Benha University Faculty of Agriculture Animal Production Dept.
EFFECT OF PHOTOPERIOD ON OVARIAN ENDOCRINE FUNCTION IN RABBITS By HUSSEIN MOSTAFA MOHAMMED EL-ZAHER
B.Sc. in Agric. Sci. (Animal Production) 1999, Faculty of Agriculture, Zagazig University (Benha Branch)
THESIS Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN ANIMAL PHYSIOLOGY Department of Animal Production Faculty of Agriculture Benha University Egypt 2011
SUPERVISION COMMITTEE
EFFECT OF PHOTOPERIOD ON OVARIAN ENDOCRINE FUNCTION IN RABBITS By HUSSEIN MOSTAFA MOHAMMED EL-ZAHER B. Sc. Agric. Sci, (Animal Production) 1999, Faculty of Agriculture, Zagazig University (Benha Branch) THESIS Submitted in partial fulfillment of the Requirement for the degree of MASTER OF SCIENCE IN )ANIMAL PHYSIOLOGY(
Under supervision of:
** Prof. Dr. Mohammed, S. Gado …………………………………. Professor of Animal Physiology, Animal Production Dept. Faculty of Agriculture, Benha University.
** Prof. Dr. Gaafar, M. El-Gendi …..……………………………...
Professor of Poultry Management, Animal Production Dept. Faculty of Agriculture, Benha University.
** Dr. Magdy Hassan, G. Ishak ….…………………………...... Assoc.Professor of Physiology and Head of Endocrinology Research Unit, Radiobiological Applied Dept. Nuclear Research Center, Atomic Energy Authority.
APPROVAL COMMITTE
EFFECT OF PHOTOPERIOD ON OVARIAN ENDOCRINE FUNCTION IN RABBITS By HUSSEIN MOSTAFA MOHAMMED EL-ZAHER B. Sc. Agric. Sci. (Animal Production) 1999, Faculty of Agriculture, Zagazig University (Benha Branch)
THESIS Submitted in partial fulfillment of the Requirements for the degree of MASTER OF SCIENCE IN ANIMAL PHYSIOLOGY
This thesis for M.Sc. degree has been approved by:
** Prof. Dr. Mohammed, S. Gado ………………………………. Professor of Animal Physiology, Animal Production Dept. Faculty of Agriculture, Benha University.
** Prof. Dr. Medhat, H. Khalil ….…………………………… Professor of Animal Physiology, Animal Production Dept. Faculty of Agriculture, Al- Azhar University. ** Prof. Dr. Ahmed, A. Radwn ...………………………..... Professor of Poultry Physiology, Animal Production Dept. Faculty of Agriculture, Benha University.
** Prof. Dr. Gaafar, M. El-Gendi .…………………………... Professor of Poultry Management, Animal Production Dept. Faculty of Agriculture, Benha University.
** Dr. Magdy Hassan G. Ishak …………………………...... Assoc. Professor of Physiology, Head of Endocrinology Research Unit, Radiobiological Applied Dept. Nuclear Research Center, Atomic Energy Authority.
Date of Examination: / / 2011.
1
ACKNOWLEDGMENT
I am greatly indebted to:
Professor Mohamed safwat Abd El-Magied Gado, supervisor of this work and Professor of Animal physiology, Department of Animal Production, Faculty of Agriculture, Benha University for planning the research work, close supervision and valuable comments, without his initiative and constructive advice, this study would not have been possible. I am also thankful for his patient and kindness.
Professor Gaafar Mohamed Ibrahim El-Gendi, Professor of Poultry Production , Faculty of Agriculture, Benha University for his helpful and effective support during the lifespan of this work.
Dr. Magdy Hassan Gamal Ishak, Assoc. Professor of Physiology and Head of Endocrinology Research Unit, Applied Radiobiological department, Nuclear Research Center, Atomic Energy Authority, Inshas Egypt and Professor Ibrahim Mohamed El-Banna Professor of Physiology, Endocrinology Research Unit, Applied Radiobiological department, Nuclear Research Center, Atomic Energy Authority, Inshas Egypt for suggesting the problem, designing the experiment work, testing the hypothesis, offering all facilities, encouragement, valuable guidance and criticism.
TABLE OF CONTENTS
Page
1- INTRODUCTION...... 1 2- REVIEW OF LITERATURE...... 7 2.1. Overview of reproduction and productivity of female rabbits 7 emphasizing photoperiod effect...... 2.2. Natural Endocrine control of reproduction...... 11 2.3. Ovulation induction...... 17 2.4. Hormonal mechanism during ovulation...... 18 2.5. Neuroendocrine control of ovulation...... 20 2.6. Mechanism of Ovarian Follicle Rupture...... 25 2.7. Biosynthesis and biological effect of Melatonin on reproduction...... 27 2.8. Effect of Photoperiod on Melatonin Biosynthesis...... 33 2.9. Pineal gland activity and the role of melatonin in transducing photoperiodic Information...... 38 2.9.1. Pineal gland activity...... 38 2.9.2. Critical features of Melatonin secretion as affected by rhythm...... 40 2.10. Role of Melatonin in reproduction...... 46 2.11. Effect of serotonin in reproduction...... 48 2.12. Effect of human chorionic gonadotropin (HCG) on the 50 mechanism of ovulation...... 2.13. Effect of sexual steroidal hormones on oogenesis and ovulation……...... 52 2.14. Effect of prolactin (PRL) on oogenesis and ovulation...... 57 2.15. Bromocriptine: an ergoline derivative acts as dopamine agonist used in Hyperprolactinaemia...... 61 2.16. Prostaglandin (PG) function on uterine contraction and ovulation……...... 63 2.17. Effect of indomethacin on the prostaglandin biosynthesis...... 66 3. MATERIALS AND METHODS...... 73 3.1. Grouping of animals and experimental design...... 73
I
3.2. Hormonal modulators applied...... 77 3.2.1. Indomethacin...... 77 3.2.2. Bromocriptine (parlodel cycloset)...... 78 3.2.3. Melatonin…………………………………………………. 79 3.2.4. Serotonin………………………………………………..... 80 3.3. Injected materials...... 81 3.4. Parameters estimated and data collection...... 82 3.4.1. Blood samples collection...... 82 3.4.2. Chemicals and reagents used in chemical analysis...... 82 3.4.3. Progesterone (P4) radioimmunoassay...... 83 3.4.3.1. Assay buffers...... 84 3.4.3.2. Assay procedure...... 85 4. RESULTS AND DISCUSSIONS...... 88 4.1. First experiment...... 88 4.1.1. Serum progesterone level in untreated does subjected to different lighting regimes applied...... 89 4.1.2. Effect of HCG...... 90 4.1.3. Effect of Melatonin...... 97 4.1.4. Effect of serotonin...... 103 4.1.5. Effect of bromocriptine...... 109 4.1.6. Effect of indomethacin...... 116 4.2. Second experiment...... 122 4.2.1. Serum progesterone level in does treated with HCG...... 122 4.2.2. Serum progesterone level in does treated with HCG in combination with melatonin...... 124 4.2.3. Serum progesterone level in does treated with HCG in combination with serotonin...... 130 4.2.4. Serum progesterone level in does treated with HCG in combination with bromocriptine...... 136 4.2.5 Serum progesterone level in does treated with HCG in combination with indomethacin...... 143 5. CONCLUSION………………………………………………………... 149 6. SUMMARY...... 150
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7. REFERENCES...... 160 8. APPENDIX TABLES...... 212 9. ARABIC SUMMARY...... ----
III
LIST OF TABLES
Table Title Page
1.a Design of the first experiment and number of animals 75 subjected to different photoperiods. Design of the second experiment and number of animals 1.b subjected to different photoperiods. 76
Protocol of injected materials during the course of 2 81 experiments
Progesterone serum levels (ng/ml) x ±SE in response to injection of HCG vs saline at short darkness photoperiod (8D/16L), equal dark 3 93 and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 4 94 collected after injection of HCG vs saline under different photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of 5 melatonin vs saline at short darkness photoperiod (8D/16L), equal dark and 99 light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 6 100 collected after injection of melatonin vs saline under different photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of 7 serotonin vs saline at short darkness photoperiod (8D/16L), equal dark and 105 light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 8 106 collected after injection of serotonin vs saline under different photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of 9 bromocriptine vs saline at short darkness photoperiod (8D/16L), equal dark 112 and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 10 collected after injection of bromocriptine vs saline under different 113 photoperiods.
IV
Progesterone serum levels (ng/ml) x ±SE in response to injection of 11 indomethacin vs saline at short darkness photoperiod (8D/16L), equal dark 118 and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 12 collected after injection of Indomethacin versus saline under different 119 photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of melatonin + 13 HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light 126 photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) collected 14 127 after injection of melatonin + HCG versus HCG under different photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of serotonin + HCG vs HCG at short darkness photoperiod (8D/16L), equal 15 132 dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) 16 collected after injection of serotonin + HCG versus HCG under different 133 photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of bromocriptine + HCG vs HCG at short darkness photoperiod (8D/16L), equal 17 139 dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Analysis of variance of data (cumulative area under curve in units) collected 18 after injection of bromocriptine + HCG versus HCG under different 140 photoperiods.
Progesterone serum levels (ng/ml) x ±SE in response to injection of indomethacin 19 + HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light 145 photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
20 Analysis of variance of data (cumulative area under curve in units) collected after injection of indomethacin + HCG versus HCG under different 146 photoperiods.
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LIST OF FIGURES
Figure Title Page
Serum progesterone level (ng/ml) in response to administration of 1 95 HCG vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 2 95 HCG vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 3 95 HCG vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 4 101 melatonin vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 5 101 melatonin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 6 101 melatonin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 7 107 serotonin vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 8 107 serotonin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 9 107 serotonin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 10 114 bromocriptine vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 11 114 bromocriptine vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 12 114 bromocriptine vs saline at long darkness photoperiod (16D/8L). Serum progesterone level (ng/ml) in response to administration of 13 indomethacin vs saline at short darkness photoperiod (8D/16L). 120
VI
Serum progesterone level (ng/ml) in response to administration of 14 120 indomethacin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 15 120 indomethacin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 16 128 melatonin + HCG vs HCG at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 17 128 melatonin + HCG vs HCG at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 18 128 melatonin + HCG vs HCG at long darkness photoperiod (16D/8L). Serum progesterone level (ng/ml) in response to administration of 19 serotonin + HCG vs HCG at short darkness photoperiod (8D/16L). 134
Serum progesterone level (ng/ml) in response to administration of 20 134 serotonin + HCG vs HCG at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 21 134 serotonin + HCG vs HCG at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 22 141 bromocriptine + HCG vs HCG at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 23 141 bromocriptine + HCG vs HCG at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 24 141 bromocriptine + HCG vs HCG at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 25 147 indomethacin + HCG vs HCG at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 26 147 indomethacin + HCG vs HCG at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 27 147 indomethacin + HCG vs HCG at long darkness photoperiod (16D/8L).
VII
LIST OF HISTOGRAMS
Histogram Title Page
Serum progesterone level (ng/ml) in response to administration of 1 96 HCG vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 2 96 HCG vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 3 96 HCG vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 4 102 melatonin vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 5 102 melatonin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 6 102 melatonin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 7 108 serotonin vs saline at short darkness photoperiod (8D/16L)
Serum progesterone level (ng/ml) in response to administration of 8 108 serotonin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 9 108 serotonin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 10 115 bromocriptine vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 11 115 bromocriptine vs saline at equal dark / light photoperiod (12D/12L).
VIII
Serum progesterone level (ng/ml) in response to administration of 12 bromocriptine vs saline at long darkness photoperiod (16D/8L). 115
Serum progesterone level (ng/ml) in response to administration of 13 121 indomethacin vs saline at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 14 121 indomethacin vs saline at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 15 121 indomethacin vs saline at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 16 129 melatonin + HCG vs HCG at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 17 melatonin + HCG vs HCG at equal dark / light photoperiod 129 (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 18 129 melatonin + HCG vs HCG at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 19 135 serotonin + HCG vs HCG at short darkness photoperiod (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 20 135 serotonin + HCG vs HCG at equal dark / light photoperiod (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 21 135 serotonin + HCG vs HCG at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 22 bromocriptine + HCG vs HCG at short darkness photoperiod 142 (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 23 bromocriptine + HCG vs HCG at equal dark / light photoperiod 142 (12D/12L).
IX
Serum progesterone level (ng/ml) in response to administration of 24 142 bromocriptine + HCG vs HCG at long darkness photoperiod (16D/8L).
Serum progesterone level (ng/ml) in response to administration of 25 indomethacin + HCG vs HCG at short darkness photoperiod 148 (8D/16L).
Serum progesterone level (ng/ml) in response to administration of 26 indomethacin + HCG vs HCG at equal dark / light photoperiod 148 (12D/12L).
Serum progesterone level (ng/ml) in response to administration of 27 indomethacin + HCG vs HCG at long darkness photoperiod 148 (16D/8L).
X
1. INTRODUCTION
Rabbits are ideal small livestock project for pre-urban and rural areas, especially in developing countries such as Egypt which is a significant proportion of citizenry living below poverty datum line. Rabbits of it well into a balanced farming system. They complement well with vegetable growing. Excess and waste from vegetable gardens and kitchen goes to feed rabbits, whereas their manure is used to fertilize gardens. Thus forming a profitable cycle and aiding to balance of mature.
The reasons for reasing rabbits are manifold. Rabbits are an important source of food particularly in Egypt. Now a day in Egypt the rabbit meat has get a high degree of popularity as protein food. So Rabbit production is very essential in improving animal protein intake. This is because rabbit is very prolific as determined by the number of its born alive at kindling and birth to weaning variability (Orunmuyi et a.l, 2006). For rabbit production to be relevant to improving protein intake in Egypt, certain factors of production must be considered.
Litter size at birth is one of such factors. The higher size, the higher the income of the farmer. Rabbit with large litter size is more likely to be selected for breeding purpose.
Introduction - 1 -
Litter size at weaning and weaning weight are also traits of economic importance in rabbit. Parity was formed to have significant effect on weaning indicating that the higher the parity the heavier the weaning weight. On the other hand litter size was also found to affect the weaning weights of rabbits. The higher the litter size at birth, the smaller the weaning weights of rabbits. (Reddy et al., 2000) reported that litter size at birth and gestation period had significant effect on weaning weight in rabbits. (Shingh, 1981) reported that litter size at first and second parities appears to be constant. From the 2nd to 6th litter size increases and start to decrease from seventh upwards. (Rouvier, 1973), however suggested that rabbits should be culled after the sixth parity for a profitable enterprise. Gestation length is normally constant within species. (Bruce and Solviter, 1957) suggested that the seasonal effects on gestation length is probably linked to seasonal fluctuations of the activity which decline with increasing temperature (Brooks and Rose, 1962) and the thyroid deficiency prior to pregnancy season and litter size on gestation usually results in prolong gestations.
Litter size at birth in rabbit has been known to be negatively correlated with individual rabbit weight at birth a good doe usually produces 6-8 kittens per litter. This number varies as some factors in
Introduction - 2 -
the influence the number of embryo that can develop into full kittens. An increase in litter size can reduce individual birth weight (Vicente et al., 1995).
It its now becomes evident that all reproductive treats related to reproductive performance in rabbit is considerably controlled by hormonal factors. The coordination between organs of hormonal cascade along the hypothalamic-hypophyseal-ovarian axis is the main important factors that affect the role of reproductive activity. In recent years, considerable progress has been made in our understanding of endocrine mechanisms that control the pattern and timing of uterine secretion of Prostaglandin F2 alpha (PGF2α) during luteolysis in mammals. (Silvia et al., 1991) stated that oxytocin may be important in establishing a pulsatile pattern of secretion. They reported that neurohypophyseal oxytocin appears to be released in a pulsatile fashion and may initiate each episode of PGF2α secretion from the uterus. Uterine PGF2α stimulates release of oxytocin from the corpus luteum. Luteal oxytocin further stimulates secretion of
PGF2α from the uterus and may induce a transient refractoriness of the uterus, to subsequent stimulation with oxytocin. They found that uterine refractoriness subsides after approximately 6 hrs. In addition a similar desensitization phenomenon occurs in response to PGF2α at the level of the corpus luteum. Together, uterine and luteal
Introduction - 3 -
refractoriness may account for the interval between pulses of PGF2α observed during luteolysis. The authors observed that uterine secretory responsiveness to oxytocin increasing at luteolysis, when endogenous, pulsatile secretion of PGF2α normally begins. This acquisition by the uterus of responsiveness to oxytocin may determine when endogenous secretion of PGF2α occurs during the estrus cycle. It becomes now well known that uterine secretory responsiveness to oxytocin develops slowly, in the presence of progesterone. Progesterone is formed to exert two types of effects that contribute to regulation of PGF2α secretion. First, prolonged exposure to progesterone appears to promote uterine accumulation of arachidonic acid, prostaglandin endoperoxide synthesis, and other substances needed for synthesis of PGF2α .Second; progesterone appears to exert a suppressive effect on secretion, which wanes after prolonged exposure. Together, these effects of progesterone ensure that PGF2α is secreted only at appropriate time to induce luteolysis. It is well known that ovarian and uterine cycles, in mammals, are controlled by chemical messengers of hormones. Gonadotropin Releasing Hormone (GnRH) which is secreted by the hypothalamus stimulates the release of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) from the pituitary (adenohypophysis). Estrogen is known as hormone of several important function one of
Introduction - 4 -
which is acting synergistically with progesterone to prepare the endometrium for implantation of the fertilized ovum and the mammary glands for milk secretion. High levels of progesterone inhibit the secretion of GnRH and LH. However a small quantity of hormone relaxin produced by the corpus luteum during consequent cycles, relaxes the uterus by inhibiting contractions .This in turn is probably to facilitate the implantation of an ovum which is perhaps more likely to occurs in a relaxed uterus. During pregnancy, the placenta produces much more relaxin and continuous to relax the uterine smooth muscle. At the end of pregnancy, relaxin also increases the flexibility of the pubic symphysis and may help dilate the uterine cervix, both of which ease delivery of the bunnies. From the previously mentioned facts it was obviously clear that hormonal regulation and hormonal coordination are found to be the more effective factors that affect the level of reproductive efficiency in rabbits. Since ovarian follicles formation and development, fertility level, the number of ova succeeded to be fertilized, the rate of pre- implantation mortality, the litter size at birth are direct affected and controlled by hormonal cascade along the hypothalamic- hypophyseal-ovarian (ovary-uterus and placenta) axis which is affected by synergetic and/or antagonistic effect of certain hormones and/or neurotransmitters. This will results in either
Introduction - 5 -
magnifying or minimizing the profit from rabbitary. Therefore this study was conducted and planned to detect the effect of some hormones and neurotransmitter on the level of progesterone hormone in the serum which is positively correlated with ovulation under various photoperiodicity.
Introduction - 6 -
2. REVIEW OF LITERATURE
2.1. Overview of reproduction and productivity of female rabbits emphasizing photoperiod effect:
Rabbits (Oryctolagus cuniculus) are induced ovulators (Ramirez and Beyer, 1988), with irregular and indefinite estrus cycles; therefore, they could be bred throughout the year when nutritious feed is abundant. Puberty occurs when age, body weight and day length interact to the level that pituitary release gonadotropins needed to initiate reproductive activity (Curtis, 1983). However, the time required to reach puberty and the number of embryos per ovulation seemed to be considerably affected by photoperiodism from weaning to puberty (Uzcategui and Johnston, 1990). Females reached puberty earlier with 17 days when reared under the long day of 18 hrs L: 6 hrs D than when raised under short daylight of 6 hrs light (L): 18 hrs dark (D) (Kamwanja and Hauser, 1983).
Berepubo et al., (1996) reported that puberty occurs earlier with a period 23 days by exposure the does to additional artificial light from 18:00hr to mid-night and 28 days when exposure the does to similar additional light and housed next to males. Such results indicate that puberty is occurs earlier with exposuring does to long daylight and this trend is more punctuated when they were housed
Review of Literature - 6 -
next to males. Doe rabbits accept mating under 16hrs /day, contrary to that occurred under 8hrs daylight exposure (Lebas et al., 1986).
Theau-Clément et al., (1990) confirmed that change in exposure to daylight from 8 to 16hrs /day before artificial insemination improve significantly the sexual receptivity of the does.
Increasing the light period artificially to 14hrs daily during summer, did not affect coitus acceptance of does or the low reproductive activity of Egyptian native White Giza rabbits (Hassanein, 1980 and Shafie et al., 1984).
Mady et al., (1990) found that receptivity of New Zealand White (NZW) doe rabbits was higher with the decrease of light period to 8 and 6hrs daily (for not more than 4 days) than for the ones exposed to extended light (14 hrs daily).
Moreover, Uzcategui and Johnston (1992) concluded that Rex rabbits in Utah need at least 14hrs of continuous light to meet their reproductive potential and that intermittent schedules of 10, 12 and 14 hrs are equally as effective as 14 hrs of continuous light in promoting doe reproduction. Gestation period was found to be affected by type of light and photoperiod (Hassanein, 1980 and Ibrahim, 1985).
Walter et al., (1968) showed that 16 hrs constant lighting/day all the year round, reduced the reproduction problems that are
Review of Literature - 7 -
normally associated with decreasing daylight periods. Exposure of domestic does to light for 16hrs light / day in Europe considerably suppressed the seasonal variations due to the combined effects of light and temperature (Lebas et al., 1986).
Particularly, Jensen and Tuxen (1981) observed higher conception rate and shorter litter interval at first parity with exposure to 14 hrs light / day than with exposure to natural daylight. Similarly, Uzcategui and Johnston (1990 and 1992) found that conception rate was significantly higher for does exposed to 14 and 12 hrs of continuous light than for those subjected to 10hrs of light. However, Maertens and Luzi (1995) found no significant difference in conception rate between 16hrs light (L): 8 hrs darkness (D) and 14 hrs light (L): 10 hrs darkness (D) (57.7 and 55.4 %, respectively).In nature, the decrease in photoperiod leads to a cessation of reproduction. Gestation is 31–33 days and, following this period, wild rabbits (or does) give birth (kindle) in a nest built inside an earthen tunnel that she digs. The nest is lined with grasses, straw, and other material items that are mixed with fur that is pulled from the doe's abdomen. This forms what is known as the maternal nest; its location at the bottom of the burrow prevents the kits from wandering off, a situation that would endanger their lives as does do not retrieve their young (Ross et al., 1959). It was shown by Cheeke, (1987) that rabbits could be mated 24 hrs after kindling since rabbits are induced
Review of Literature - 8 -
ovulators. McNitt et al., (1996) also reported that does are fertile 24 hrs after kindling and can be rebred at this time. Post-partum re- mating intervals at 1 and 9 days Mendez et al., (1986), 9 days Rebollar et al., (1992), 1, 5 and 10 days Yamani et al., (1992) 4 and 11 days, Nicodemus et al., (2002) have been reported in the temperate regions. On the other hand, Xiccato (1996) observed that 10 days rebreeding can be applied to maximize production and this is also close to the peak receptivity at 9 days post-partum as observed by Diaz et al., (1987). However, short re-mating intervals such as 1, 4, 9 and 10 days after kindling may not allow for adequate recovery of the body reserve of the does. As a consequence, there may be a decrease of fertility, milk production, litter weight at weaning and an increase in kid mortality. Moreover, the practice of rebreeding at 24 hrs (1day) after kindling was condemned by animal welfare group (Harkness, 1988).
In the United States, majority of producers re-breed at 14 days or 35 days post-partum (Harris et al., 1982).
Re-mating interval most commonly adopted by Spanish farmers is 11 days post-partum (Rafel, 2001). Also Partridge et al., (1984) reported that in United Kingdom, does in large scale production units were usually re-mated 14 to 21 days after kindling, given an optimal output of 7 to 8 litters per doe per year.
Review of Literature - 01 -
Performance levels obtained in developed countries for rabbits are reported to be 45 to 60 kits reared per doe per year from approximately six to eleven litters (Lebas, 1983; Partridge et al., 1984 and Rajadevan et al., 1986). This level of production is not easily achieved under tropical conditions (Rajadevan et al., 1986). In most tropical and developing countries, the current practice is weaning kits at the age of 6 - 8 weeks and rabbit does are re-mated thereafter. Under tropical conditions, long rebreeding intervals of 30 to 60 days or more are observed to reduce the number of kits raised per doe per year, also in the tropics, one of the problems in the management of domestic rabbits is the selection and adoption of a suitable time of mating after parturition especially under intensive system of production. In most tropical countries have been to find ways of increasing the number of kits per doe per year probably by reducing weaning age. Thus, early re-mating at 14, 21 and 28 days postpartum in a tropical environment, was studied by Iyeghe- Erakpotobor et al., (2005).
2.2. Natural Endocrine control of reproduction:
Harris (1952) indicated that the hypothalamo-hypophyseal axis is related to the reproductive organs in two ways. Firstly via the hypothalamo-hypophyseal nerve tract which innervates and controls the secretion of the neurohypophysis (posterior pituitary gland), and
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secondly via the hypophyseal portal vessels through which the hypothalamus maintains and regulates the secretion of the adenohypophysis (anterior pituitary gland).
In different animal species, prolonged caloric restriction inhibits pulsatile Luteinizing Hormone (LH) secretion and induces a condition of anoestrous by depressing the Gonadotropin Releasing Hormone (GnRH) pulse generator within the hypothalamus (Wade and Jones, 2004). The nutritional status of rabbit does decreases both fertility and sexual receptivity with mechanisms not yet well understood (Brecchia et al., 2006). Peripheral plasma estradiol-17β and GnRH-induced LH secretion were much lower in 48 hrs fasted does than in rabbits fed ad libitum (Brecchia et al., 2006). In principle, the fault LH surge may be due to a decreased synthesis of LH by the pituitary gonadotroph cells and/or to a reduced sensitivity for GnRH caused by a scarce density of GnRH Receptors (GnRH-R) on their plasma membrane. An increasing body of evidence indicates that the expression of GnRH-R in the pituitary is regulated by GnRH itself and several other factors, including gonadal hormones (Rispoli and Nett, 2005 and Hapgood et al., 2005). Among them, estradiol- 17β, acting via its receptor (ER), is a likely candidate for targeting at different hypothalamic loci both local pre-motor and GnRH neurons to modulate the GnRH pulse pattern (Bauer-Dantoin et al., 1995 and Pau and Spies, 1997) as well as pituitary gonadotropes to
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modify their responsiveness to GnRH through modulation of its cognate receptor. Shigeto and Charles (1963) reported that estrogen acts on the posterior median eminence-basal tuberal area to stimulate production but not release of prolactin and, simultaneously, to inhibit synthesis of LH.
Gonadotropin releasing hormone (GnRH) from hypothalamus controls the secretion of LH and Follicle Stimulating Hormone (FSH) from the anterior pituitary gland by which the ovaries are controlled. It is unlikely that very much hypothalamic GnRH reaches the ovary because of dilution in the peripheral circulation and its rapid degradation by enzymes (Tsafriri and Adashi, 1994). Therefore it affects follicular growth through an effect on pituitary with the end result of LH and FSH synthesis. The amount of gonadotrophin secreted is a function of the amount of GnRH reaching the pituitary gland and the responsiveness of anterior pituitary gland to GnRH (Baird and McNeilly, 1981 and Kumar et al., 1997).Several metabolites, including glucose and Non-Esterified Fatty Acids (NEFA), and hormones, such as insulin and Insulin like Growth Factor (IGF-I) regulate ovulation rate, follicle development and embryo survival (Ashworth et al., 1999; Comin et al., 2002 and Ferguson et al., 2003).
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Recently, also leptin has been implicated in several key points of the mammalian reproductive functions (Cunnigham et al., 1999 and Brecchia et al., 2006). Blood level of leptin above a minimal threshold is necessary to activate the hypothalamic-hypophyseal- gonadal axis, trigger puberty and maintain normal reproductive function (Zerani et al., 2005).
Insulin was found to directly influence LH release by the anterior pituitary in rats (Weiss et al., 2003) and glucose-induced insulin response is related to milk production (Sartin et al., 1985).There is a hormonal antagonism between lactation and fertility, including receptivity, ovulation rate, and litter size (Beyer and Rivaud, 1969; Theau-Clément and Mercier, 2005 and Theau-Clément and Roustan,1992). This effect is strongest on days 4–5 of lactation, so breeding during this period is generally not successful in situations where intensive production is desired. It has been suggested that the primary hormonal action involved in this effect is PRL, which blocks the responses of LH and FSH to GnRH (Rodríguez et al., 1989). As a result, commercial producers generally rebreed does 7–11 days postpartum with the litters being weaned at 4 to 5 weeks of age. In commercial situations, it is advantageous to have groups or batches of does kindling at the same time (Maertens and Luzi, 1995). This allows the organization of work schedules on a weekly basis and allows for” all-in all-out”
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management of the rabbit units. To do this, it is necessary to synchronize sexual receptivity so groups of lactating does can mate and ovulate at the same time. Five daily injections of PRL prior to insemination produced receptivity in 74% of the does (i.e., they accepted service from the buck) within three days and another 15% on days four and five after cessation of the treatment. Of the does accepting the male mount; 99% ovulated (Torres and Cotton, 1976).
Injecting prostaglandin F2α on gestation day 29 to induce kindling at each pregnancy, across 1 year, increased the proportion of does that were receptive on lactation days 6–9 from 43.0% (control group) to 70.4% (Ubilla and Rodríguez, 1988).Receptivity, conception rate and prolificacy can also be improved in lactating, non-receptive does by intra-muscular injection of 30 IU Pregnant Mare Serum Gonadotrophin PMSG 48 hrs before Artificial Insemination (AI) (Bourdillon et al., 1992).Yet, there is little improvement in non-lactating or receptive does (Maertens and Luzi, 1995). Perhaps the most severe problem with PMSG is its antigenicity, which provokes immune responses in many of the does receiving the treatment. Actually, a progressive loss of reproductive efficiency has been reported after using PMSG to induce estrus in seven or more successive inseminations (Boiti et al., 1995).
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The percentage of affected does can vary among studies. Only one third of the does were affected according to Lebas et al. (1996). Artificial insemination (AI) is the breeding method of choice for synchronized breeding systems. It allows greater potential for genetic improvement and provides adequate semen for breeding a large number of does at one time. Because rabbits are reflex (induced) ovulators, hormonal stimulation must be applied for ovulation to occur. This can be accomplished by intra-muscular injections of LH, Human Chorionic Gonadotrophin (HCG) or GnRH (Castellini, 1996).
LH analogues induce ovulation in a manner comparable to the mating effect (Morton et al., 2005) but the problem also exists for development of immunity and subsequent lack of response likewise PMSG. The injection of either of two synthetic GnRH compounds (0.2ml) caused no increase in ovulation frequency and fertility in receptive does following AI, but did cause improvements in non-receptive does when compared to a control group that was naturally mated (Theau-Clément et al., 1990).
Other studies have confirmed that, in most does, ovulation at AI can be easily obtained with an intra-muscular injection of synthetic GnRH (Castellini, 1996; Mollo et al., 2003).
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2.3. Ovulation induction:
It has been ago shown by Heape (1905) that ovulation in the rabbit only takes place after copulation. Ovulation has been directly observed in the anaesthetized rabbit. The follicle ruptures, on the average, about 10 hrs after coitus but there is some variation and all follicles do not rupture simultaneously.
Ovulation is not the invariable consequence of the normal growth of the follicles and, although confined to those which are mature, is independent of the actual size attained. Ovulation is accompanied by increased vascularity of the follicle. The principal cause of rupture is the rapid distension of the follicle by marked secretory activity. The ovum is carried out in the flow of this secretion.
Ovulation in the rabbit is initiated by some component of the orgasm which accompanies coitus rather than by the mechanical stimulus of the penis or the presence of semen in the vagina. Corpora lutea are not formed if the follicle is ruptured artificially and this effect is not due to operative trauma but to the absence of some effect of coitus necessary for the formation of the luteal tissue.
Artificial rupture or ablation of ripe follicles is followed by an immediate compensatory growth of new follicles. Blood follicles formed from those artificially ruptured do not inhibit this compensatory growth (Walton and Hammond, 1928).
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Domestic does periodically become sexually attracted to male in intervals of 4-6 days (Meyers and Poole, 1958).However; Mayeres and Poole (1962) reported that intervals of 7day cycles have been reported in wild rabbit (Oryctolagus Cuniculus). Variation in reproductive activity was found to be a function of many factors. Strain and age of rabbit, nutritional, environmental conditions and exposure to male were reported as the most important factors in this aspect (Fox and Krinsky, 1968).
Studies with New Zealand white and Californian white rabbits, disclosed a steep decline in sexual desire 12-24 hrs post coitum (Beyer and Rivaud, 1969). Although unfertilized ova may remain viable for 8-9 hrs after ovulation (Adams and Chang 1962), maximum fertilization can be achieved 2 hrs after ovulation (Hafez, 1970).
2.4. Hormonal mechanism during ovulation:
Gonadotropin releasing hormone (GnRH) and its agonistic analogs exert direct inhibitory and stimulatory effects on ovarian function in mammals (Knecht et al., 1985). Stimulatory responses to GnRH ,including ovulation and prostaglandin (PG) production have been observed in hypophysectomized rats at proestrus in vivo( Ekholm et al., 1982) and in rats granulose cell in vitro (Clark, 1982).
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During ovulation the pituitary gland of the rat is highly responsive to LH-RH with respect to the release of FSH, for which secretory changes in the ovary after an ovulating dose of HCG may be responsible. Kazuyoshi and Shuji , (1980) and Yoshimura and Wallach (1987) found that GnRH analog at all dosage tested increased prostaglandins synthesis by the perfused rabbit ovaries with similar stimulatory effects, whereas each GnRH analog induced meiotic maturation in a dose related manner.
Bergland and Page; (1979) and Oliver et al., (1977) reported that, FSH and LH implanted into medial basal hypothalamus (MBH) of the rat inhibit gonadotropin secretion from the anterior pituitary. Hypothalamic Norepinephrine (NE) synthesis and metabolism is enhanced when FSH is administered to hypophysectomized gonadectomized animals.
LH produced through the preovulatory surge of FSH and steroid secretion, is quickly bound to high affinity receptors in the theca and granulose layers of the ripe follicle and to interstitial cells of the ovarian stroma. Although the duration of the LH surge may be as long as 48 hrs it is clear that the mature follicle are adequately stimulated , to ovulate by gonadotropin during the first hours of LH surge (Bullock and Kappauf, 1973).
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Inmaculada et al., (1994) reported that estradiol to progesterone ratio improves during the early luteal phase when ovulation is induced with LH, and that estradiol and progesterone concentrations could play a role in determining oocyte and embryo quality.
2.5. Neuroendocrine control of ovulation:
Halász, et al., (1988) reported that Pituitary gonadotrophin function is controlled to a great extent by the central nervous system and by the feedback action (positive and negative) of sex steroids. Neural structures involved in this mechanism may be divided into two levels. The first level is represented by the nervous structures releasing GnRH in a pulsatile manner into the portal circulation. The gene encoding the precursor protein for GnRH has been described recently. The precursor protein appears to be composed of 92 amino acids, in which the GnRH decapeptide is preceded by a signal peptide and followed by a peptide termed GAP for GnRH-associated peptide. The GnRH-synthesizing neurons and the nervous structures synchronizing the GnRH discharge (pulse generator) in the monkey, and probably also in the human, reside in the medial basal hypothalamus, which appears to have a highly integrated structure. The GnRH pulse generator is influenced by nervous structures outside the medial basal hypothalamus (second level of control) as
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well as by ovarian and other hormones. These influences probably impinge directly or indirectly on the hypothalamic oscillator. Concerning the chemical nature of the substances mediating the action, a large number of neurotransmitters and neuropeptides have been reported to influence GnRH secretion.
Bakker and Baum (2000) reported that in females of induced ovulating species, including rabbits, ferrets, cats, and camels, the preovulatory release of GnRH, and the resultant preovulatory LH surge, is induced by the receipt of genital somatosensory stimuli during mating. Induced ovulators generally do not show "spontaneous" steroid-induced LH surges during their reproductive cycles, suggesting that the positive feedback actions of steroid hormones on GnRH release are reduced or absent in these species. By contrast, mating-induced preovulatory surges occasionally occur in some spontaneously ovulating species.
Most research in the field of GnRH neurobiology has been performed using spontaneous ovulators including rat, guinea pig, sheep, and rhesus monkey.
Neuroendocrine mechanisms controlling GnRH biosynthesis and release in females of several induced ovulating species and whenever possible it contrasts the results with those obtained for spontaneously ovulating species. In females of induced ovulating
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species estradiol acts in the brain to induce aspects of proceptive and receptive sexual behavior. The primary mechanism involved in the preovulatory release of GnRH among induced ovulators involves the activation of midbrain and brainstem noradrenergic neurons in response to genital-somatosensory signals generated by receipt of an intromission from a male during mating. These noradrenergic neurons project to the MBH and, when activated, promote the release of GnRH from nerve terminals in the median eminence.
The hypothesis that episodic release of GnRH into the portal vasculature is responsible for basal, pulsatile, non surge secretion of LH has been supported by measurement of hypothalamic GnRH release in rats (Levine and Duffy, 1988), sheep (Levine et al., 1982 and Clarke and Cummins, 1982), and rabbits (Pau and spies 1986).
The control of preovulatory surge secretion of LH has been investigated in the rat (Sutton et al., 1988), sheep (Clarke et al., 1987) and there have been reported an increase in GnRH secretion at the time of the LH surge.
Afferent impulses produced by coitus reach the hypothalamus from many directions since complete surgical differentiation is required to block ovulation in the rabbit (Volochin and Gallardo, 1976).
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Immunohistochemical studies have shown that GnRH (Barry, 1977) and catecholamine (Bensch et al., 1978) containing cell bodies and nerve terminals are present in both the anterior and mediobasal hypothalamic areas. While several neurotransmitters (Barrachough and selmanoff, 1984) and neuropeptides (Kalra and Kalra, 1986) are capable of modulating GnRH neuronal activity. In the rabbits, coitus triggers a surge release of LH and FSH a neuroendocrine reflex which is believed to require a neurohumoral signal transmitted by the hypothalamus and received by the pituitary (Ramirez and Beyer, 1988). This coitally induced LH surge triggers ovulation and thus ensures the simultaneous presence of both male and female gametes in the female reproductive tract. In rodents, norepinephrine (NE) pathways terminating in the hypothalamus appear to exert a facilitatory influence on GnRH release. NE nerve terminals are concentrated in those hypothalamic nuclei that contain GnRH (Fuxe et al., 1978).
Pau and Spies (1986) reported that in the rabbit, either coitus or intra- ventricular administration of norepinephrine (NE) induces gonadotropin release and ovulation. It is hypothesized that ovulation induced with these manipulations involves activation of neuronal pathways that include catecholaminergic and peptidergic neurons.
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Histamine activates pituitary-ovarian function by stimulating central noradrenergic elements and that NE has more of the physiological-pharmacological characteristics of a natural central nervous activator of luteinizing hormone-releasing hormone than has epinephrine (Sawyer and Radford , 1978) .
Pau et al., (1997) demonstrated that in mammalian species, ovulation occurs following a massive release of hypothalamic gonadotropin-releasing hormone (GnRH). Several chemicals, including norepinephrine (NE) and neuropeptide Y (NPY), are responsible for the initiation and/or magnitude and duration of this pre-ovulatory GnRH surge. In the central nervous system, norepinephrine neural cell bodies are located in the brain stem; some are co-localized with neuropeptide Y neurons and/or co-express the norepinephrine transporter (gene which dictates norepinephrine transporter protein production. The activity of norepinephrine transporter at norepinephrine terminals is critical for synaptic norepinephrine function. In the rabbit, coitus induces a hypothalamic NE release which precedes the GnRH surge. The coital stimulus is transmitted to the brain stem and transformed and integrated into GnRH-stimulating signals via NE, (NET) norepinephrine transporter and/or (NPY) neuropeptide Y. However, very little is known about the distribution of cells expressing NET, NPY and tyrosine hydroxylase, the rate-limiting enzyme of NE synthesis in this species.
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Therefore, the sensitive in situ hybridization technique was utilized to identify the presence of these messages in conjunction with the location of NE cells, the latter being marked by dopamine β- hydroxylase, and the specific enzyme for norepinephrine synthesis.
Furthermore , fluorescence histochemical and immunofluoresence studies have shown that the NE nerve terminals within the lateral portion of the subependymal layer of the median eminence and medial preoptic area of the hypothalamus are activated during the critical period of the ovarian cycle in some mammals when GnRH pathways are activated (Fuxe et al., 1977). In addition, the role of dopamine (DA), the catecholamine precursor of NE, in the control of GnRH secretion is the subject of considerable debate .Evidence for a stimulatory and an inhibitory role has been presented. Dopamine (DA), like NE, neurons has cell bodies in the hypothalamus but has been shown to be distinct from GnRH containing neurons (Fuxe et al., 1978).
2.6. Mechanism of Ovarian Follicle Rupture:
The fundamental process of ovulation and follicle rupture has long been a matter of intense debate but the intra-ovarian mechanism of rupture is still essentially unknown. Two main ideas have prevailed in various modifications: (1) follicle rupture is caused by increased follicular pressure; (2), follicle rupture is caused by the
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action of enzymes on the follicular wall (Espey and Rondell, 1968; Rondell, 1970a, b and Lipner, 1973). In 1963 measurements of intra- follicular pressure were reported both in rats (Blandau and Rumery, 1963) and rabbits (Espey and Lipner, 1963).The records showed a constant pressure during the preovulatory period; instead of an increase. Some records even displayed a slight fall in pressure just before rupture. This of course meant a renewed interest in the enzymic theory and in Espey and Lipner (1965) demonstrated that some proteolytic enzymes including bacterial collagenase have the capacity to digest the rabbit follicle wall. Espey (1970) published additional results indicating that a variety of proteolytic agents has the ability to decompose the follicular connective tissue and effectively reduce the tensile strength of the follicle wall.
Rondell (1970a) discussed the physical aspects of ovulation in detail. Concluded that already after a relatively small increase in distensibility of the follicular wall tissue, rupture can occur at normal intra-follicular pressure. Any hypothesis on follicle rupture must take these facts into consideration. Furthermore, it remains to determine the source of such possible proteolytic agents and to explain what induces their appearance and activity and why rupture occurs in a well circumscribed region at the apex of the follicle.
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2.7. Biosynthesis and biological effect of Melatonin on reproduction:
Melatonin was discovered by Lerner et al., (1958) with exquisite potent effects on melanophore pigment dispersion. It had been known for many years (McCord and Allen, 1917), that pineal gland extracts could lighten the skin color of tadpoles, which is an aggregating action of melanosomes. More than 250,000 bovine pineal glands were processed chromatographically to identify the bioactive compound, N-acetyl-5- methoxytryptamine (Lerner et al., 1958).). This novel compound was named melatonin because it could affect melanin pigmentation in frog skin and was chemically related to serotonin (Lerner, 1999). Melatonin produced in the pineal gland, which is outside the blood – brain barrier, acts as an endocrine hormone since it is released into the blood, in human, 90% of melatonin is cleared in a single passage through the liver, and a small amount is excreted in urine (Buscemi, 2004). It is also known chemically as N- acetyl-5- methoxytryptamine (http://www. Sleepdex. Org / melatnin . htm ). Melatonin may also be produced by a variety of peripheral cells such as bone marrow cells, lymphocytes and epithelial cells, usually, the melatonin concentration in these cells is much high than that found in the blood but it does not seem to be regulated by photoperiod (Maestroni , 2001 and conti et al., 2000).
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The biosynthetic pathways of melatonin have been reviewed Wurtman et al., (1968), Simonneaux and Ribelayga (2003).The precursor of melatonin is the essential amino acid L-tryptophan, which is taken up from the bloodstream and converted to serotonin in two steps, tryptophan is first metabolized into5-hydroxy-tryptophan by tryptophan hydroxylase in the pineal mitochondria, and is then transformed into serotonin in the pineal cytosol by an aromatic amino acid decarboxylase. This transformation of tryptophan into serotonin occurs during both day and night. Serotonin concentrations in the pineal gland are higher during the day than at night as a result of the nocturnal transformation of serotonin into melatonin. Serotonin is first acetylated by arylalkylamine-N-acetyl-transferase (AANAT) into-acetyl-serotonin that is then O-methylated by hydroxyindole-O- methyltransferase (HIOMT) to form melatonin. The activity of AANAT displays marked day/night variations [>100-fold nocturnal increase (Simonneaux and Ribelayga, (2003) and (Roseboom et al., 1996), whereas that of HIOMT only increases by about 50% at night (Axelrod et al., 1965).
Therefore AANAT is considered the “rate-limiting” enzyme for melatonin synthesis in the pineal gland. Because melatonin is a lipophilic molecule, it is not stored within the pinealocytes and is released into pineal interstitial space immediately.
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Norepinephrine (NE) is the primary neurotransmitter of the sympathetic fibers, and it is released in large quantities at the onset of darkness; the nocturnal release is about 100-fold as high as that in daytime (Drijfhout et al., 1996). Numerous approaches have shown the critical role of this increase in NE release to stimulate melatonin synthesis (Simonneaux and Ribelayga, 2003). NE acts upon α1- and β-adrenergic receptors in the pinealocyte membrane (Klein, 1985). Activation of the β-adrenergic receptors appears as the major way by which NE stimulates melatonin biosynthesis during darkness. In vivo injections of the β-adrenergic receptor agonist, isoproterenol, during the day stimulates AANAT activity to levels observed at night time, whereas an in vivo injection of the antagonist, propranolol, inhibits the nocturnal increase in AANAT (Simonneaux and Ribelayga, 2003). The β-adrenergic effects of NE are mediated via the protein and lead to an activation of the adenylate cyclase, which leads to a 10-fold increase in cyclic adenosine mono phosphate (cAMP) levels (Strada et al., 1972). The maximal increase in cAMP is actually reached when the α1-adrenergic receptors are activated at the same time (Vanecek et al., 1985).
Stimulation of α1-adrenergic receptors alone has no effect on cAMP accumulation but causes a large increase in the intra-cellular concentration of free calcium ions, Ca2+, and activation of protein kinase C (Sugden and Klein, 1987).The regulation of AANAT
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activity by day/night changes in cAMP levels differs greatly between two groups of species: (a) rodents and (b) ungulates and humans (Stehle, 2001). In rodents, cAMP activates cAMP-dependent protein kinase, which phosphorylates cAMP response element binding protein (Maronde et al., 1999), and in turn binds to cAMP response element sites in the AANAT promoter to activate transcription (Baler, Covington and Klein, 1999), thereby inducing a large increase in AANAT mRNA expression (Roseboom et al., 1996).
A negative feedback mechanism is responsible for the fall in AANAT mRNA expression at the end of the night. Phosphorylated cAMP response element binding protein stimulates synthesis of the inducible cAMP early repressor that reaches a peak in the pineal gland during the second part of the night (Stehle et al., 1993).And shuts down transcription of AANAT (Foulkes et al., 1996). This transcriptional control is supplemented by posttranscriptional mechanisms leading to a degradation of the AANAT protein by cAMP-dependent changes in proteasomal proteolysis (Gastel et al., 1998.), this proteolysis allows for a more rapid switch-off in AANAT protein than transcriptional processes. As a consequence of light exposure, melatonin synthesis decreases very rapidly because the enzyme is degraded with a halftime of 3.5 minutes (Klein and Weller, 1972).
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In ungulates and primates, posttranscriptional mechanisms are more important to explain the rhythmic melatonin biosynthesis as the mRNA for AANAT fluctuates only marginally. In sheep and primates, the nocturnal increase in melatonin and AANAT activity is accompanied by a minimal increase in AANAT mRNA expression (Coon et al., 1995). In addition, in bovine pinealocytes, treatment with NE does not change constitutively elevated AANAT mRNA levels but does increase AANAT protein and activity. Furthermore, the NE- induced rise in protein and activity is blocked by a protein synthesis inhibitor but not by a transcriptional inhibitor (Schomerus et al., 2000). It is thus proposed that in these species AANAT protein is constantly synthesized but instantly degraded by proteasomalproteolysis during the day (absence of cAMP). At night, increased levels of cAMP protect the protein from degradation and thereby enhance AANAT activity (Schomerus et al., 2000). This model, which emphasizes proteasomalproteolysis as the dominant switch-on mechanism of AANAT activity, may explain the rapid increase in melatonin secretion at the onset of the night in ungulates and primates by a continuous availability of the AANAT mRNA. HIOMT activity is also regulated. However, in contrast to AANAT, which displays rapid changes inactivity in response to both light signals and NE stimulation thereby providing an ON or OFF switch for melatonin secretion, HIOMT appears to be regulated on a longer term basis to modulate the level of
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nocturnal melatonin secretion (Simonneaux and Ribelayga, 2003).HIOMT activity is regulated over several days or weeks by the nocturnal noradrenergic stimulation of the pineal gland. Exposure of rat to continuous light or superior cervical ganglionectomy’s induces a large decrease in HIOMT activity, an amount comparable with animals kept in Dark/Dark (DD). This effect is reversed by daily injections of a noradrenergic agonist (Yang et al., 1976).
This regulation of HIOMT activity on a long term basis is due in part to the high stability of the protein half-life which is longer than 24hrs (Sugden and Klein, 1987).
The regulation of HIOMT activity may be relevant to the seasonal regulation of the amplitude of the melatonin secretory rhythm in several species (Simonneaux and Ribelayga, 2003). For instance, in the Siberian hamster, pineal HIOMT activity is twice as high in short days as in long days, and this increase parallels a twofold increase in the amplitude of the nocturnal melatonin peak (Ribelayga et al., 2000). These changes in HIOMT activity couldresult from changes in norepinephrine as suggested in the rat (Ribelayga et al., 1999). In addition to melatonin, the pineal gland synthesizes other 5-methoxyindoles from serotonin, such as 5- methoxytryptophol and 5-methoxytryptamine, in a changing way according to photoperiod and season (Raynaud et al., 1991).
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2.8. Effect of Photoperiod on Melatonin Biosynthesis:
In animals, circulating levels of melatonin vary in a daily cycle, thereby regulating the circadian rhythms of several biological functions (Altum and ugur. Altum, 2007). Many biological effects of melatonin are produced through activation of melatonin receptors (Boutin et al., 2005), while others are due to its role as a pervasive and powerful antioxidant (Hardeland, 2005), with a particular role in the protection of nuclear and mitochondrial DNA (Reiter et al., 2001).
Many animals use the variation in duration of melatonin production each day as seasonal clock (Lincoln et al., 2003).
In animals and humans, the profile of melatonin synthesis and secretion is affected by the variable duration of night in summer as compared to winter. The change in duration of secretion thus serves as biological signal for organization of day length, dependent (photoperiodic) seasonal functions such as reproduction , behavior in seasonal animals (Arendt and Skene , 2005) . In seasonal breeders which not have long gestation periods and which mate during longer daylight hours, the melatonin signal controls the seasonal variation in their sexual physiology, and similar physiological effects can be induced by exogenous melatonin in animals including mynah birds. (chaturvedi, 1983) and hamsters (chen , 1981).
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Production of melatonin by pineal gland is inhibited by light and permitted by darkness. Secretion of melatonin as well as its level in the blood, peaks in the middle of night and gradually falls during the second half of the night, with normal variation in timing according to an individuals chronotype. Even low light levels inhibit melatonin production (Brainard et al., 2001).
In virtually all species studied, melatonin is synthesized and secreted during the dark phase of the day. This is true whether species are active at night or during the day. The lack of storage of melatonin in pinealocytes on the one hand and the rapid hepatic destruction of melatonin on the other cause a very close relationship between melatonin synthesis and presence in biological fluids.
The twenty four hours (1 day) profile of melatonin secretion varies greatly between species, and a classification into three groups has been proposed by Reiter (1991). Melatonin levels remain low during the first part of the night; and increase only in the second half of the night and decrease to daytime levels before light onset. This pattern is the least common and has been described in Syrian hamster, Mongolian gerbil, and house mouse. Soon after night onset, melatonin levels increase slowly to reach a peak at mid-darkness; during the second part of the night, melatonin falls to reach daytime levels near the time of light onset; this pattern has been described,
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among other species, in rat and humans. Melatonin levels reach a plateau soon after the onset of the night 10–30 minutes in sheep (Ravault et al., 1999), remain elevated throughout the entire night, and decrease soon before or around the time of light onset; this pattern has been described, among other species, in sheep and Siberian hamsters. It is important to note that, regardless of the type of pattern, the duration of elevated melatonin secretion increases when the night is lengthened. The magnitude of the night time rise in melatonin varies greatly between species. In the same individual, it is highly reproducible in amplitude, details of the profile, and in timing in a synchronized environment, almost like a hormonal fingerprint (Arendt, 1998). In addition, between individuals there are large differences in amplitude .For example, in sheep kept in the same environment, circulating blood nighttime levels may vary from 50 to 1000 pg/ml between individuals of similar age and origin (Zarazaga et al., 1998). This variability is under strong genetic control because the heritability coefficient (heritable part of the phenotypic variance) was found to be 0.53 in humans and 0.45 in sheep (Wetterberg et al., 1983). From a physiological point of view, this variability does not result from differences in melatonin catabolism but from melatonin secretion (Zarazaga et al., 1998). Variability in secretion is related to the size of the pineal and the number of pinealocytes that it contains rather than to the activity of enzymes of the biosynthetic
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pathways. This difference in size is established as early as 1 week of age in sheep (Coon et al., 1999). The genes involved in the control of such variability probably act during development in utero or very early at postnatal life.
The light–dark cycle controls the 24hrs melatonin secretory in two ways, entrainment of the circadian rhythm of melatonin secretion and inhibition of melatonin secretion by light. The diurnal cycle of melatonin release is the expression of a circadian rhythm as it free- runs with a period close to 24 hrs in constant darkness (Maywood et al., 1993). This rhythm is entrained with melatonin secretion occurring at night by light–dark cycle characterized by a 24hrs period; this entrainment is the primary effect of light in natural conditions. The SupraChiasmatic Nuclei (SCN) is involved in generating and transmitting to the pineal gland circadian information, but it now clearly evident that the SCN is also a photoperiodic structure and is involved in the integration of seasonal information that it relays to the pineal gland (Schwartz et al., 2001).
Several approaches have allowed investigators to demonstrate that the functional state of the SCN depends on photoperiod. When sheep are transferred from natural photoperiod to continuous darkness, melatonin secretion reflects the state of the clock in the absence of any direct influence of light. Using this paradigm, the
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duration of detectable melatonin in the plasma is shorter in summer than in winter, indicating that seasonal information is stored in the SCN (Matthews et al., 1992).Induction occurs gradually (about 3 weeks).Importantly, this photoperiodic dependence of c-fos induction is not pineal dependent because it persists in pinealectomized animals (Sumova and Illnerová, 1996).. This photoperiodic effect persists when animals are maintained in continuous dim red light (Nuesslein- Hildesheim et al., 2000). Also, multi-unit neuronal activity rhythm in slices of the Syrian hamster SCN depends on photoperiod, and this capacity persists in vitro (Mrugala et al., 2000). This encoding of photoperiodic information in the SCN is responsible for driving the pineal and shaping differentially the pattern of melatonin secretion between long and short days. However, light can also influence melatonin secretion by overriding the signal transmitted by the clock and exerting an acute inhibitory effect on melatonin synthesis and release irrespective of circadian time; this second effect of light causes remarkably rapid changes by inhibition of AANAT activity (Ganguly et al., 2002). In addition to entrainment, it is the second possible mechanism of light to determine the characteristics of melatonin secretory function by inhibiting melatonin secretion at the beginning or at the end of the period when the endogenous clock dictates secretion by the pineal (Arendt, 1995).
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The relative importance of these two mechanisms, entrainment and inhibition, in shaping the melatonin rhythm according to photoperiod appears to differ among species. In rodents, the entraining effect is dominant as illustrated by the slow and gradual lengthening of the duration of melatonin secretion when animals are changed from long to short days where 3 to 4 weeks are necessary for a full adjustment (Illnerova and Vanecek, 1988). In contrast, in ungulates, the inhibitory effect of light is important to gate the melatonin peak at both ends of the melatonin peak. When day length is changed abruptly, the duration of melatonin secretion changes as soon as the first night and is fully adapted after 3 or 4 days (Bittman; Dempsey and Karsch, 1983). Also, when animals are exposed to constant darkness after being in long days, the duration of melatonin secretion expands within the first day of transfer to constant darkness (Picazo and Lincoln, 1995).
2.9. Pineal gland activity and the role of melatonin in transducing photoperiodic information
2.9.1. Pineal gland activity:
The importance of the pineal gland as a transducer of photoperiodic information was demonstrated by numerous experiments showing that the effect of photoperiod on seasonal reproduction is profoundly altered after removal or denervation of the
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pineal gland (Goldman, 2001). In Syrian hamsters, pinealectomy prevents the gonadal regression normally brought about by experimental or natural short photoperiod (Girod et al., 1964). In mink, (a short day breeder) a remarkable effect of pinealectomy was observed in a 3-year experiment: After removal of the gland, animals display sexual rest and remain permanently reproductively inactive (Boissin-Agasse et al., 1988). In sheep, the demonstration of the role of the pineal was made more difficult because the early pinealectomy experiments failed to show a significant effect of the removal of the gland on the seasonal pattern of reproduction (Kennaway et al., 1984). The reasons for this failure to show a role of the pineal gland in the timing of the breeding season are now better understood. First, because there is an endogenous rhythm of reproduction in this species, the persistence of alternations between breeding and anestrous is expected even though the external signal is removed. Second, this endogenous cycle can be indirectly synchronized in pinealectomized ewes through social signals transmitted by their pineal-intact nonspecific (male or female) (Wayne et al., 1989).
A clear demonstration of the role of the pineal gland came from studies in which animals were tested for their ability to respond to controlled photoperiodic changes. Soay rams in which the innervation of the pineal was removed by superior cervical ganglionectomy were neither sensitive to the inhibitory effects of
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long days nor responsive to the stimulatory effects of short days (Lincoln, 1979). Similarly, pinealectomy makes rams or ewes unable to display synchronized changes in reproductive activity or in gonadotropin secretion in response to alternations between long and short days (Barrell and Lapwood, 1979).
2.9.2. Critical features of Melatonin secretion as affected by rhythm:
The critical secretory product of the pineal gland involved in transuding photoperiodic information is melatonin. Timed injections of melatonin reverse the effect of pinealectomy on gonadotropin secretion and cause gonadal regression in hamsters (Goldman et al., 2004).
This early work on the role of the pineal gland and melatonin in long day breeders, mainly in hamster, led to the notion of the anti- gonadal action of melatonin. However, although this term still appears occasionally in the literature, more recent data, particularly those obtained in short day breeders, have led to a more generalized concept of the role of melatonin in the control of seasonal rhythms. Specifically, pinealectomy suppresses responses to both short and long photoperiod, and melatonin, depending on its specific pattern, reinstates both these responses (Bartness et al., 1993). Thus, the role of melatonin is to provide an endocrine code for day length. This role
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was well illustrated by replacement studies in pinealectomized animals. In the short day breeding sheep, melatonin delivered into the peripheral circulation to mimic short day or long day profiles can reproduce the stimulatory and inhibitory effects on LH secretion of short and long days, respectively (Bittman, Karschand and Hopkins, 1983).
In the long day breeding hamster, on the other hand, short duration melatonin infusions are inductive of reproductive activity, whereas long duration infusions are inhibitory (Carter and Goldman, 1983).The characteristic of the melatonin secretory rhythm that conveys the photoperiodic information to affect seasonal rhythms has been a matter of controversy. The rhythm of melatonin secretion is characterized by three features that could code for day length: amplitude, duration (length of time with elevated levels), and phase (presence of melatonin at a given time of day to coincide with a period of sensitivity to melatonin). Amplitude received some attention because in some wild species, such as European hamster, red deer (Cervus elaphus), reindeer (Rangifer tarandus), and muskoxen, strong seasonal changes in the amplitude of nocturnal secretion of melatonin have been reported Vivien-Roels et al., (1997).
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However, amplitude is unlikely to be important because it does not consistently differ between short and long days in most species and it is highly variable among individuals (Chemineau et al., 2002).
In addition, in sheep, the characteristics of the breeding season do not differ between high and low melatonin secretors (Zarazaga; Malpaux and Chemineau, 2003)., and in hamsters, experimental manipulation of the amplitude of the night time rise does not alter the reproductive response to melatonin (Bartness et al., 1993).Although, it is not possible to exclude that mean concentration of nocturnal melatonin may modulate reproductive response to photoperiod, it is not important in the discrimination between long and short days. The amplitude of the nocturnal elevation of melatonin must simply exceed a minimum level that is necessary for a nocturnal rise to be registered. Evidence for the phase hypothesis was obtained from studies in which single injections of pharmacological doses of melatonin administered daily in late afternoon to Syrian hamsters caused gonadal regression. Injections at other times of the day, except for a brief interval immediately before lights-on, were ineffective (Stetson and Watson-Whitmyre, 1983).
A serious limitation of these studies resides in the fact that using daily injections of melatonin means virtually that it is
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impossible to control either the duration or the amplitude of the elevation of melatonin in the blood. In addition, in pineal-intact animals, the exogenous hormone given at the end of the light phase could have been effective because it added to the endogenous secretion to produce a single daily signal of long duration. When physiological doses of melatonin are infused into pinealectomized Siberian hamsters or sheep for fixed durations but at different times relative to the light–dark cycle, the response varies only with the duration of the infusion, not with time of the day (Wayne et al., 1988). For instance, in pinealectomized sheep exposed to long days, melatonin is equally effective in causing an inhibition of gonadotrophic activity whether it is infused for 8 hours every night or in the middle of the 16-hour day (Wayne et al., 1988).
Furthermore, when melatonin signals are delivered at experimental periodicities of less than 24 hours and therefore scan across the prevailing light–dark cycle, short day like responses still occur in Syrian hamsters (Maywood et al., 1990). However, all these studies supporting the duration hypothesis present a limitation in that infusions are given on a regular and predictable basis. It therefore remains possible that a rhythm of sensitivity to melatonin is established by the melatonin signals themselves. One of the most convincing pieces of evidence in favor of duration being critical is therefore the observation that long melatonin pulses infused
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randomly at one of three different phases of the day induce the short day gonadal response in pinealectomized Syrian hamster (Grosse et al., 1993).
These results and others obtained in several species led to the concept that the nocturnal duration of the melatonin elevation is the critical feature of the melatonin signal for its action on photoperiodic physiological functions. This is also in keeping with the fact that the duration of melatonin secretion is always positively correlated to the length of the night. However, this issue remains controversial, and two observations are not consistent with the duration hypothesis. First, 2-2.5 hrs infusions of melatonin produce a reproductive inhibition in Syrian hamsters, suggesting that melatonin does not need to be present continuously to be read as a short day. One interpretation is that the first infusion of melatonin synchronizes a rhythm of sensitivity and that the second infusion occurs during this period of sensitivity (Pitrosky et al., 1995).These data would be fully conclusive once it is demonstrated that melatonin disappears from the target tissue during the 3-hour inter infusion interval. Second, 1hr infusion of melatonin produces a short day response in pinealectomized Siberian hamsters but only if it is given immediately after lights-off and not at other times of the daily cycle (Gunduz and Stetson, 2001). This suggests that responsiveness to melatonin varies throughout the day, but the physiological significance of the presence
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of melatonin for 1hr has to be evaluated. The definition of duration of melatonin is relative, not absolute. As discussed above, the interpretation of the duration of a given melatonin pattern depends on the photoperiodic history of the animal. A change in day length is always accompanied by a similar change in the duration of melatonin secretion (Robinson and Karsch, 1987).Therefore, the response to a given melatonin duration depends on the previous pattern of change in duration, and the ability to perceive gradual changes in photoperiod is the reflection of mechanisms allowing the discrimination of similar changes in the duration of melatonin secretion. This was tested directly in Siberian a hamster in which melatonin was infused with gradual duration changes. The pattern of change in melatonin duration determined the testicular response. The central nervous system appears to compare the duration of a given melatonin signal with those that preceded it (Gorman and Zucker, 1997).
Animals must therefore encode a representation of prior durations of melatonin signals. In Siberian hamsters, the memory of prior photoperiods persists for at least 6.5 weeks and is absent after 20 weeks, suggesting that only information about recent melatonin signals is stored (Prendergast et al., 2000).
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2.10. Role of Melatonin in reproduction:
Photoperiod induces changes in reproductive activity through modifications in the secretion of LH and FSH. In the male Syrian hamster, short photoperiod induced testicular atrophy is accompanied by decreases in pituitary and serum LH and FSH, leading to testicular regression and lowered levels of circulating testosterone (Steger et al., 1985). Similar changes are observed in Soay rams transferred from short to long days (Lincoln, G. A., and Short, R. V., 1980). In intact females, seasonal variations in gonadotropin secretion are not so clear because of the changes occurring during the ovarian cycle at the time of the breeding season. Photoperiod regulates the secretion of gonadotropins by means of two complementary mechanisms: one independent of and the other dependent on gonadal steroids (Steger et al., 1985).
The steroid-independent effects of photoperiod are observed in gonadectomized animals. In castrated rams and ovariectomized ewes, the secretion of LH is lower during inhibitory long days or the anestrusseason than during short days or the breeding season, being one pulse every hour or every 30 minutes, respectively, in ovariectomized ewes (Montgomery et al., 1985). This difference between LH secretion during long days and short days is greatly increased in the presence of estradiol or testosterone, which
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demonstrates a steroid-dependent effect of photoperiod. In ovariectomized ewes treated with estradiol implant mimicking mid- follicular phase levels, an LH pulse is observed either every 12 to 24hrs or every 30 minutes depending on whether animals are experiencing long or short days (Pelletier and Ortavant, 1975). Melatonin can suppress libido by inhibiting secretion of Luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the adenohypophysis, especially in mammals that have a breeding season when daylight hours are long. The reproduction of long day breeders is suppressed by melatonin and the reproduction of short day breeders is stimulated by melatonin (http://en.wikipedia.org/wiki/Melatonin).
Bellipanni et al., (2005) reported that melatonin supplementation in the evening in perimenopausal women produces an improvement in thyroid function and gonadotrophin levels , as well as restoring fertility and menstruation and preventing depression associated with the menopause melatonin also lowers FSH levels , it is believed that the normal changes could in some cases impair fertility (http://www.sleepdex.org/melatonin.htm) .
A similar dual action of photoperiod on gonadotropin secretion is demonstrated in other species (Steger et al., 1985). For example, short day exposed hamsters exhibit a considerable increase in sensitivity to the negative feedback actions of testosterone, and the
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levels of gonadotropins are also higher in long day castrates than in short day castrates (Turek et al., 1975).
The steroid independent effect of photoperiod on gonadotropin secretion is not dependent on adrenal steroids as the difference between short days and long days persist after both ovariectomy and adrenalectomy (Montgomery and Hawker, 1987). Melatonin mediates both the steroid dependent and independent effects of photoperiod on gonadotropin secretion (Bittman, 1985). How a seasonal variation in the mechanisms controlling pulsatile LH secretion account for the seasonal variations in ovulatory activity has been particularly well studied in the ewe.
2.11. Effect of serotonin in reproduction:
Serotonin or 5- hyroxytryptamin (5-HT) is a monoamine transmitter biochemically derived from tryptophan that is primarily found in gastrointestinal (GI) tract, platelets and central nervous system of humans and animals (king 2009).
Approximately 80 percent of the human body's total serotonin is located in the enter chromaffin cells in the gut (GI) where it is used to regulate intestinal movements. The remainder is synthesized in serotonergic neutrons in the central nervous system where it has various functions including regulation of mood, appetite, sleeping
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muscle contraction and some cognitive fuetins including memory and learning (Berger et al., 2009).
The released serotonin activates the muscles used for feeding (Niacaris and Avery, 2003), serotonin diffuses to serotonin sensitive neurons, which control the animal's perception of nutrient availability, when humans smell food, and dopamine is released to increase the appetite. Serotonin released while consuming activates 5- HT2C receptors on dopamine producing cells, this halts their dopamine release and thereby serotonin decrease appetite (Stahl et al, 2009).
Artificial depletion of serotonin or increase of octopamine suppresses mating and formation of ovarian follicle in female animals, similarly serotonin is necessary for normal male mating behavior (Loer and Kenyon, 1993). Human serotonin can act as growth factor directly. Liver damage increases cellular expression of 5- HT2a and 5-HT2B receptors (Lesurtel et al., 2006). Serotonin present in the blood then stimulates cellular growth to repair liver damage (Matondo et al., 2009). 5-HT2B receptors also activate osteoblasts, which build up bone (collet et al., 2008) however, serotonin also activates asteoclast, which degrade bone (Yadav et al., 2008).
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2.12. Effect of Human Chorionic Gonadotropin (HCG) on the mechanism of ovulation:
Human Chorionic Gondotropin or Human Chorionic Gonadotrophin (HCG) is known as glycoprotein hormone produced in pregnancy that is made by the developing embryo of the conception and later by the syncytiotrophoblast which is a part of placenta. Its role is believed to prevent the disintegration of the corpus luteam of the ovary and thereby maintain progesterone production that is critical for pregnancy. HCG may have additional functions. It is thought to affect the immune tolerance of the pregnancy (Cole, 2009).
Human chorionic gonadotrophin interacts with Luteinizing Hormone/ChorioGonadotropin Receptor (LHCGR) and promotes the maintenance of the corpus luteum during the beginning of pregnancy , causing it to secret the hormone progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus . It has also been hypothesized that (HCG) may be placental link for the development of local maternal immunotolerance (Kayisly et al., 2003). Because of its similarity to LH, HCG can also be used clinically to induce ovulation. As the most abundant biological source in women who are presently
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pregnant, some organizations collect urine from pregnant women to extract HCG for use in fertility treatment (Cole, 2009).
Human chorionic gonadotophin is extensively used parentrally as ovulation inducer in lieu of luteinizing hormone. In the presence of one or more mature ovarian follicles. Ovulation can be triggered by administration of HCG. As ovulation will happen 24-36 hrs after injection of HCG, procedures can be scheduled to take advantage of this time sequence (WWW.IVF.COM).
Bjersing and Cajander, (2004) reported that a prominent follicular oedema appeared in the theca interna region after 4 hrs from HCG injection in rabbit, after 10 hrs from injection and later there was an obvious oedema in all layers between granulosa membran and the surface epithelium. The connective tissue cells were apparently disrupted and widely separated already after 4 hrs from injection. The granulosa cells were slightly dissociated after 8 hrs and later after injection. Most distinct changes were found in the germinal epithelium prior to ovulation. The surface cells of the preovulatory follicles increased in size after 4 hrs from injection and onwards they showed an increasing number of dense and large intra- cytoplasmic bodies. Such intra-cellular alterations in the germinal epithelium have not been described before. It is suggested that they may be causally related to follicle rupture.
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Human chorionic gonadotropin (HCG) serves as preovulatory stimulus, ovulation characteristically occurs approximately 6 hrs after treating with 50 IU HCG and it was successfully possible to fertilize the ovulated ova. This could be used to establish normal pregnancies in the adequately prepared host rabbit (Kobayashi et al., 1981).
Kobayashi et al., (1981) reported positive effects of 0, 5, 50, and 100 IU of HCG on ovulatory efficiency, the time interval between HCG administration and follicle rupture, the stage of maturity of ovulated and un ovulated ova, the relationship between ovum maturity and time of ovulation, and ovarian edema in both in situ and in vitro-perfused ovaries. Ovulation did not occur in ovaries without gonadotropin stimulation. Ovulatory efficiency, the percentage of mature follicles that proceed to rupture by 12 hrs after HCG treatment, was similar after treating with either 50 or 100 IU of HCG (78.7% and 77.9%, respectively) and did not differ in right and left ovaries of the same animal.
2.13. Effect of sexual steroidal hormones on oogenesis and ovulation:
The formation of rabbit oocytes includes several steps which basically occur in most domestic animals as generation of primordial germ cell, migration of primordial germ cell to the respective gonads, colonization of the gonads by primordial germ cell, differentiation of
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primordial germ cell oogonia, proliferation of oogonia, initiation of meiosis, and arrest at the diplotene stage of prophase I of meiosis as suggested by (Van den Hurk and Zhao, 2005).
Oogenesis in rabbit begins early in embryonic development with differentiation of the primordial germ cell (Zuckerman and Baker, 1977). It is completed during the first 2 weeks of neonatal life simultaneously with growth of the primordial follicles (Gondos, 1969). However, the majority of oocytes degenerate during their initial meiotic activities. The 4th to 8th week- old rabbit ovaries already contain follicles at progressively more mature stages of early development (Lee and Dunbar, 1993 and Lee et al., 1996)
LH is absolutely required to initiate corpus luteum formation in rabbits; however progesterone production by corpora lutea (CL) and CL maintenance in rabbits requires estrogen and not LH (Bill and Keyes, 1983 and Hunzicker-Dunn and Miller, 1983). Estradiol is therefore considered to be the principal luteotrophic hormone in rabbits because it is sufficient for maintenance of serum progesterone concentrations in hypophysectomized pseudopregnant and pregnant rabbits and withdrawal causes rapid luteal regression (Keyes and Nalbandov, 1967; Rennie, 1968; Spies et al., 1968; Holt et al., 1975 and Bill and Keyes, 1983). The rabbit CL in fact appears to be a typical estradiol target tissue, exhibiting specific, high-affinity and low-capacity estrogen receptors (Yuh and Keyes,
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1979; Drake and Cook, 1979 and Miller and Toft, 1983) which appear with the physiological dependence of CL function on estrogen (Mills and Osteen, 1977 and Miller and Keyes, 1978) and disappear with luteal demise (Miller and Toft, 1983). However, an acute and strong steroidogenic response to estradiol has not been demonstrated. Steroidogenesis in virtually all other tissues is thought to be regulated by trophic hormones via surface membrane receptors and a cAMP-mediated process. Rabbit CL, like other steroidogenic tissues; also exhibit the initial responses for LH- and catecholamine- directed functions. These include LH and catecholamine surface receptors (Abramowitz et al., 1982 and Miller et al., 1986), an adenylate cyclase responsive to both effectors (Hunzicker-Dunn and Birnbaumer, 1976 and Hunzicker- Dunn, 1982) and type 1 and II forms of cAMP-dependent protein kinase (Hunzicker-Dunn and Jungmann, 1978). Changes in these measures appear to correlate with luteal function. Progesterone is produced by the corpus luteum after formation of corpus luteum, the ability to secrete progesterone for several days without a requirement for pituitary hormones (Salisbury et al., 1978).It is known as the hormone of pregnancy, because it causes thickness of the endometrium and development of uterine glands prior to implantation of the fertilized ovum ( Frandson, 1986).
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The output of progesterone in the female rabbits rose rapidly after implantation on day 7-8 post coitus to reach peak values between days 14 and 18 and thereafter remained high until the final week of pregnancy, and just prior to parturition (day 30-33) progesterone output dropped to the basal level (Hilliard et al., 1974).
Drill (1958) showed that ovulation is physiologically inhibited during pregnancy. Progesterone produced by the corpus luteum of pregnancy, and later by the placenta responsible for this effect. Similarly, the administration of the naturally occurring steroids, such as estradiol and progesterone can inhibit ovulation. However, he stated that these steroids are unsatisfactory for clinical use as they are not regularly effective and secondly, the parenteral administration of progesterone is painful. The male hormone, testosterone, can also inhibit ovulation but cannot be used routinely clinically because of androgenic side effects. Thus, a search of other steroid derivatives was undertaken in attempts to find a satisfactory compound for human use.
Rondell, (1974) suggested that, LH stimulates follicular synthesis of progesterone, which in turn may lead to increased enzymatic activity of the follicle wall and thereby to an increase in its distensibility. Recent data obtained from in vitro cultured rabbit follicle model have demonstrated that ovulation can be inhibited by
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suppression of steroid production through the use of aminoglutethimide phosphate (AGP) which is considered as an inhibitor of cholesterol side-chain cleavage (Testart et al., 1983). On the other hand, progesterone may induce the release of LH required for ovulation by stimulating the hypothalamus (Fraps and Dury, 1943; Ralph and Fraps, 1959, 1960 and Wilson and Sharp, 1976).The rise in progesterone that normally occurs immediately after the LH surge is not a prerequisite for ovulation in the rabbit. However, progesterone may have a modifying effect on LH-induced follicle rupture when at a pharmacologically high level (Holmes et al., 1985). Measurements of plasma LH and progesterone concentrations during the ovulatory cycle seem to support this suggestion (Peterson and Common, 1971; Kappauf and Van Tienhoven, 1972; Furr et al., 1973; Haynes et al., 1973; Wilson and Sharp, 1973; Laguë et al., 1975; Shahabi, et al., 1975 and Shodono et al., 1975). In addition, Harper, (1961) found no ovulation by 9 hrs, 50% ovulation between 10:30 to 10:45 hrs. and 88% ovulation by 13 hrs after the injection of LH in rabbits .
Furr et al., (1973) reported that the rise in progesterone always preceded or occurred simultaneously with the LH increase. By contrast, (Williams and Sharp, 1978) found that the preovulatory release of LH was accompanied by a large increase in the secretion of androgen and progesterone. It has also been reported that
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corticosteroids are involved in the ovulation (Van Tienhoven, 1961; Soliman and Huston, 1974; Etches and Cunningham, 1976; Etches, 1977 and Wilson and Lacassagne, 1978).
2.14. Effect of prolactin (PRL) on oogenesis and ovulation:
Prolactin released from pituitary lactotropes in an episodic fashion, with ~14 major pulses every day. A major nocturnal peak occurs shortly after sleep onset. Regulation of prolactin secretion differs from most other pituitary hormones as under primarily negative control from the hypothalamus (via dopamine) and not under long loop feedback control as it produces no known target organ hormone. Prolactin secretion is inhibited by dopamine from hypothalamic neurons, via Dopamine (D2) receptors and involving decreases in adenylyl cyclase and cAMP. Inhibition of secretion is the predominant control mechanism for prolactin secretion. Synthesis and secretion of dopamine is itself enhanced by prolactin, which in turn decreases prolactin secretion, providing a short loop negative feedback. Enhanced by Thyrotrophic Releasing Hormone (TRH) from the hypothalamus. Other hypothalamic factors may also be important in stimulating prolactin release. Stimulated immediately by suckling via a neuroendocrine reflex arc. The lactational hyperprolactinaemia may contribute to postpartum an ovulation and infertility. Enhanced during pregnancy, probably by the actions of
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estrogen. Estrogen increases the number of lactotropes and stimulates prolactin expression. It also enhances secretion of prolactin by augmenting TRH stimulated secretion and blunting dopamine inhibition of secretion. Finally, it increases expression of prolactin receptors, so that target tissue sensitivity is enhanced. Prolactin secretion is enhanced by many factors that also stimulate GH release (e.g. stress, exercise, sleep, and hypoglycemia) (Medical Pharmacology, 2003).
Prolactin is a polypeptide hormone that is synthesized in and secreted from specialized cells of the anterior pituitary gland, the lactotrophs (Riddle et al., 1933).
Biological actions are not limited solely to reproduction because it has been shown to control a variety of behaviors and even play a role in homeostasis. Prolactin releasing stimuli not only include the nursing stimulus, but light, audition, olfaction, and stress can serve a stimulatory role, although it is well known that dopamine of hypothalamic origin provides inhibitory control over the secretion of prolactin, other factors within the brain, pituitary gland, and peripheral organs have been shown to inhibit or stimulate prolactin secretion as well (Marc et al., 2000). Prolactin has many effects including disruption of pituitary gonadotophin secretion leading to hypogonadism (Mancini et al., 2008 ), regulating lactalin , orgasms
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and stimulating proliferation of oligodendocyte precursor cells (Nelson , 2005).
Djiane et al., (1979) reported that PRL is a major hormone implicated in mammogenesis and lactogenesis, as well as mammary tumor development. Receptors for PRL are abundant in mammary glands and can be maintained in organ culture.
Lactogenic responses can be elicited by estrogen-sensitive neurons in the amygdala acting via the stria terminals on the preoptic area and/or basal hypothalamus to cause the release of prolactin, and the amygdala may play a role in the normal animal, via the hypothalamo-hypophyseal system, in the modulation of the secretion of prolactin by the anterior pituitary (Tindal, 1967).
The hormone contracts the effect of dopamine which responsible for sexual arousal. This is thought to cause the sexual refractory period (Utama et al., 2006). Pituitary prolactin secretion is regulated by neuroednocrine neurons in the hypothalamus , the most important ones being the neurosecretroy tuberoinfundibulun (TIDA) neurons of the arcuate nucleus , which secrete dopamine to act on the dopamine -2- Receptors of lactotrops , causing inhibition of prolactin secretion theyotropin releasing factor has a stimulatory effect on prolactin release (Kulick et al., 2005).
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High prolactin levels tend to suppress the ovulatory cycle by inhibiting the secretion both follicle – stimulating hormone (FSH) and gonadotopic – releasing hormone (GnRH) (Mancini et al., 2008).
It have been reported that the mechanism of PRL inhibition appears to reside in the ability of prolactin (PRL) to inhibit its own release when implanted into the Median Eminence in the area of the tuberoinfundibular dopaminergic neurons, in addition ,in mammals, hypothalamic extracts initially stimulate PRL release in vitro , suggesting the presence of a releasing factor(PRF). The leading candidates for prolactin realizing factor (PRF) are thyrotrophic releasing hormone (TRH), NE and serotonin (Enjalbert et al., 1977).
Mena et al., (1982) found that a single injection of 1 mg bromocriptine (PRL inhibitor) given to rabbits 30 min before suckling on days 11 or 31 caused a significant reduction in milk yield after approximately 8hrs, they added that depressant effect of the drug was then maintained over the next 24–36 hrs. The recovery accelerated more in the rabbits in the early lactating group. Attainment of the maximal stimulatory effect occurred by 24 hrs after prolactin injection during both early and late lactation.
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Keiichi et al., (1993) stated bromocriptine induces a reversible lysosome change and could inhibit gene transcription of prolactin and growth hormone (GH).
2.15. Bromocriptine: an ergoline derivative acts as dopamine agonist used in hyperprolactinaemia:
Prolactin is solely secreted by the lactotroph cells of the pituitary gland. Hyperprolactinaemia therefore either results from hyper-secretion of lactotroph cells or decreased clearance. The regulation of secretion of prolactin differs from other pituitary hormones in that it is mainly suppressive in nature by the dopamine pathway. Hyperprolactinaemia can cause hypogonadotropic hypogonadism and is closely related to the reproductive axis. However, the management of hyperprolactinaemia relies heavily on the underlyingcauses and extends beyond its biochemical correction (Molitch, 1992).
Plasma Prolactin (PRL) concentrations are controlled via a classical negative feedback loop (Freeman et al., 2000). PRL is under predominantly inhibitory control by the catecholamine dopamine synthesized by the tuberoinfundibular dopaminergic (TIDA) neurons, most of which are localized to the dorsomedial arcuate nucleus of the hypothalamus. These neurons project to the median eminence where dopamine is released into hypophyseal
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portal blood, and after reaching the anterior pituitary acts upon dopamine D2 receptors on lactotrophs to inhibit the synthesis and release of PRL. In turn, PRL acts in a short loop negative feedback manner to regulate the synthesis and secretion of dopamine by the TIDA neurons. Many studies have shown that the activity of the TIDA neurons (as determined by the rate of dopamine synthesis, dopamine turnover, or dopamine concentrations in hypophyseal portal blood) is increased when plasma PRL levels are increased by pharmacological manipulation (Moore, 1987) and (Ben-Jonathan and Hnasko, 2001). This negative short loop feedback between PRL and dopamine is well recognized and is responsible for PRL homeostasis in most physiological states. However, the mechanisms by which PRL exerts negative feedback are relatively unknown, although PRL receptors are expressed on TIDA neurons (Arbogast and Voogt, 1997); (Lerant and Freeman, 1998) and Grattan, (2001), suggesting a direct effect.
Hyperprolactinaemia is a well established cause of infertility in both male and female mammals. Elevated prolactin may impact reproduction through an action on the GnRH neurons of the hypothalamus and/or on the pituitary gland to affect secretion of the gonadotrophins, LH, and FSH (Evans et al., 1982 and McNeilly, 2001).
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(Ferland et al., 1979) Bromocriptine (2-bromo-a- ergocryptine, a dopamine agonist) has been used to examine the neuroendocrine mechanism of dopamine that controls prolactin secretion in vivo. In addition, dopamine agonist therapy for pituitary prolactinomas results in the reduction of prolactin secretion and tumor regression (Chiodini et al., 1981 and Tindall et al., 1982).
2.16. Prostaglandin (PG) function on uterine contraction and ovulation:
The prostaglandins have been characterized as unsaturated fatty acids. It has been shown that arachidonic acid (5, 8, 11, 14- eicosatetraenoic acid) is the precursor of prostaglandin (PGF1 and PGF2) (Van Dorp et al., 1964 and Bergstrom et a l., 1964) .PGF2α is luteolytic in the rabbit when administered in large, supra- physiological doses (Gutknecht et al., 1972 and Bruce and Hillier, 1974). The observation that arachidonic acid, the fatty acid precursor of PGF2α, is also luteolytic indicates that uterine arachidonic acid released into the systemic circulation might act as the physiological stimulus for CL regression by conversion to PGF2α at the CL (Hoffman, 1974 and Carlson and Gole, 1978).
Prostaglandins are found in most tissues and organs they are produced by all nucleated cells except lymphocytes. They are autocrine and paracrine mediators that act up on platelets
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endothelium, uterine and mast cells. They are synthesized in the cell from the essential fatty acids (EFA) (Dorland's medical Dictionary). In addition prostaglandins are potent but have a short half – life before being inactivated and excreted. Therefore they secreted only paracrine (locally active) or autocrine "(acting on the same cell from which it is synthesized) signal (Gado , 1996).
Prostaglandins are believed to act as mediators. They have a variety of strong physiological effects such as regulating the contraction and relaxation of smooth muscle tissue (Nelson, 2005). Although, they are technically hormones, they are rarely classified as such.
There are currently ten known prostaglandin receptors on various cell types (komoto et al., 2004) the diversity of receptors means that prostaglandin act on an array of cells and have a wide variety of effect such as, causing contactor of the uterus and have serum hormonal regulation effect especially progesterone, antagonist, so it induce parturition or abortion (Gado, 1996).Prostaglandins of the E and F series have been implicated in uterine function by several observations. They are highly potent in causing uterine contraction in several species (Bergstrom et al., 1968) and are released by distension of the guinea-pig uterus (Poyser et al., 1971).
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The increases in prostaglandin concentrations in human blood and amniotic fluid (Karim and Devlin, 1967) during labour suggest that they may play a part in the expulsion of the uterine contents at parturition.
The uterine luteolytic factor is assumed to be prostaglandin PGF2α (Pharriss, 1971; McCracken, 1971; McCracken et al., 1972 and Horton and Poyser, 1973) and its effect depends on an intact utero ovarian vascular system (Baird and Land, 1973) for the veno-arterial transfer of the luteolytic factor to the ovaries (Hixon and Hansel, 1974). The inhibitory effect of PGF2α on the progesterone secretion of the corpus luteum may be connected with the constriction of the ovarian blood vessels, thereby decreasing ovarian blood flow (Pharriss, 1971 and McCracken, 1971). However, Baird (1974) reports that PGF2α reduces progesterone secretion in sheep but does not reduce ovarian blood flow. In pseudopregnant rabbits, PGF2α increased local blood flow in the stroma while that to the corpus luteum was reduced (Novy and Cook, 1973; Janson et al., 1975). In the bitch, PGF2α is known to reduce ovarian progesterone secretion (Jِochle et al., 1973), but ovarian blood flow has not been determined.
Prostaglandins (PGs) play an important role in the regulation of uterine function such as parturition during the reproductive
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process. An increased level of prostanoids such as PGE2 and PGF2α causes the myometrial contractility that initiates labor at term (Mitchell et al., 1995 and Novy and Liggins, 1980).
Aikn (1972) and Chester et al., (1972) confirms this suggestion by showing that parturition is delayed and prolonged in rats when prostaglandin production is prevented by prostaglandin synthetase inhibitors such as aspirin and indomethacin. We have used indomethacin and meclofenamate, both' potent inhibitors of prostaglandin biosynthesis (Flower et al., 1972) to study the contribution of intra-mural prostaglandin production to spontaneous and drug-induced contractions of the rat isolated uterus. Contractions of the non-pregnant isolated uterus from the rat elicited by oxytocin were antagonized by indomethacin whereas those evoked by PGF2α and acetylcholine were not. Spontaneous contractions of pregnant rat isolated uteri were also blocked by indomethacin and there was a consistent decline in prostaglandin output. Oxytocin induced contractions of pregnant uteri were not antagonized by indomethacin. (Vane and Williams, 1972 and Williams, 1973).
2.17. Effect of indomethacin on the prostaglandin biosynthesis:
Indomethacin is known to decrease urinary protein excretion and renal blood flow and to enhance water and sodium retention (Usberti et al., 1975) .This action on kidney function is explained by its inhibition of
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the synthesis of renal prostaglandins (PGs) (Lee, 1980). With regard to the relationship among indomethacin, PGs and mesenteric circulation, Maher demonstrated that intra-peritoneal administration of PGE2 in rabbits on peritoneal dialysis increases the peritoneal clearance of urea and creatinine, whereas similar administration of PGF2α significantly decreased peritoneal clearances. Furthermore, oral administration of mefanamic acid or indomethacin did not change peritoneal clearances (Maher et al., 1980 and Maher et al., 1981), probably because changes in prostaglandin activity did not produce sufficiently large changes in peritoneal blood flow. It has been reported that bovine mesenteric arteries and veins (Terragno et al., 1975), and rabbit mesenteric vessels (Blumberg et al., 1975) have the capacity to synthesize prostaglandins.
Anti-inflammatory agents such as indomethacin and aspirin inhibit prostaglandin synthesis by all cells and tissues so far examined (Vane, 1971 and Humes et al., 1977). This has been attributed to a direct effect on the cyclooxygenase that converts arachidonic acid to prostaglandins (Smith and Lands 1971 and Lands and Rome, 1976). However, indomethacin and related compounds that are known to inhibit numerous enzymes (Flower, 1974) may also act on other steps in the synthesis of prostaglandins. Because the regulation of prostaglandins under physiological conditions must include the repletion of the exceedingly small intra- cellular pool of unesterified arachidonic acid by the action of
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lipolytic enzymes, we entertained the possibility that indomethacin might also act on the synthesis of prostaglandins by an effect on phospholipases of inflammatory cells.
Ovulation induced by mating or with exogenous gonadotropins, was blocked by indomethacin given during a critical time period. A single concomitant injection of 20 mg/kg was sufficient to block LH-induced ovulation. Additional treatment (8 mg/kg every 12 hrs for 48 hrs) was necessary if HCG were administered instead of LH, probably reflecting the longer half-life of HCG. Caitus-induced ovulation was not affected if indomethacin (20 mg/kg) was given at the time of mating, but a single subcutaneous injection of 8 mg/kg given 8 hrs after mating inhibited ovulation. The block was presumably at the ovarian level since in all cases where indomethacin was effective; the ovaries were characterized by large hemorrhagic follicles in which the ova were retained. Considerable luteinization of the follicles was observed in association with normal progesterone levels, suggesting that indomethacin affected only the physical process of ovulation. Indomethacin treatment was also found to lengthen pseudopregnancy to the same extent as produced by hysterectomy, and when administered from the day of mating, indomethacin was associated with fetal resumption in pregnant animals (Patrick et al., 1972).
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The levels of prostaglandin F (PGF) and prostaglandin E (PGE) were determined by radioimmunoassay in follicles obtained from rabbits at estrous and at 5 or 9 hrs after an ovulatory injection of HCG, LH or mating. The ovulatory stimuli produced a marked increase in follicular prostaglandin levels (PGF and PGE). The increases produced by HCG and LH, or mating were completely abolished by the intra-venous injection of indomethacin (20 mg per kg) 30 minutes prior to the gonadotropin treatment or at the time of mating (Norman et al., 1973).
Indomethacin, an inhibitor of prostaglandin biosynthesis, was 100% effective in blocking luteinizing hormone (LH)-induced ovulation when administered via micro injection directly into 22 follicles in twelve rabbits, 5 hrs after intra-venous injection of the gonadotropin. Ovulation was similarly blocked in 24 of 25 follicles injected with antiserum prepared against prostaglandin F2α.
Antiserum against prostaglandin E2, at the same dosage (100 μg lyophilized serum per follicle), was considerably less effective, preventing ovulation in only 6 of 14 follicles. Control follicles injected with the phosphate buffer vehicle, or with normal rabbit serum, underwent normal ovulation and luteinization. LH injection caused a striking increase in concentration of F-type prostaglandins in follicles shortly before ovulation, an increase which was prevented
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by intra-venous or intra-follicular injection of ovulation-blocking blocking dosages of indomethacin (David et al. 1974).
Several studies have convincingly shown that prostaglandins (PGs) are involved in the process of ovulation (Behrman, 1979; Owman et al., 1979; LeMaire et al., 1980; Armstrong, 1981 and Dennefors et al., 1983). Systemic administration of prostaglandin (PG) synthetase inhibitors, e.g., indomethacin and aspirin, block ovulation in rats (Armstrong and Grinwich, 1972 and Orczyk and Behrman, 1972).
Intra-follicular injections of indomethacin into mature follicles of the rabbit, as well as of antisera to PGE2 and especially PGF2α, prevent luteinizing hormone (LH)-induced ovulations, indicating that the PGs exert their action at the follicular level and that PGF2α is the most important PG for ovulation in the rabbit (Armstrong et al., 1974a).
The intra-follicular increase in PGE2 and PGF2α after LH stimulation has been verified by perfusion of rabbit ovaries in vitro (Koos et al., 1983). Both PGF and its stable metabolite, 13, 14- dihydro, 15 keto PGF2α, have been measured in the perfusion medium, and their increase has been correlated with both ovulatory efficiency and the total number of ovulations (Schlaff et al., 1983).
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Moreover, it has been shown that the blockade of gonadotropin-induced ovulations by indomethacin can be overcome by exogenous PGF2α (Hamada et al., 1978 and Holmes et al., 1983). And that PGF2α can induce ovulations even in the absence of gonadotropins (Holmes et al., 1983).
Schmidt et al., (1986) reported that the effects of prostaglandin E2 (PGE2) on the ovulation process were studied in a recirculating perfusion model using ovaries from virgin rabbits. Ovulation frequency, time of ovulation, and progesterone release from the ovaries were examined after the addition of PGE2, either alone or with luteinizing hormone (LH) in the presence or absence of indomethacin. The stimulatory effect of LH on ovulation was totally blocked if the ovaries were exposed to indomethacin at the same time. Ovaries treated with PGE2 alone did not ovulate, and PGE2 was unable to restore indomethacin-blocked ovulation. Conversely, the frequency of ovulation was reduced in ovaries treated with PGE2 and LH compared with controls receiving only LH. There was no measurable difference in the pattern of progesterone release between ovaries simultaneously treated with LH and indomethacin and LH- treated controls. Ovaries exposed to PGE2 alone showed only a slight increase of progesterone release in the medium, while those treated with PGE2 in combination with LH in the perfusate showed a smaller progesterone release than those treated with LH alone. The results confirm the blocking effect of indomethacin on ovulation. PGE2
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could not reverse this effect, but instead reduced the number of LH- induced follicular ruptures. Indomethacin had no effect on progesterone levels, while PGE2 (which alone showed a slight stimulating effect on the steroid concentration) together with LH counteracted the effect of LH on progesterone release.
Heigo et al., (1983) suggested that progesterone can substitute the action of prostaglandins injected serially in the first half of the preovulatory process. Mori et al., (1980) concluded that prostaglandins may mediate the action of HCG on ovulation through two mechanisms which operate at different stages of the preovulatory process.
The peak of prostaglandins (PGs) synthesis after HCG treatment related closely to the timing of ovulation; the steroidogenic response to HCG was not blocked by doses of indomethacin sufficient to inhibit synthesis of PGE2 and PGF2α by more than 80%, (Katz et al., 1989). Furthermore it have been reported that Indomethacin blocks ovulation in human chorionic gonadotropin-stimulated rabbits (Munalulu et al., 1987). Holmes et al., (1983) suggested that Ovaries treated in perfusion experiments with PGF2α alone had very low steroid levels compared to the ovaries treated with LH. .
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3. MATERIALS AND METHODS
The field study of this experiment was carried out at rabbit colony established in Endocrinology Research Unit, Applied Radiobiology Department, Nuclear Research Center, Atomic Energy Authority, Inshas, Egypt. The hormonal radioimmunological assays were performed in the laboratories of the same Research Unit. It was planned to point out the effect of treating adult does exposed to three regimes of photoperiodicity with different hormonal treatments including HCG, Melatonin, Serotonin, Bromocriptine (anti prolactin) and Indomethacin (anti prostaglandins) alone or HCG combined with the same previously mentioned drugs: (Melatonin, Serotonin, Bromocriptine and Indomethacin).
3.1. Grouping of animals and experimental design:
A total number of 300 young adult female New Zealand white does purchased from rabbit farm belonging to Faculty of Agriculture, Cairo University, Giza, Egypt. Selected does aged 3-4 months and weighed 2-3 Kg. All animals were individually caged and kept in an environmental chamber under controlled ambient temperature of 250C, isolated from outdoors day light and well ventilated. Does subjected to study were kept under these condition for pre- experimental period leaved two weeks then divided into three groups each of 100 does. Animals of these three groups were kept under
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certain photoperiod regime (Light-Dark cycle) indicated as follows:
Photoperiod regime (hrs daily) Group Light Dark
First 16 8
Second 12 12
Third 8 16
Electronic timer was used to control lightening cycle. Illumination was adjusted using neon lamps to give 150 lux as measured by luminometer Sinometer (MS - 8209). Lamps were thus equally arranged surrounding all rabbits, when blood sampling was done at complete darkness a dark light bulb was used. The light emitted was below detection limits of the measuring of the lux meter. Water was permanently offered by means of water system feeding through nipples. Animals were fed ad lib balanced concentrate diet in the form of pellets formulated as shown in the following table:
In order to study in delineation the interaction of photoperiod with endocrine events linked to induced ovulation, rabbits were injected stimulatory and inhibitory factors to mimic long or short photoperiod effects on induced ovulation. Induced ovulation as culminated in elevated blood levels of progesterone is the main criterion in this study. This work was accomplished in two separate experiments spaced with two weeks rest to adapt the animals for the photoperiod regime. Thus animals of each experiment in each
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photoperiod cycle were divided into ten treatment contains10 does. The final number of does in each experiment at the end differed greatly from that at the start of the experiment as shown in (Table1). This was due to mortality of does along the experimental period. The collected data from these does were used in statistical analysis. Actual number of does in groups (photoperiod) and (treatments) that still survived at the end of the first experiment (injected drugs only) and second experiment (injected drug materials followed by HCG) as shown in Table (1a and 1b).
First experiment
Design of the first experiment and number of animals subjected to various lighting regimes is indicated in the following Table:
Table (1a): Design of the first experiment and number of animals subjected to different photoperiods.
Photoperiod (hrs. daily) Injected materials 8D/16L 12D/12L 16D/8L
Control (Saline) 6 8 6
HCG 3 4 4
Melatonin 3 4 4
Serotonin 6 4 4
Bromocriptine 5 4 5 Indomethacin 3 3 3
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Control animals received 1ml saline/animal and treated animal injected drug materials as in (Table 2).
Second experiment
Design of the second experiment and number of animals subjected to various lighting regimes is indicated in the following Table:
Table (1b): Design of the second experiment and number of animals subjected to different photoperiods.
Photoperiod (hrs. daily) Injected materials 8D/16L 12D/12L 16D/8L
Control (HCG) 3 4 4
Melatonin+HCG 3 3 3
Serotonin+HCG 3 3 3
Bromocriptine+HCG 3 3 6
Indomethacin+HCG 2 3 3
Control animals received 167 IU HCG/1ml /animal and treated groups received drug in double dose to achieve complete effective drug fulfillment of the physiological effect followed by the HCG ovulation induction dose (167 IU HCG / ml / animal). A summary of the followed scheme is depicted in (Table2).
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3.2. Hormonal modulators applied: 3.2.1. Indomethacin
Also called indomethacin is a non-steroidal anti – inflammatory drug, commonly used to inhibit the production of prostaglandins . It is marketed under many trade names, including Indocin , indocid , indochron E-R and indocin SR. Indometacin has a systemic name 2- {1-(4- chlorophenyl) carbonyl) 5-methoxy -2- methyl -1H – indol -3- yl. ) acetic acid, having chemical formula -1 C19H16CINO4 with molecular mass 354 .787 g. mol . It is mobilized at liver and have half – life 4.5hrs and exerted renal 60% and faecal 33%.
Inodmethacin is nonselective inhibitor of cyclooxygenase (COX) 1 and 2, enzymes that participate in prostaglandin synthesis from arachidonic acid. Prostaglandins are hormone – like molecules normally found in the body of animals, where have a wide variety effect some of which lead to pain, fever, and inflammation. Prostaglandins also cause uterine contractions in pregnant animals. Indomethacin is an effective locolytic agent able to delay premature labor by reducing uterine contractions through inhibition of prostaglandin synthesis in the uterus and possibly through calcium channel blockade. Indomethacin has two additional modes of actions with clinical importance: inhibits motality of polymorphonunclear leukocytes, similar to colchicines and uncouples oxidative phosphorylation in cartilaginous (and hepatic) mitochondria, like
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salicylates these additional effects account as well for the analgesic and anti – inflammatory properties. Indomethacin readily crosses the placenta, and can reduce fetal urine production to treat polyhydramnios by reducing renal vascular resistance, possibly by enhancing the effects of vasopressin on the fetal kidneys.
3.2.2. Bromocriptine (parlodel cycloset)
Bromocriptine has systematic name Ergotaman -3,6,8 trione, 2- bromo -12 hydroxy -3'- (1- methylethyl) – 5' alpha – (2- methlpropyl). Its chemical formula C32 H4O BrN5O5 with molecular mass 654. 595 . its half – life 12 -14 hrs and secreted in faeces . It is ergoline derivative which is a dopamine agonist that used in the treatment of pituitary tumers, Parkinson”disease, Hyperprolactinaemia, neuroroleptic malignant syndrome and type 2 diabetes.
Bromocriptine is commonly used for treating amenorrhea, female infertility, galactorrhea, hypogonadism. In 2009 bromocriptine mesylate was approved by FDA for treatment of type 2 diabetes under trade name cycloset. It is currently unknown how this drug, improve glycemic control.
Hyper prolactinaemia can be a part of normal body changes during pregnancy and suckling. It can also be caused by diseases affecting the hypothalamus and pituitary gland. It can also be caused by disruption of the normal regulation of prolactin levels by drugs and
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heavy metals. Hyper prolactinaemia may also be the result of disease of the organ such as the liver, kidneys, ovaries and thyroid.
3.2.3. Melatonin
Melatonin, also known chemically as N-acetyl-5- methoxytryptamine, is a naturally occurring compound found in animals, plants, and microbes. In animals, circulating levels of the hormone melatonin vary in a daily cycle, thereby allowing the entrainment of the circadian rhythms of several biological functions. Many biological effects of melatonin are produced through activation of melatonin receptors, while others are due to its role as a pervasive and powerful antioxidant, with a particular role in the protection of nuclear and mitochondrial DNA. In mammals, melatonin is secreted into the blood by the pineal gland in the brain. Known as the "hormone of darkness", it is secreted in darkness in both day-active (diurnal) and night-active (nocturnal) animals.
Melatonin acts as an endocrine hormone since it is released into the blood. By contrast, melatonin produced by the retina and the gastrointestinal (GI) tract acts as a paracrine hormone. It also exerts a powerful antioxidant activity. Melatonin interacts with the immune system it might be useful fighting infectious disease.
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3.2.4. Serotonin
Serotonin or 5-Hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal (GI) tract, platelets, and in the central nervous system (CNS) of animals including humans. It is a well-known contributor to feelings of well-being; therefore it is also known as a "happiness hormone" despite not being a hormone. 80 percent of serotonin is located in the enterochromaffin cells in the gut, where it is used to regulate intestinal movements. The remainder is synthesized in serotonergic neurons in the CNS where it has various functions. These include the regulation of mood, appetite, sleep, as well as muscle contraction. Serotonin also has some cognitive functions, including in memory and learning. Modulation of serotonin at synapses is thought to be a major action of several classes of pharmacological antidepressants.
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3.3. Injected materials: As shown in (Table2) (Table 2): Protocol of injected materials during the course of experiments.
Groups Subcutaneous injection dose
First experiment
1 Saline 1ml / animal
2 HCG 167 IU / 1ml saline / animal.
3 Melatonin 300 µg / 1ml saline / animal.
4 Serotonin 1 mg / 1ml saline /animal.
5 Bromocriptine 3 mg / 1ml saline / animal.
6 Indomethacin 20 mg / 1ml saline / animal.
Second experiment
7 HCG 167 IU / 1ml saline / animal.
Melatonin in double dose 2hrs 8 Same dose as in group 3 and 7. separated followed by HCG
Serotonin in double dose 2hrs 9 Same dose as in group 4 and 7. separated followed by HCG
Bromocriptine in double dose 10 2hrs separated followed by Same dose as in group 5 and 7. HCG
Indomethacin in double dose 11 2hrs separated followed by Same dose as in group 6 and 7. HCG
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3.4. Parameters estimated and data collection Serum blood progesterone level was estimated for each doe within each subgroup at 0,1,2,4,8,16 hrs, after the last injection.
3.4.1. Blood samples collection: Blood samples were individually collected directly by puncture marginal ear vein using a sterile needle. No anticoagulant material was applied. Sampling was facilitated using a glass device designed especially for collection of rabbit blood samples. This device was attached to an air vacuum pump to facilitate driving of blood from the incised marginal ear vein. Vacuum was reduced using an on line regulator valve to prevent painful excitation of rabbits. All collected blood samples were allowed to clot overnight at 4oC. Samples were then centrifuged at 2500 rpm for 30 min and sera collected for storage at -200C till assay time.
3.4.2. Chemicals and reagents used in chemical analysis:
All chemicals and organic solvents used in this study were Analar (A.R) grade and/or high performance liquid chromatography (HPLC) quality. Human chorionic gonadotropin (HCG) was purchased from I.F. Serono, (Roma, Italy).Melatonin was purchased from (Across Organics, new jersey, USA). Bromocriptine, Indomethacin, Serotonin, Dexamethasone (DXM), pig skin gelatin,
(Nacl) sodium chloride and sodium azide (NaN2) were purchased from (Sigma Chemicals Co, St. louis, USA). Mono sodium dehydrate
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(NaPo4.2H2O) extra pure was from (Merck Darmstadt, Germany).
Disodium hydrogen phosphate anhydrous (Na2Po4.H2O) was from (Panreac, Barcelona-Spain). Ethlene diamine tetra acetic acid disodium salt dihydrate pure (EDTA) was from (Colnbrack Bucks,England). Progesterone for preparation of standard curve was obtained from Steraloids (New Hampshire USA).Progesterone tracer 3 3 [1, 2, 6, 7- H] P4- H was from Amersham BioScience, (Buckinghamshire, England). Normal rabbit serum (NRS) was prepared by bleeding male rabbit ,separating normal serum by centrifugation and was preserved at 200C.Precipitating second antibody (ARGG) was produced in sheep by immunization using purified rabbit gamma globulin ( Sigma chemicals , Stlouise Mo. USA ) and was prepared previously at Endocrinology Research Unit Lab.
3.4.3. Progesterone (P4) radioimmunoassay: Radioimmunological assays for serum progesterone were performed in the laboratories of the Endocrinology Research Unit, Applied Radiobiological Department, Applied Radioisotope Division, Nuclear Research Center, Atomic Energy Authority.
Micro-radioimmunoassay system was adapted set up the assay (Elbanna and Gamal, 1986). Slight modifications were introduced due to complement effect. The increase in complement concentration interfered with antibody-antigen interaction.
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3.4.3.1. Assay buffers: Assay buffer phosphate buffer saline /gelatin (PBS-G), used was (NaPo4.2H2O/ Na2Po4.H2O) 0.1M pH 7.4 containing 0.9 %
NaCl, 0.1% pig skin gelatin and 0.1 %( NaN2 -PBS). Buffers were prepared and stored 40C.Antibody was diluted (1:1000) in PBS (0.1
M, pH 7.4) containing EDTA 0.05M, 0.01% NaN2 and 1:600 normal rabbit serum (NRS). While antirabbit gamma globulin (ARGG), was diluted (1:32) in PBS (0.1 M, pH 7.4) containing EDTA 0.05M,
0.01% (NaN2).Standard curve set points, p4 tracer and dexamethasone (DXM) blocking agent (300 ng/ 50ul/RIA tube) were prepared in assay buffer PBS-G (0.1 M, pH 7.4).
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3.4.3.2. Assay procedure: All assay procedure in (6x 35 mm) polystyrene tubes for standard curves, quality control (QC) points; total count (TC) and total binding (B0) were performed in quadruplicates. Unknown samples and nonspecific binding
(NSB) were performed in duplicates in 350 µl reaction volume. All additions arrangement are indicated as follows.
C
o
G 3
- H
4
QC
Sple PBS
STD
NRS
P
HCL
DXM
Hisafe
PBS
1ST Ab
2ND Ab 2ND
♂R.H.F.S
TC ______50 ______350µl 10µl 250µl
NSB-B 150 50 ____ 50 100 ______350µl 10µl 250µl
C in cooled room in cooled C o NSB-S 50 50 ____ 50 100 ____ 50 ______50 350µl 10µl 250µl
BO-B 150 ____ 50 50 100 ______350µl 10µl 250µl
BO-S 50 ____ 50 50 100 4 at incubation hours 4 wait hen ____ 50 ______50 350µl 10µl 250µl
STD ______50 50 100 50 50 ______50 350µl 10µl 250µl
QC 50 ____ 50 50 100 ______50 ____ 50 350µl 10µl 250µl
Centrifugation 3000 rpm for 45 minute 45 for 3000 rpm Centrifugation
Incubation overnight at 4 at overnight Incubation
Sple 50 ____ 50 50 100 ______50 50 350µl 10µl 250µl
Mixed by vortex & t & vortex by Mixed
Abbreviations ; TC = total count , NSB(B) = non-specific binding in buffer , NSB(S) = non-specific binding in serum , B0(B) = 0 binding in buffer , B0(S) = 0 binding in serum , STD= Standard curve set points , QC = quality control points , Sple = sample , PBS-G = 3 phosphate buffer saline gelatin , NRS = normal rabbit serum, 1St Ab = first antibody, P4 H = progesterone tritiated (tracer) , 2nd Ab = second antibody, ♂ RHFS = male rabbit hormone free serum , DXM = dexamethasone, PBS = Phosphate buffer saline , HCL = hydrochloric acid , Hisafe = liquid scintillation cocktail.
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All tubes were vortexes carefully and incubated overnight at +4o c to ensure complete equilibration and binding between antibody and tracer, 350µl of cold washing buffer (PBS 0.1 M, pH7.4) was added and followed by centrifugation at 3000 rpm for 45 min. to separate bound from free fractions. Supernatant was poured off by decantation and the rims of RIA tubes were washed and let drain to dry. 10 µl of HCl (6N) was added to all tubes. 250µl of counting scintillation fluid (Hisafe) was dispensed to each tube; mixed by vortex and incubated overnight at +4o c to ensure complete extraction of tritiated tracer. The tubes were counted for 1 min. in liquid scintillation counter (LSC) Wallac, 1409(wallac Turuk, Finland). All tubes were counted for 1 min. in liquid scintillation counter. Wallac, 1409(wallac oy Turuk, Finland). Radioactivity as count /minute (cpm) for progesterone standard points were calculated as percent of total bound radioactivity (Bo %). These percentage values were plotted on logit / log paper to obtain a liner standard curve. The unknown samples were calculated using the same procedure using the regression equation .The progesterone concentration in each unknown sample was calculated as ng/ml serum for data analysis. Area under curve (X axis representing time pre and after injection and Y axis representing P4 serum levels, ng/ml) were calculated using the Origin-8 program (origin Lab corporation one roundhouse plaza, Northampton, MA 01060 USA). These values (area units)
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were used for analysis of variance .Area under curve values represents collective ovulation response to injected material over the whole experimental period rather than individual samples. The data were analyzed according to analysis of variance model with unequal subclass numbers (Steel and Torrie, 1980).
The statistical model applied for this study is:-
Yijk = µ +αi +βj + (αβ)ijk +εijk
µ = the total overall mean.
αi = standard for fixed rows (injection ) .
βj = standard for fixed columns (photoperiod ) .
(αβ)ijk = interaction between (injection and photoperiod).
εijk = Error term
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4. RESULTS AND DISSCUSSION
The presented data are graphically represented as mean values (ng/ml serum) along with table values. Moreover individual curves for each rabbit within a specific cell of all experiment was transformed to area under curve in order to be more indicative to cumulative effect of injected drug materials over the 16 hrs of each experiment. These areas under curve data were graphically exhibited and used for analysis of variance under each photoperiod (short darkness, equal dark / light and long darkness).
4.1 First experiment:
First experiment was planned and conducted as a trial to detect the possibility for improving the reproductive performance of does by magnifying the hormonal coordination related to productive activity with relation to different light regimes tested, to fulfill this aim applying human chorionic gonadotropin as well as other chemical compounds that thought to have considerable effect on hormonal regulation of reproduction. Melatonin as an important regulator of the circadian rhythms of several biological functions including reproduction , serotonin a neurotransmitter that effects growth and reproduction , bromocriptine which is considered as a dopamine agonist that is used in the treatment of hyperprolactinaemia, and indomethacin as antiprostaglandin drug. The effects of these
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hormones and drugs on the level of serum progesterone are discussed as follows:
4.1.1. Serum progesterone level in untreated does subjected to different lighting regimes applied:
Table 3; Figures 1, 2 and 3 and Histograms 1, 2 and 3 shows that the variation in serum progesterone level due to either lighting regimes or experimental intervals. Averages of progesterone level were almost higher in does exposed to short lighting regime (16 hours (hrs) darkness / 8 hours (hrs) light) when compared to other lighting regimes, on the other hands, the higher level of serum progesterone were detected after two hours .This was quite true in all lighting regimes applied. Variation in serum progesterone level for does treated much saline solution due to estimation period may be attributed to the individual variation existed the reproductive activity rather than due to injected saline solution since the high serum progesterone levels were detected almost at the second hour after injection . The higher levels of serum progesterone found along the experimental period for does exposed to short lighting regime (16 hrs darkness and 8 hrs lighting) may lead to conclude that surge of LH hormone needs at least 8 hrs lighting to show its biological effect of ovulation in the animals of induced ovulation pattern like rabbits. Thus, the photoperiod regime shows its significant effect on ovulation mechanism in accordance
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with the time of ovulation which occurred at approximately at the same time in all experimental animals.
4.1.2. Effect of HCG:
Data listed in Table (3) represented in Figures (1, 2 and 3) and Histograms (1 , 2 and 3) show averages of serum progesterone level in ( ng/ml) in does exposed to different lighting regimes and injected with HCG compared with those of control group (injected with saline solution along 16 hrs after injection) .
Data obtained revealed an increase in serum progesterone levels as a result of injecting does with HCG, The rate of increment differed obviously according to the lighting regimes applied. Applying long lighting regime (8 hrs darkness and 16 hrs lighting) resulted in gradual increase in serum progesterone level reaching its lightest level (6.9526 ng /ml) at the 4th hr after injection then decreased up to the end of experimental period. Applying short lighting regime (16 hrs darkness (D) / 8 hrs light (L)) increased serum progesterone level with greater rate up to 4th hr after injection reaching highest level (13.0508 ng/ml) at the 2nd hr and (14.0844 ng /ml) at 4th hr then decreased steadily to reach lowest level at the 16th hr after injection . The same trend was found in does subjected to equal lighting regime (12 hrs D / 12 hrs L) but with lowest magnitude serum progesterone level increased after injection to reach its highest level (3.2797 ng/ml) after 2 hrs of
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injection then decreased sharply and steadily towards the 16th hr after injection to reach the lowest level at this time (0.1384 ng/ml). Results obtained go in agreement with those of Hilliard and Eaton (1971) and Lau et al., (1978), who reported that, peaks of progesterone and 20α-dihydroprogesterone in rabbit have been observed at 2 and 6 hrs after HCG injection respectively.
The obtained results concerning that, treating rabbit with HCG increased the level of serum progesterone during short photoperiod (16 hrs D/8 hrs L) compared to long photoperiod (8 hrs D/16 hrs L). Previously mentioned result may lead to conclude that, heat stress during long photoperiod (summer season) might result in higher glucocorticoid (Roman – Ponce et al., 1977) and prolactin levels (Peters and Tucker, 1977) resulting in negative feedback effect on luteutropic hormone and consequently reduced progesterone secretion rates.
Analysis of variance for obtained data (Table 4) revealed significant variation in serum progesterone level due to lighting regime , injection and the interaction between them (P < 0.01) .
Obtained data agreed with results obtained by kayisly et al., (2003) who stated that human chorionic gonadotropin interacts with the LHCG receptors and promotes the maintenance of corpus luteum during the beginning of pregnancy, causing it to secret the hormone progesterone. Progesterone enriches the uterus with thick
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lining of blood vessels and capillaries so that it can sustain the growing fetus. He added that due to highly negative charge, HCG might repel the immune cells of the mother, protecting the fetus during the early pregnancy period. It has also been hypothesized that HCG may be placental link for the development of local maternal immunotolerance. The authors suggested that HCG may be a link in the development of peritrophoblastic immune tolerance and may facilitate the trophoblast invasions, which is known to expedite fetal development in the endometrium. Results obtained agree with those reported by Caillol et al., (1986) who found that, progesterone concentrations are undetectable before mating or HCG treatment. However, transient rise in progesterone secretion was observed in the 24 hrs following mating or HCG injection which, occurs after the LH surge induced. Batra et al., (1979) also reported that elevation in the level of progesterone in non-pregnant rabbits resulted from increased ovarian output through HCG injection has a pronounced and immediate depressing effect on the character of myometrial activity which is indicative of a shift from non-pregnant to pregnant type. These results are in accordance with Browning et al., (1980) and Dharmarajan et al., (1989), who reported that the magnitude of estradiol (E2) and progesterone (P4), rise occurred in response to HCG injection.
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Table (3): Progesterone serum levels (ng/ml) x ±SE in response to injection of HCG vs saline at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time Saline HCG after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.3518±0.0448 0.5169±0.1248 0.8723±0.1633 0.0973±0.0516 0.2005±0.0821 0.1867±0.0371
1hr 0.6303±0.1729 0.3871±0.0445 0.7767±0.2260 0.1925±0.1273 1.2733±0.2450 0.2626±0.0610
2hr 0.6034±0.0897 1.4128±0.9812 1.2750±0.4254 5.3860±5.0900 3.2797±0.9767 13.0508±3.3939
4hr 0.5303±0.0887 0.5035±0.2030 0.6457±0.0937 6.9526±5.3762 0.8559±0.4248 14.0844±3.4214
8hr 0.4546±0.1422 0.2775±0.0173 0.5748±0.1137 1.3982±0.9838 0.2573±0.0884 6.0789±1.1813
16hr 0.2927±0.0323 0.4057±0.1030 0.6742±0.2112 1.1544±0.62064 0.1384±0.1768 1.4430±0.3942
Results and Discussion - 93 -
Table (4): Analysis of variance of data (cumulative area under curve in units) collected after injection of HCG vs saline under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 9079.57501 4539.7875 12.6317**
Injection 1 14258.2824 14258.28 39.673**
Interaction 2 10125.6967 5062.85 14.087**
Residual 25 8984.92118 359.39684
Total 30 42448.47529 1414.9493
** Significant at level of 0.01
Results and Discussion - 94 -
Figure (1) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at short darkness photoperiod (8D/16L).
Figure (2) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at equal dark / light photoperiod (12D/12L).
Figure (3) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 95 -
Histogram (1) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at short darkness photoperiod (8D/16L).
Histogram (2) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at equal dark / light photoperiod (12D/12L).
Histogram (3) Serum progesterone level (ng/ml) in response to administration of HCG vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 96 -
4.1.3. Effect of Melatonin:
Data listed in Table (5) represented in Figures (4, 5 and 6) and Histograms (4, 5 and 6) show serum progesterone level in does subjected to different lighting regime applied and treated with melatonin.
Results obtained showed that treating experimental does with melatonin. Increase serum progesterone level at all intervals of estimation when compared with the corresponding values of does treated with saline solution. However, the rate of increase varied according to either lighting regime applied or interval of estimation after injection. The highest progesterone level was estimated after 1 hr of injection does subjected to long lighting regime (8 hrs D/16 hrs L) (14.5463 ng /ml) which sharply decrease to (7.1431 ng /ml) at 2 hrs after injection and still decrease up to 16 hrs after injection to reach the value of (1.7013 ng /ml).
Different pattern of change along the whole intervals of estimation was found in does subjected to other two lighting regimes. Serum progesterone level increased in does subjected to equal lighting regime (12 hrs D /12 hrs L) to reach its highest value at 2 hrs after injection (1.7679 ng /ml) then steadily decrease to reach a value of (0.8685 ng /ml) at 16 hrs after injection. Same trend of variation along all times of estimation was found in does subjected to short lighting regime (16 hrs D /8 hrs L) but with lower magnitude when compared to the other photo regimes applied.
Results and Discussion - 97 -
The obtained results may lead to conclude that melatonin has been implicated as a key factor in the photoperiod reproductive cycles (Thorpe and Herbert, 1976). Dodge and Badura (2002a) reported that short day (SD) (16 hrs D/8 hrs L) exposure in female Syrian hamster reduces serum concentrations of LH and progesterone. Maximum differences in basal and peak of progesterone levels may have been observed between short day (16 hrs D/8 hrs L) and long day (8 hrs D/16 hrs L) (Beasley et al., 1981). Turek et al., (1975) indicated that the reproductive system of photoperiodic mammals is more likely to be responsive to exogenous melatonin.
However , analysis of variance for obtained data (Table 6) revealed in significant variation in serum progesterone level due to either lighting regime , injection or the interaction between them .Obtained results were scientifically logic since melatonin vary in daily cycle , there by regulating the circadian rhythms of several biological functions (Altum and Agur-Altum , 2007). Many biological effects of melatonin are produced through activation of melatonin receptors (Boutin et al., 2005) while other are due to its role as a pervasive and powerful anti-oxidant (Hardeland, 2005) with a particular role in protection of nuclear and mitochondrial DNA (Reiter et al., 2001). Finally, Terzola (1993) stated that melatonin also Lowes FSH levels, these hormonal changes could impair fertility.
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Table (5): Progesterone serum levels (ng/ml) x ±SE in response to injection of melatonin vs saline at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time Saline Melatonin after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.3518±0.0448 0.5169±0.1248 0.8723±0.1633 0.6828±0.0357 0.6810±0.1737 0.4703±0.0616
1hr 0.6303±0.1729 0.3871±0.0445 0.7767±0.2260 14.5463±6.2482 0.8314±0.1629 0.7528±0.3248
2hr 0.6034±0.0897 1.4128±0.9812 1.2750±0.4254 7.1431±1.9315 1.7679±1.2901 0.8729±0.1802
4hr 0.5303±0.0887 0.5035±0.2030 0.6457±0.0937 7.8530±1.3296 0.8750±0.3565 0.8863±0.2503
8hr 0.4546±0.1422 0.2775±0.0173 0.5748±0.1137 5.6212±2.5341 0.8742±0.2927 0.5104±0.0589
16hr 0.2927±0.0323 0.4057±0.103 0.6742±0.2112 1.7013±0.8089 0.8685±0.2342 0.5959±0.1875
Results and Discussion - 99 -
Table (6): Analysis of variance of data (cumulative area under curve in units) collected after injection of melatonin vs saline under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 3498.59945 1749.2995 3.3389
Injection 1 5817.320466 5817.32 11.03677
Interaction 2 647.87987 323.93995 0.6183
Residual 25 13097.73335 523.9092
Total 30 23061.53314 768.7177
Results and Discussion - 100 -
Figure (4) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at short darkness photoperiod (8D/16L).
Figure (5) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at equal dark / light photoperiod (12D/12L).
Figure (6) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 101 -
Histogram (4) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at short darkness photoperiod (8D/16L).
Histogram (5) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at equal dark / light photoperiod (12D/12L).
Histogram (6) Serum progesterone level (ng/ml) in response to administration of melatonin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 102 -
4.1.4. Effect of serotonin:
Data related to the effect of injection serotonin on serum progesterone level in does subjected to the different lighting regime applied are listed in Table (7) represented in Figures (7, 8 and 9) and Histograms (7, 8 and 9). Analysis of variance for data obtained is presented in Table (8).
It was obviously clear that injected serotonin lowered the level of progesterone level in does subjected to all lighting regime applied. However, the rate of this decrement differed according the ratio between the periods of darkness to that of lighting. It was found that the decreasing rate of serum progesterone level increased as period of darkness increased. Does subjected to long lighting regime of (8 hrs D /16 hrs L) had higher progesterone level in this serum followed by those subjected to equal lighting regime (12 hrs D /12 hrs L). However, the lowest averages of serum progesterone levels were observed in does of short lighting regime (16 hrs D /8 hrs L). This trend of variation in serum progesterone level was true at all estimation intervals.
The previously mentioned results may lead to conclude that the secretion of GnRH is controlled by neuroendocrine signals that include: serotonin, norepinephrine, epinephrine, dopamine, acetylcholine, gamma amino butyric acid, opioid peptides, and glutamate (Arias et al., 1990 and Fernández-Fernandez et al., 2005). Serotonin may affect
Results and Discussion - 103 -
GnRH secretion by either direct effect on GnRH neurons or indirectly by acting through other neurotransmitters, such as noradrenaline. An interrelationship between noradrenaline and GnRH neurons exists in the Preoptic area and median eminence (Smith and Jennes, 2001).
Serotonin had an adverse affect on ovulation which in closely affected by the level of LH surge which is effected by the length of daily lighting period from one side and the mating behavior of animals (specially of males) which correspondingly affect the incidence of ovulation in animals of inducing ovulation. Serotonin was found to be necessary for normal male mating behavior (Loer and Kenyon, 1993) and the inclination to leave food to reach for a mate (Lipton et al., 2004).
In mammals though insulin regulates blood sugar and insulin live growth factor IGF regulates growth, serotonin controls the release of both hormones so that serotonin suppresses insulin release from the beta cells in pancreas (Paulmann et al., 2009) and exposure to selective serotonin reuptake inhibitor increase the extracellular level of serotonin and increase the level of serotonin in the synaptic cleft that becomes available to bind to post synaptic receptor. In addition, serotonin in mammals can also act as growth factor directly (Wikipedia encyclopedia).
Results and Discussion - 104 -
Table (7): Progesterone serum levels (ng/ml) x ±SE in response to injection of serotonin vs saline at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Saline Serotonin Time after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.3518±0.0448 0.5169±0.1248 0.8723±0.1633 0.1056±0.0391 0.0188±0.0001 0.0188±0.0001
1hr 0.6303±0.1729 0.3871±0.0445 0.7767±0.2260 0.0227±0.0030 0.0371±0.0183 0.0283±0.0095
2hr 0.6034±0.0897 1.4128±0.9812 1.2750±0.4254 0.0574±0.0251 0.0188±0.0001 0.0380±0.0164
4hr 0.5303±0.0887 0.5035±0.2030 0.6457±0.0937 0.0496±0.0120 0.0188±0.0001 0.0341±0.0113
8hr 0.4546±0.1422 0.2775±0.0173 0.5748±0.1137 0.0345±0.0108 0.0270±0.0082 0.0203±0.0009
16hr 0.2927±0.0323 0.4057±0.1030 0.6742±0.2112 0.0383±0.0171 0.0191±0.0003 0.0193±0.0005
Results and Discussion - 105 -
Table (8): Analysis of variance of data (cumulative area under curve in units) collected after injection of serotonin vs saline under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 51.3809392 25.6905 47.0169**
Injection 1 541.8625145 541.8625 991.6775**
Interaction 2 244.7032583 122.352 223.9198**
Residual 28 15.2995939 0.54641
Total 33 853.2463059 25.8559
** Significant at level of 0.01
Results and Discussion - 106 -
Figure (7) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at short darkness photoperiod (8D/16L).
Figure (8) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at equal dark / light photoperiod (12D/12L).
Figure (9) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 107 -
Histogram (7) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at short darkness photoperiod (8D/16L).
Histogram (8) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at equal dark / light Photoperiod (12D/12L).
Histogram (9) Serum progesterone level (ng/ml) in response to administration of serotonin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 108 -
4.1.5. Effect of bromocriptine:
Data concerning the effect of injection experimental rabbits, subjected to three different lighting regimes, with bromocriptine are tabulated in Table (9) represented in Figures (10, 11 and 12) and Histograms (10, 11 and 12), analysis of variance for obtained data is shown in (Table 10).
Highly significant effect on serum progesterone level was found due to injection applied and the interaction between lighting regime and injection (p<0.01). While lighting regime showed significant effect (P<0.05).
From results obtained, it was obviously found that injection bromocriptine lowered the level of serum progesterone. However, the rate of decrease differed according to lighting regime and the time intervals after injection. The greatest decrease level was observed when applying animals to short lighting regime (16 hrs D /8 hrs L), while the lowest rate of decrease was found in the group of animals subjected to long lighting regime (8 hrs D /16 hrs L).Serum progesterone level in rabbits subjected to short lighting regime (16 hrs D /8 hrs L) decreased after injection bromocriptine and remained approximately constant along the whole experimental period with value mentioned (0.1665 ng/ml) (Table 9). Different pattern of variation was found in rabbits subjected to either long lighting regime (8 hrs D/16 hrs L) or regime of equal dark and light
Results and Discussion - 109 -
period (12 hrs D /12 hrs L).Serum progesterone level in rabbits subjected to long lighting regime increased at 1 hr after injection then decreased thereafter up to the 4th hr after injection, after that serum progesterone level increased at the 8th hr, then decreased sharply at the 16 hrs. However, serum progesterone level slightly and almost increased up to the end of the experimental period (Table 9). Obtained results agreed with the scientific facts concerning the relationship between bromocriptine biological action as dopamine agonist from one side and the biological action of dopamine as a primary neuroendocrine inhibitor of secretion of prolactin from the pituitary gland (Browman et al., 2005). It is well known that bromocriptine is an ergoline derivative, which act as dopamine agonist used in the treatment of hyperprolactinaemia (Boyed, 1995). Hyperprolactinaemia may stimulate hypothalamus negative feedback effects on gonadal steroids (McNeilly, 1980). It is suggested that bromocriptine exerts its effect through the hypothalamic- hypophyseal-ovarian axis, which influence the processes of ovarian follicle formation and ovulation. This is tern affect the number of corpora lutea and correspondingly affect the serum progesterone level. Rabbit, which is induced ovulating animal, has a complex noradrenergic coital stimulus which is interrelated to gonadotropic releasing hormones (Kaynard et al., 1990). Obtained results go in agreement with those of Yoshimura et al., (1990) who stated that prolactin in rabbits was found to
Results and Discussion - 110 -
interfere directly with mechanical events within the ovary, thus preventing rupture of mature follicles via inactivation of plasminogen activator system. On the other hand inhibition of prolactin via bromocriptine injection in rabbits leads to stimulate ovulatory process ending with corpora lutea formation and progesterone secretion. Therefore, noradrenergic and dopaminergic neurons interrelated in hypothalamus is expected as a dopamine agonist (bromocriptine), which might result in enhancing the HCG – ovulation – induced rise in progesterone. This effect might have resulted from lifting the prolactin inhibitory effect on ovarian function (McNeilly et al., 1982).
Inspection of data revealed that, effect of bromocriptine injection on progesterone levels increased during long photoperiod (8D/16L) compared to short photoperiod (16D/8L), where neuropeptide Y was found to stimulate GnRH release, which in turn is mediated via α1- adrenergic receptors. These receptors could be stimulated by increased levels of dopamine resulting from bromocriptine injection (Berria et al., 1991). Therefore, priming rabbit with dopamine further potentiates the effect of LH releasing hormone as manifested with higher progesterone levels (Yoshimura et al., 1990).
Results and Discussion - 111 -
Table (9): Progesterone serum levels (ng/ml) x ±SE in response to injection of bromocriptine vs saline at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time Saline Bromocriptine after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.3518±0.0448 0.5169±0.1248 0.8723±0.1633 0.1665±0.0001 0.1665±0.0001 0.1672±0.0007
1hr 0.6303±0.1729 0.3871±0.0445 0.7767±0.2260 0.3444±0.1775 0.1704±0.0039 0.1665±0.0001
2hr 0.6034±0.0897 1.4128±0.9812 1.2750±0.4254 0.2058±0.0655 0.1712±0.0048 0.1665±0.0001
4hr 0.5303±0.0887 0.5035±0.2030 0.6457±0.0937 0.1950±0.0689 0.1928±0.0263 0.1665±0.0001
8hr 0.4546±0.1422 0.2775±0.0173 0.5748±0.1137 0.3493±0.2268 0.1665±0.0001 0.1665±0.0001
16hr 0.2927±0.0323 0.4057±0.103 0.6742±0.2112 0.1332±0.0333 0.2183±0.0518 0.1665±0.0001
Results and Discussion - 112 -
Table (10): Analysis of variance of data (cumulative area under curve in units) collected after injection of bromocriptine vs saline under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 17.07983412 8.53992 4.3383*
Injection 1 223.429331 233.4293 118.5829**
Interaction 2 279.115395 139.5577 70.89578**
Residual 28 55.1177544 1.968491071
Total 33 574.0948941 17.39681515
*Significant at level of 0.05 **Significant at level of 0.01
Results and Discussion - 113 -
Figure (10) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at short darkness photoperiod (8D/16L).
Figure (11) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at equal dark / light photoperiod (12D/12L).
Figure (12) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 114 -
Histogram (10) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at short darkness photoperiod (8D/16L).
Histogram (11) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at equal dark / light photoperiod (12D/12L).
Histogram (12) Serum progesterone level (ng/ml) in response to administration of bromocriptine vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 115 -
4.1.6. Effect of indomethacin:
Data tabulated in Table (11) represented in Figures (13, 14 and 15) and Histograms (13, 14 and 15) show that means of serum progesterone levels in experimental rabbits subjected to different lighting regimes applied and treated with indomethacin (indometacin) drug that antagonist prostaglandin through its effect to inhibit its production. Analysis of variance for data obtained is listed in ANOVA Table (12). Detecting obtained data related to the previously mentioned concern revealed insignificant variation in serum progesterone level in animals injected with indomethacin due to photoperiod applied. This could be considered as a logic result since photoperiod may affect surge of gonadotrophic hormone (especially LH hormone) rather than the level of progesterone in blood serum which is considered as a function of number of ovulated follicles and correspondingly number of corpora lutea succeeded to be developed of the ovulation and persistency for biosynthesizing and secreting progesterone hormone along the pregnancy period.
On the other hand, significant variation (P<0.05) was observed in serum progesterone level due to injecting indomethacin. Rabbits treated with indomethacin had almost higher level of progesterone along the time passed after
Results and Discussion - 116 -
injection. However, the rate of increment differed according to lighting regimes applied and estimation period. This was insured by the highly significant interaction effect between photoperiod and injection (P<0.01).
The previously mentioned results go in a harmony with many of scientific statement .It is well known now that indomethacin (or indometacin) is non-steroid drug which is non selective inhibitor of cyclooxygenase (COX) 1 and 2, enzymes, that participate in prostaglandin synthesis from arachidonic acid. So indomethacin is an effective tocolytic agent, able to delay premature labor by reducing uterine contractions through inhibition of PG synthesis in the uterus and possibly through calcium channel blockade. ( Giles and Bisits, 2007). In addition, treating pregnant does with indomethacin and or any of tocolytic medication may result in increasing the litter surge. On the other hand injection of indomethacin prior to LH inhibited the ovulatory process in the rabbit (Grinwich et al., 1972; O’Grady et al., 1972a) and indomethacin inhibited coitus induced ovulation in the rabbit (O’Grady et al., 1972a).
Results and Discussion - 117 -
Table (11): Progesterone serum levels (ng/ml) x ±SE in response to injection of indomethacin vs saline at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time Saline Indomethacin after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.3518±0.0448 0.5169±0.1248 0.8723±0.1633 0.3869±0.2407 0.5996±0.2647 0.0860±0.032
1hr 0.6303±0.1729 0.3871±0.0445 0.7767±0.2260 0.7817±0.2021 0.5606±0.4232 0.2430±0.1337
2hr 0.6034±0.0897 1.4128±0.9812 1.2750±0.4254 0.4369±0.0945 0.2506±0.1109 0.1477±0.0531
4hr 0.5303±0.0887 0.5035±0.2030 0.6457±0.0937 1.0858±0.6363 0.2898±0.1499 0.5623±0.2919
8hr 0.4546±0.1422 0.2775±0.0173 0.5748±0.1137 0.4270±0.1301 0.5804±0.1614 0.2974±0.2030
16hr 0.2927±0.0323 0.4057±0.1030 0.6742±0.2112 0.2390±0.0409 0.1367±0.0136 0.1441±0.0790
Results and Discussion - 118 -
Table (12): Analysis of variance of data (cumulative area under curve in units) collected after injection of indomethacin versus saline under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 14.087074 7.043535 1.9693
Injection 1 16.823256 16.82326 4.7036*
Interaction 2 330.8706334 165.4353 46.254**
Residual 23 82.263366 3.576668
Total 28 444.0433 14.0687
*Significant at level of 0.05 **Significant at level of 0.01
Results and Discussion - 119 -
Figure (13) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at short darkness photoperiod (8D/16L).
Figure (14) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at equal dark / light photoperiod (12D/12L).
Figure (15) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 120 -
Histogram (13) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at short darkness photoperiod (8D/16L).
Histogram (14) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at equal dark / light photoperiod (12D/12L).
Histogram (15) Serum progesterone level (ng/ml) in response to administration of indomethacin vs saline at long darkness photoperiod (16D/8L).
Results and Discussion - 121 -
4.2. Second experiment:
The second experiment was planned and conducted to point out the magnitude of improving the hormonal pattern in does subjected to different lighting regimes by treating animals with HCG alone or in combination with pharmaceutical preparations (melatonin, serotonin, bromocriptine, indomethacin) that are thought to have considerable effect on efficiency of biosynthesize and secretion of progesterone along a period of 16 hrs after injection.
4.2.1. Serum progesterone level in does treated with HCG:
Data presented in Table (13) represented in Figures (16, 17 and 18) and Histograms (16, 17 and 18) show the level of serum progesterone level in does subjected to different lighting regimes and treated with HCG ( subcutaneous Injection). From data obtained it is obviously clear that serum progesterone level increased as time after injection passed up to 2 or 4 hrs after injection then decreased sharply thereafter to reach its minimum level after 16 hrs. However, the rate of increase or decrease differed according to the lighting regimes applied. The higher rate of increase occurred during the first 4 hrs after injection was found in does subjected to short lighting regime (16 hrs D/8 hrs L). It increased from (0.18665 ng/ml) at pre injection (0 hr) to (13.0508 ng/ml) at the 2nd hr and to (14.0844 ng/ml) at the 4th hr after injection .Similar trend of
Results and Discussion - 122 -
increase, but with lower rate was found in does subjected to long lighting regime (8 hrs D/16 hrs L). Serum progesterone level in does of this group increased from (0.0973 ng/ml) at pre injection time (0hr) to (6.9526 ng/ml) at the 4th hr after injection quite different pattern of change in serum progesterone level was observed in does subjected to a lighting regime characterized by an equal light and dark periods (12 hrs D/12 hrs L). In this case, lower rate of increase was observed after 2hrs from injection time serum progesterone level decreased from (0.2005 ng/ml) before injection time (0hr) to (3.2797 ng/ml) after 2hrs from injection then decreased sharply up to the 16th hr.
From previously mentioned result, it could be concluded that progesterone biosynthesis and/or surge is directly affected by lighting regime. It seems that short lighting regime (16hrs darkness and 8hrs lighting) is the most convenient lighting regime for progesterone biosynthesis and/or surge by corpus luteum. In addition, results obtained go in harmony with various statements concerning the biological function of HCG. It worth to mention that HCG interacts with LHCG receptor and promotes the maintenance of corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone (Cole, 2009). In addition it was suggested that HCG may be a link in the development of peritrophoblastic immune tolerance, and may facilitate the
Results and Discussion - 123 -
trophoblast invasion, which is known to expedite embryonic development in the indomethacin (Kayisly et al., 2003).Because of its similarity LH, HCG can also be used clinically to induce ovulation in the ovaries (www.IVF.com).
4.2.2. Serum progesterone level in does treated with HCG in combination with melatonin:
Data concerning the level of serum progesterone in does subjected to applied lighting regimes and injected with HCG in combination with melatonin are tabulated in Table (13) represented in Figures (16, 17 and 18) and Histograms (16, 17 and 18). Analysis of variance for obtained data is shown in Table (14).
From data obtained it could be reported that injecting does with HCG in combination with melatonin decreased the level of progesterone in blood serum when compared with injecting animals with HCG only. The rate of decrement differed according to the lighting regime applied. The higher decreasing rate occurred when applying short lighting regime (16 hrs D / 8 hrs L) followed by applying lighting regime of equal light-dark period (12 hrs L / 12 hrs D). However, less rate of decrease was observed when applying long lighting regime (8hrs D / 16 hrs L). On the other hand serum progesterone level increased as time of injecting HCG in combination with melatonin passed reaching its maximum level at the 8th hr in short lighting regime (16 hrs D / 8 hrs L) and long
Results and Discussion - 124 -
lighting regime (8 hrs D / 16 hrs L) after injection .This was quite true in all lighting regimes applied but with great variation within them. The higher increasing rate was observed in does of long lighting period (8 hrs D/16 hrs L). In this case serum progesterone level increased from (1.2092 ng/ml) at 0hr to (7.4325 ng/ml) at the 8th hr after injection. The rate of increase was very low when applying short lighting regime. It increases from (0.1334 ng/ml) at 0hr to (0.4351 ng/ml) at the 8th hr after injection. However, the higher serum progesterone level was observed at the 4th hr after injecting does subjected to lighting regime of equal Light /Dark period (12 hrs D/12 hrs L). Analysis of variance for obtained data (Table 14) shows significant variation in serum progesterone level due to lighting regime applied only (P < 0.05). Results obtained were scientifically logic and agree with different statements related to the biological action of either HCG or melatonin. It is well known that melatonin lowers FSH levels but it improves the thyroid function (Bellipanni et al., 2005).
Results and Discussion - 125 -
Table (13): Progesterone serum levels (ng/ml) x ±SE in response to injection of melatonin+HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time HCG Melatonin+HCG after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.0973±0.0516 0.2005±0.0821 0.18665±0.0371 1.2092±0.2311 0.0680±0.0492 0.1334±0.0950
1hr 0.1925±0.1273 1.2733±0.2450 0.2626±0.0610 6.1484±1.9946 0.1515±0.1327 0.0354±0.0083
2hr 5.3860±5.0900 3.2797±0.9767 13.0508±3.3939 6.7445±3.2104 0.4652±0.3526 0.0356±0.0102
4hr 6.9526±5.3762 0.8559±0.4248 14.0844±3.4214 4.3127±1.8072 2.0006±1.5144 0.0662±0.0242
8hr 1.3982±0.9838 0.25727±0.0884 6.0789±1.1813 7.4325±2.8376 0.5933±0.3604 0.4351±0.2040
16hr 1.1544±0.62064 0.1384±0.1768 1.443±0.3942 3.4128±0.3831 0.0757±0.0458 0.0447±0.0447
Results and Discussion - 126 -
Table (14): Analysis of variance of data (cumulative area under curve in units) collected after injection of melatonin+HCG versus HCG under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 13879.29693 6939.65 4.5709*
Injection 1 1215.96854 1215.969 0.80092
Interaction 2 10133.342 5066.67 3.3373
Residual 14 21255.0175 1518.2157
Total 19 46483.62497 2446.5063
*Significant at level of 0.05
Results and Discussion - 127 -
Figure (16) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at short darkness photoperiod (8D/16L).
Figure (17) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at equal dark / light photoperiod (12D/12L).
Figure (18) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 128 -
Histogram (16) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at short darkness photoperiod (8D/16L).
Histogram (17) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at equal dark / light photoperiod (12D/12L).
Histogram (18) Serum progesterone level (ng/ml) in response to administration of melatonin+HCG vs HCG at long photoperiod (16D/8L).
Results and Discussion - 129 -
4.2.3. Serum progesterone level in does treated with HCG in combination with serotonin:
Data concerning the level of serum progesterone level in does subjected to different lighting regimes and treated with HCG in combination with serotonin are tabulated in Table (15) represented in Figures (19, 20 and 21) and Histograms (19, 20 and 21). Analysis of variance for this data is presented in Table (16).
Obtained data revealed that injecting experimental does with HCG in combination with serotonin resulted into decreasing serum progesterone level when compared by those injected with HCG alone. However, the rate of decrease differed within the various lightening regime applied. Higher serum progesterone levels (0.1983 ng/ml) were observed at the 4th hr after injection in does subjected to long lighting regime (8 hrs D/16 hrs L) and to (1.8238 ng/ml) in does subjected to equal lighting regime (12hrs D/12 hrs L). However, higher progesterone levels in blood serum was observed at the 8th hr after injection for does subjected to short lighting regime (16hrs D / 8hrs L) that mounted (2.5732 ng/ml). However, no characterized pattern was found in variation of serum progesterone level as time after injection passed.
The decrease in serum progesterone level due to treatment with HCG in combination with serotonin may attribute to the effect of serotonin, which acts as growth factor directly. In addition,
Results and Discussion - 130 -
serotonin was found to suppress insulin release from beta cells in the pancreas (Paulmann et al., 2009). Correspondingly affect the energy supply needed to reproductive activity in general and development of corpora lutea .This may be reflected as decrease in serum progesterone level.
Results and Discussion - 131 -
Table (15): Progesterone serum levels (ng/ml) x ±SE in response to injection of serotonin+HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time HCG Serotonin +HCG after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.0973±0.0516 0.2005±0.0821 0.18665±0.0371 0.1599±0.0686 0.2270±0.1357 0.3116±0.0812
1hr 0.1925±0.1273 1.2733±0.2450 0.2626±0.0610 0.1284±0.0263 0.1439±0.0526 0.3111±0.1970
2hr 5.3860±5.0900 3.2797±0.9767 13.0508±3.3939 0.1180±0.0120 1.1678±0.8608 0.3903±0.2827
4hr 6.9526±5.3762 0.8559±0.4248 14.0844±3.4214 0.1983±0.1070 1.8238±0.8977 0.7255±0.4929
8hr 1.3982±0.9838 0.25727±0.0884 6.0789±1.1813 0.1652±0.0547 0.4613±0.1910 2.5732±2.2926
16hr 1.1544±0.62064 0.1384±0.1768 1.443±0.3942 0.1606±0.0346 0.2016±0.1103 0.6513±0.4200
Results and Discussion - 132 -
Table (16): Analysis of variance of data (cumulative area under curve in units) collected after injection of serotonin +HCG versus HCG under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 12969.05293 6484.525 16.3403**
Injection 1 8457.804431 8457.804 21.3127**
Interaction 2 11713.83551 5856.92 14.7588**
Residual 14 5555.809439 396.8435
Total 19 38696.50231 2036.6579
**Significant at level of 0.01
Results and Discussion - 133 -
Figure (19) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at short darkness photoperiod (8D/16L).
Figure (20) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at equal dark / light photoperiod (12D/12L).
Figure (21) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 134 -
Histogram (19) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at short darkness photoperiod (8D/16L).
Histogram (20) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at equal dark / light photoperiod (12D/12L).
Histogram (21) Serum progesterone level (ng/ml) in response to administration of serotonin+HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 135 -
4.2.4. Serum progesterone level in does treated with HCG in combination with bromocriptine:
Table (17) represented in Figures (22, 23 and 24) and Histograms (22, 23 and 24) shows averages of serum progesterone level in experimental does exposed to different lighting programs and treated with either HCG alone or in combination with bromocriptine (Dopamine agonist drug that blocks D2 receptor in adenohypophysis to inhibit prolactin release).
Results obtained revealed that injecting HCG in combination with bromocriptine mostly increase the level of serum progesterone than did injecting HCG alone. This was quite true in does of all lighting regimes applied but with different increment magnitudes. The higher increment rate was obviously found in does subjected to short lighting program (16hrs D/ 8hrs L). When compared with those subjected to other two lighting regimes applied (8hrs D/16hrs L) or (12 hrs D /12 hrs L). Approximately similar trend of variation in serum level of progesterone was observed in does of these two lightening regimes mentioned.
Serum progesterone level in does injected with HCG in combination with bromocriptine increased as time after injection passed. It reached its highest level at the 3rd hr after injection. At this time average of serum progesterone level weighed (3.12616 ng/ml , 3.5317 ng/ml and 28.7236 ng/ml) in does subjected to
Results and Discussion - 136 -
lighting program of (8 hrs D/16 hrs L , 12 hrs D/12 hrs L and 16 hrs D/8 hrs L), respectively (Table 17). Analysis of variance for data obtained is listed in ANOVA Table (18).
It could be concluded that injecting does with HCG in combination with bromocriptine becomes more efficient in increasing the level of serum progesterone. This may be due to the inhibiting action of bromocriptine on prolactin that may stimulate LH surge, which has positive effect on maintenance of corpora lutea, and correspondingly increase progesterone secretion. It may be also suggested that adding bromocriptine to HCG improve either the rate of follicular formation and development by diminishing the antagonist action of prolactin against gonadotrophic hormones in general and luteinizing hormone in particular. This LH surge triggers ovulation thereby not only release the egg, but also initiating the conversion of residual follicle into a corpus luteum that, in turn, produces progesterone to prepare indomethacin for a possible implantation. In addition, LH is necessary to maintain luteal function along the gestation period. Obtained results go in agreement with those of Lafond et al., (1986), who reported that inhibition of prolactin via bromocriptine treatment potentiates the HCG stimulatory effect. Furthermore, bromocriptine (a dopamine agonist) administration elicits a dopamine rise in blood levels of the neurotransmitter. High dopamine levels inhibits prolactin secretion
Results and Discussion - 137 -
(buys et al., 1990), whereas elevated concentrations of blood prolactin were found to directly inhibit ovarian functions and decrease progesterone levels (McNeilly et al., 1982). It could be concluded that inhibition of prolactin via bromocriptine treatment would further potentiates the effect of HCG in ovulation induction and subsequent corpus luteum function.
Results and Discussion - 138 -
Table (17): Progesterone serum levels (ng/ml) x ±SE in response to injection of bromocriptine +HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
HCG Bromocriptine +HCG Time after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.0973±0.0516 0.2005±0.0821 0.18665±0.0371 1.5770±0.3026 1.3418±0.4245 3.1927±0.5687
1hr 0.1925±0.1273 1.2733±0.2450 0.2626±0.0610 1.4760±0.2359 1.6364±0.2426 7.6859±2.4423
2hr 5.3860±5.0900 3.2797±0.9767 13.0508±3.3939 1.5848±0.5753 1.7510±0.4316 18.8177±6.1949
4hr 6.9526±5.3762 0.8559±0.4248 14.0844±3.4214 3.1262±0.5344 3.5317±0.6712 28.7236±9.2569
8hr 1.3982±0.9838 0.25727±0.0884 6.0789±1.1813 1.9163±0.6560 6.2311±1.3188 13.1143±1.7458
16hr 1.1544±0.62064 0.1384±0.1768 1.443±0.3942 1.4406±0.5183 4.7947±2.1990 6.8879±1.7410
Results and Discussion - 139 -
Table (18): Analysis of variance of data (cumulative area under curve in units) collected after injection of bromocriptine +HCG versus HCG under different photoperiods.
S.O.V d.f S.S M.S F
Photoperiod 2 119607.4 59803.7 2455.5705**
Injection 1 48359.28 48359.28 1985.6567**
Interaction 2 44374.86 22187.43 911.0272**
Residual 17 414.0231 24.3543
Total 22 212655.499 9670.7045
**Significant at level of 0.01
Results and Discussion - 140 -
Figure (22) Serum progesterone level (ng/ml) in response to administration of bromocriptin+HCG vs HCG at short darkness photoperiod (8D/16L).
Figure (23) Serum progesterone level (ng/ml) in response to administration of bromocriptin+HCG vs HCG at equal dark / light photoperiod (12D/12L).
Figure (24) Serum progesterone level (ng/ml) in response to administration of bromocriptin+HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 141 -
Histogram (22) Serum progesterone level (ng/ml) in response to administration of bromocriptine+HCG vs HCG at short darkness photoperiod (8D/16L).
Histogram (23) Serum progesterone level (ng/ml) in response to administration of bromocriptine+HCG vs HCG at equal dark / light photoperiod (12D/12L) .
Histogram (24) Serum progesterone level (ng/ml) in response to administration of bromocriptine +HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 142 -
4.2.5. Serum progesterone level in does treated with HCG in combination with indomethacin:
Averages of serum progesterone level in does exposed to three lighting programs and treated with either HCG only or in combination with indomethacin are tabulated in Table (19) represented in Figures (25 , 26 and 27) and Histograms (25, 26 and 27). Indomethacin was applied as medication inhibit production of prostaglandins and therefore inhibit uterus contraction .Inspecting the obtained data, it was found that injection HCG in combination with indomethacin increased the level of serum progesterone level when compared with injection HCG alone. However, the rate of increase differed according lighting regime applied the greater increment rate was found in does subjected to long darkness photoperiod of (16 hrs D and 8 hrs L). Does of long lighting period regime (8 hrs D / 16 hrs L) shows lowest rate of serum progesterone level. While does of an equal dark and light regime (12 hrs D /12 hrs L) shows intermediate serum progesterone level values.
The level of serum progesterone increased during the early period of the injection then decreased there after reaching its lowest level at the 16th hr after injection. The highest serum progesterone level was found at the 4th hr after injecting does subjected to short lighting regime (16 hrs D/ 8 hrs L), it
Results and Discussion - 143 -
mounted (201.6806 ng/ml) However, the highest values were found at the second hour after injecting does subjected to equal lighting regime (12 hrs D/ 12 hrs L) (35.1933 ng/ml) and at the 16th hr after injecting does subjected to long lighting regime (8 hrs D/ 16 hrs L) (30.5246 ng/ml). Analysis of variance of obtained data showed highly significant variation in average serum progesterone level due to lighting regime and injection (P<0.01) (ANOVA Table 20). Increasing the average of serum progesterone level due to injecting does with HCG in combination with indomethacin may be attributed to cumulative biological function of HCG and indomethacin. HCG, which interacts with LHCG receptor and promotes the maintenance of corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone (Kayisly et al., 2003). In addition, indomethacin works by inhibiting the production of prostaglandin and inhibit the uterine contraction (Ferreia et al., 1971).
Results and Discussion - 144 -
Table (19): Progesterone serum levels (ng/ml) x ±SE in response to injection of indomethacin +HCG vs HCG at short darkness photoperiod (8D/16L), equal dark and light photoperiod (12D/12L) and long darkness photoperiod (16D/8L).
Time HCG Indomethacin +HCG after injection 8D/16L 12D/12L 16D/8L 8D/16L 12D/12L 16D/8L
0hr 0.0973±0.0516 0.2005±0.0821 0.1867±0.0371 1.5653±0.1089 8.2714±1.1120 1.5488±0.2956
1hr 0.1925±0.1273 1.2733±0.2450 0.2626±0.0610 27.6202±14.6004 9.1089±2.5677 21.5399±5.4167
2hr 5.3860±5.0900 3.2797±0.9767 13.0508±3.3939 15.1631±0.8003 35.1933±13.3733 70.7313±17.0880
4hr 6.9526±5.3762 0.8559±0.4248 14.0844±3.4214 12.8135±2.7953 30.7422±7.0919 201.6806±46.5550
8hr 1.3982±0.9838 0.25727±0.0884 6.0789±1.1813 19.4920±2.4440 23.1776±3.9866 86.9393±20.5270
16hr 1.1544±0.62064 0.1384±0.1768 1.443±0.3942 30.5246±10.6894 16.9155±1.7647 46.1649±9.8367
Results and Discussion - 145 -
Table (20): Analysis of variance of data (cumulative area under curve in units) collected after injection of indomethacin+HCG versus HCG under different photoperiods.
S.O.V d.f S.S M.S F
2 1186364.181 Photoperiod 593182 7.22**
Injection 1 2469703.965 2469704 30.05**
Interaction 2 113514.2532 56757.1266 0.6905347
Residual 13 1068509.133 82193.01023
Total 18 4838091.532 268782.89
**Significant at level of 0.01
Results and Discussion - 146 -
Figure (25) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at short darkness photoperiod (8D/16L).
Figure (26) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at equal dark / light photoperiod (12D/12L).
Figure (27) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 147 -
Histogram (25) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at short darkness photoperiod (8D/16L).
Histogram (26) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at equal dark / light photoperiod (12D/12L).
Histogram (27) Serum progesterone level (ng/ml) in response to administration of indomethacin +HCG vs HCG at long darkness photoperiod (16D/8L).
Results and Discussion - 148 -
5. CONCLUSION
From the previously mentioned result it could be concluded that reproductive efficiency in rabbits is mainly dependant on a very close relationship to not only the hypothalamic, hypophyseal and gonadal hormonal integration, but also is affected by neural and neurohormonal coordination. This may result in optimizing various control systems regulating the reproductive activity. In addition the photoperiodicity plays an important role in modifying these effects.
Conclusion - 149 -
6. SUMMARY
The present work was carried out at rabbit colony established in Endocrinology Research Unit, Applied Radiobiology Department, Nuclear Research Center, Atomic Energy Authority, Inshas, Egypt. The hormonal radioimmunological assays were performed in the laboratories of the same Research Unit. It was planned to point out the effect of treating adult does subjected to three regimes of photoperiodicity )8 hrs D/16 hrs L, 12 hrs D/12 hrs L and 16 hrs D/8 hrs L(, with different hormonal treatments including HCG and pharmaceutical preparations including Melatonin, Serotonin, Bromocriptine (anti prolactin), Indomethacin (anti prostaglandins) alone or in combined with HCG. A total number of 300 young adult female New Zealand white does aged 3-4 months and weighed 2-3 Kg were individually caged and kept in an environmental chamber under controlled ambient temperature of 250C, isolated from outdoors day light and well ventilated This work was accomplished in two separate experiments spaced with two weeks rest to adapt the animals for the photoperiod regime, does subjected to study were kept under these condition for pre-experimental period leaved two weeks then divided into three groups (photoperiods) each of 100 does.
Summary - 150 -
The first experiment: was planned and conducted to detect the possibility for improving the reproductive performance of does by magnifying the hormonal coordination related to productive activity with relation to different lighting regimes tested, to fulfill this aim applying human chorionic gonadotropin as well as other chemical compounds that thought to have considerable effect on hormonal regulation of reproduction (Melatonin, Serotonin, Bromocriptine (anti prolactin) and indomethacin (anti prostaglandin)). The second experiment: Was planned and conducted to point out the magnitude of improving the hormonal pattern in does subjected to different lighting regimes by treating animals with HCG alone or in combination with pharmaceutical preparations in double dose 2hrs separated that are thought to have considerable effect on efficiency of biosynthesize and secretion of progesterone along a period of 16 hrs after injection.
Results could be summarized as follows: The first experiment 1-Serum progesterone level in untreated does subjected to different lighting regimes applied: Averages of progesterone level were almost higher in does exposed to short lighting regime (16 hrs Darkness (D) / 8 hrs Light (L)) when compared to others. On the other hands, the higher levels of serum progesterone were detected after two hours .This was quite
Summary - 151 -
true in all lighting regimes applied. The higher levels of serum progesterone found along the experimental period for does exposed to short lighting regime (16 hrs D and 8 hrs L), may lead to conclude that surge of LH hormone needs at least 8 hrs lighting to show its biological effect of ovulation in the animals of induced ovulation pattern, like rabbits. Thus the photoperiod regime show its significant effect on ovulation mechanism in accordance with the time of ovulation which occurred at approximately at the same time in all experimental animals. 2- Effect of HCG: Data obtained revealed an increase in serum progesterone levels as a result of injecting does with HCG, The rate of increment differed obviously according to the lighting regimes applied. Does subjected to long lighting regime (8 hrs darkness and 16 hrs lighting) resulted in gradual increase in serum progesterone level reaching its lightest level at the 4th hr after injection then decreased up to the end of experimental period. Subjected does to short lighting regime (16 hrs darkness (D) / 8 hrs light (L)) increased serum progesterone level with greater rate up to 4th hr after injection reaching highest level at the 2nd hr and at 4th hr then decreased steadily to reach lowest level at the 16th hr after injection. The same trend was found in does subjected to equal lighting regime (12 hrs D / 12 hrs L) but with lowest magnitude serum progesterone level increased after injection to reach its highest level after 2 hrs of injection then decreased
Summary - 152 -
sharply and steadily towards the 16th hr after injection to reach the lowest level at this time. Analysis of variance for obtained data revealed significant variation in serum progesterone level due to lighting regime , injecting material and the interaction between them (P < 0.01) 3-Effect of melatonin: Treating experimental does with melatonin increased serum progesterone level at all intervals of estimation. However, the rate of increase varied according to either lighting regime applied or interval of estimation after injection. The highest progesterone level was estimated after 1 hr of injecting does subjected to long lighting regime (8 hrs D/16 hrs L) which, sharply decrease at the second hr after injection and still decrease up to 16th hr after injection.
Different pattern of change along the whole intervals of estimation was found in does subjected to other two lighting periods. Serum progesterone level increased in does subjected to equal lighting regime (12 hrs D /12 hrs L) to reach its highest value at two hours after injection then steadily decrease till 16 hrs after injection. Same trend of variation along all times of estimation was found in does subjected to short lighting regime (16 hrs D /8 hrs L) but with lower magnitude when compared to the other photo regimes applied. However, analysis of variance for obtained data revealed insignificant variation in serum progesterone level due to either lighting regime, injection or the interaction between them.
Summary - 153 -
4-Effect of serotonin: Injecting serotonin lowered the level of progesterone level in does subjected to all lighting regimes applied. However, the rate of this decrement differed according the ratio between the periods of darkness to that of lighting. It was found that the decreasing rate of serum progesterone level increased as period of darkness increased. However, the lowest averages of serum progesterone level were observed in does subjected to short lighting regime (16 hrs D /8 hrs L). This trend of variation in serum progesterone level was true at all estimation intervals. Analysis of variance of obtained data showed highly significant variation in average serum progesterone level due to lighting regime, injecting material and interaction between them (P<0.01). 5-Effect of bromocriptine: Highly significant effect on serum progesterone level was found due to injecting material applied and the interaction between lighting regime and injection (P<0.01). While lighting regime showed significant effect (P<0.05). Injecting bromocriptine lowered the level of serum progesterone. However, the rate of decrease differed according to lighting regime and the time intervals after injection. The greatest decrease level was observed when subjected does to the lighting regime of (16 hrs D /8 hrs L) while, the lowest rate of decrease was found in the group of animals subjected to long lighting regime (8 hrs D /16 hrs L). Serum progesterone level in rabbits subjected to short lighting regime (16 hrs D /8 hrs L) decreased after injecting bromocriptine and remained approximately constant along the whole
Summary - 154 -
experimental period with value mentioned. Different pattern of variation was found in rabbits subjected to either long lighting regime and regime of equal dark and light period (12 hrs D /12 hrs L). Serum progesterone level in rabbits subjected to long lighting regime increased at 1 hr after injection then decreased thereafter up to the 4th hr after injection. However, serum progesterone level increased at the 8th hr after injection and then decreased sharply at the 16th hr. Serum progesterone level slightly and almost increased up to the end of the experimental period. 6-Effect of indomethacin: Insignificant variation in serum progesterone level in animals injected with indomethacin due to photoperiod applied. On the other hand, significant variation (P<0.05) was observed in serum progesterone level due to injecting indomethacin. Rabbits treated with indomethacin had almost higher level of progesterone along the time passed after injection. However the rate of increment differed according to lighting regimes applied and estimation period. This was insured by the highly significant interaction effect between photoperiod and injection (P<0.01). The second experiment 1-Serum progesterone level in does treated with HCG: As mentioned before treated does with HCG increase serum progesterone level to reach its highest level at 4 hr after injection. Subjected does to short lighting regime (16 hrs D / 8hrs L) increased serum progesterone level when compared with does subjected to long and equal
Summary - 155 -
light / dark lighting regimes. Subjected does to long lighting regime (8 hrs D and 16 hrs L) resulted in gradual increase in serum progesterone level reaching its highest level at the fourth hour after injection then decreased up to the end of experimental period. The same trend was found in does subjected to (12 hrs D / 12 hrs L) but with lowest magnitude. Analysis of variance for obtained data revealed highly significant variation in serum progesterone level due to lighting regime , injecting material and the interaction between them (P<0.01) . 2-Serum progesterone level in does treated with HCG in combination with melatonin: Injecting does with HCG in combination with melatonin decreased the level of progesterone in blood serum when compared with does injecting HCG only. The rate of decrement differed according to the lighting regimes applied. The higher decreasing rate occurred when subjected does to short lighting regime of (16 hrs D / 8 hrs L) followed by subjected does to lighting regime of equal lighting-darkness period (12 hrs L / 12 hrs D). However, less rate of decrease was observed when subjected does to long lighting regime of (8hrs D / 16 hrs L). On the other hand serum progesterone level increased as time of injection passed reaching its maximum level at the 8th hr after injection .This was quite true in long lightening regime and short lightening regime applied but with great variation within them. The higher increasing rate was observed in does subjected to long lighting period (8 hrs D/16 hrs L).
Summary - 156 -
3- Serum progesterone level in does treated with HCG in combination with serotonin: Injecting experimental does with HCG in combination with serotonin resulted into decreasing the level serum progesterone when compared by those injected with HCG alone. However, the rate of decrease differed within the various lightening regimes applied. Higher serum progesterone levels were observed at the 4th hr after injection in does subjected to long lighting regime (8 hrs D/16 hrs L) and to equal lighting regime (12hrs D/12 hrs L). However, higher progesterone levels in blood serum was observed at the 8th hr after injection for does subjected to short lighting program (16hrs D / 8hrs L). However, no characterized pattern was found in variation of serum progesterone level as time after injection passed. Highly significant effect on serum progesterone level was found due to injecting material applied, the interaction between lighting regime and injection and lighting regime (P<0.001). 4- Serum progesterone level in does treated with HCG in combination with bromocriptine: Injecting HCG in combination with bromocriptine mostly increase the level of serum progesterone than did injecting HCG alone. This was quite true in does subjected to all lighting regimes applied but with different increment magnitudes. The higher increment rate was obviously found in does subjected to short lighting program (16 hrs D/ 8 hrs L). When compared with those subjected to other two lighting regimes applied (8 hrs
Summary - 157 -
D/16 hrs L) or (12 hrs D /12 hrs L). Approximately similar trend of variation in serum level of progesterone was observed in does subjected to these two lightening regimes. Serum progesterone level in does injected with HCG in combination with bromocriptine increased as time after injection passed, it reached its highest level at the 4 hr after injection in does subjected to either long or short lighting regimes. It reached its highest level at the 4 th hr to 8 th hr after injection in does subjected to equal dark and light lighting regime (12 hrs dark / 12 hrs light). Analysis of variance of obtained data showed highly significant variation in average serum progesterone level due to lighting regime, injecting material and interaction between them (P<0.01). 5- Serum progesterone level in does treated with HCG in combination with indomethacin: Injecting HCG in combination with indomethacin increased the level of serum progesterone level when compared with injection HCG alone. However, the rate of increase differed according lighting regimes applied, the greater increment rate was found in does subjected to photoperiod of (16 hrs D and 8 hrs L). Does subjected to long lighting regime (8 hrs D/ 16 hrs L) shows lowest rate of serum progesterone level. While does subjected to an equal dark and light periods (12 hrs D /12 hrs L) shows intermediate serum progesterone level values. The level of serum progesterone increased during the 4th hr of the injection then decreased there after reaching its lowest level at the 16th hr after injection. The highest serum progesterone level was found at the 4th hr
Summary - 158 -
after injecting does subjected to short lighting regime (16 hrs D/ 8 hrs L) and equal dark and light lighting regime (12 hrs dark / 12 hrs light). However, serum progesterone level increase in does subjected to long lighting regime (8 hrs D /16 hrs L) to reach its highest value at 1hr after injection then decrease steadily to increase again at 16 hr after injection. Analysis of variance of obtained data showed highly significant variation in average serum progesterone level due to lighting regime and injecting material (P<0.01).
Summary - 159 -
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8. APPENDIX
Table (1): Mean values of area under curve (units) for each experimental animal in response to saline and HCG injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L Saline 6.12 4.17 9.81 6.33 5.02 10.28 5.68 4.62 19.15 11.16 4.47 9.54 7.08 7.63 9.80 6.84 6.03 9.89 0 12.02 0 0 17.22 0
Mean ±SE 5.401±1.32 7.65±1.65 8.56±2.19
11.39 HCG 7.46 67.18 16.2 6.19 104.08 98.97 11.93 85.48 0 18.25 160.98 31.64±22.69 Mean ±SE 10.9575±2.72 104.43±20.29
Appendix - 212 -
Table (2): Mean values of area under curve (units) for each experimental animal in response to saline and melatonin injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L Saline 6.12 4.17 9.81 6.33 5.02 10.28
5.68 4.62 19.15
11.16 4.47 9.54
7.08 7.63 9.8
6.84 6.03 9.89
0 12.02 0
0 17.22 0
Mean ±SE 5.40±1.322 7.65±1.65 8.56±2.19
Melatonin 112 15.95 16.46 87.89 12.51 10.02
101.5 13.85 10.02
0 22.87 6.94
Mean ±SE 75.35±25.59 16.29±2.30 10.86±2.00
Appendix - 213 -
Table (3) Mean values of area under curve (units) for each experimental animal in response to saline and serotonin injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L Saline 6.12 4.17 9.81 6.33 5.02 10.28
5.68 4.62 19.15
11.16 4.47 9.54
7.08 7.63 9.8
6.84 6.03 9.89
0 12.02 0
0 17.22 0 Mean ±SE 5.40±1.32 7.65±1.65 8.56±2.19
Serotonin 0.81 0.32 0.51 0.74 0.32 0.47
0.44 0.32 0.39
0.37 0.6 0.32
0.41 0 0
1.42 0 0
Mean ±SE 0.69±0.16 0.26±0.09 0.28±0.09
Appendix - 214 -
Table (4): Mean values of area under curve (units) for each experimental animal in response to saline and bromocriptine injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L Saline 6.12 4.17 9.81 6.33 5.02 10.28 5.68 4.62 19.15 11.16 4.47 9.54 7.08 7.63 9.8 6.84 6.03 9.89 0 12.02 0 0 17.22 0 Mean ±SE 5.401±1.32 7.65±1.65 8.56±2.19
Bromocriptine 3.21 2.83 2.83 2.14 4.02 2.83 2.83 2.83 2.83
9.34 2.83 2.83
3.9 0 2.83 Mean ±SE 4.28±1.29 2.50±0.67 2.83±0.0001
Appendix - 215 -
Table (5): Mean values of area under curve (units) for each experimental animal in response to saline and indomethacin injection under different photoperiods.
Photoperiod Injection 8D /16L 12D/12L 16D/8L
Saline 6.12 4.17 9.81
6.33 5.02 10.28
5.68 4.62 19.15
11.16 4.47 9.54
7.08 7.63 9.8
6.84 6.03 9.89
0 12.02 0
0 17.22 0 Mean ±SE 5.401±1.32 7.65±1.65 8.56±2.19
Indomethacin 6.06 5.33 3.82
14.4 10.1 1.13
6.81 6.34 8.98
Mean ±SE 9.09±2.66 7.26±1.45 4.64±2.30
Appendix - 216 -
Table (6): Mean values of area under curve (units) for each experimental animal in response to HCG and melatonin+HCG injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L
HCG 11.39 7.46 67.18
16.2 6.19 104.08
98.97 11.94 85.48
0 18.25 160.98
Mean ±SE 31.64±22.69 10.96±2.72 104.43±20.29
Melatonin+HCG 94.97 10.33 4.28
94.38 18.51 0.58
108.68 3.69 4.71
Mean ±SE 99.34±4.67 10.84±4.28 3.19±1.31
Appendix - 217 -
Table (7): Mean values of area under curve (units) for each experimental animal in response to HCG and serotonin+HCG injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L
HCG 11.39 7.46 67.18
16.2 6.19 104.08
98.97 11.94 85.48
0 18.25 160.98
Mean ±SE 31.64±22.69 10.96±2.72 104.43±20.29
Serotonin+HCG 2.72 8.027 2.84
3.51 17.48 6.31
2.91 9.23 56.14
Mean ±SE 3.05±0.24 11.58±2.97 21.76±17.22
Appendix - 218 -
Table (8): Mean values of area under curve (units) for each experimental animal in response to HCG and bromocriptine+HCG injection under different photoperiods.
Photoperiod
Injection 8D /16L 12D/12L 16D/8L 7.46 67.18 HCG 11.39
6.19 104.08 16.2
11.94 85.48 98.97 18.25 160.98 0 Mean ±SE 31.64±22.69 10.96±2.72 104.43±20.29
Bromocriptine+HCG 41.16 77.18 255.93
35.11 70.94 202.2
25.48 75.17 312.43
0 0 280.24
0 0 282.45
0 0 84.83
Mean ±SE 16.96±7.85 37.22±16.66 236.347±33.87
Appendix - 219 -
Table (9): Mean values of area under curve (units) for each experimental animal in response to HCG and indomethacin+HCG injection under different photoperiods.
Photoperiod
Injection 16L/8D 12L/12D 16D/8L
11.39 HCG 7.46 67.18
16.2 6.19 104.08
98.97 11.94 85.48
0 18.25 160.98
Mean ±SE 31.64±22.69 10.96±2.72 104.43±20.29
Indomethacin+HCG 395.65 436.73 1390.63
337.42 242.29 1684.44
0 458.5 1323.88
Mean ±SE 244.36±123.33 379.17±68.73 1466.32±110.75
Appendix - 220 -
Table (10): progesterone serum levels (ng/ml) in response to injection of saline at short darkness photoperiod (8D/16L) in does.
Saline
ANIM P4(ng /ml) µ± SE NO.
R40 R41 R42 R43 R44 R45
0hr 0.2602 0.2901 0.4361 0.5317 0.2602 0.3327 0.3518 ±0.0448
1hr 0.2602 1.3504 0.2602 0.705 0.3826 0.8239 0.6303 ±0.1729
2hr 0.5561 0.4265 0.6499 0.9964 0.3771 0.6148 0.6034 ±0.0897
4hr 0.7659 0.5317 0.542 0.3095 0.2602 0.7729 0.5303 ±0.0887
8hr 0.2602 0.2602 0.2602 1.1201 0.5668 0.2602 0.4546 ±0.1422
16hr 0.2602 0.2602 0.2602 0.2602 0.4547 0.261 0.2927 ±0.0323
Appendix - 221 -
Table (11): progesterone serum levels (ng/ml) response to injection of saline at neutral photoperiod (12D/12L) in does.
Saline
P4 (ng/ml) µ± SE ANIM
NO. R1 R2 R3 R4 R5 R6 R7 R8
0hr 0.2751 0.3664 0.5084 0.2602 0.2602 0.8333 0.3794 1.2525 0.5169 ±0.1248
1hr 0.2602 0.3009 0.5946 0.2602 0.344 0.3411 0.4902 0.5057 0.3871 ±0.0445
2hr 0.2602 0.2602 0.2602 0.2602 0.2602 0.2602 1.552 8.1897 1.4128 ±0.9812
4hr 0.2602 0.3085 0.2602 0.3617 0.2602 0.2602 1.9187 0.3989 0.5035 ±0.2030
8hr 0.2602 0.2602 0.2602 0.2602 0.2602 0.3989 0.2602 0.2602 0.2775 ±0.0173
16hr 0.2602 0.2602 0.2602 0.2602 1.1049 0.416 0.424 0.2602 0.4057 ±0.1030
Appendix - 222 -
Table (12) progesterone serum levels (ng/ml) response to injection of saline and at long darkness photoperiod (16D/8L) in does.
Saline
P4(ng/ml) µ± SE ANIM
NO. R77 R78 R79 R80 R81 R82
0hr 0.4182 1.0357 1.3421 1.2554 0.4461 0.7368 0.8723 ±0.1633
1hr 0.5143 0.6392 0.3147 0.7185 0.6028 1.8712 0.7767 ±0.226
2hr 0.5515 1.1074 1.0101 0.4802 3.3168 1.184 1.2750 ±0.4254
4hr 0.7944 0.5433 0.906 0.8333 0.345 0.4523 0.6457 ±0.0937
8hr 0.8059 0.5499 1.0132 0.3609 0.3181 0.4011 0.5748 ±0.1137
16hr 0.2602 0.6319 1.6606 0.7023 0.2602 0.5301 0.6742 ±0.2112
Appendix - 223 -
Table (13): progesterone serum levels ( ng /ml ) in response to injection of HCG at short darkness photoperiod (8D/16 L) in dose.
HCG
µ± SE ANIM NO. P4(ng/ml)
R63 R64 R65
0hr 0.1975 0.0694 0.0252 0.0973 ±0.0516
1hr 0.0838 0.0474 0.4463 0.1925 ±0.1273
2hr 0.1021 0.4928 15.5644 5.386 ±5.0900
4hr 0.1452 3.1484 17.5644 6.9526 ±5.3762
8hr 0.1743 0.6759 3.3446 1.3982 ±0.9838
16hr 2.3934 0.4697 0.6001 1.1544 ±0.6206
Appendix - 224 -
Table (14): progesterone serum levels (ng/ml) response to injection of HCG at neutral photoperiod (12D/12L) in does.
HCG
P4(ng/ml) µ± SE ANIM NO. R24 R25 R26 R27
0hr 0.1769 0.1296 0.4362 0.0596 0.2005 ±0.0821
1hr 1.6139 0.6316 1.6967 1.1513 1.2733 ±0.2450
2hr 2.1164 1.1477 4.6836 5.1712 3.2797 ±0.9767
4hr 0.4268 0.12 0.8213 2.0555 0.8559 ±0.4248
8hr 0.1622 0.4399 0.0593 0.3677 0.2573 ±0.0884
16hr 0.0819 0.1928 0.0446 0.2344 0.1384 ±0.1768
Appendix - 225 -
Table (15) Progesterone serum levels (ng/ml) in response to injection of HCG at long darkness photoperiod (16D/8L) in does.
HCG
P4(ng/ml) µ± SE
ANIM NO. R100 R101 R102 R103
0hr 0.0942 0.2322 0.2593 0.1609 0.1866 ±0.0371
1hr 0.3963 0.1721 0.1471 0.3351 0.2626 ±0.0610
2hr 7.4249 8.4143 14.1558 22.2083 13.0508 ±3.3939
4hr 6.41 16.7776 11.011 22.139 14.0844 ±3.4214
8hr 4.3224 6.2256 4.3996 9.3681 6.0789 ±1.1813
16hr 2.6097 0.8716 1.1335 1.1573 1.4430 ±0.3942
Appendix - 226 -
Table (16): progesterone serum levels (ng/ml) response to injection of melatonin at short darkness photoperiod (8D/16L) in does.
Melatonin
P4(ng/ml) µ± SE ANIM NO. R46 R47 R48
0hr 0.6473 0.7543 0.6469 0.6828±0.0357
1hr 12.6566 26.1891 4.7934 14.5463 ±6.2482
2hr 10.9599 5.7514 4.7182 7.1431 ±1.9315
4hr 9.9599 8.2047 5.3945 7.8530 ±1.3296
8hr 3.7471 2.4801 10.6365 5.6212 ±2.5341
16hr 3.2992 1.1223 0.6824 1.7013 ±0.8089
Appendix - 227 -
Table (17) progesterone serum levels (ng/ml) response to injection of melatonin at neutral photoperiod (12D/12L) in does.
Melatonin
µ± SE ANIM NO. P4 (ng/ml) R9 R10 R11 R12
0hr 0.7738 1.1336 0.4084 0.4084 0.6810 ±0.1737
1hr 0.6973 0.7815 1.2982 0.5488 0.8314 ±0.1628
2hr 0.4585 0.43 0.5456 5.6377 1.7679 ±1.2902
4hr 0.4084 0.5066 0.5233 1.902 0.8351 ±0.3565
8hr 1.7313 0.4084 0.6678 0.6893 0.8742 ±0.2927
16hr 0.6274 1.5295 0.465 0.8522 0.8685 ±0.2342
Appendix - 228 -
Table (18) progesterone serum levels (ng/ml) response to injection of melatonin at long darkness photoperiod (16D/8L) in does.
Melatonin
P4(ng/ml) µ± SE ANIM NO. R83 R84 R85 R86
0hr 0.6548 0.4084 0.4084 0.4084 0.4700 ±0.0616
1hr 1.727 0.438 0.438 0.4084 0.7528 ±0.324792
2hr 1.2886 0.8973 0.8973 0.4084 0.8729 ±0.180222
4hr 1.5918 0.7725 0.7725 0.4084 0.8863 ±0.25034
8hr 0.4084 0.6125 0.6125 0.4084 0.5104 ±0.05892
16hr 1.1584 0.4084 0.4084 0.4084 0.5959 ±0.1875
Appendix - 229 -
Table (19): progesterone serum levels (ng/ml) in response to injection of serotonin at short darkness photoperiod (8D/16L) in does.
Serotonin
P4(ng/ml) µ± SE
ANIM NO. R49 R50 R51 R52 R53 R54
0hr 0.0675 0.0443 0.2418 0.0486 0.0188 0.2127 0.1056 ±0.0391
1hr 0.0372 0.0243 0.0188 0.0188 0.0188 0.0188 0.0227 ±0.003
2hr 0.0188 0.1784 0.0235 0.0452 0.0188 0.06 0.0574 ±0.0251
4hr 0.0975 0.0508 0.0188 0.0188 0.0492 0.0626 0.0496 ±0.012
8hr 0.0489 0.0188 0.0188 0.0188 0.0188 0.0829 0.0345 ±0.0108
16hr 0.0253 0.0242 0.0188 0.0188 0.0188 0.124 0.0383 ±0.0171
Appendix - 230 -
Table (20): progesterone serum levels (ng/ml) in response to injection of serotonin at neutral photoperiod (12D/12L) in does.
Serotonin
ANIM NO. P4 (ng/ml) µ± SE
R13 R14 R15 R16
0hr 0.0188 0.0188 0.0188 0.0188 0.0188 ± 0.0001
1hr 0.0188 0.0188 0.0188 0.0922 0.0371 ± 0.0183
2hr 0.0188 0.0188 0.0188 0.0188 0.0188 ± 0.0001
4hr 0.0188 0.0188 0.0188 0.0188 0.0188 ± 0.0001
8hr 0.0188 0.0188 0.0188 0.0519 0.027 ± 0.0082
16hr 0.0188 0.0188 0.0188 0.0202 0.0191 ± 0.0003
Appendix - 231 -
Table (21) progesterone serum levels (ng/ml) in response to injection of serotonin at long darkness photoperiod (16D/8L) in does.
Serotonin
ANIM NO. µ± SE P4(ng/ml)
R87 R88 R89 R90
0hr 0.0188 0.0188 0.0188 0.0188 0.0188 ±0.0001
1hr 0.0188 0.0569 0.0188 0.0188 0.0283 ±0.0095
2hr 0.0188 0.0869 0.0275 0.0188 0.038 ±0.0164
4hr 0.0667 0.0188 0.0322 0.0188 0.0341 ±0.0113
8hr 0.0225 0.0188 0.0213 0.0188 0.0203 ±0.0009
16hr 0.0188 0.0208 0.0188 0.0188 0.0193 ±0.0005
Appendix - 232 -
Table (22): progesterone serum levels (ng/ml) in response to injection of bromocriptine at short darkness photoperiod (8D/16L) in does.
Bromocriptine µ± SE ANIM NO. P4(ng/ml)
R55 R56 R57 R58 R59
0hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
1hr 0.1665 1.0547 0.1665 0.1665 0.1682 0.3444 ±0.1775
2hr 0.3496 0 0.1665 0.1665 0.3464 0.2058 ±0.0655
4hr 0.2028 0 0.1665 0.1748 0.4313 0.195 ±0.0689
8hr 0.1665 0 0.1665 1.2474 0.1665 0.3493 ±0.2268
16hr 0.1665 0 0.1665 0.1665 0.1665 0.1332 ±0.0333
Appendix - 233 -
Table (23): progesterone serum levels (ng/ml) in response to injection of bromocriptine at neutral photoperiod (12D/12L) in does.
Bromocriptine
µ± SE ANIM NO. P4 (ng/ml) R17 R18 R19 R20
0hr 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
1hr 0.1665 0.1823 0.1665 0.1665 0.1704 ±0.0039
2hr 0.1665 0.1855 0.1665 0.1665 0.1712 ±0.0047
4hr 0.1665 0.2717 0.1665 0.1665 0.1928 ±0.0263
8hr 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
16hr 0.1665 0.3738 0.1665 0.1665 0.2183 ±0.05183
Appendix - 234 -
Table (24): progesterone serum levels (ng/ml) in does response to injection of bromocriptine at long darkness photoperiod (16D/8L) in does.
Bromocriptine
µ± SE P4(ng/ml) ANIM NO. R91 R92 R93 R94 R95
0hr 0.1665 0.1665 0.1665 0.1709 0.1665 0.1672 ±0.0007
1hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
2hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
4hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
8hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
16hr 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 ±0.0001
Appendix - 235 -
Table (25): progesterone serum levels (ng/ml) in response to injection of indomethacin at short darkness photoperiod (8D/16L) in does.
Indomethacin
µ± SE ANIM NO. P4 (ng/ml)
R60 R61 R62
0hr 0.1785 0.1153 0.8671 0.3869 ±0.2407
1hr 0.9704 0.3778 0.9971 0.7817 ±0.2021
2hr 0.2887 0.6128 0.4093 0.4369 ±0.0945
4hr 0.7994 2.3029 0.1551 1.0858 ±0.6363
8hr 0.2036 0.6544 0.4232 0.4270 ±0.1301
16hr 0.1901 0.2066 0.3204 0.2390 ±0.0409
Appendix - 236 -
Table (26): progesterone serum levels (ng/ml) in response to injection of indomethacin at neutral photoperiod (12D/12L) in does.
Indomethacin
ANIM NO. P4 (ng/ml) µ± SE R21 R22 R23 0hr 1.0872 0.5347 0.1769 0.5996 ±0.2647
1hr 0.2199 1.4021 0.06 0.5606 ±0.423241
2hr 0.2678 0.4335 0.0506 0.2506 ±0.110866
4hr 0.5877 0.1707 0.111 0.2898 ±0.149944
8hr 0.2854 0.6147 0.8413 0.5804 ±0.161385
16hr 0.1113 0.1579 0.1409 0.1367 ±0.013615
Appendix - 237 -
Table (27): progesterone serum levels (ng/ml) in response to injection of indomethacin at long darkness photoperiod (16D/8L) in does.
Indomethacin
µ± SE ANIM NO. P4(ng/ml)
R97 R98 R99
0hr 0.0534 0.0555 0.1492 0.086 ±0.032 1hr 0.5095 0.1282 0.0914 0.243 ±0.1337 2hr 0.2274 0.1686 0.0471 0.1477 ±0.0531
4hr 0.4882 0.0979 1.1009 0.5623 ±0.2919
8hr 0.1584 0.0374 0.6966 0.2974 ±0.203
16hr 0.0959 0.0383 0.2982 0.1441 ±0.079
Appendix - 238 -
Table (28): progesterone serum levels (ng/ml) in does response to injection of (melatonin+HCG) at short darkness photoperiod (8D/16L).
Melatonin+HCG
P4(ng/ml) µ± SE ANIM NO. R66 R67 R68
0hr 1.6532 1.0982 0.8762 1.2092 ±0.23106
1hr 9.8166 5.6722 2.9563 6.14836 ±1.9946
2hr 12.9955 4.8897 2.3484 6.7445 ± 3.2104
4hr 3.764 7.6811 1.4931 4.3127 ±1.8072
8hr 5.8387 3.5123 12.9466 7.4325 ±2.8376
16hr 2.9489 4.173 3.1167 3.4128 ±0.3831
Appendix - 239 -
Table (29): progesterone serum levels (ng/ml) in does response to injection of (melatonin+HCG) at neutral photoperiod (12D/12L).
Melatonin+HCG
ANIM NO. P4 (ng/ml) µ± SE
R28 R29 R30
0hr 0.0188 0.0188 0.1664 0.0680 ±0.0492
1hr 0.0188 0.417 0.0188 0.1515 ±0.1327
2hr 0.1397 1.1698 0.0863 0.4652 ±0.3526
4hr 0.6914 5.0206 0.2899 2.0006 ±1.5144
8hr 1.3116 0.1831 0.2844 0.5933 ±0.3604
16hr 0.028 0.0318 0.1675 0.0757 ±0.0458
Appendix - 240 -
Table (30): progesterone serum levels (ng/ml) in does response to injection of (melatonin+HCG) at long darkness photoperiod (16D/8L).
Melatonin+HCG
P4(ng/ml) µ± SE ANIM NO. R104 R105 R106
0hr 0.322 0.0188 0.0594 0.1334 ±0.0950
1hr 0.0445 0.0188 0.0431 0.0354 ±0.0083
2hr 0.0341 0.0188 0.054 0.0356 ±0.0102
4hr 0.0188 0.0819 0.0981 0.0662 ±0.0242
8hr 0.6264 0.0273 0.6517 0.4351 ±0.2040
16hr 0.0437 0.0188 0.0717 0.0447 ±0.0447
Appendix - 241 -
Table (31): progesterone serum levels (ng/ml) in does response to injection of (serotonin+HCG) at short darkness photoperiod (8D/16L).
Serotonin+HCG
ANIM NO. P4(ng/ml) µ± SE
R69 R70 R71
0hr 0.0913 0.2972 0.0913 0.1599 ±0.0686
1hr 0.1795 0.0913 0.1146 0.1284 ±0.0263
2hr 0.1254 0.1342 0.0946 0.118 ±0.012
4hr 0.0913 0.0913 0.4123 0.1983 ±0.107
8hr 0.1323 0.2721 0.0913 0.1652 ±0.0547
16hr 0.194 0.1965 0.0913 0.1606 ±0.0346
Appendix - 242 -
Table (32): progesterone serum levels (ng/ml) in does response to injection of (serotonin+HCG) at neutral photoperiod (12D/12L).
Serotonin+HCG
ANIM NO. P4 (ng/ml) µ± SE
R31 R32 R33
0hr 0.4984 0.0913 0.0913 0.227 ±0.1357 1hr 0.2491 0.0913 0.0913 0.1439 ±0.0526
2hr 2.8879 0.371 0.2445 1.1678 ±0.8608
4hr 0.3184 3.4238 1.7294 1.8238 ±0.8977
8hr 0.1143 0.7735 0.4963 0.4613 ±0.1910
16hr 0.0913 0.4222 0.0913 0.2016 ±0.1103
Appendix - 243 -
Table (33) progesterone serum levels (ng/ml) in does response to injection of (serotonin+HCG) at long darkness photoperiod (16D/8L).
Serotonin+HCG
ANIM NO. P4(ng/ml) µ± SE
R107 R108 R109
0hr 0.4582 0.2991 0.1776 0.3116 ±0.0812 1hr 0.0913 0.7035 0.1386 0.3111 ±0.197
2hr 0.0913 0.1243 0.9553 0.3903 ±0.2827
4hr 0.0913 0.389 1.6962 0.7255 ±0.4929
8hr 0.0913 0.4753 7.1531 2.5732 ±2.2926 16hr 0.2674 0.1961 1.4906 0.6513 ±0.4200
Appendix - 244 -
Table (34): progesterone serum levels (ng/ml) in does response to injection of (bromocriptine+HCG) at short darkness photoperiod (8D/16L).
8D/16L
Bromocriptine+HCG
P4(ng/ml) µ± SE ANIM NO. R72 R73 R74 0hr 1.9807 0.9845 1.7658 1.577 ±0.3026 1hr 1.5906 1.0223 1.8151 1.476 ±0.2359 2hr 1.617 0.5726 2.5649 1.5848 ±0.5753 4hr 2.8764 4.1512 2.3509 3.1262 ±0.5344 8hr 3.2288 1.3143 1.2058 1.9163 ± 0.656 16hr 1.1997 2.4342 0.6879 1.4406 ±0.5183
Appendix - 245 -
Table (35) progesterone serum levels (ng/ml) in does response to injection of (bromocriptine+HCG) at neutral photoperiod (12D/12L).
Bromocriptine +HCG
ANIM. NO. P4 (ng/ml) µ± SE
R34 R35 R36 0hr 0.71 2.1488 1.1666 1.3418 ±0.4245 1hr 1.5631 1.2577 2.0886 1.6364 ±0.2426 2hr 2.0886 0.8942 2.2703 1.7510 ±0.4316 4hr 2.8371 2.8843 4.8739 3.5317 ±0.6712 8hr 7.752 3.6044 7.3369 6.2311 ±1.3188 16hr 3.7735 9.01 1.6008 4.7947 ±2.1990
Appendix - 246 -
Table (36) progesterone serum levels (ng/ml) in does response to injection of (bromocriptine+HCG) at long darkness photoperiod (16D/8L).
Bromocriptine+HCG
ANIM NO. P4(ng/ml) µ± SE
R110 R111 R112 R113 R114 R115
0hr 4.5666 3.0954 1.8504 2.4884 1.9632 5.1923 3.1927 ±0.5687
1hr 4.0385 6.2433 5.1026 19.7549 4.6002 6.3764 7.6859 ±2.4423
2hr 28.9844 20.3806 14.5294 42.2537 2.6147 4.1436 18.8177 ±6.1949
4hr 20.2131 23.8192 65.6819 16.9031 44.0744 1.65 28.7236 ±9.2569
8hr 16.4142 13.6009 11.5206 16.4594 15.4077 5.2835 13.1143 ±1.7458
16hr 9.3554 1.8504 2.6607 10.9539 11.5206 4.9869 6.8879 ± 1.7410
Appendix - 247 -
Table (37): progesterone serum levels (ng/ml) in does response to injection of (indomethacin+HCG) at short darkness photoperiod (8D/16L).
Indomethacin+HCG
ANIM NO. P4 (ng/ml) µ± SE
R75 R76 0hr 1.4563 1.6742 1.5653 ± 0.1089
1hr 13.0198 42.2206 27.6202 ±14.6004
2hr 14.3628 15.9635 15.1631 ±0.8003
4hr 15.6089 10.0181 12.8135 ±2.7953
8hr 21.936 17.048 19.4920 ±2.444
16hr 41.2141 19.8352 30.5246 ±10.6894
Appendix - 248 -
Table (38) progesterone serum levels (ng/ml) in does response to injection of (indomethacin+HCG) at neutral photoperiod (12D/12L).
Indomethacin+HCG
P4 (ng/ml) ANIM NO µ± SE R37 R38 R39
0hr 9.5478 6.0559 9.2106 8.2714 ±1.1120 1hr 11.783 3.9751 11.569 9.1089 ±2.5676 2hr 41.043 9.666 54.871 35.1933 ±13.3733 4hr 34.036 17.148 41.043 30.7422 ±7.0919 8hr 26.408 15.25 27.875 23.1776 ±3.9866 16hr 20.168 16.476 14.102 16.9155 ±1.7647
Appendix - 249 -
Table (39) progesterone serum levels (ng/ml) in does response to injection of (indomethacin+HCG) at long darkness photoperiod (16D/8L).
indomethacin+HCG µ ± SE P4(ng/ml) ANIM. NO. R116 R117 R118
0hr 1.9865 0.9856 1.6743 1.5488 ±0.2956 1hr 12.1409 30.9048 21.574 21.5399 ±5.4167 2hr 39.6996 98.6465 73.848 70.7313 ±17.088 4hr 127.9906 287.8154 189.2359 201.6806 ±46.555 8hr 126.7191 75.8405 58.2585 86.9393 ± 20.527 16hr 32.7581 40.3993 65.3374 46.1649 ±9.8367
Appendix - 250 -
.
HCG)
.
º
الملخص العربى - 1 -
:
الملخص العربى - 2 -
LH
الملخص العربى - 3 -
:
الملخص العربى - 4 -
:
:
الملخص العربى - 5 -
: -6
الملخص العربى - 6 -
:
( )
( )
:
الملخص العربى - 7 -
:
الملخص العربى - 8 -
:
4
الملخص العربى - 9 -
5 :
الملخص العربى - 11 -
تأثير فترة اإلضاءة علً وظائف المبيض كغدة صماء فً األراوب
رسالت مقدمت مه حسيه مصطفً محمد الظاهر
بكالىريىش فً العلىم السراعيت )إوتاج حيىاوً( كليت السراعت – جامعت السقازيق / فرع بىها )9111( للحصىل علً
درجت الماجستير فً العلىم السراعيت
)فسيىلىجي الحيىان( لجىت اإلشراف العلمً: ** أ.د / محمد صفىث عبد المجيد جادو ...... أستبذ الفسيىلىجى – قسم اإلنتبج الحيىانى كليت الززاعت – جبمعت بنهب.
** أ.د / جعفر محمىد الجىدي ...... أستبذ زعبيت الدواجن– قسم اإلنتبج الحيىانى كليت الززاعت – جبمعت بنهب. ** أ.م/ مجدي حسه جمال إسحق ...... أستبذ مسبعد الفسيىلىجى- زئيس وحدة بحىث الغدد الصمبء - قسم التطبيقبث البيىلىجيت – مسكز البحىث النىويت – هيئت الطبقت الرزيت.
حأثير فخرة اإلضاءة عهً وظائف انمبيض كغدة صماء فً األراوب
رسانت مقدمت مه حسيه مصطفً محمد انظاهر بكانىريىش فً انعهىو انسراعيت )إوخاج حيىاوً( – كهيت انسراعت – جامعت انسقازيق / فرع بىها)9111( نهحصىل عهً درجت انماجسخير فً انعهىو انسراعيت )فسيىنىجً حيىان(
وقد حمج مىاقشت انرسانت و انمىافقت عهيها: انهجىـــــــــت: ** أ.د / محمد صفىث عبد انمجيد جادو .……...... أستبذ الفسيىلىجى – قسم اإلنتبج الحيىانى كليت الصزاعت – جبمعت بنهب.
** أ.د / مدحج حسيه خهيم ...... أستبذ الفسيىلىجى – قسم اإلنتبج الحيىانى كليت الصزاعت – جبمعت األشهس.
** أ.د / أحمد أبى انسعىد رضىان ……...... أستبذ فسيىلىجى الدواجن – قسم اإلنتبج الحيىانى كليت الصزاعت – جبمعت بنهب.
** أ.د / جعفر محمىد انجىدي ……...... أستبذ زعبيت الدواجن– قسم اإلنتبج الحيىانى كليت الصزاعت – جبمعت بنهب.
** أ.و / مجدي حسه جمال إسحق ...…...... أستبذ مسبعد الفسيىلىجي – زئيس وحدة بحىث الغدد الصمبء – قسم التطبيقبث البيىلىجيت – مسكص البحىث النىويت – هيئت الطبقت الرزيت.
جايعت بُها كهيت انعهىو انسراعيت قسى اإلَتاج انحيىاَى
تأثير فترة اإلضاءة عهى وظائف انًبيض كغدة صًاء فى األراَب
رسانت يقديت يٍ حسيٍ يصطفى يحًد انظاهر
بكانىريىش فى انعهىو انسراعيت )إَتاج حيىاَى( – كهيت انسراعت جايعت انسقازيق – فرع بُها )9111(
كجسء يكًم نًتطهباث انحصىل عهى درجت انًاجستير فى انعهىو انسراعيت )فسيىنىجي انحيىاٌ(
قسى اإلَتاج انحيىاَى كهيت انسراعت - جايعت بُها جًهىريت يصر انعربيت 1199