physiological studies on ovarian functions in …
TRANSCRIPT
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