Cairo University

Faculty of Veterinary Medicine

Department of Physiology

PHYSIOLOGICAL STUDIES ON OVARIAN

FUNCTIONS IN BUFFALOES AND CATTLE

A thesis presented By

Seham Samir Soliman Awad Alla

(B.V.SC., Menoufia University, 2011)

In partial fulfillment of the requirements for the master

degree in Veterinary Science (Physiology)

Under Supervision of

Dr. Mahmoud Zaghloul Attia

Professor and Head of Physiology Department

Faculty of Veterinary Medicine

Cairo University

Dr. Nahed El-Sayed El-Toukhey

Professor of Physiology

Faculty of Veterinary Medicine

Cairo University

Dr. Ahmed Sabry Abdoon

Professor of Physiology Veterinary Research Division

National Research Centre

2016

Cairo University

Faculty of Veterinary Medicine

Department of Physiology

Supervision sheet

Dr. Mahmoud Zaghloul Attia

Professor and Head of Physiology Department

Faculty of Veterinary Medicine

Cairo University

Dr. Nahed El-Sayed El-Toukhey

Professor of Physiology

Faculty of Veterinary Medicine

Cairo University

Dr. Ahmed Sabry Abdoon

Professor of Physiology Veterinary Research Division

National Research Centre

2016

Name: Seham Samir Soliman Awad Alla

Date and place of birth: 14/11/1988 (Menoufia).

Nationality: Egyptian

Specialty: Physiology

Degree: M.V.Sc.

Title of thesis: “Physiological studies on ovarian functions in

buffaloes and cattle”

Supervisors:

Prof. Dr. Mahmoud Zaghloul Attia

Prof. Dr. Nahed El- Sayed El-Toukhey

Prof. Dr. Ahmed Sabry Abdoon

Abstract

Hot seasons are involved in the control of ovarian functions. This thesis aimed

to study the influence of season on ovarian and physiological parameters mediating

fertility in buffalo and cattle. This study was conducted on 400 buffaloes and 128

cattle slaughtered at Al-Warak slaughterhouse, Egypt over one year. The obtained

results shown an increase in the incidence of smooth inactive ovaries than cyclic one

in hot season than the cold season. The number of the follicles was increased in late

luteal and estrus stages than in early and mid-luteal stages. The oocyte numbers and

quality were increased in cold season than hot season. Maturation rate was increased

in cold season than hot season. Estrogen, progesterone, anti Mullerian

hormone(AMH) and thyroid hormones were decreased in hot than cold season. There

is a positive correlation between AMH level and the percentage of antral follicles in

buffalo and cattle, and this first trial in Egypt made to isolate the preantral follicles

from buffaloes and cattle ovaries by the mechanical or enzymatic methods, and their

percentage decreased in hot season than cold season. Also, the number of preantral

was higher in cattle than buffalo ovaries.

In conclusion, the current study threw the light on the effect of season on ovarian

functions in buffalo and cattle which may be of value in understanding their role in

reducing the fertility cases in the hot season and help in improvement of fertility in

buffalo and cattle through the application of assisted reproductive technologies and

advanced management systems. Such as AMH can predict the number of the antral

follicles and expected the fertility in buffalo and cattle.

Key words: Buffaloes, Cattle, Season, Reproductive status, Follicle number, Oocyte

number and quality, Maturation rate, Hormone levels, Preantral follicles.

Acknowledgement

Firstly, and forever, my deep thanks to “ALLAH” who gave

me the ability and patience to finish this work.

Also, I would like to express my sincere gratitude to

Dr. Mahmoud Zaghloul Attia Professor and Head of physiology

Department, Faculty of Veterinary Medicine, Cairo University for

his kind supervision, useful suggestions, direct guidance and help

during the course of this study and during writing and preparing

this thesis.

My deep thanks are also to Dr. Nahed El-Toukhey Professor

of Physiology, Faculty of Veterinary Medicine, Cairo University

for her kind supervision and her helpfulness during this study and

during the writing.

My thanks, deepest gratitude and respect to Dr. Ahmed S.

Abdoon, Professor of Physiology, Veterinary Division, National

Research Centre for wise planning of this study, kind supervision,

continuous help and for teaching me all techniques used in the

present study.

My sincere gratitude and thanks to Dr. Omaima M. Kandil,

Professor of Theriogenology, National Research Centre, for her

great help and encouragement during the course of this work. She

always pushed me to take everything a step further and taught me

to think like a scientist.

My sincere thanks to Prof. Dr. Hussein A. Sabra Professor

of Reproductive physiology, National Research Center, for his

indispensable support, wrathful advices and generous help

throughout the work.

I wish to express my thanks to all the staff members,

Department of Animal Reproduction and Artificial Insemination,

National Research Centre and Department of Physiology, Faculty

of Veterinary Medicine, Cairo University, for their valuable help,

support and encouragement.

I will dedicate this study to my parents for their understanding and for their overwhelming support and helping me to succeed. Thank you for every unconditional love and guidance.

Furthermore, dedication to my husband, (Dr. Emad Saad) who inspired me to be strong despite many obstacles in life.

DEDICATION

Contents

Items Page

1. Introduction…………………………………………. 1

2. Review of literature………………………………… 4

3. Material and Methods……………………………… 31

4. Results………………………………………………... 45

5. Discussion …………………………………………… 86

6. Conclusion ………………………………………….. 106

7. Summary……………………………………………. 108

8. References…………………………………………… 113

9. List of abbreviations…………………………........... 149

..…………………………………………الملخص العربي .10

المستخلص العربي ............................................................. .11

List of Tables

No. Title Page

1. Effect of season on reproductive

status in buffalo.

48

2. Effect of season on reproductive status

in cattle. 51

3. Effect of reproductive status on ovarian

follicular number in buffalo and cattle. 52

4. Effect of season on follicle number in

buffalo and cattle. 54

5. Effect of season on recovery rate of

oocyte in buffalo and cattle. 56

6. Effect of season on oocyte quality in

buffalo. 61

7. Effect of season on oocyte quality in

cattle. 63

8. Effect of season on maturation rate of

oocyte in buffalo. 64

9. Effect of season on maturation rate of

oocyte in cattle. 65

10. Effect of season on cumulus cell

expansion in buffalo 70

11. Effect of season on cumulus cell (COCs)

expansion in cattle 72

12. E2 hormone level in mid luteal and estrus

stages in buffalo and cattle 74

13. P4 hormone level in mid luteal and estrus

stages in buffalo and cattle 74

14. AMH hormone level in mid luteal and

estrus stages in buffalo and cattle 75

15. T3 hormone level in mid luteal and estrus

stages in buffalo and cattle 75

16. T4 hormone level in mid luteal and estrus

stages in buffalo and cattle 76

17. Correlations between level of AMH and

total number of follicle in buffalo and

cattle

77

18. Effect of season on preantral follicles

number in buffalo. 79

19. Effect of season on preantral follicles

number in cattle. 80

20. Number of preantral follicle in histological

section in buffalo and cattle. 84

List of Figures

No. Title Page

1. Elisa reader 41

2. Tissue homogenizer (Mechanical method) 41

3. The daily low (blue) and high (red) temperature during 2015. 45

4. Reproductive status in buffaloes and cattle. 47

5. Effect of season on reproductive status in buffalo 49

6. Effect of season on reproductive status in cattle 49

7. Effect of reproductive status on ovarian follicular population in

buffalo. 53

8. Effect of reproductive status on ovarian follicular population in

cattle. 53

9. Effect of season on follicle number in buffalo 55

10. Effect of season on follicle number in cattle 55

11. Effect of season on oocyte recovery rate of oocyte in buffalo 57

12. Effect of season on oocyte recovery rate of oocyte in cattle 57

13. Oocyte quality in buffalo (40X) 59

14. Oocyte quality in cattle (40X) 60

15. Effect of season on oocyte quality in buffalo 62

16. Effect of season on oocyte quality in cattle 62

17. Nuclear maturation of buffalo oocyte (200X) 65

18. Nuclear maturation of cattle oocyte (200X)

65

19. Effect of season on maturation rate of oocyte in buffalo 66

20. Effect of season on maturation rate of oocyte in cattle 66

21. Expansion degree of buffalo cumulus cell (70X). 68

22. Expansion degree of cattle cumulus cell (70X). 69

23. Effect of season on cumulus cell expansion in buffalo. 71

24. Effect of season on cumulus cell expansion in cattle. 71

25. Correlations between level of AMH (ng/mL) and total number of

follicle in buffalo 78

26. Correlations between level of AMH (ng/mL) and total

number of follicle in cattle 78

27. Pre antral follicle (Mechanical method) (100X) 80

28. Preantral follicle (Enzymatic method) (100X) 80

29. Effect of seasons on preantral follicles number in buffalo 81

30. Effect of seasons on preantral follicles number in cattle 81

31. Preantral follicle in histological section in buffalo ovaries.

A: Primordial, B: Primary, C: Secondary, D: Tertiary 83

32. Number of pre antral follicle in histological section in buffalo

ovaries 83

33. Number of pre antral follicle in histological section in cattle

ovaries 85

34. Percentage of preantral follicle in histological section in buffalo

ovaries (%). 85

35. Percentage of preantral follicle in histological section in

cattle ovaries (%). 85

149

List of abbreviations

Abbreviation

Scientific meaning

AFC Antral follicle count

AMH Anti Mullerian hormones

BSA Bovine Serum Albumin

C L Corpus Lutum

COC Cumulus oocyte complex

E2 Estradiol

ELISA Enzyme linked immune sorbent assay

FF Follicular fluid

FSH Follicular stimulating hormones

(G0) Grade zero

(G1) Grade one

(G2) Grade two

(G3) Grade three

GV-stage Germinal vesical stage

HS Heat stress

IGFBP Insulin like growth factor binding

protein

IVC In vitro culture

IVEP In Vitro Embryo Production

IVF In vitro fertilization

IVM In vitro maturation

IU International Unit

LH Luteinizing hormone

M I Metaphase I

ng Nano gram

150

P4 Progesterone

PBS Phosphate Buffer Saline

PFs Preantral follicles

T4 Thyroxine

T3 Triiodothyronine

TSH Thyroid-stimulating hormone

µg Micro gram

µl Micro liter

1

Introduction

Buffalo and cattle are an important worldwide species in terms of

meat and milk production. Relating to milk production, buffalo is the

second most essential dairy species in the world. The cattle similarly

have a very significant role. They are meat producing animals and ideal

for increasing fattening (Terzano et al., 2012 and Soumya et al., 2016).

Buffalo spread out in 43 countries in the world. However, solitary four

countries, India, China, Pakistan and Egypt are producing more than

98% of the world buffalo milk and around 73% of the world buffalo

meat (Soliman and Bassiony, 2011).

Egypt is a tropical country facing a foremost thermal stress

throughout the hottest period of the year (Abdoon et al., 2014).

Buffalo are sensitive to higher temperature, because of their metabolic

and physiology heat created (Das and Khan, 2010). This is for buffalo

bodies absorb excessive solar radiation because of their dark skin and

sparse hair. They have a less effective evaporative cooling system due to

their rather poor sweating ability (Marai and Habeeb, 2010). When

dairy cattle exposure to humid and hot conditions in the summer cannot

dissipate enough body heat to prevent a rise in body temperature (West,

2003). High environmental temperature through the summer is a major

limitation for the production and fertility of farm animals (Abdoon et

al., 2014). This effect is severe once the high temperature is

accompanied by high ambient humidity (Marai and Habeeb, 2010).

Exposure to high seasonal temperature evokes a series of severe

changes in the biological functions of the animals that include despair in

2

feed efficiency and consumption disorder in metabolism of energy,

water, protein, mineral, balances, enzymatic reactions, blood metabolites

and hormonal secretions. These changes bring about harm of both

reproductive and productive performance (Marai and Habeeb, 2010).

Low reproductive efficiency in female buffaloes, counting

distinct seasonal reproductive pattern, inherent late maturity, poor

conception rate, silent estrus or poor estrus expression in summer,

prolonged inter-calving intervals and higher rates of follicular atresia

(Singh et al., 2000 and Manik et al., 2002).

Ovarian function is the basis to each reproductive problem and

enhancement of reproductive performance, therefore it needs a better

understanding of the mechanisms controlling ovarian follicle quantity

and maturation (Singh et al., 2013).

Reproductive similarities among bubaline (Bubalus bubalis) plus

bovine (Bos Taurus and Bos indicus) reproduction let reproductive

controlling technologies advanced in buffalo to be applied to cattle

(Drost, 2007). Up to now, there is no exact literature accessible

investigating the mechanisms by which season can be able to make

such effects on bovine ovarian function. Therefore, it is obligatory to

describe the cellular and physiological mechanisms that identify

possible local effects of season on bovine ovarian functions such as

cumulus oocyte complexes (COCs) yielding and oocytes' maturation

rate.

Deeper knowledge is the requirement to permit plan of more

suitable methods to improve such harmful effects. Hence, the present