ADIPOKINES, METABOLIC HORMONES AND BLOOD METABOLITES IN HIGH AND MEDIUM BODY CONDITION TRANSIENT CROSSBRED COWS DURING WINTER AND SUMMER SEASONS

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1 ADIPOKINES, METABOLIC HORMONES AND BLOOD METABOLITES IN HIGH AND MEDIUM BODY CONDITION TRANSIENT CROSSBRED COWS DURING WINTER AND SUMMER SEASONS THESIS SUBMITTED TO THE ICAR- NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF VETERINARY SCIENCE IN ANIMAL PHYSIOLOGY BY BIKASH DEBNATH B.V.Sc. & A.H. DAIRY CATTLE PHYSIOLOGY DIVISION ICAR-NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) KARNAL (HARYANA), INDIA 2015 Regn. No. 13-M-AP-01

2 ADIPOKINES, METABOLIC HORMONES AND BLOOD METABOLITES IN HIGH AND MEDIUM BODY CONDITION TRANSIENT CROSSBRED COWS DURING WINTER AND SUMMER SEASONS By BIKASH DEBNATH THESIS SUBMITTED TO THE ICAR - NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF VETERINARY SCIENCES IN ANIMAL PHYSIOLOGY Approved by: ~l Dr. ANJALI AGGARWAL MAJOR ADVISOR & CHAIRMAN (GUIDE) Members of Advisory Committee Dr. O.K. Hooda Head, DCP Division Dr. Parveen Kumar Principal Scientist, DCP Division Dr. Sudarshan Kumar Scientist, ABTC Division Dr. Veena Mani ~~~ ( Principal Scientist, DCN Division Jt. Director Nominee / \ ') ~2.),~~)' ~I~

3 Division of Dairy Cattle Physiology ICAR - National Dairy Research Institute (Deemed University) Karnal (Haryana), India Dr. ANJALI AGGARWAL Principal Scientist CERTIFICATE This is to certify that the thesis entitled, "ADIPOKINES, METABOLIC HORMONES AND BLOOD METABOLITES IN HIGH AND MEDIUM BODY CONDITION TRANSIENT CROSSBRED COWS DURING WINTER AND SUMMER SEASONS" submitted by BIKASH DEBANATH towards the partial fulfilment of the award of the degree of MASTER OF VETERINARY SCIENCE in ANIMAL PHYSIOLOGY of the ICAR NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY), Kamal (Haryana), India, is a bonafide research work carried out by him under my supervision, and no part of the thesis has been submitted for any other degree or diploma. Dated: 16 th JULY, 2015 (ANJALI AGGARWAL) MAJOR ADVISOR & CHAIRMAN (GUIDE)

4 DEDICATE TOM BELOVED PARENTS

5 ACKNOWLEDGEMENTS Emotions can t be adequately expressed in words hence my acknowledgement is much more and deep beyond the words used here. Acknowledgement for a few might be just a trifle written on a piece of paper, but, in its true essence, it gives one opportunity to remember and express one`s feeling for those whom one loves and respect a lot. Here, I get a chance to express my token of thanks to people who are not enough to express my feeling for them, yet these lines are not exaggeration, but feelings, which come straight from my heart. As a matter of eternal compliance, above all I would like to thank soulfully, the Almighty God who bestowed me with his blessings to accomplish this investigation. With all the experiences of joys and sorrow God has always been kind to me and helped me in every step of my life to make me understand the value of life. I thank God Almighty for His Grace and Mercy upon me during my entire work. With deep reverence, I express my profound gratitude to my guide Dr Anjali Aggarwal, Principal Scientist, DCP Division for her guidance, inspiration, sustained encouragement and moral support during the execution of the present piece of research work, I am extremely thankful for her immense affection and exceptional manners to make the scholars more comfortable to work in a team with high spirits. I would like to express my sincere gratitude to Director-cum-Vice Chancellor, NDRI, Dr. A. K. Shrivastava for providing all the necessary facilities and funds for successful completion of the project. Financial assistance from NDRI, in the form of JRF is gratefully acknowledged. I am thankful to Dr. R.C. Upadhyay former head of the division for his suggestions, encouragement and allowing me to access to all the required facilities during my research work Dr. R.C Upadhyay was always ready to do anything for my work right from my synopsis. I deeply admired and honoured his brilliancy and simple attitude towards me and other students. And through him I got the financial support for my work from the biggest project NICRA (National Initiative on Climate Resilience Agriculture ) became one of the most resourceful funds I received during my entire work. My heartfelt gratitude to all the Advisory Committee Members-, Dr. O.K.Hooda, Dr. (Mrs.) Veena Mani, Dr. Parveen Kumar and Dr. Sudarshan Kumar for their timely suggestions, guidance and advice during the entire research work. I am also thankful to Mr. Gian Singh for his help in analyzing my data. I would be failing in my duties if I do not express my appreciation to the persons who helped throughout my research work, especially Suresh kumar who always encourage me to complete my research work and my lab mate Dr. Suresh kapoor who always with me all the time in every step of work and all my

6 batchmates Dr. Jadhav Vyankat Gangadhar, Dr., Dr. Rachana Sharma, Dr. Purnima Singh, Dr. Ravi Kumar Soni, Dr. Anjali Shrivastava for giving me help whenever sought. I also feel indebted to my ever helping seniors, Dr. BS Bharat, Dr. Ramendra das, Dr. Asif Ahammed Saikh, Dr. Ovias Aarif for their abiding interest and timely help. Memorable time spent with these seniors, class mates and juniors would always be cherished. My special thanks go to my UG batchmates Dr Amaresh Nath, Dr. Ratan Das, Dr. Arunava Sarkar for helping me in final compilation of thesis. A special thanks goes to dairy workers Prem, Jitender, Sonu who despite their fixed time schedule, always spared time to assist me to examine farm animals. All the words in the lexicon will be futile and meaningless, if I fail to divulge my extreme sense of regards to adorable parent s father, Mr. Nripendra Chandra Debnath and mother, Mrs. Kanak Debnath. The selfless help, moral support, blessings and sacrifices rendered by my parents will always remain evergreen in my mind at each step of my life. Words sound hollow and phrases lose their meaning, as I find myself unable to put forth my warmest love and thanks to my elder brothers Biswajit Debnath and Biplab Debnath, Sister Sukla Debnath and my other relatives who have always cared for my happiness cannot be acknowledged by mere words. Finally, I would like to thank everybody who was important to the successful realization of this thesis, as well as expressing my apology that i could not mention personally one by one. Date: 16/07/2015 (Bikash Debnath)

7 LIST OF ABBREVIATIONS % : Percentage eq/l : Microequivalent per litre mol : Micromol mol/l : Micromol per litre C : Degree centigrade / Celsius µg/l : Microgram per litre µg/ml : Microgram per millilitre µiu/ml : Micro international unit per millilitre µl : Microlitre a.m. : Ante meridien ADP : Adiponectin ANOVA : Analysis of variance APPs : Acute phase proteins BCS : Body condition score bpm : Beats per minutes bpm : Breaths per minutes BT : Body temperature CNS : Central nervous system CORT : Cortisol CSSRI : Central Soil Salinity Research Institute DLC : Differential leukocyte count E : Evening E : East EAT : Effective ambient temperature EDTA : Ethylene Diamine Trichloro Acetic Acid ELISA : Enzyme linked immunosorbent assay fl : Femtolitre gm/dl : Gram per decilitre GOD-POD : Glucose oxidase peroxidase h : hour Hb : Hemoglobin HBC : High body condition

8 Hct : Haematocrit HRP : Horse redish peroxidise IAEC : Institutional Animal Ethics Committee IGF-1 : Insulin like growth factor 1 IL-6 : Interleukin 6 INS : Insulin IRS-1 : Insulin receptor substrate 1 kda : Kilo Dalton KF : Karan-Fries Kg : Kilogram Km : Killometre LEP : Leptin LFI : Liver Functility Index M : Morning MBC : Medium body condition MCH : Mean corpuscular haemoglobin MCHC : Mean corpuscular haemoglobin concentration MCV : Mean corpuscular volume mg/dl : Milligram per decilitre min : Minute(s) ml : Millilitre mm : Millimol mm : Millimeter mmol/l : Millimol per litre mrna : Messenger ribonucleic acid mu/l : Milli international unit per litre N : North NEB : Negative energy balance NEFA : Non esterified fatty acids ng : Nanogram ng/dl : Nanogram per decilitre ng/ml : Nanogram per millilitre nm : Nanometre nmol/l : Nanomol per litre O.D : Optical Density

9 O 2 : Oxygen PBS : Phosphate buffer saline PCV : Packed cell volume pg : Picogram pg/ml : Picogram per millilitre PR : Pulse rate RBC : Red Blood Cells RETN : Resistin RH : Relative humidity rpm : Revolution per minute RR : Respiration rate RT : Rectal temperature S.E.M : Standard Error Mean SD : Standard deviation SOD : Superoxide Dismutase ST : Skin temperature T 3 : Triiodothyronin T 4 : Thyroxin TAG : Tri-acyl glyceride TCA : Tricarboxylic acid cycle TEC : Total Erythrocytic Count THI : Temperature Humidity Index TLC : Total Leukocyte Count T max : Maximum Ambient Temperature TMB : Tetra methyl benzedene T min : Minimum Ambient Temperature TNF-α : Tumour Necrosis Factor Alpha Tr : Rectal temperature UV : Ultra violet WBC : White Blood Cells wk : Week β-hba : Beta hydroxyl butyric acid CONTENTS

10 Chapter No. Title Page No. 1.0 Introduction Review of literature Body condition score (BCS) Body condition score at the time of calving Body condition score during early lactation Adipokines in transition cows during winter and summer seasons Leptin Adiponectin Resistin Tumour necrosis factor- α (TNF-α) Interleukin-6 (IL-6) Metabolic hormones Cortisol Insulin Insulin like growth fator-1 (IGF-1) Thyroid Hormones Heamatological parameters Blood metabolites Glucose Non-Esterified Fatty Acids (NEFA) β-hydroxybutyrate (β-hba) Physiological responses Rectal Temperature Respiration rate Pulse rate Skin temperature Materials and Methods Location of the farm Experimental animals Housing of the animals Feeding of the animals Body condition score Ethical permission Plan of work Winter Experiment (December - February) Summer Experiment (March - May) Meteorological conditions Physiological Parameters Rectal temperature (RT) 32

11 Chapter No. Title Page No Respiration rate (RR) Pulse Rate (PR) Skin temperature (ST) Biochemical parameter Blood Profile Estimation of blood metabolites Estimation of Glucose Estimation of Non-Esterifies Fatty Acids (NEFA) Estmation of β- Hydroxy Butyric Acids (βhba) 3.12 Estimation of Adipokines in plasma Estimation of Leptin (LEP) Estimation of Adiponectin (ADP) Estimation of Resistin (RETN) Estimation of Tumor Necrosis Factor Alpha (TNF-α) Estimation of Interleukin 6 (IL-6) Estimation of metabolic hormones in plasma Estimation of Cortisol (CORT) Estimation of Insulin (INS) Estimation of Insulin Like Growth Factor 1 (IGF-1) Estimation of Triiodothyronine (T 3 ) Estimation of Thyroxine (T 4 ) Results and Discussion Body condition score during winter and summer 54 seasons 4.2 Plasma adipokines Leptin Adiponectin Resistin Tumour Necrosis Factor (TNF-α) Interleukin Metabolic Hormones Cortisol Insulin Insulin like growth factor-1 (IGF-1) Tri-iodothyronin (T3) 62-63

12 Chapter No. Title Page No Thyroxin Blood metabolites Glucose Non esterified fatty acid (NEFA) Beta hydroxy butyrate acid (β-hba) Heamatological Parameters Total leucocyte count (TLC) Total erythrocyte count (TEC) Granulocyte Lymphocyte Hemoglobin (g %) Mean Corpuscular Haemoglobin (MCH) Mean Corpuscular Haemoglobin Concentration (MCHC) Mean Corpuscular Volume (MCV) Physiological parameters Rectal temperatures ( 0 C) Respiration rate (breaths/minute) Pulse rate (beats/minute) Skin temperature ( 0 C) Summary and Conclusions Bibliography i-xviii LIST OF TABLES

13 Table Page Title No. No. 3.1 Details of the experimental animals during winter Details of the experimental animals during summer Environmental variable recorded during different months of 31 the year ( Evaluation of body condition score in cows during winter After Evaluation of body condition score in cows during summer After Average milk yield (liters) in HBC and MBC cows during After 55 winter and summer seasons 4.4 Plasma Leptin level (ng/ml) in medium (n=6) and high body After 65 condition (n=6) KF cows during winter and summer seasons 4.5 Plasma adiponectin level (ng/ml) in medium (n=6) and high After 65 body condition (n=6) KF cows during winter and summer seasons 4.6 Plasma resistin level (ng/ml) in medium (n=6) and high After 65 body condition (n=6) KF cows during winter and summer seasons 4.7 Plasma TNF-α level (pg/ml) in medium (n=6) and high After 65 body condition (n=6) KF cows during winter and summer seasons 4.8 Plasma IL-6 (ng/ml) level in medium (n=6) and high body After 65 condition (n=6) KF cows during winter and summer seasons 4.9 Plasma cortisol level (ng/ml) in medium (n=6) and high After 65 body condition (n=6) KF cows during winter and summer seasons 4.10 Plasma insulin level (ng/ml) in medium (n=6) and high After 65 body condition (n=6) KF cows during winter and summer seasons 4.11 Plasma IGF-1 level (ng/ml) in medium (n=6) and high body After 65 condition (n=6) KF cows during winter and summer seasons 4.12 Plasma T 3 level (nmol/l) in medium (n=6) and high body After 65 condition (n=6) KF cows during winter and summer seasons 4.13 Plasma T 4 level (nmol/l) in medium (n=6) and high body After 65 condition (n=6) KF cows during winter and summer seasons 4.14 Plasma glucose level (mg/dl) in HBC (n=6) and MBC (n=6) After 67 Karan-Fries cows during winter and summer seasons 4.15 Plasma NEFA (µmol/l) level in medium (n=6) and high After 67 body condition (n=6) KF cows during winter and summer seasons 4.16 Plasma β-hba level (µmol/l) in medium (n=6) and high After 67 body condition (n=6) KF cows during winter and summer seasons 4.17 Total Leukocyte count (TLC) (10 3 /µl) in medium (n=6) and After 71

14 Table Title No. high body condition (n=6) KF cows during winter and summer seasons 4.18 Total Erythrocyte Count (10 6 /µl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.19 Granulocyte count (10 3 /µl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.20 Lymphocyte Count (x10 3 ) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.21 Hemoglobin (g %) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.22 Mean Corpuscular Hemoglobin (pg) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.23 Mean Corpuscular Haemoglobin Concentration (g/dl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.24 Mean Corpuscular Volume (fl /cell) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.25 Rectal temperatures ( 0 C) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.26 Respiration rate (counts/minute) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.27 Pulse rate (counts/minute) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.28 Skin temperatures ( 0 C) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons 4.29 Correlation of adipokines, metabolic hormones, blood metabolites, blood and physiological parameters with body condition score of high and medium body condition in Karan-Fries cows during winter 4.30 Correlation of adipokines, metabolic hormones, blood metabolites, blood and physiological parameters with body condition score of high and medium body condition Karan- Fries cows during summer LIST OF FIGURES Page No. After 71 After 71 After 71 After 71 After 71 After 71 After 71 After 73 After 73 After 73 After 73 After 73 After 73

15 Figure No Title Body condition score (BCS) in high (n = 6) and medium body condition (n =6) KF cows during winter and summers PAGE No. After 55 Plasma Leptin level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma adiponectin level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma resistin level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma TNF-α level (pg/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma IL-6 level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma cortisol level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma insulin level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma IGF-1 level (ng/ml) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma T 3 level (nmol/l) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma T 4 level (nmol/l) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 65 seasons Plasma glucose level (mg/dl) in HBC (6) and MBC (6) After 67 Karan-Fries cows during winter and summer seasons Plasma NEFA level (µmol/l) in medium (n=6) and high body condition (n=6) KF cows during winter and summer After 67 seasons Plasma β-hba level (µmol/l) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons After Total Leukocyte count (TLC) (x10 3 /µl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons After 67

16 Figure No Title Total Erythrocytic Count(x10 6 /µl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Granulocyte count (x10 3 /µl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Lymphocyte Count (x10 3 ) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Haemoglobin (g %) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Mean Corpuscular Haemoglobin (pg) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Mean Corpuscular Haemoglobin Concentration (g/dl) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Mean Corpuscular Volume (fl /cell) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Respiration rate (breaths/minute) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Pulse rate (beats/minute) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Rectal temperatures ( 0 C) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons Skin temperatures ( 0 C) in medium (n=6) and high body condition (n=6) KF cows during winter and summer seasons PAGE No. After 71 After 71 After 71 After 71 After 71 After 71 After 71 After 73 After 73 After 73 After 73 ABSTRACT

17 SCHOLAR MAJOR ADVISOR DIVISION Bikash Debnath Dr. Anjali Aggarwal Dairy Cattle Physiology ABSTRACT The present investigation was carried out to monitor changes in adipokines, metabolic hormones and blood metabolites in high and medium body condition transient crossbred cows during winter and summer seasons. For this, twelve Karan- Fries cows, six each of medium body condition (MBC) and high body condition (HBC) were selected in both the seasons. Blood samples were collected on 21 st, 15 th and 7 th day prepartum, on day of calving and on 7 th, 15 th and 21 st days postpartum. Various parameters like hematological, biochemical, hormonal and metabolic profile were estimated. In winter season, plasma level of insulin and IGF-1 were higher in MBC cows, whereas, leptin, adiponectin, resistin, TNF-α, IL-6, cortisol, T 3, T 4, glucose, NEFA and β-hba were higher in HBC cows. Physiological responses were numerically lower in MBC cows as compared to HBC cows in both seasons. While in summer season, plasma level of insulin and glucose were higher in MBC, whereas, leptin, adiponectin, resistin, TNF-α, IL-6, cortisol, IGF-1, T 3, T 4, glucose, NEFA and β-hba were higher in HBC cows. In winter, body condition score of both HBC and MBC cows were positively correlated with resistin, TNF-α, IGF-1, glucose and NEFA and negatively with T 3, T 4 and β-hba, while, MBC cows were further correlated positively with leptin, insulin and cortisol and related negatively with adiponectin, whereas, reverse trend was observed in HBC cows. In summer, body condition score of both HBC and MBC positively correlated with leptin, adiponectin, resistin, insulin and glucose, while negatively correlated with TNF-α, IL-6, cortisol, IGF-1 and T 3 while MBC cows were further positively correlated with β-hba and negatively correlated with T 4 and NEFA, whereas, reverse trend was observed in HBC cows. Plasma concentrations of leptin (p=0.03) and IGF-1 (p<0.0001) significantly differed between HBC and MBC cows during winter. Plasma cortisol was numerically lower in MBC cows as compared to HBC cows in winter (3.57 ± 0.54: HBC and 2.19 ± 0.71 ng/ml: MBC) as well as in summer (6.84 ± 0.45: HBC and 5.28 ± 0.29 ng/ml: MBC). In summer, plasma NEFA and β-hba differed significantly between HBC and MBC cows. The average milk yield of MBC cows was numerically higher (18.84 liters: MBC vs liters: HBC) in winter as well as summer (15.53 liters: MBC vs liters: HBC). MBC cows lost less body condition as compared to HBC cows in both seasons. Thus, it may be concluded that MBC cows perform better than HBC cows in transition period. SCHOLAR MAJOR ADVISOR

18 ( ) 6,,,

19 CHAPTER 1 Introduction

20 Introduction 1. INTRODUCTION Body s thermoregulatory system adjusts a variety of physiological mechanisms through a combination of dry heat exchange and evaporative heat loss to attain a balance between the heat produced within the body and the heat lost to the environment. Heat and cold outside the thermoneutral zone forces an animal to utilize compensatory mechanism to maintain its body temperature within normal limits. During hot summer months there is accumulation of additional heat load in the animal body. The effects of elevated environmental temperatures which has been seen in dairy cattle depends on the degree and duration of heat stress. According to the seasonal variations in environmental conditions in post-partum cows, some biochemical and physiological changes which affect the productive efficiency of farm animals may occur. Transition period is the most stressful period in dairy cows both physiologically and metabolically. Due to abrupt reproductive changes at parturition as well as high metabolic demands to initiate lactation impose great stress on the cows. Profound alterations at the cellular level also occur upon heat stress. These alterations encompass changes in gene expression and biochemical adaptive responses, characterized by the impairment of major cellular functions and reprogramming of cellular metabolism. Leptin, a 16 kda protein is synthesized by adipose tissue is involved in regulation of feed intake, energy balance, fertility and immune functions. Energy deficit of periparturient cows causes a sustained reduction in plasma leptin. A linear relationship was demonstrated in well-fed lactating dairy cows between plasma leptin and body condition score. The plasma concentration of leptin was positively correlated with plasma concentrations of insulin and glucose and negatively correlated with plasma concentrations of growth hormone, cortisol and NEFA (Block et al., 2001). Adiponectin plays an important role in energy homeostasis, with involvement in regulating glucose concentrations through reductions in insulin resistance and fatty acid breakdown, whereas leptin regulates food intake and energy expenditure. The adipose tissue of obese animal produces more pro-inflammatory adipokines (TNF-α, IL6, leptin) and less anti-inflammatory adipokines (adiponectin). The most important consequences of the altered secretion of adipokines, as seen in obesity, are the induction of a pro-inflammatory state, cardiovascular damage and insulin resistance of the adipose tissue, the liver and the skeletal muscle (Cornier et al., 2008). Adiponectin and leptin together normalize 1

21 Introduction insulin action in severely insulin-resistant animals that have very low levels of adiponectin and leptin due to lipoatrophy. Two adipokines, leptin and adiponectin, are metabolically relevant in coordinating energy homeostasis. Leptin affects energy homeostasis by decreasing food intake and by up-regulating fatty acid oxidation and down-regulating lipogenesis in peripheral tissues (Rabe et al., 2008) and also increases insulin sensitivity. Adiponectin regulates feeding behaviour through peripheral and central mechanisms and serves as a starvation signal (Hoyda et al., 2011). Increased insulin activity under heat stress has been demonstrated to promote the intracellular uptake of glucose and to reduce the circulating nonesterified fatty acids (NEFA). The result is an improvement in energy metabolism through the increased uptake of glucose by the cells. Obesity increases activation of stress kinases, which then increase serine phosphorylation of the insulin receptor substrate 1 (IRS-1), and this ultimately reduces the functionality of the insulin signaling pathway. Cows with high BCS (Body condition score) ( 3.5) have higher TNF-α (an inflammatory cytokine) expression than cows with medium BCS which may partially explain their increased disease susceptibility. Plasma non esterified fatty acids (NEFA) levels were significantly higher in high BCS cows than medium BCS cows. Maximum milk production is associated with BCS of 3.0 to 3.5 at calving. Cows with medium body condition produce more milk as compared to cows of high body condition and better reproductive efficiency. The activity of the thyroid gland has an important role in determining cell metabolism, metabolism of lipids and carbohydrates and the functions during the transition period (Nikolic et al., 1997). A positive correlation has been observed between serum thyroid hormones and energy balance (Reist et al., 2003). There is increase in plasma concentration of cortisol, corticosterone and less frequently an increase in plasma epinephrine and norepinephrine concentration in heat stressed animals (Minton, 1994). Objectives: 1) To evaluate plasma levels of adipokines, metabolic hormones and blood metabolites in crossbred cows during winter and summer seasons. 2) To find out relation of plasma level of adipokines, metabolic hormones and blood metabolites with body condition of crossbred cows. 2

22 CHAPTER 2 Review of Literature

23 Review of Literature 2. REVIEW OF LITERATURE Animal environment is a broad term which includes both physical and biological components (Gates, 1968). An external factor having a positive or negative impact on growth, lactation or reproduction is generally included in the term environment include photoperiod, sound, altitude, effective ambient temperature (EAT), contaminants, physiological restraint and management systems (Gwazdauskas,1985). The homeotherms can maintain their body temperature within a narrow range. Most of the large domestic animals are able to maintain equilibrium between the heat production and heat loss under thermoneutral environmental conditions. In stressful conditions, a number of physiological and behavioural responses vary in intensity and duration in relation to the animal condition. Due to parturition stress cows are less resistant to infectious diseases. 2.1 Body condition score (BCS) Body condition score is a subjective means of estimating fat stores in dairy cows independent of the animal s frame size and body weight (Waltner et al., 1993). Body condition determines the metabolizable energy reserves in the adipose tissues. It is also a valuable tool in predicting the productive and reproductive performance in many domesticated animals. The BCS is determined by visual or tactile evaluation of the body fat at defined locations (Wildman et al., 1982; Butler and Smith, 1989; Domecq et al., 1997) by appearance and palpation, examined the thoracic and lumber regions of the vertebral column (chine, loin and rump), spinous processes (loin), anterior coccygeal vertebrae (tail head), tuber sacral (hook bones) and tuber Ischia (pin bones). Prasad (1994) developed a practical body condition scoring chart for Karan-Fries cows based on the thickness of fat cover and prominence of bone where he mentioned low, medium and high body condition of cows. He observed the flesh covering at the spinous processes of vertebral column (chin, loin, and rump), their prominence and sharpness, depression between backbone and pins as well as between pin and hook bones and flesh covering over the ribs. Block et al. (2001) reported that relative to parturition there is change in body condition score which was 3.6, 3.7 (at 4 week and 1 week before parturition) and 3.4, 3.0, 2.9 (at 1 week, 3 week and at 8 week) after parturition. 3

24 Review of Literature Body condition score at the time of calving Dairy cows that are over conditioned at calving are much more likely to experience several of the important transition cow disorders. They have a greater chance of developing fatty liver, ketosis, retained placenta, dystokia and milk fever as well as reduced feed intake in early-lactation, which disturbs energy balance and will predispose them to fertility problems Body condition score during early lactation During early-lactation there is loss of BCS in lactating cows. Buckley et al. (2003) reported that dairy cows losing excess BCS are less fertile than those that maintain BCS well. This problem of BCS loss is due to negative energy balance when the cow exports more energy from the body in milk. There are a number of important factors for minimizing this BCS loss in early lactation. One of the most important factor to consider is that cows with a high BCS at calving loss more BCS in early-lactation. Buckley et al. (2003) reported that from total cow of his experiment, only 30% of cows with a BCS of 3.25 at calving lost more than 0.5 units of BCS in early-lactation, while 50% of cows with a BCS of 3.5 at calving lost 0.5 units of BCS or more in early lactation. It means the higher the BCS at calving, the more BCS will loss in early-lactation. The loss of BCS is due to inability of liver to metabolize large quantities of non-esterified fatty acids as well as fat. Valde et al. (2007) reported more cases of postparturient diseases, including mastitis, among fat cows than among thin cows. It has been observed that high BCS cows lose higher level of BCS from days 7 ± 3 before and after calving as compared to medium BCS group which was 1.87 unit for high BCS and 1.27 units for medium BCS cows. High rates of body condition score (BCS) loss in the early postpartum period are associated with a severe negative energy balance status, alterations in blood metabolites and hormone profiles (Wathes et al., 2007). Cows losing more than one BCS unit during early lactation were at the greater risk for reduced conception rates (Butler, 2003). Fratrić et al. (2013) reported that cows with high ( ) and moderate BCS ( ) had similar BCS at 7 ± 3 days after calving (2.69 ± 0.67 for high BCS cows and 2.62 ± 0.27 for medium BCS cows). 4

25 Review of Literature 2.2 Adipokines in transition cows during winter and summer seasons Leptin Leptin, a protein hormone which is produced and secreted primarily by white adipocytes. It was discovered in 1994 and has been shown to be a potential regulator (inhibitor) of feed intake and could influence the hypothalamo-pituitarygonadal axis. Leptin may play a role as a signal to the CNS indicating energy status of the animal, with its most important function is conservation of energy during times of nutrient deprivation (Block et al., 2001). Villalobos et al. (2007) found that leptin is positively correlated with body condition score of cows. Evidence of leptin having direct effects on the reproductive tract have come from the discovery of leptin receptor mrna found in the anterior pituitary, hypothalamus and within the ovary of several species (Spicer, 2001). Circulating leptin levels increased from early to mid pregnancy and remained elevated until late pregnancy in cattle (Liefers et al., 2003; Villalobes et al., 2007). These elevations were both due to an increase in adiposity as an increase in leptin mrna expression in adipose tissue (Thorn et al., 2008). Block and colleagues (2001) reported that in periparturient dairy cows leptin level were highest in late pregnancy, whereas, during postpartum period the level dropped by about 50%. Leptin levels began to decline between weeks 1 and 2 prepartum from an average of 5.8 to 5.5 ng/ml. In early lactation due, to negative energy balance and reduction in synthesis of leptin by adipocytes the level reached to it s lowest concentration. They found average leptin level was 3.0, 3.2 and 2.9 ng/ml at 1, 3 and 8 weeks of lactation, respectively. Plasma leptin levels continued to be depressed when the energy balance was improved by week 8 postpartum. It has been observed that plasma leptin levels are positively correlated with body fat, glucose and insulin and negatively to NEFA levels. The reason for reduced leptin during early lactation could be that as lactation progresses and body condition has not improved, mobilization of adipose tissue has led to considerable depletion of the cow s supply of white adipocytes, the source of leptin synthesis (Block et al., 2001; Kadokawa et al., 2000). Soliman and colleagues (2002) reported that leptin concentrations did not differ significantly over a 50-day period in both pregnant and non-pregnant cows. Serum leptin levels remained relatively constant, averaging between 5.9 to 9.2 ng/ml over the periparturient period. Chilliard and colleagues (2001) reported that the length of photoperiod altered circulating concentrations of leptin in the ewe. Longer day length increased both mrna in adipose tissue as well 5

26 Review of Literature as plasma leptin concentrations. Leon et al. (2004) reported that in heifers plasma concentrations of leptin were positively correlated with weight gain periods as well as with BCS. Plasma concentrations of leptin decreased during nutritional restriction (P < 0.01) as BCS decrease and IGF-1 also decrease (P < 0.01). During weight gain IGF-I increased significantly (P < 0.01) with every unit change in body condition up to BCS of 4. Insulin concentrations did not change during nutritional restriction when BCS decreased from 3 to 1. Insulin increased among heifers at BCS 4 to 6. Leptin was positively correlated (P < 0.01) with both IGF-I and insulin. Bernabucci (2009) reported that leptin mrna expression was higher at 41 0 C as compared to 39 0 C when adipocytes were exposed to heat. Lemor et al. (2009) reported that in high yielding dairy cows plasma leptin level decreased postpartum (4.64 ± 2.24ng/ml) as compared to antepartum period (7.83 ± 2.84ng/ml). Villalobos et al. (2007) reported that plasma leptin levels in early lactating cows of 30 and 60 days in milk was 2.9 (0.42), 4.98 (0.49) ng/ml, respectively. Vadiya (2012) reported least square mean values of leptin in Sahiwal and Karan Fries; summer and winter season and in high and low yielding groups of both breeds were 5.02 ± 0.15 and 4.81 ± 0.15 ng/ml; 4.87 ± 0.12 and 1.23 ± 0.08 ng/ml and 1.09 ± 0.07 and 1.15 ± 0.05 ng/ml respectively. The least square mean levels of plasma leptin during summer season decreased to 1.6% compared to winter season. In high yielder groups the least square mean levels of plasma leptin decreased to 5.7% compared to low yielder groups Adiponectin Adiponectin is an adipocytokine secreted by adipose tissue of various species which involve in glucose and fatty acid metabolism. Adiponectin gene expresses in adipose tissues and differentiated adipocyte derived from bovine stromal-vascular cells in ruminants (Sun et al. 2009) and was isolated from bovine serum (Wang et al. 2004). Kubota et al. (2007) reported that concentration of adiponectin increases with fasting and when the animals are fed then the concentration reaches to its normal level. Plasma adiponectin concentrations have recently been investigated in dairy cows. Adiponectin, like leptin, increases insulin sensitivity in various species. Lemor et al. (2009) reported that plasma leptin concentrations and the levels of two adiponectin receptors (AdipoR1 and AdipoR2) in subcutaneous adipose tissue were lower one week before calving than three weeks post partum. Bernabucci (2009) reported that adiponectin mrna expression was lower at 41 0 C as compared to 39 0 C 6

27 Review of Literature when adipocytes were exposed to heat. Ohtani et al. (2012) reported that serum adiponectin concentration (µg/ml) in primiparous cows were 0.21 ± 0.03, 0.21 ± 0.04, 0.22 ± 0.05, 0.19 ± 0.03, 0.21 ± 0.04, 0.24 ± 0.05, 0.27 ± 0.05, 0.36 ± 0.10, 0.32 ± 0.07 at 14, 7 (days prior to calving), day of calving, at 1, 2, 3, 7, 14, 21 days postpartum respectively but in multiparous cows the concentration were 0.23 ± 0.06, 0.20 ± 0.06, 0.18 ± 0.04, 0.18 ± 0.03, 0.21 ± 0.04, 0.23 ± 0.06, 0.24 ± 0.05, 0.24 ± 0.03, 0.28 ± 0.05 µg/ml. Singh et al. (2014) reported that the concentrations of adiponectin in milk (0.61 ± 0.03 µg/ml) were about 92% lower than the mean plasma adiponectin concentrations (32.1 ± 1.0 µg/ml). Decreasing adiponectin concentrations is important for accomplishing the adaptation to the rapidly increasing metabolic rates in early lactation. Astessiano et al. (2014) reported that concentrations of adiponectin were high in thin cows than moderate cows (152 vs.106 ± 18 ng/ml, respectively) and were affected by day postpartum. Serum adiponectin increased from - 49 to 21 days postpartum, decreased from -21 to 21 days post partum, and remained stable through 49 days postpartum. Mielenz et al. (2013) reported that in multiparous Holstein-Friesian dairy cows, plasma adiponectin concentration decreased from day 21 antepartum, reaching a trough at day 1, and increasing thereafter, with the highest values attained on day 14 postpartum. Koltes and Spurlock (2012) and Saremi et al. (2014) observed a decrease of the adiponectin mrna in subcutaneaous adipose tissue throughout the transition period. Kabara et al. (2014) reported that plasma adiponectin levels in periparturient cattle are inversely correlated with the concentrations of plasma NEFA Resistin Resistin is an adipokine which is related to regulation of energy metabolism in rodents but has been little studied in dairy cows. Reverchon et al. (2014) reported that mean plasma resistin concentration in dairy cows were ng/ml from 4 week before calving to 22 week postpartum. They also reported plasma resistin level was ng/ml at 4 to 2 wk prior to calving, ng/ml, ng/ml, ng/ml, ng/ml and ng/ml at 1 to 2 wk, 4-6 wk, 8-10 wk, wk and wk post partum. Plasma resistin concentration was low before calving, subsequently increasing and reaching peak value at 1 wk post partum and then decreasing slowly to reach pre-calving levels at 6 wk postpartum. Cools et al. (2013) reported that resistin hormone concentration gradually increases throughout the periparturient 7

28 Review of Literature period. They found the resistin concentration in fat sow and lean sow was 496 pg/ml and 524 pg/ml over the peripartum period Tumor necrosis factor- α (TNF-α) Boyle et al. (2006) reported that during mid-lactation cows with a BCS of > 3.5 use to have higher plasma TNF-α concentration than cows with a normal BCS of They found plasma level of TNF-α was 0.53 ± 0.05 ng/ml in HBC cows and 0.41 ± 0.05 ng/ml in normal BCS cows. They also examined that high BCS cows have a significantly lower overall antioxidant potential when compared with the normal BCS cows so high BCS cows use to suffer from oxidative stress without alteration of energy status. Due to changes in the oxidative state (pro-oxidant state) of over conditioned cows there is significantly higher expression of TNF-α which possibly be a contributing factor to the enhanced susceptibility to disease in high BCS dairy cows. Keswan et al. (2012) reported that in high and low yielding cows TNF-α concentration in plasma was highest on the day of calving (1.52 ng/ml) and it declined significantly in low yielding cows up to 45 th day (0.44 ng/ml), but it declined non-significantly in high yielding cows. TNF-α was observed higher in concentration during the first day (2.09 ng/ml) of mastitis which later reduced by the fifth day (0.59 ng/ml). Sordillo et al. (1995) reported that isolated mononuclear cells of periparturient dairy cows produced significantly higher levels of TNF-α than mid to late lactating dairy cows regardless of tissue location. Chandra et al. (2012) reported that TNFα concentration in summer control group at -20, -10, -5 days prior to calving, at the day of calving (0 day), 5, 10, 20 days postpartum was 21.30, 14.02, 46.33, 55.05, 20.94, 23.89, pg/ml, whereas, for winter control group at -20, -10, -5 days prior to calving, at the day of calving, 5, 10, 20 days postpartum was 19.40, 22.57, 37.86, 50.05, 23.60, 24.28, pg/ml. They found that plasma TNF-α level was increased towards calving but were higher at the day of calving, after that levels decreased up to day 20 postpartum in all the groups. They also found in summer control group the concentration was higher at the day of calving as compared to winter calving group Interleukin-6 (IL-6) Interleukin-6 is known to be a cytokine produced by T-cells. IL-6 has a major effect on the hepatic synthesis of acute phase proteins (APPs) and is also critical in controlling the extent of acute local and systemic inflammation, particularly in 8

29 Review of Literature decreasing the level of proinflamatory cytokines by exerting a protective effect against potential damage and favouring anti-inflammatory activity. Ishikawa et al. (2004) registered that in Holstein-Friesian cows before parturition, the IL-6 concentrations changed in the range of 2.1 ± 0.5 to 2.7 ± 0.4 ng/ml. The IL-6 concentrations before parturition were significantly higher than those after parturition. They also reported that IL-6 concentration differ between animals in the range of 1.4 to 3.5 ng/ml 60 days before parturition. It gradually decreases until the day of parturition. Keswan et al. (2012) reported that in high and low yielding cows IL-6 concentrations were observed to be higher during the first day (26.2 ng/ml) of mastitis which later reduced by the fifth day (13.91 ng/ml). Trevisi et al. (2012) reported that liver functionality index (LFI) depends on production of acute phase protein. When LFI value goes down it indicates high inflammatory response and vice versa. Low LFI cows show higher IL-6 concentration. The greater IL-6 levels were correlated with higher ceruloplasmin and lower lysozyme serum concentration. 2.3 Metabolic hormones Cortisol The domestic animals have a complex system of stressors and plasma cortisol concentration has been used as physiological markers of stress. The major stress hormone produced by the adrenal glands in the ruminant is the glucocorticoid, cortisol. Protein and lipid metabolism are regulated by glucocorticoid hormone. In liver they have anabolic effect but in skeletal muscle and adipose tissue they have catabolic effect. Heat and cold exposure, exercise, burns etc were comes under physical stress (Orth et al., 1992). Due to fluctuation in climatic or environmental temperature there is variation in the plasma cortisol levels in the animal. At C with relative humidity (RH) 44%, the cortisol levels were 5.5 ng/ml in guernsey heifers and increased to 9.1 ng/ml at C (Abilay et al., 1975). Lee et al. (1976) studied the effect of temperature on cortisol function in bovines and it was reported that under cool, intermediate and hot condition the cortisol levels were 42.3, 36.3 and 22.8 ng/ml, respectively. Aggarwal (2004) reported 4.35 ± 0.25 ng/ml of cortisol in hot-humid season in Karan-Fries cows. El-Nouty et al. (1989) found lower plasma cortisol levels in buffaloes as compared to Holstein cows. According to Yousef and Johonson (1967) and Stuart and Wiersma (1971) the higher concentrations of cortisol is due to catabolic hormone, may have been produced by heat stressed 9

30 Review of Literature cows to maintain milk production. Plasma cortisol may increase within 20 min of exposure to acute heat stress, and reach a plateau within 2 h (Christison and Johnson, 1972). Habeeb et al. (1992) reported that plasma cortisol rises markedly when cattle are acutely exposed to high environmental temperatures and decreases during the chronic phase. Muller et al. (1994) found that shaded cows under South African mediterranean summer conditions maintained lower plasma cortisol concentration, along with lower rectal temperatures and respiration rates, during periods of peak heat stress. Dhami et al. (2006) showed rising trend of cortisol concentration with increase in heat stress from May to June-July, but then decreased significantly in all three groups due to two spells of heavy rains at monthly interval, and then again rose with increase in THI. Francisco et al. (1992) reported significant increase in plasma cortisol concentration in heat stressed cows as compared to unstressed cows (12.7 vs 9.4 ng/ml) and the effect as still pronounced with BST treatment during summer (cortisol level 16.1 vs 10.0 ng/ml). Seasonally, blood cortisol levels increased significantly (P < 0.01) due to increase in ambient temperature from C during February to C during July. The cortisol values were 9.07 and ng/ml during February and July, respectively, in Egyptian buffaloes (Marai and Habeeb, 2010). Leining et al. (1980) reported that acute heat exposure ( C and RH %) of young (aged 6 months) and old buffalo calves (aged 12 months) induced increase in plasma cortisol concentration. The values were 408% and 213% in young and old calves, respectively (Nessim, 2004). Friesian calves, plasma cortisol concentration also increase from 11 to 29 ng/ml when exposed to direct solar radiation of the hot summer (Yousef, 1985) and increased from 3.8 to 6.5 ng/ml when ambient temperature in climatic chamber increased from 24 to 38 0 C during 9 h heat exposure (Habeeb et al., 2001). The overall average value of cortisol was 4.33 ± 0.16 and 2.04 ± 0.32 ng/ml (Aggarwal and Singh, 2010). Seasonally, blood cortisol levels increased significantly (P < 0.01) due to increase in ambient temperature from C during February to C during July. Block et al. (2001) reported that in periparturient dairy cows cortisol levels began to decline between weeks 1 and 2 prepartum from an average of 2.6 to 2.4 ng/ml. Trevisi et al. (2012) found that during an acute stress concentration of circulating plasma cortisol reduces with inflammation and metabolic changes in the body. Brunet and Sebastian (1991) found that the cortisol concentration in pregnant ewes was highest up to first 60 days of pregnancy and then declined until the day of 10

31 Review of Literature parturition, when there was a significant but transient increase thereafter. Lactating female shows higher cortisol level after suckling. The decline in cortisol in the heat stressed lactating buffaloes during July month may be responsible for decline in milk components (Marai and Habeeb, 2010). Alameen et al. (2012) reported that serum cortisol level increased in response to summer heat load and it was higher during late pregnancy in both seasons Insulin The pancreatic hormone insulin is involved in the delivery of glucose to cells for energy and is a potential regulator of feed intake. It is an important regulator of feed intake and body weight, acting endogenously on the ventromedial nucleus of the hypothalamus. Levels of insulin are normally greatly elevated during the last 3 weeks of gestation in cows. Some studies have seen a decrease in insulin prepartum with levels steeply rising at calving up to about 16 mu/l. Levels of insulin following calving then dropped back down to about 5 mu/l and gradually rose up to 10 mu/l after 100 days into lactation (Kunz et al., 1985; Grum et al., 1996). Plasma insulin levels have also been reported to be low (300 pg/ml) up until day 14 of lactation, when levels began increasing (Smith et al., 1997). Taylor et al. (2003) reported a postpartum decline in plasma insulin up until week 4 in primiparous cows, followed by a gradual increase until the end of the study at week 20. Itoh et al. (1998) reported that basal insulin level (µu/ml) in four lactating cows following exposure to thermoneutral (18 C) and hot (28 C) environments was 13.2 and They also observed a depression in milk yield in this group versus thermally neutral cows. Fratrić et al. (2013) reported that blood insulin level in both high and medium body condition cow use to reduce from the early dry period towards early lactation. They observed insulin level was significantly lower in high BCS cows at 7 ± 3 days before calving (16.26 ± 4.60 miu/l) as compared to medium BCS cows (20.18 ± 4.96 miu/l) while at 7 days after parturition the concentration was miu/l in case of HBC cows and miu/l in case of MBC cows. Block et al. (2001) reported that in periparturient dairy cows insulin levels began to decline between weeks 1 and 2 prepartum from an average of 0.8 to 0.7 ng/ml. They found average insulin level was 2.3, 2.8 and 3.1ng/ml at 1, 3 and 8 weeks of lactation respectively. Villalobos et al. (2007) reported that mean with standard error of plasma insulin levels in early lactating cows (30 day in milk) of control, 0.5 kg fat and1 kg fat 11