DOCTOR OF PHILOSOPHY IN ANIMAL PHYSIOLOGY BVSc. & AH, MVSc.

236 Pages·2016·2.99 MB·English

LEPTIN AND HSP72 RESPONSES IN LOW AND HIGH PRODUCING
ZEBU AND CROSSBRED COWS DURING METABOLIC AND
THERMAL STRESS

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY
IN
ANIMAL PHYSIOLOGY

BY
VAIDYA MANGESH MAHADEO
B.V.Sc. & A.H., M.V.Sc.

DIVISION OF DAIRY CATTLE PHYSIOLOGY
NATIONAL DAIRY RESEARCH INSTITUTE
(I.C.A.R.)
KARNAL-132001 (HARYANA), INDIA
2012

Regn. No. 1090902
Dedicated To

My beloved parents

&

Respected guide

ACKNOWLEDGEMENTS

With great pleasure, I extend my sincere thanks to Dr. Sohan Vir Singh, Principal
Scientist, DCP Division, NDRI, Karnal “Mangesh, one day goes and then another, months, years
are passing without realizing, don’t waste your time make the best out of it”, Sir’s most
encouraging and realistic words to me, Dr. Sohan Vir Singh, Principal Scientist, DCP Division who
has become my major guide never failed to show me the value of time and its importance. I will
forever endure all his teachings, care, support, understanding and patient guidance to make me
become what I am today. There were times when things ran out of control but he always wanted
to make the best for me.
My profound reverence and gratitude to the Director Dr. Anil Kumar Srivastava for
providing me all the necessary facilities and financial support in the form of senior research
fellowship during the entire period of my studies.
I am thankful to Dr. R.C. Upadhyay head of the division and Dr. B.S. Prakash ADG ICAR
as a ex Head of Division for their suggestions, encouragement and allowing me to access to all
the required facilities during my research work.
My heartfelt gratitude to all the Advisory Committee Members- Dr R.C. Upadhyay,
Principal Scientist & Head, DCP Division, Dr. Anjali Aggarwal, Senior Scientist, DCP Division, Dr.
S.S.Kundu, Principal Scientist & Head, DCN Division, Dr. S. De, Senior Scientist, ABTC Division,
and Dr. Shiv Prasad Head, LPM Division for their timely suggestions, guidance and advice during
the entire research work.
Whenever I need help related to my research work I always go to Dr. R. C. Upadhyay
and Dr. O. K. Hooda, their timely help, suggestions and support are so numerous that I am
running out of words to express my gratitude. They are one of the most caring scientists I have
come across. Dr. R.C Upadhyay was always ready to do anything for my work right from my
synopsis till the final binding of my thesis. 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.
I thank all the scientists of DCP division for their advice and suggestions given
whenever I needed namely Dr. Parveen Kumar Principal Scientist, Dr. Mahendra Singh Principal
Scientist, Dr. Ashutosh Senior Scientist, Dr. Manju Ashutosh Senior Scientist, Dr. Sujata
Pandita Principal Scientist, Dr. Anandlaxmi Senior Scientist, Dr A.K.Dang Senior Scientist and
Dr. B.K.Roy Principal Scientist for the timely support
I thanks for encouraging words from Dr. S.K Sirohi, Senior Scientist DCN and allowing
me to use all the instruments in the lab that i required from time to time
I am highly thankful to ICAR, New Delhi for providing me financial support in the form of
S.R.F., without which it was not possible to complete my study
I am also thankful to Dr. R.K Malhotra and Mr. Gian Singh for their kind help in Statistical
analysis of data, without their cooperation it was not possible to interpretate my results.
I have no words to express my deep gratitude to Dr. Deepak Bannerji Sir and Dr. Anil
Kumar Singh without their help I can’t achieve this.

CONTENTS
Page
Chapter No. Title
No.
1.0 Introduction 1-3
2.0 Review of literature 4-30
2.1 Physiological reactions in response to thermal stress 4
2.1.1 Heat storage 8
2.1.2 Importance of temperature humidity index 8
2.2 Heat shock proteins 9
2.2.1 Functions of heat shock proteins 9
2.2.2 Heat shock proteins and stress 10
2.2.3 Heat Shock Protein 72 mRNA expression 10
2.2.4 Heat Shock Protein and energy metabolites 11
2.3 Leptin 11
2.3.1 Leptin level, body weight and feed intake 12
2.3.2 Circulatory leptin 13
2.3.3 Leptin and lactation 14
2.3.4 Levels of leptin during dry period and early lactation 14
Levels of leptin in relation to insulin and cortisol
2.3.5 15
during dry period and early lactation
2.3.6 Leptin and milk components 16
2.3.7 Leptin and negative energy balance (NEBAL) 16
2.3.8 Leptin and energy homeostasis 17
2.3.9 Relationship between leptin and energy metabolites 18
2.3.10 Relationship between leptin with HSP72 19
2.4 Stress and Energy metabolites 19
2.4.1 Non esterified fatty acids (NEFA) 19
2.4.2 Glucose 23
2.5 Stress and hormones level 25
2.5.1 Plasma cortisol 25
2.5.2 Plasma thyroid hormones 27
2.5.3 Plasma insulin 28
2.6 Role of enzymes during stress condition 29
2.6.1 Alkaline phosphatase 29
2.6.2 Lactate dehydrogenase 29
3.0 Materials and Methods 31-46
3.1 Location of the study 31
3.2 Selection of animals 31
3.3 Housing and management of the animals 31
3.4 Experimental protocol 31
3.5 Recording of body weight 32
3.6 Sampling and analysis of milk 32
Recording of climatic variables and temperature
3.7 32
humidity index
3.8 Physiological responses measurements 33
3.8.1 Respiration rate (RR) 33
3.8.2 Pulse Rate (PR) 33
3.8.3 Rectal temperature (T ) 33
re
3.8.4 Skin temperature (T ) 33
skin
3.8.5 Body heat storage 33
3.9 Estimation of energy metabolites in plasma samples 33
3.9.1 Estimation of glucose 33
3.9.2 Estimation of plasma NEFA 34
Estimation of biochemical hormones in plasma
3.10 35
samples
3.10.1 Assay for Plasma insulin 35
3.10.2 Assay for Plasma cortisol 36
3.10.3 Triiodothyronine (T ) 37
3
3.10.4 Thyroxine (T ) 38
4
3.11 Estimation of biochemical enzymes in plasma samples 39
3.11.1 Alkaline phosphatase 39
3.11.2 Lactate dehydrogenase 40
3.12 Estimation of leptin and Hsp72 in plasma samples 40
3.12.1 Assay for plasma leptin 40
3.12.2 Assay for plasma HSP72 41
3.13 Molecular study 43
3.13.1 Preparation of RNAse free plastic wares 43
3.13.2 RNAse free surfaces 43
3.13.3 RNA extraction from blood 43
Assessment of integrity of RNA by Agarose gel
3.13.4 44
electrophoresis
3.13.5 Quantitation and evaluation of purity of RNA 44
3.13.6 Methods of first strand cDNA synthesis 44
3.13.7 Primers for leptin and HSP72 gene 45
Standard curves for target and housekeeping
3.13.8 45
(reference) genes
3.13.9 Reaction mixture for Real-Time PCR 45
3.13.10 Reaction programme 45
3.13.11 Real-time data analysis 46
3.14 Statistical analysis 46
4.0 Results and Discussions 47-84
4.1 Biochemical parameters 47
4.1.1 Plasma hormones 47
4.1.1.1 Plasma cortisol 47
4.1.1.2 Plasma insulin 48
4.1.1.3 Plasma triiodothyronine 50
4.1.1.4 Plasma thyroxine 52
4.1.1.5 Plasma leptin 54
4.1.1.6 Plasma HSP72 56
4.1.2 Energy metabolites 57
4.1.2.1 Plasma glucose 57
4.1.2.2 Plasma NEFA 59
4.1.3 Liver enzymes 62
4.1.3.1 Plasma alkaline phosphatase activity 62
4.1.3.2 Plasma lactate dehydrogenase activity 64
4.2 Physiological responses 65
4.2.1 Respiration rate 65
4.2.2 Pulse rate 67
4.2.3 Rectal temperature 68
4.2.4 Skin temperature 70
4.2.5 Heat storage 72
4.3 Molecular study 73
4.3.1 HSP72 mRNA expression in lymphocytes 73
4.3.2 Leptin mRNA expression in lymphocytes 76
4.4 Lactational parameters 78
4.4.1 Milk yield 78
4.4.2 Milk fat 79
4.4.3 Milk protein 80
4.4.4 Milk lactose 81
4.4.5 Milk solid not fat (SNF) 82
5.0 Summary and Conclusions 85-93
5.1 Plasma hormones & proteins 85
5.2 Energy metabolites 88
5.3 Liver enzymes 89
5.4 Physiological responses 90
5.5 HSP72 and leptin mRNA expression 91
5.6 Lactational parameters 92
6.0 Bibliography i-xxv
LIST OF TABLES
Table Title After
No. Page
3.1 Details of the experimental animals used for study during 32
summer season
3.2 Details of the experimental animals used for study during winter 32
season
3.3 Environmental parameters recorded during summer and winter 32
season
4.1 Plasma cortisol levels (ng/ml) in periparturient Sahiwal and 48
Karan Fries cows during summer and winter season
4.1.1 Plasma cortisol levels (ng/ml) during periparturient period with 50
respect to breed, season and groups
4.2 Plasma insulin levels (ng/ml) in periparturient Sahiwal and 50
Karan Fries cows during summer and winter season
4.2.1 Plasma insulin levels (ng/ml) during periparturient period with 50
respect to breed, season and groups
4.3 Plasma triiodothyronine levels (ng/ml) in periparturient Sahiwal 52
and Karan Fries cows during summer and winter season
4.3.1 Plasma T levels (ng/ml) during periparturient period with 52
3
respect to breed, season and groups
4.4 Plasma thyroxine (T ) levels (ng/ml) in periparturient Sahiwal 52
4
and Karan Fries cows during summer and winter season
4.4.1 Plasma T levels (ng/ml) during periparturient period with 52
4
respect to breed, season and groups
4.5 Plasma leptin levels (ng/ml) in periparturient Sahiwal and 56
Karan Fries cows during summer and winter season
4.5.1 Plasma leptin levels (ng/ml) during periparturient period with 56
respect to breed, season and groups
4.6 Plasma HSP72 levels (ng/ml) in periparturient Sahiwal and 56
Karan Fries cows during summer and winter season
4.6.1 Plasma HSP72 levels (ng/ml) during periparturient period with 56
respect to breed, season and groups
4.7 Analysis of variance of different hormones in periparturient 56
Sahiwal and Karan Fries cows during summer and winter
season
4.8 Plasma glucose levels (mg/dl) in periparturient Sahiwal and 58
Karan Fries cows during summer and winter season
4.8.1 Plasma glucose levels (mg/dl) during periparturient period with 62
respect to breed, season and groups
4.9 Plasma NEFA levels (μM/L) in periparturient Sahiwal and 62
Karan Fries cows during summer and winter season
4.9.1 Plasma NEFA levels (μM/L) during periparturient period with 62
respect to breed, season and groups
4.10 Analysis of variance of different energy metabolites in 62
periparturient Sahiwal and Karan Fries cows during summer
and winter season
4.11 Plasma ALP activity (KA Unit) in periparturient Sahiwal and 64
Karan Fries cows during summer and winter season