., 2007; Krys et al., 2009; Weiss et al., 2010; Karkoodi et al., 2012) are trying to figure out, what effect do mineral additives have on level of microelements and on body wellness; however, the results are diverse. This indicates, that the entire food chain, associated with germ of life, growth, development; and various factors, influencing the nutritional needs of the body, are not fully disclosed. 6

7 Lack of microelements for the cattle was noticed worldwide: Belgium (Guyot et al., 2009), Pakistan (Khan et al., 2009), Check Republic (Podhorsky et al., 2007), New Zealand (Grace 2010), Cuba (Grace et al., 2010a), Iran (Noaman 2013(a)). A few researchers (Yokus and Cakir 2006; Blanco Penedo et al., 2009; Soch et al., 2010) have assessed effect of season and location on the change of level of microelements in the blood serum of cows. Other (Meglia et al., 2004; Kincaid et al., 2003; Ulutas et al., 2003; Slavik et al., 2006; Van Ryn, 2009; Bahram et al., 2011) determined what effect health condition of the body has on level of microelements. There are no generalizing data on how level of microelements change in the body of a cow during parturition, calving and after calving in our country. It is not known, what influence on level of microelements have productivity, morbidity of milk fever after parturition, age and other factors. It is also not clear, how different sources of mineral additives affect level of microelements of cattle blood. The evaluation of the main reasons, predetermining change of level of microelements in the body, can ease the balance of rations, maintenance of animals health and welfare. Rationale Microelements are such elements, which are necessary for the body in quantities of milligrams and micrograms. Any imbalance of these substances in the body disturbs normal body function. Cattle are especially vulnerable to deficiency of microelements. The lack of minerals in the body is evidenced by changes of coat, skin wounds, nail ulcers, mastitis, metritis, infertility and other disorders of the body function. All these factors reduce the utility of herd, and cattle breeders are experiencing severe economic losses. To ensure the health of cattle and to improve features of exploitation, it is necessary to evaluate all factors, that determine the change of microelements level in the animal body. This requires evaluation of microelements in soil, fodder and water. A well in order to determine microelements level in the body, it is necessary to consider the condition of health of the cattle, their productivity, age, breed and climate conditions. Deficiency of microelements is spread worldwide. Intensive farming, use of various fertilizers, pesticides and herbicides exhausts the soil and modifies biochemical features of the soil and plants growing in it, and at the same time it modifies microelements level in the body. Effect of mineral additives that are used along with the fodder on the 7

8 body varies depending on their chemical form, balance and composition of microelements. Most of the data, found in the literature, is about the macro elements and their biological significance on the animal body. In Lithuania there is a lack of data on microelements level in the animal body. It is not known, how microelements level change in the blood serum of cattle, depending on parturition, productivity, age and what influence on level of microelements has morbidity of parturient paresis. There is also no evidence on how the mineral additives used along with the fodder effect microelements level in the blood serum of cattle. Objective and Tasks Objective: The aim of this thesis is to determine the changes of microelements' (Mn, Zn, Cu, Co, I, Fe and Mo) level in the blood serum of cows, depending on pregnancy, productivity, morbidity paresis after parturition, age and to evaluate the efficiency of mineral additives of different chemical composition in stabilizing microelements' level. Tasks: 1. Determine the dynamics of manganese, zinc, copper, cobalt, iodine, iron and molybdenum level in cows' blood serum before calving, at calving and 1, 2, 6 and 10 days after calving and to evaluate periods, during which the level of microelements decreases the most. 2. Determine and compare the level of Mn, Zn, Cu, Co, I, Fe and Mo in the blood serum of healthy cows of different productivity and cows with paresis after parturition. 3. Determine and compare the level of Mn, Zn, Cu, Co, I, Fe and Mo in the blood serum of heifers (1 2 years) and cows of different ages (3 5 and 6 8 years old). 4. Evaluate and compare the efficiency of different mineral additives (inorganic and mixed) on stabilizing the level of microelements (Mn, Zn, Cu, Co, I) in the blood serum of cows. Scientific novelty and practical significance In Lithuania this kind of work was performed for the first time, and the knowledge obtained during this research is very important when analyzing imbalance of the microelements in the body of the cattle. This research 8

9 provided new information on peculiarities of dynamics of microelements level in the blood serum of cows before calving, at calving and 1, 2, 6 and 10 days after calving. It was determined which days the microelements level decreases the most. Also, effect of age, productivity, and morbidity with paresis after parturition on level of microelements in the blood serum of cows was evaluated. Based on the study results, in the laboratory of experimental and clinical pharmacology of LUHS VA, were developed new products mineral additives of different chemical composition (inorganic and mixed) (see Table 2), the main use purpose of which is to find out the effectiveness of mineral additives of different chemical composition by stabilizing the level of microelements in cows blood serum. The knowledge of during which periods the microelements level in the blood serum of cows is the lowest, will give the opportunity to protect the body from possible illnesses, which may occur due to deficiency of microelements, by improving ration of cows with effective mineral additives. RESEARCH MATERIAL AND METHODS Researches have been performed in accordance with Law of Republic of Lithuania on animal care, keeping and use No , dated 06-Nov-1997, ( Valstybės žinios, 28-Nov-1997, No. 108), new edit of the Law, Law of the Republic of Lithuania on animal welfare and protection No. XI 2271, 03-Oct-2012 (Zin., 2012, No (20-Oct-2012)), as well as EU Directive 2010/63/EU Regarding protection of animals used for scientific purposes, dated 22 August 2010, Order of the Minister of Environment No. 517, dated 22 October 2001 (Zin., 2001, No ), EU Directive 86/609/EEC and EC Recommendation 2007/526 EC Animal use and keeping for experimental and other purposes. Work was conducted in in Practical Training and Testing Centre of Veterinary Academy, Experimental and Clinical Pharmacology Laboratory, Department of Non-Infectious Diseases of Veterinary Academy of Lithuanian University of Health Sciences, Environmental and Health Research Laboratory of Biomedical Research Institute of Kaunas University of Medicine and Chemical Metrology Laboratory of Semiconductor Physics Institute. The research was performed on 270 cattle, which consisted of 30 heifers and 240 cows. The research was done in the Giraitės farm, the Practical 9

10 Training and Testing Centre of VA. Groups of cows were formed on analogues basis, in accordance with the time of parturition, productivity, morbidity paresis after parturition, age and supplement of rations with minerals of different chemical structure. Prior to groups formation and during the trials tentative cows were clinically examined. Overall condition of the animal was observed, the pulse was counted, temperature was measured, rumen contractions were calculated, and animal s rumination, diuresis, defecation were observed. The samples of blood were collected from Lithuanian and German Black and White cows and heifers. During the stable period, cows were kept loose and during the grazing period were spent in folds. During the stable period the cows and heifers were fed with perennial haylage of grass and during the period of pasture they were fed with cultural gramma grass. Research was divided into two stages: Research was performed in in Department of Non-Infectious Diseases of Veterinary Academy of Lithuania, Practical Training and Testing Centre of VA, the farm of Giratė. 210 cattles (30 units of heifers and 180 cows) were selected for the trial. The cows and heifers were divided into four groups in accordance with their in-calf features, age, morbidity parturient paresis and productivity: group I: cows in calf, which were tested on 210; 140; 2; 1 day before calving, at calving and on 1; 2; 6; 10 days after calving), n=30 group II: cows, which have paresis following parturition, n=30 group III: cows of different productivity: Cows with an average productivity of 22.4 ± 2.23 l/day of milk are named as less productive cows, which average day of lactation is 62.6 ± 5.37 days, n=30 Cows with an average productivity of 32.6 ± 1.02 l/day of milk, named as more productive cows, which average day of lactation is ± 5.2 days, n= 30 group IV: Heifers and cows of various age: Heifers (1 2 years), n=30 Cows (3 5 years), n= 30 Cows (6 8 years), n=30 Blood draw for biochemical tests was performed in cows of group I when 210; 140; 2; 1; 0 days were left before parturition and 1; 2; 6; 10 days after parturition. Group II was formed of cows with parturient paresis, which yield of colostrums after calving ranged from 15 to 28 liters per day. Paresis after 10

11 parturition was identified based on clinical signs (cows were apathetic, had no appetite, have been observed the disturbed movements of hind legs, tachycardia (heart rate beats/min), subnormal body temperature (ranged from 36.1 to 37.3 ºC), the estrangement of extremity) and biochemistry tests of blood (the level of Ca ranged from 1.6 to 2 mmol/l, compared with standard of calcium mmol/l (Stundžienė et al., 2000)). Blood from cows of group III was collected during the start of lactation (the average day of lactation day). Stage I RESEARCH ON FACTORS, INFLUENCING ON DYNAMICS OF MICROELEMENTS LEVEL IN THE BLOOD SERUM OF COWS Assessment of microelements level dynamics before calving, at calving and 1, 2, 6 and 10 days after calving Evaluation of microelements level in the blood serum of healthy cows of different productivity and cows with paresis after parturition Evaluation of microelements level in the blood serum of heifers and cows of different age Stage II EVALUATION OF EFFICIENCY OF MINERAL ADDITIVES (inorganic and mixed) Evaluation of microelements (Mn, Zn, Cu, Co and I) level in the blood serum of cows before and after administration of mineral additives 11

12 Evaluation of microelements' (Mn, Zn, Cu, Co, I, Fe and Mo) level in the blood serum of cows, with regard to pregnancy, productivity, morbidity paresis after parturition and age The blood samples from cows and heifers of IV group were collected during the grazing season (July August months.) Microelements level, determined in all tested groups, was compared with recommended physiological standards of microelements in cow s blood serum, according to references data (Table 1). Table.1. Microelements level in the blood serum of cattle, according to references data Microelements Standard Deficiency Excess Optimal level in 1 kg of forage DM Manganese, µmol/l 0.4 < 0.4 up to 1.0 mg/kg References: NRC, 2001; Pechová et al Zinc, µmol/l < mg/kg References: Pavlata et al., 2005; Fisher, 2008; Baranauskas et al., 2009; Cope et al., 2009; Spolders et al.,2010 Copper, µmol/l < mg/kg References: Kincaid, 2000; Underwood, Suttle, 2001; Pavlata et al., 2004; Öhlschläger, 2006; Baranauskas et al., 2009 Cobalt, µg/l 0.3 < mg/kg References: Stangl et al., 2000; Tiffany et al., 2001; Kincaid et al., 2003; Kincaid, Socha, 2004; Iodine, µg/l 105 < mg/kg References: Kincaid, 1999; Randhawa, Randhawa, 2001; NRC, 200; Fisher, 2008 Iron, µmol/l < 23 > mg/kg References: Ulutas et al., 2003; Bredon, Dugmore, 2005; Hesketh et al., 2007 Molybdenum, µg/l mg/kg References: NRC, 2001; Raisbeck et al., 2006; Hesketh et al., 2007 Evaluation of microelements (Mn, Cu, Zn, Co and I) level in the blood serum of cows before and after administration of mineral additives Research was performed in August November of 2009 with 60 Lithuanian and German Black and White cows in LVA practical training and testing center and Department of Non-Infectious Diseases of 12

13 Veterinary Academy of Lithuania. Based on the results of the first stage of research, different chemical composition mineral additives have been developed in the Experimental and clinical pharmacology laboratory, Department of Non-Infectious Diseases of Veterinary Academy of Lithuanian University of Health Sciences (Table 2). Table 2. Additives foddered at once, g Group I. Components of inorganic additive Group II. Components of mixed additive Calcium chloride, g 30.0 Calcium lactate, 19 %, g Magnesium chloride, g 5.00 Magnesium chloride, g 5.00 Sodium dihydrogen phosphate, g 4.00 Sodium dihydrogen phosphate, g 4.00 Potassium chloride, g 0.50 Potassium chloride, g 0.50 Cobalt carbonate, g 0.03 Cobalt gluconate, %, g 0.03 Copper sulphate, g 0.48 Copper gluconate, %, g 0.85 Zinc sulphate, g 1.66 Zinc gluconate, %, g 3.86 Manganese sulphate, g 1.66 Manganese sulphate, g 1.66 Potassium iodide, stabilized, g 0.02 Potassium iodide, stabilized, g 0.02 Sodium selenate, g 0.01 Sodium selenate, g 0.01 The study was performed in healthy cows. Depending on their feeding features, the investigative and control groups were formed. Each following group contains 20 cows: group I: cows receiving fodder with inorganic mineral additives (n=20); group II: cows receiving fodder with mixed (inorganic + organic) mineral additives (n=20); group III: control cows, which did not receive mineral additives (they have received lumpy salt licks) (n=20). Cows from group I and II were given mineral additives in the morning before milking (inorganic mineral additives were given in the amount of 43.3 g and mixed mineral additives were given in the amount of g) every second day for 3 months (August November) together with flour. Doses of mineral additives were set in accordance with received results from the first research and recommended physiological microelement standards in the blood serum of cows, provided in the literary sources (Table 1). 13

14 Cows from control group received lumpy salt licks, consisting of 90% of sodium chloride (NaCl), the remaining part consisted of forming substance, instead of mineral additives. Number of lumpy salt licks given to cows was unlimited. Blood of cows for biochemical tests was drawn in August November, i.e. before administration of mineral additives (0 in August) and after month 1 (1 in September), month 2 (2 in October), month 3 (3 in November) after administration of mineral additives, in the mornings. Biochemical test of cows blood Blood samples were collected from the jugular vein into disposable tubes Venoject (Terumo Europe NV, Belgium) without anticoagulant. Blood samples were delivered to the laboratory within one hour and centrifuged for five minutes with speed of revolutions per minute. The tubes filled with serum were frozen in refrigerator compartment at 20 ºC temperature. We have determined microelements level when all tubes with blood serum were thawed simultaneously. Microelements (Mn, Zn, Cu, Fe and Mo) level in the blood serum were determined in Institute for Biomedical Research of Kaunas University of Medicine, in accordance with modified method (Schlemmer, 1989), using Perkin - Elmer (USA) electrical thermographic atomic absorption spectrophotometer Zeeman We have used Medisafe Metalle S, Level 1, E (manufacturer Medichem, Germany) standards. Level of cobalt was determined by using the method of GF-AAS (Graphite furnace atomic absorption spectrometry). I concentration in the blood samples was determined by Inductively-Coupled Plasma Mass Spectrometer Element-2 (ThermoFinnigan AB) in Chemical Metrology Laboratory of Semiconductor Physics Institute using method of Inductively Coupled Plasma Mass Spectrometry (ICP-MC). Statistic data assessment Research results and statistic data were calculated using computer program Statistica 10 (StatSoft Inc., USA), as well as standard programs Windows and Excel. Also arithmetic medium (M), standard deviation (SD) and Pearson correlation coefficient (r) were calculated of received data. Student s multiple comparison method was applied to determine the difference between group significance criterion (P). The difference was considered significant, if P <

15 RESULTS OF THE RESEARCH Changes of microelements (Mn, Zn, Cu, Co, I, Fe and Mo) level in the blood serum of cows before calving, at calving and after 1, 2, 6 and 10 days following calving Obtained results show, that Mn level in the blood serum of tested cows varied from 0.4 to 1.1 µmol/l. Mn level in the blood serum of cows reduced during calving (0.58 ± µmol/l, P<0.05) and on 1 day (0.55 ± µmol/l, P<0.05), 2 days (0.53 ± µmol/l, P<0.05), and 6 days (0.58 ±0.139 µmol/l, P<0.05) after calving, comparing to element level, determined at 210 and 140 days before parturition. After ten days following calving Mn level in the blood serum of cows significantly increased (up to 0.75 ± 0.14 µmol/l, P<0.05), comparing to 210, 2 and 1 day before calving (Fig. 1). μmol/l 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0, Days before and after parturition Mn Mn (standard) Fig. 1. The changes of manganese level in the blood serum of cows before and after parturition Note: Mn (standard) Recommended physiological standard of manganese in the blood serum of cows 0.4 µmol/l (Pechová et al. 2008). Zn level in the blood serum of cows varied from 11.3 µmol/l to 21.7 µmol/l. Insignificant Zn deficiency ( µmol/l) was determined in eight cows on 1, 2, 6 days after calving and in three cows on 10 day after 15

16 calving, comparing to physiological zinc standard (12.7 µmol/l), recommended by scientists in cows blood serum. Zn level significantly decreased at calving (15.45 ± 0.8 µmol/l, P<0.05) and on 1 (12.3 ± 0.56 µmol/l, P<0.05), 2 (12.57 ± 0.62 µmol/l, P<0.05), 6 (12.26 ± 0.52 µmol/l, P<0.05) and 10 (12.75 ± 0.6 µmol/l, P<0.05) days after calving, comparing to the element level, determined on 210, 140 and 2 days before calving (Fig. 2). µmol/l Days before and after parturition Zn Zn standard Fig. 2. The changes of zinc level in the blood serum of cows before and after parturition Note: Zn (standard) Recommended physiological standard of zinc in the blood serum of cows 12.7 µmol/l (Cope et al., 2009). Cu level in the blood serum of tested cows varied from 9.35 µmol/l to µmol/l. Cu deficiency ( µmol/l) was noticed in one cow on day 210 before calving, in three cows on day 140 before calving, in four cows after 1 day following calving, in twelve cows after 2 days following calving, in seventeen cows after 6 days following calving and in all cows after 10 days following calving, comparing to physiological Cu standard (12 µmol/l), recommended by scientists in cows blood serum. Cu level in the blood serum of cows significantly decreased on 1 (13.19 ± 0.9 µmol/l, P<0.05), 2 (12.13 ± 0.8 µmol/l, P<0.05), 6 (11.91 ± 0.74 µmol/l, P<0.05) and 10 (10.2 ± 0.46 µmol/l, P<0.05) days after calving, comparing to this element level, determined on 210, 140, 2, and 1 day before calving (Fig. 3). 16

17 µmol/l Days before and after parturition Cu Cu (standard) Fig. 3. The changes of copper level in the blood serum of cows before and after parturition Note: Cu (standard) Recommended physiological standard of copper in the blood serum of cows 12 µmol/l (Öhlschläger, 2006). µg/l 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0, Days before and after parturition Co Co (standard) Fig. 4. The changes of cobalt level in the blood serum of cows before and after parturition Note: Co (standard) recommended physiological standard of cobalt in the blood serum of cows 0.3 µg/l (Kincaid, Socha, 2004). 17

18 We have determined, that concentration of Co in the blood serum of cows was above ( µg/l), than physiological Co standard (0.3 µg/l), recommended by scientists in cows blood serum. However, concentration of this element was less, comparing to Co level, determined on 210 and 2 days before calving, during calving (0.53 ± 0.04 µg/l, P<0.05) and on 1 (0.54 ± 0.04 µg/l, P<0.05), 2 (0.53 ± 0.03 µg/l, P<0.05), 6 (0.52 ± 0.03 µg/l, P<0.05) and 10 (0.51 ± 0.03 µg/l, P<0.05) days after calving (Fig. 4). I level in the blood serum of cows ranged from 63.4 to 110 µg/l. Insignificant I level deficiency ( µg/l) was determined in all cows on 1 day before calving, during calving and after it, comparing to physiological iodine standard (105 µg/l), recommended by scientists. Significantly decreased level of I was determined at calving (92.9 ± 3.17 µg/l, P<0.05) and on 1 (85.9 ± 3.72 µg/l, P<0.05), 2 (72.6 ± 5.16 µg/l, P<0.05), 6 (75.3 ± 5.64 µg/l, P<0.05) and 10 (72.2 ± 5.45 µg/l, P<0.05) days after calving, comparing to this element level, determined on 210, 140, 2 and 1 day before calving (Fig. 5) µg/l Days before and after parturition I I (standard) Fig. 5. The changes of I level in the blood serum of cows before and after parturition Note: I (standard) Recommended physiological standard of iodine in the blood serum of cows µg/l (Randhawa, Randhawa, 2001; NRC, 2001). 18

19 Fe level in the blood serum of cows ranged from 18.3 to 33.1 µmol/l. Fe level deficiency ( µmol/l) was determined in fifteen cows on 1 day before calving, in twenty three cows - at calving, in twelve cows - on 1 day after calving and in nine cows - on 2 days after calving, comparing to physiological iron standard (23 µmol/l), recommended by scientists in cows' blood serum. Fe level in the blood serum of cows decreased significantly on 1 day before calving (23.3 ± 2.09 µmol/l, P<0.05), at calving (22.1 ± 1.88 µmol/l, P<0.05) and on 1 (23.5 ± 2.11 µmol/l, P<0.05), 2 (24.3 ± 2.12 µmol/l, P<0.05) and 6 (27.4 ± 1.33 µmol/l, P<0.05) days after calving, comparing to Fe level, determined on 210, 140 and 2 days before calving (Fig. 6) µmol/l Days before and after parturition Fe Fe (standard) Fig. 6. The changes of Fe level in the blood serum of cows before and after parturition Note: Fe (standard) Recommended physiological standard of iron in the blood serum of cows 23.0 µmol/l (Ulutas et al., 2003; Hesketh et al., 2007). Mo level in the blood serum of cows ranged from µg/l. This element decreased significantly on 1 day after calving (up to 2.41 ± 0.26 µg/l), comparing to Mo level, determined on 210, 140 and 2 days before calving. After 6 and 10 days following calving the level of molybdenum in the blood serum of cows increased significantly ( 2.68 ± 0.33 µg/l and 2.76 ± 0.33 µg/l, P<0.05), comparing to Mo level, determined on 1 day before calving (Fig. 7). 19

20 3,0 2,5 2,0 µg/l 1,5 1,0 0,5 0, Days before and after parturition Mo Mo (standard) Fig. 7. The changes of molybdenum level in the blood serum of cows before and after parturition Note: Mo (standard) Recommended physiological standard of molybdenum in the blood serum of cows 1.0 µg/l (Hesketh et al., 2007; Miranda et al., 2006). The negative correlation between Zn and Cu level was determined on 210 days before calving and on 10 days after calving (r= and r= -0.49). Positive correlation between I and Zn level (r= 0.39) was noticed on 140 days before calving. Negative correlation between I and Co level (r= -0.4) and between Zn and Co (r= -0.37) were noticed on 1 day before calving. The Cu level negatively correlated with Co (r= -0.61) and I level correlated positively with Zn level (r= 0.37) on 1 day after calving. Negative correlation between Mn and Mo level (r= -0.52; r= and r= -0.39) was noticed at calving and on 1 and 2 days after calving. Microelements (Mn, Zn, Cu, Co, I, Fe and Mo) level in the blood serum of healthy cows of different productivity and cows with paresis after parturition Mn level in the blood serum of healthy cows of different productivity and cows with paresis after parturition varied from µmol/l. The lowest level of Mn (0.63 ± 0.16 µmol/l, P<0.05) was determined in the 20

21 blood serum of more productive cows, comparing to other groups of cows. Mn was lower by 0.1 µmol/l (P<0.05) in the blood serum of cows with paresis, comparing to less productive cows (Fig. 8). 1,2 1,0 0,8 µmol/l 0,6 0,4 0,2 0,0 Less productive More productive Cows Paresis Mn Mn (standard) Fig. 8. Manganese level in the blood serum of healthy cows of different productivity and cows with paresis after parturition 13,5 12,0 10,5 9,0 µmol/l 7,5 6,0 4,5 3,0 1,5 0,0 Less productive More productive Cows Paresis Zn Zn (standard) Fig. 9. Zinc level in the blood serum of healthy cows of different productivity and cows with paresis after parturition 21

22 Zn level in the blood serum of healthy cows of different productivity and cows with paresis after parturition varied from µmol/l. The lowest level of Zn (11.3 ± 1.19 µmol/l, P<0.05) was determined in the blood serum of cows with paresis after parturition, comparing to other groups of cows. Zinc was lower by 0.57 µmol/l (P<0.05) in the blood serum of more productive cows, comparing to less productive cows. Level of zinc, determined in the blood serum of more productive cows and cows with paresis after parturition was significantly lower (12.09 ± 1.21 µmol/l and 11.3 ± 1.19 µmol/l, P<0.05) than recommended physiological zinc standard (12.07 µmol/l), recommended by scientists in the blood serum of cows (Fig. 9). The level of Cu in the blood serum of healthy cows of different productivity and cows with paresis after parturition varied from µmol/l. The lowest level of Cu (10.32 ± 1.14 µmol/l, P<0.05) was determined in the blood serum of cows with paresis after parturition, comparing to other groups of cows and physiological Cu standard (12 µmol/l), recommended by scientists. Cu was lower by 0.42 µmol/l (P<0.05) in the blood serum of more productive cows, comparing to less productive cows; however, more reliable reading was not determined (Fig. 10) µmol/l Less productive More productive Cows Paresis Cu Cu (standard) Fig. 10. Copper level in the blood serum of healthy cows of different productivity and cows with paresis after parturition 22

23 The level of cobalt in the blood serum of tested cows ranged from µg/l. The lowest level (0.49 ± µg/l, P<0.05) of cobalt was determined in the blood serum of cows with paresis after parturition, comparing to other cows. Cobalt level in the blood serum of more productive cows was 0.02 µg/l (p>0.05) less, comparing to cobalt level, determined in the blood serum of less productive cows (Fig. 11). 0,7 0,6 0,5 µg/l 0,4 0,3 0,2 0,1 0,0 Less productive More productive Cows Paresis Co Co (standard) µg/l Fig. 11. Cobalt level in the blood serum of healthy cows of different productivity and cows with paresis after parturition Less productive More productive Cows Paresis I I (standard) Fig. 12. Iodine level in the blood serum of healthy cows of different productivity and cows with paresis after parturition 23

24 Level of iodine in the blood serum of healthy cows of different productivity and cows with paresis after parturition varied from 47.7 to 108 µg/l. The lowest level of iodine (47.7 ± 7.47 µg/l, P<0.05), was determined in the blood serum of cows with paresis after parturition, comparing to other groups of cows. I was lower by µg/l (P<0.05) in the blood serum of more productive cows, comparing to less productive cows. Iodine level, determined in the blood serum of all tested cows was significantly lower, than physiological iodine standard (105 µg/l), recommended by scientists in the blood serum of cows (Fig. 12). Fe level in the blood serum of healthy cows of different productivity and cows with paresis after parturition ranged from 20.4 to 33.2 µmol/l. The lowest level of Fe (24.16 ± 2.38 µmol/l, P<0.05) was determined in the blood serum of more productive cows, comparing to other groups of cows. Fe was lower by 1.62 µmol/l (P<0.05) in the blood serum of cows with parturient paresis, comparing to less productive cows (Fig. 13) µmol/l Less productive More productive Cows Paresis Fe Fe (standard) Fig. 13. Iron level in the blood serum of healthy cows of different productivity and cows with paresis after parturition Level of Mo in the blood serum of healthy cows of different productivity and cows with paresis after parturition varied from 1.8 to 3.2 µg/l. The lowest Mo level (2.33 ± 0.3 µg/l) was determined in the blood serum of more productive cows, however the reliable data were obtained only by comparing to less productive cows (Fig. 14). 24

25 3,0 2,5 2,0 µg/l 1,5 1,0 0,5 0,0 Less productive More productive Cows Paresis Mo Mo (standard) Fig. 14. Molybdenum level in the blood serum of healthy cows of different productivity and cows with paresis after parturition Negative correlation between Co and Mo level (r= -0.5) and positive correlation between Fe and Co (r= 0.41), Fe and Mn (r= 0.44) and negative correlation between Fe and Mo (r= -0.46) were noticed in the blood serum of less productive cows. Negative Fe correlation with Zn (r= -0.38) and with Mo (r= -0.39) was noticed in the blood serum of more productive cows. Positive Zn correlation with Cu (r= 0.43) and positive Fe correlation with Mo (r= 0.47) were noticed in the blood serum of cows with paresis after parturition. Microelements (Mn, Zn, Cu, Co, I, Fe and Mo) level in the blood serum of heifers and cows of different age Mn level varied from 0.4 to 1.2 µmol/l in the blood serum of heifers and cows of different age. Mn level in the blood serum of heifers and cows of 3 5 years old was almost equal (0.81 ± 0.14 µmol/l and 0.80 ± 0.14 µmol/l). Concentration of Mn in the blood serum of cows of 6 8 years old was significantly lower (0.58 ± 0.18 µmol/l, P<0.05), comparing to heifers and cows of 3 5 years old (Fig. 15). Zn level in the blood serum of heifers and cows of different age, varied from µmol/l. Significantly lowered Zn level was determined in the blood serum of cows of 3 5 years old (0.80 ± 0.14 µmol/l, P<0.05) 25

26 and 6 8 years old (11.98 ± 1.15 µmol/l, P<0.05), comparing to zinc level, determined in the blood serum of heifers. Zinc level in the blood serum of cows of 6 8 years old was significantly lower, than physiological zinc standard (12.7 µmol/l), recommended by scientists (Fig. 16). µmol/l 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 1-2 yr. 3-5 yr. 6-8 yr. Experimental groups Fig. 15. Manganese level in the blood serum of heifers and cows of different age Mn Mn (standard) µmol/l yr. 3-5 yr. 6-8 yr. Experimental groups Zn Zn (standard) Fig. 16. Zinc level in the blood serum of heifers and cows of different age 26

27 Concentration of Cu in the blood serum of heifers and cows of different age varied from 7.69 to µmol/l. Cows of 6 8 years old had the lowest level of Cu (10.33 ± 1.45 µmol/l, P<0.05), comparing to heifers and cows of 3 5 years old. Concentration of copper in the blood serum of heifers was 2.21 µmol/l less (P<0.05) in comparison with Cu level, determined in the blood serum of cows of 3 5 years old. Level of Cu, determined in the blood serum of heifers and cows of 6 8 years old, was significantly less (11.33 ± 1.53 µmol/l and ± 1.45 µmol/l, P<0.05), than physiological Cu standard (12 µmol/l), recommended by scientists (Fig. 17) µmol/l yr. 3-5 yr. 6-8 yr. Experimental groups Cu Cu (standard) Fig. 17. Copper level in the blood serum of heifers and cows of different age Concentration of Co in the blood serum of heifers and cows of different age ranged from 0.36 to 0.77 µg/l. Co level in the blood serum of cows of 6 8 years old was significantly less (0.5 ± 0.08 µg/l, P<0.05), comparing to heifers and cows of 3 5 years old. Cows of 3 5 years old had 0.07 µg/l, (P<0.05) less cobalt, comparing to cobalt level, determined in heifers blood serum (Fig. 18). Concentration of I in the blood serum of heifers and cows of different age ranged from µg/l. The lowest level of I (66.2 ± 8.61 µg/l, P<0.05) was determined in the blood serum of heifers, comparing to cows of different age. Cows of 3 5 years old had 9.2 µg/l less iodine (P<0.05), comparing to iodine level, determined in the blood serum of cows of 6 8 years old. Level of iodine in the blood serum of all tested cows and heifers 27

28 was reliably less, comparing to physiological I standard (105µg/L) recommended by scientists (Fig. 19). µg/l 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 1-2 yr. 3-5 yr. 6-8 yr. Experimental groups Co Co (standard) 120 Fig. 18. Cobalt level in the blood serum of heifers and cows of different age µg/l yr. 3-5 yr. 6-8 yr. Experimental groups I I (standard) Fig. 19. Iodine level in the blood serum of heifers and cows of different age 28

29 Level of Fe in the blood serum of heifers and cows of different age varied from 21.4 to 32.8 µmol/l. The lowest Fe level (24.63 ± 2.11 µmol/l, P<0.05) was determined in the blood serum of cows of 6 8 years old, comparing to heifers and cows of 3 5 years old. Fe level in the blood serum of cows of 3 5 years old was 1.37 ± 0.58 µmol/l (P<0.05) less, comparing to Fe level, determined in the blood serum of heifers (Fig. 20) µmol/l yr. 3-5 yr. 6-8 yr. Experimental groups Fe Fe (standard) 3,0 Fig. 20. Iron level in the blood serum of heifers and cows of different age 2,5 2,0 µg/l 1,5 1,0 0,5 0,0 1-2 yr. 3-5 yr. 6-8 yr. Experimental groups Mo Mo (standard) Fig. 21. Molybdenum level in the blood serum of heifers and cows of different age 29

30 Mo level in the blood serum of heifers and cows of different age varied from 1.8 to 3.4 µg/l. The lowest Mo level (2.48 ± 0.42 µg/l, p>0.05) was determined in the blood serum of heifers, comparing to other groups of cows. Mo level in the blood serum of cows (3 5 years old and 6 8 years old) was similar (2.6 ± 0.4 µg/l and 2.58 ± 0.32 µg/l) (Fig. 21). Positive Mn correlation with I was noticed in the blood serum of heifers (r= 0.4). Positive Zn correlation with Cu (r= 0.5), negative Mn correlation with Fe (r= -0.41) and positive I correlation with Mo (r= 0.37) were noticed in the blood serum of cows of 6 8 years old. Effect of mineral additives on the level of microelements (Mn, Zn, Cu, Co, I) in the blood serum of cows Mn level in the tested cows group increased insignificantly after two months, and increased significantly (up to 0.86 ± µmol/l, P<0.05) after three months following administration of mineral additives. The highest level of Mn (0.86 ± µmol/l) in the blood serum of cows from this group was determined after three months following administration of additives (Fig. 22). Mn, µmol/l 0,96 0,94 0,92 0,90 0,88 0,86 0,84 0,82 0,80 0,78 0,76 0,74 0-Aug. 1-Sept. 2-Oct. 3-Nov. Months I gr. II gr. III gr. Fig. 22. Manganese level in the blood serum of cows before and after administration of mineral additives 30

31 After two and three months following mixed mineral additives administration, Mn level in the blood serum of tested cows was significantly higher (0.85 ± 0.06 µmol/l and 0.91 ± µmol/l, P<0.05), in comparison to ration before administration of additives and to control group. After three months following mixed mineral additives administration, Mn level in the blood serum of cows was increased by 5.49 % (P<0.05), comparing to cows, which have received inorganic mineral additives (Fig. 22). Mn level in the blood serum of cows from control group varied from 0.6 to 1 µmol/l. After three months from start of trial, the level of Mn in this group of cows was 0.80 ± µmol/l (Fig. 22). Zn level in the blood serum of tested cows significantly increased after two and three months following administration of inorganic mineral additives (15.93 ± 0.64 µmol/l and ± 0.71 µmol/l, P<0.05). The highest level of Zn (15.93 ± µmol/l) in the blood serum of cows from this group was determined after two months following administration of inorganic mineral additives (Fig. 23). Zn, µmol/l 16,6 16,4 16,2 16,0 15,8 15,6 15,4 15,2 15,0 14,8 0-Aug. 1-Sept. 2-Oct. 3-Nov. Months I gr. II gr. III gr. Fig. 23. Zinc level in the blood serum of cows before and after administration of mineral additives Zn level in the blood serum of tested cows was significantly higher after one (15.69 ± µmol/l, P<0.05), two (15.91 ± µmol/l, P<0.05) and three (16.07 ± µmol/l, P<0.05) months following administration of mixed mineral additives, comparing to ration before 31

32 administration of additives and to control group. After three months from the start of the trial Zn level in the blood serum of cows receiving mixed mineral additives was increased by 1.24 % (P<0.05), comparing to cows receiving inorganic mineral additives (Fig. 23). After three months from the start of the trial Zn level in the blood serum of cows from control group was ± µmol/l (Fig. 23). Cu level in the blood serum of tested cows significantly increased after one, two and three months following administration of inorganic additives (13.17 ± 0.69 µmol/l, ± 0.66 µmol/l and ± 0.64 µmol/l, P<0.05), comparing to ration before administration of mineral additives. The highest level of Cu (13.46 ± µmol/l) in the blood serum of cows from this group was determined after three months following administration of additives (Fig. 24). Level of Cu in the blood serum of cows receiving mixed mineral additives ranged from to µmol/l. After two and three months following administration of mixed mineral additives Cu level has significantly increased (13.41 ± µmol/l and ± µmol/l, P<0.05) in the blood serum of tested cows, comparing to ration before administration of additives and to control group. After three months following administration of mixed mineral additives Cu level in the blood serum of cows has increased by 1.68 % (P<0.05), in comparison to cows receiving inorganic mineral additives (Fig. 24). Cu, µmol/l 14,2 14,0 13,8 13,6 13,4 13,2 13,0 12,8 12,6 12,4 0-Aug. 1-Sept. 2-Oct. 3-Nov. Months I gr. II gr. III gr. Fig. 24. Copper level in the blood serum of cows before and after administration of mineral additives 32

33 Cu level in the blood serum of cows from control group varied from to µmol/l. After three months from the start of the trial level of Cu in the blood serum of cows from this group was ± µmol/l (Fig. 24). Co level in the blood serum of tested cows increased significantly after one, two and three months following administration of inorganic mineral additives (0.55 ± 0.07 µg/l, 0.57 ± 0.07 µg/l and 0.58 ± 0.07 µg/l, P<0.05), comparing to ration before administration of additives and to control group. The highest level of Co (0.58 ± µg/l) in the blood serum of cows from this group was determined after three months following administration of additives (Fig. 25). After two and three months following mixed mineral additives Co level has significantly increased (0.58 ±0.071 µg/l and 0.60 ± µg/l, P<0.05) in the blood serum of tested cows, comparing to ration before administration of additives and to control group. After three months following administration of mixed mineral additives Co level in the blood serum of cows increased by 3.3 % (P<0.05), comparing to cows receiving inorganic mineral additives (Fig. 25). Co, µg/l 0,64 0,62 0,60 0,58 0,56 0,54 0,52 0,50 0,48 0,46 0-Aug. 1-Sept. 2-Oct. 3-Nov. Months I gr. II gr. III gr. Fig. 25. Cobalt level in the blood serum of cows before and after administration of mineral additives After three months from the start of the trial level of Co in the blood serum of cows from control group was 0.53 ± µg/l (Fig. 25). 33

34 Iodine level in the blood serum of tested cows increased significantly after two and three months following administration of inorganic mineral additives (80.97 ± 4.26 µg/l and ± 5.29 µg/l, P<0.05), comparing to rations before administration of mineral additives and to control group. The highest level of I in the blood serum of cows from this group was determined (90.14 ± 5.29 µg/l) after three months following administration of additives (Fig. 26). After two and three months following mixed mineral additives I level has significantly increased (82.12 ± 3.6 µg/l and ± 3.7 µg/l, P<0.05) in the blood serum of tested cows, comparing to ration before administration of additives and to control group. After three months following administration of mixed mineral additives I level in the blood serum of cows increased by 2.86 % (P<0.05), comparing to cows receiving inorganic mineral additives (Fig. 26). I, µg/l Aug. 1-Sept. 2-Oct. 3-Nov. Months Fig. 26. Iodine level in the blood serum of cows before and after administration of mineral additives I gr. II gr. III gr. After three months from the start of the trial level of I in the blood serum of cows from control group was ± µg/l (Fig. 26). The negative Co correlation with Zn (r= -0.47) was noticed in the blood serum of cows before administration of inorganic mineral additives. The negative Cu correlation with I (r= -0.49; r= -0.45; r= -0.58; r= -0.62) was noticed in the blood serum of control cows, before trial and after 1, 2 and 3 months of trial. 34

35 CONCLUSION 1. Tendency of microelements level decrease was determined at calving and 1 10 days after calving. Determination of microelements level in the blood serum of cows before calving, at calving and after 1, 2, 6 and 10 days following calving allows to assess deficiency of microelements (Zn, Cu, I and Fe) in the early stage. 2. Microelements are significantly lower in the blood serum of more productive cows (32.6 ± 1.02 l of milk per day) and cows with paresis after parturition, comparing to less productive cows (22.4 ± 2.23 l of milk per day). Determined Zn, Cu and I levels did not reach physiological standards of microelements (12.7 µmol/l; 12 µmol/l; 105 µg/l), recommended by scientists, P< The lowest level of microelements was determined in the blood serum of older cows (6 8 years old), comparing to younger cows (3 5 years old) and heifers (1 2 years old), P<0.05. With reference to recommended physiological standard of microelements (Zn 12.7 µmol/l; Cu 12 µmol/l), Zn and Cu deficiency was determined in the blood serum of older cows, P< Mineral additives are effective in maintaining microelements level in the blood serum of cows. Use of mixed mineral additives (43.93 g every second day) in the blood serum of cows determined significantly higher level of Mn (5.49%), Zn (1.24%), Cu (1.68%), Co (3.3%) and I (2.86%), comparing to inorganic mineral additives (43.36 g every second day) (P<0.05). Publications: LIST OF HANDOUT AND PUBLICATIONS 1) Vytautas Špakauskas, Vaida Jokubauskienė, Algimantas Matusevičius, Algirdas Černauskas, Modestas Ružauskas, Irena Klimienė. Effect of mineral additives on level of microelements in the blood serum of milch cows (Lith. Mineralinių priedų įtaka mikroelementų kiekiui melžiamų karvių kraujo serume). Vet. Med. Zoot (73). P ) Vaida Jokubauskienė, Vytautas Špakauskas, Algimantas Matusevičius, Irena Klimienė, Modestas Ružauskas, Monika Žilinskaitė. Effect of various factors on Mn, Mo, Fe levels in the blood serum of incalf and milch cows (Lith. Įvairių veiksnių įtaka mangano, molibdeno, 35