INVESTIGATIONS ON FACTORS AFFECTING IN VITRO GONADOTOPINS SECRETIONS FROM BUFFALO ADENOHYPOPHYSIS

By

KALEEM IQBAL M.Sc Hons. (UAF)

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY IN THERIOGENOLOGY

DEPARTMENT OF THERIOGENOLOGY FACULTY OF VETERINARY SCIENCE UNIVERSITY OF AGRICULTURE FAISALABAD, PAKISTAN 2013

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To, Controller of Examinations University of Agriculture Faisalabad.

We the members of supervisory committee of Mr. Kaleem Iqbal 91-ag-763, student of Doctor of Philosophy in Theriogenology, are satisfied by the manuscript of his research, moreover, the manuscript is also recommended by the external examiners for award of degree.

Supervisory committee

i. Dr. Nafees Akhtar (Chairman) ______

ii. Prof. Dr. Nazir Ahmad (Member) ______

iii. Prof. Dr. Sajjad-ur-Rahman (Member) ______

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DECLARATION

I hereby declare that the contents of the thesis, “Investigations on factors affecting in vitro gonadotopins secretions from buffalo adenohypophysis” are product of my own research and no part has been copied from any published source (except the references, standard mathematical equation/formula or protocols etc.). I further declare that this work has not been submitted for award of any other diploma/degree. The university may take action if the information provided is found inaccurate at any stage.

(Kaleem Iqbal) 91-ag-763

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DEDICATED TO

MY BELOVED PARENTS

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ACKNOWLEDGEMENTS

Words are bound and knowledge is limited to praise ALLAH. The omnipotent, the beneficent, the merciful, Who, created the universe and bestowed mankind with knowledge and ability to think into His secrets. Peace and blessings of ALLAH be upon Holy Prophet Muhammad (peace be upon him), the everlasting source of guidance and knowledge for humanity. I always feel honored being under the supervision of Dr. Nafees Akhter (Associate Professor of Theriogenology, University of Agriculture Faisalabad) as he always guided, helped and suggested me during period of my research work. I learnt salient aspects of teaching and conducting research following his mode of magnetic personality. I am thankful to Prof. Dr. Najeeb-ur-Rehman (Chairman, Department of Theriogenology University of Agriculture Faisalabad) for his support and guidance during my research work. I always appreciate Prof. Dr. Nazir Ahmad (Member supervisory committee) for his support in completion of my research work. I would like to highlight the name of Prof. Dr. Sajjad-ur-Rehman (Institute of Microbiology, UAF) for his continuous guidance and help during my research work. His suggestions proved to be much useful whenever I suffered from technical hindrance in performing experiments relating to cell cultures. I would like to mention Dr. Muddassar Zafar for his sincere company being my sweet friend throughout my stay at University of Agriculture Faisalabad. He colored the simple moments of my life and made them really pleasurable. I am obliged to Dr. Shujait Ali for his guidance in my practical research work. I strongly appreciate him for his precious time he reserved for accompanying me during many of the experiments. I am especially thankful to my friend Dr. Muhammad Ramzan because his companionship was always a ladder towards my each success in my student life and I can’t enjoy any moment of life without him. I always feel myself incomplete without my parents, brothers and sisters as their prayers are always behind my each success. Finally the errors, which remain, are mine only.

KALEEM IQBAL

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CONTENTS

TITLE Page No

THESIS TITLE I

ACKNOWLEDGMENTS V

CONTENTS VI

LIST OF TABLES VIII

LIST OF FIGURES X

LIST OF PLATES XI

ABBREVIATIONS XII

ABSTRACT 1

INTRODUCTION 2

REVIEW OF LITERATURE 5

EXPERIMENT-1 CULTURE OF BUFFALO ADENOHYPOPHYSIS AND MEASUREMENT OF 35

MATERIALS AND METHODS 35

RESULTS 45

DISCUSSION 55

EXPERIMENT-2 EFFECT OF IN VITRO PRODUCED GONADOTROPINS (FSH/LH) ON 62 FEMALE RABBITS

MATERIALS AND METHODS 62

RESULTS AND DISCUSSION 65

EXPERIMENT-3 EFFECT OF IN VITRO PRODUCED FSH ON SERUM FSH, LH, 97 AND PROGESTERONE CONCENTRATIONS AND ON OVARIAN BIOMETRY IN BUFFAOES

MATERIALS AND METHODS 97

RESULTS 99

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DISCUSSION 109

CONCLUSION 115

SUGGESTIONS 116

SUMMARY 117

REFERENCES 119

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LIST OF TABLES

Table Page No TITLE No.

1 Amount of salts used to prepare 1000 ml of PBS (pH 7.4) 36

2 Treatment to pituitary cells in the culture Medium RPMI-1640 40

Mean (±SD) activity of FSH and LH in the extract drawn from culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 3 48 mg (T2); progesterone 2.5 mg (T1), 5.0 mg (T2); and in un-treated culture (control) determined by ELISA.

Mean (±SD) activity of FSH and LH in the extract drawn from culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 4 50 mg (T2); progesterone 2.5 mg (T1), 5.0 mg (T2); and in un-treated culture (control) determined by HPLC.

Estimated amount of hormone separated from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2) 5 54 and progesterone 2.5 mg (T1), 5.0 mg (T2), compared with standards of FSH and LH.

Mean concentration of in vitro produced FSH and LH from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 6 54 mg (T2); medroxy-progesterone 2.5 mg (T1), 5.0 mg (T2) along with Insulin.

Mean (±SD) body weight of experimental rabbits before and after treatment 7 67 with in vitro produced FSH, LH and placebo.

Mean (±SD) red blood cell count (x106/µL) in the blood of pre-pubertal 8 rabbits before and after treatment with in vitro produced FSH, LH and 69 placebo.

Mean (±SD) white blood cell count (x103/µL) in the blood of pre-pubertal 9 73 rabbits treated with in vitro produced FSH, LH and placebo.

Mean (±SD) haemoglobin concentration (g/dL) in the blood of pre-pubertal 10 75 rabbits treated with in vitro produced FSH, LH and placebo.

Mean (±SD) packed cell volume (%) in the blood of pre-pubertal rabbits 11 77 treated with in vitro produced FSH, LH and placebo.

Mean (±SD) ovarian length (cm) of pre-pubertal rabbits treated with in 12 80 vitro produced FSH, LH and placebo.

Mean (±SD) ovarian width (cm) of pre-pubertal rabbits treated with in vitro 13 81 produced FSH, LH and placebo.

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Mean (±SD) weight (mg) of ovaries collected from pre-pubertal rabbits 14 83 treated with in vitro produced FSH, LH and placebo.

Mean (±SD) volume (ml3) of ovaries collected from pre-pubertal rabbits 15 84 treated with in vitro produced FSH, LH and placebo.

Mean (±SD) number of Graafian follicle on the ovaries of pre-pubertal 16 86 rabbits treated with in vitro produced FSH, LH and placebo.

Mean (±SD) serum FSH and LH (mIU) in the experimental rabbits treated 17 91 with in vitro produced FSH.

Mean (±SD) concentration of FSH (ng/ml) in the serum of experimental 18 100 buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Mean (±SD) concentration of LH (ng/ml) in the serum of experimental 19 102 buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Mean (±SD) concentration of estrogen (pg/ml) in the serum of 20 experimental buffaloes treated with in vitro produced FSH, Folltropin-V 104 and control.

Mean (±SD) concentration of progesterone (ng/ml) in the serum of 21 experimental buffaloes treated with in vitro produced FSH, Folltropin-V 106 and control.

22 Correlation among serum LH, FSH, estrogen and progesterone. 106

Group wise mean ovarian length and width of experimental buffaloes 23 before and after treatment with in vitro produced FSH, Follitropin-V and 108 control.

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LIST OF FIGURES Figure Page No TITLE No.

Chromatogram derived from commercially available standard gonadotropins by gradient HPLC/UV-visible detector, showing hormone activity in the form of 1. 50 peak-1 for FSH and peak-2 for LH at mean retention time of 1.827±0.4 and 4.120±0.4 (min) and peak area of 34.8 and 65.2%, respectively.

Chromatogram of extract drawn from cell culture of buffalo adenohypophysis kept free from any treatment (Control) by gradient HPLC/UV-visible detector, showing 2. 51 low hormone activity in the form of a line for FSH and LH at retention time of 2.087 to 3.853 (min). Peak area for FSH and LH were 5.8 and 9.4%, respectively.

Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol (0.5mg/100ml of Medium-1640). Peaks-1 3. represents low FSH activity at 1.947 (min) and peak area was 23.1%. Other peaks 51 of variable heights are also present, indicating presence of some other hormones or factors having similar activity.

Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol (1.0mg/100ml of Medium-1640). Peaks-2 represents FSH at 2.093 (min) and peak-3 represents LH at 3.967 (min) and peak 4. 52 area was 20.1 and 5.5%, respectively. Other peaks of variable heights are also present, indicating presence of some other hormones or factors having similar activity.

Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with medroxy-progesterone 2.5 mg (T1). Peaks-2 represents LH at 5. retention time 3.847 (min) and peak area is 18.0%. Other peaks of variable heights 52 are also present, indicating presence of some other hormones or factors having similar activity.

Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with medroxy-progesterone 5 mg (T2). Peaks-2 represents FSH and LH at 2.04 and 3.133 (min). Peak area for FSH and LH were 11.2 and 71.642%, 6. 53 respectively, indicating high activity of LH. Other peaks of variable heights are also present, indicating presence of some other hormones or factors having similar activity.

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LIST OF PLATES

Plate TITLE Page No No.

1 Photomicrograph shows variable sized viable and dead 46 gonadotrophs in a smear prepared from cell culture of buffalo adenohypophysis after 24 hours of incubation (320 X).

2 Photomicrograph shows viable and enlarged mono-nucleated and 46 poly-nucleated pituitary cells in a smear prepared from cell culture of buffalo adenohypophysis after 48 hours of incubation (320 X).

3 Ovaries of a rabbit in control group showing plane surface without 88 Graafian follicles (encircled).

4 Ovaries of a rabbit treated with FSH showing multiple Graafian 88 follicles (arrows).

5 Ovaries of rabbit from group B treated with FSH showing multiple 89 Graafian follicles (arrows).

6 Photomicrograph of an ovary collected from a rabbit treated with in 93 vitro produced FSH, showing multiple growing Graafian follicles.

7 Photomicrograph of an ovary collected from a rabbit treated with 93 placebo (control) showing small non developing GF.

8 Photomicrograph of a liver collected from a rabbit treated with in 95 vitro produced FSH showing normal parenchyma.

9 Photomicrograph of a kidney collected from a rabbit treated with in 95 vitro produced FSH showing normal muscular and secretory tissue.

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ABBREVIATIONS

AA: Arachidonic acid C-18-ODS: C-18 Octadecyl Silicate Ca: Calcium CL: Corpus luteum DAG: Diacyl-glycerol DHT: Dihydrotestosterone DNA: Deoxyribonucleic acid E: Estrogen E2: 17 beta ECG: Equine chorionic gonadotrophin ELISA Enzyme-linked immunosorbent assay ET: Embryo transfer FCS: Fetal calf serum FSH: Follicle stimulating hormone FSHb Follicle stimulating hormone beta GnRH: -releasing hormone GnRHR: GnRH-R receptor GnRH-TT: Gonadotropins releasing hormone, conjugated to Tetanus toxoid Hb: Hemoglobin hCG: Human chorionic gonadotrophin HPLC: High pressure liquid chromatography IGF: Insulin like growth factor IGFBP: Insulin like growth binding factor IGF-I: Insulin-like growth factor-I IGF-II: Insulin-like growth factor II iIGF: Immune reactive IGF-I IVF: in vitro fertilization KCL: Potassium chloride LH: Lutenizing hormone MAPK: Mitogen-activated protein kinase MCH: Mean corpuscular hemoglobin MCV: Mean corpuscular volume NaCl: Sodium chloride P4: Progesterone PACAP: Pituitary adenyl cyclase-activating PBS: Phosphate buffered saline PCV: Packed cell volume pFSH: Porcine follicle stimulating hormone PGF2α: Prostaglandin F2 alpha PKC: Protein kinase C PLA-2: Phospholipase A-2 PL-C: Phospholipase-C PLD: Phospholipase-D PVP: Polyvinylpyrolidone RBC: Red blood cell rFSH: Recombinant FSH RNA: Ribonucleic acid SOT: Super ovulation technology T: Testosterone

xii uFSH: Urinary follicle stimulating hormone WBC: White blood cell

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ABSTRACT

This project was planned to investigate effects of estrogen and progesterone along with insulin in optimizing the in vitro production of pituitary gonadotropins from buffalo adenohypophysis. Attempts were also made to monitor bioactivity of in vitro produced FSH and LH in prepubertal female rabbits and buffaloes. In the first experiment, gonadotropins i.e. FSH and LH were produced by treating in vitro culture of buffalo adenohypophysis with two levels of estrogen (0.5, 1.0 mg) and progesterone (2.5, 5.0 mg). Insulin and liver extract were added in the culture medium as growth promoting factors. The amounts of FSH and LH produced were estimated by ELISA and HPLC techniques. Results showed significant (P<0.05) increase in the secretion of FSH and LH in cultures treated with estrogen and progesterone, respectively compared with control. The FSH and LH obtained were estimated to be 33.0 IU/ml and 68.67 IU/ml, respectively, in proportionate to standard of FSH being 100.0 IU/ml and LH being 1500.0 IU/ml. Experiment was repeated and sufficient amount of FSH and LH was obtained. Repeated procedure proved that the technique can be used for further production of FSH and LH. In the second experiment four groups of pre-pubertal female rabbits were treated with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5 and 85 IU (s/c), twice daily in divided dose at an interval of 12 hours for five days. Similarly, one group of rabbits was treated with placebo as control-1 and one was untreated kept as control-2. There was no treatment-related mortality; no hypersensitivity or toxic sign was seen in any of the experimental rabbit. None, of the experimental rabbits showed change in general behavior, aggressiveness/lethargy and weight. The in vitro produced FSH≈4.0, 40 IU, LH≈8.5 and 85 IU did not affect body weight and most of the blood parameters i.e. RBC and WBC count, PCV and hemoglobin (Hb) concentration of female rabbits. FSH≈4.0 and 40 IU resulted (P<0.05) on increased number of GF’s along with increased mean length, width, weight and volume of ovaries compared with rabbits treated with LH≈8.5, 85.0 IU and control. Moreover, treating rabbits with in vitro produced FSH≈40 IU, LH≈85.0 IU resulted in significantly (P<0.001) increased serum FSH (260-470 Vs 4-6 mlU/ml) and serum LH (28-48 Vs 4-5 mlU/ml), compared with rabbits of control group. In third experiment, two groups A and B (three in each group) of healthy but acyclic dairy buffaloes were treated with in vitro produced FSH≈500 IU and Folltropin-V≈5mg (s/c), twice daily and buffaloes in group C were treated with placebo/control. Treating buffaloes with in vitro produced FSH and Folltropin-V resulted in significantly (P<0.01) higher serum FSH, LH and estrogen concentration at day 5 to 6, whereas progesterone concentration was decreased (P<0.01). Day to day increase in serum FSH was also significant (P<0.01). Increase in serum LH was observed at day 5 to 6. Though the size of ovaries was increased showing numerous small GF’s, however, no one attained ovulatory capability. Hormones obtained from the culture materials showed bioactivity in the experimental animals without any toxicity.

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INTRODUCTION

Pituitary gonadotropins i.e. follicle stimulating hormone (FSH) and luteinizing (LH) hormone are required for normal reproductive functions in all mammals. In females of all species, FSH and LH support folliculogenesis and ovulation (Richards and Hedin, 1988) and in males, these hormones initiate and maintain spermatogenesis (Moudgal et al., 1992; Antonio et al., 1999; Hafez, 2000). Use of gonadotropins has been proved effective for the treatment of true anestrus in mature females and stimulation of ovarian functions in young females to attain puberty. This not only reduces the long calving intervals and enhance calf crop and milk production but in adult animals, also brings early puberty in young females (Morrow, 1986; Hafez, 2000). Exogenous gonadotropins i.e. FSH and LH are the hormones which may be used to enhance the ovarian function in various animal species (Morrow, 1986; Driancourt, 2001) and can help to reduce reproductive problems. Moreover, advancement in animal and human reproduction largely depends upon understanding reproductive endocrinology. At present, it is well established that successful in vitro fertilization (IVF) and embryo transfer (ET) both require stimulation of the ovary and suppression of the pituitary (Balasch et al., 2001). Thus, exogenous gonadotropins are the key hormones required to maximize IVF success (Barbieri and Hornstein, 1999) and super ovulation technology (SOT) in various animals (Morrow, 1986).

As a result of intensive research, FSH has been derived from pituitary extracts, purified preparations from animals and human cadavers, eventually evolving to their production by cell culture and recombinant DNA technology. However, the process of evolution has been constantly driven by the need to make gonadotropin products safe, pure and effective. Reliable batch-to-batch consistency is also needed to reduce the variability in the results of treatments (Lunenfeld, 2004). Various factors like GnRH, ovarian steroids, blood insulin, vitamins and their interactions have been reported to increase the release of gonadotropins from in vitro cell culture of adenohyphosis (Ortmann et al., 1999; Baker et al., 2000; Looper et al., 2003; Stormshak and Bishop, 2008). Narasimhan and Anderson (1981) demonstrated the use of estradiol monobenzoate for the production of FSH and medroxy progesterone for the production of LH. They further stated that large amount of pituitary hormones can be obtained in short period of time through using these compounds.

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Much work has been reported to explore the gonadotropin producing cells of anterior pituitary gland in various species including rat, rabbit and mice (Child et al., 1994). Buffalo adenohypophysis has been successfully used to develop in vitro culture and secretions of FSH and LH have been obtained in the culture medium (Akhtar and Rehman, 2005; Chand et al., 2005). Moreover, Umer et al. (2009) reported that no toxicity was observed when culture extracts of buffalo adenohypophysis containing FSH and LH were injected to pre-pubertal female rabbits. Akhtar and Rehman (2005, 2007 and 2009) reported sequential secretions of FSH (3.89 mIU/mL) and LH (3.75 mIU/mL) in the cell culture extracts of buffalo adenohypophysis and bioactivity of FSH and LH using pre pubertal female rabbits which resulted in uterine stimulation and multiple follicle maturation. However, these workers could not conduct experiments on large animals like buffaloes.

Measurement of FSH is required for clinical and diagnostic purposes. The definition of “FSH”, its measurable property i.e. biological activity and the corresponding measurement process must be related to the purpose for which the measurement is required. One of the most important requirements for the measurement of FSH is to determine the potency of therapeutic products (Wang, 1988). Steelman and Pohley (1953) reported increased ovarian weight and follicle size in immature or hypophysectomized rats by using hCG and test samples containing unknown quantity of FSH. Experiments in buffaloes proved that the use of gonadotropins alone or in combination showed better results than any other therapies (Samad et al., 1996). However, the availability of gonadotropins is scared and is expensive. Production of gonadotropins locally in Pakistan will serve the purpose of their inexpensive availability, which may help in enhancing the early maturity of young buffalo females and increase milk and calf production in adults.

FSH and LH secretion in the cell culture of bovine adenohypophysis needs thorough bioassay for their biological activity and assessment of their economical production. The cheaper extractions of FSH will be helpful to meet the requirement of local farmers in solving problem of late maturity and various types of infertility treatments in dairy animals. Production of FSH and LH by in vitro culture of pituitary cells is now practicable; however, the quantities to be produced need to be enhanced. Therefore, this project was designed to:

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1. Investigate effects of estrogen and progesterone along with insulin for optimizing the in vitro production of pituitary gonadotropins from buffalo adenohypophysis. 2. Estimate bioactivity present in the in vitro produced hormones from culture of buffalo adenohypophysis using pre-pubertal female rabbits and buffaloes.

3. Explore the toxicological effects if any of in vitro produced hormones from culture of buffalo adenohypophysis on hematology and visceral organs of rabbits.

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REVIEW OF LITERATURE

Gonadotropins (FSH and LH) are heterodimeric, heterogeneous glycoproteins which have been identified in most vertebrate species. They play a key role in development and functions of testicular and ovarian function and are widely used both diagnostically and therapeutically in reproductive medicine (Rose and Gaines-Das, 1998; Rose et al., 2000). They are synthesized and secreted by gonadotrophs of the anterior pituitary in response to various hypothalamic and gonadal factors (McLachlan et al., 1989). FSH binds to gonadal receptors, leading to effect on steroidogenesis and gametogenesis (Chappel and Howles, 1991; Kristine and Stone, 1992; Simoni and Nieschlag, 1995; Rulli and Huhtaniemi, 2005). These hormones are central to the complex endocrine system that regulates normal growth, sexual development, and reproductive functions (Bousfield and Dias, 2011).

Advancement in animal and human reproduction largely depends upon understanding the reproductive endocrinology. At present, it is well established that successful in vitro fertilization (IVF) and embryo transfer (ET) both require stimulation of the ovary and suppression of the pituitary (Balasch et al., 2001). Exogenous gonadotropins i.e. FSH and LH are the key hormones required to maximize IVF success (Barbieri and Hornstein, 1999) and super ovulation technology (SOT) in various animals (Morrow, 1986).

Synthesis and secretion of FSH is regulated by a variety of neuroendocrine, endocrine, paracrine, and autocrine factors, the best studied of which include GnRH, activins/inhibins/follistatins, and sex steroids. Each of these factors, alone and in combination, regulate FSHb transcription. However, interspecies differences may help explain observable differences in FSH secretion (e.g. the high-amplitude/short- duration secondary FSH surge in rodents versus the low-amplitude/long-duration follicular-phase FSH increase in humans) (Bernard et al., 2010). The secretion of LH and FSH diverges under a range of physiological and experimental conditions: the turnover rate (including synthesis and secretion) of FSH is greater than that for LH (Apfelbaum and Taleisnik, 1976); the magnitude of stimuli-induced FSH release is smaller than that for LH (Muyan et al., 1994), LH and FSH appear to be packaged into morphologically distinct secretory granules (Childs, 1986).

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Various sized gonadotrophs respond to GnRH and steroids (E2 and P4) treatment in different pattern in terms of the amounts and proportions of LH and FSH secretion, with larger gonadotrophs secrete more FSH than LH after exposure to GnRH. Pituitaries collected from pro-estrus rats showed well developed gonadotrophs. Moreover, in pro-estrus rats small pituitary cells increased their proportion of FSH in response to GnRH alone, or with estrogen. However, these responses depended on the amount of stimulus (Childs, 2006). Childs (2006) also stated that proestrous female rat population had larger cells possibly reflecting the increasing size or density of LH and FSH cells.

FSH and LH are glycoproteins and production of these hormones occurs in either the pituitary or the chorion of a developing foetus. These hormones are heterodimeric, consisting of a common α-subunit and a unique β-subunit and they are also glycosylated. Therefore, their production requires coordination of transcriptional regulation of the subunit genes, translation of mRNA, post-translational processing, assembly, packaging and secretion (Bousfield and Dias, 2011).

The hypothalamic control of gonadotropin secretion is mediated by episodic basal secretion and mid-cycle surges of GnRH, which binds to specific plasma membrane receptors in pituitary gonadotrophs and stimulates phosphoinositide hydrolysis, calcium signaling, and secretion of LH and FSH (Stojilkovic et al., 1994). Synthesis and secretion of LH and FSH are under the control of GnRH, and gonadal steroids (estrogen, progesterone and testosterone) and , including inhibin and activin. GnRH is a decapeptide, which is a product of hypothalamic neuro secretion and is carried by the hypothalamo-hypophysial portal system to the gonadotropic cells of the anterior pituitary. Binding of GnRH to receptors on the pituitary gonadotrophs causes the release of both FSH and LH (Bremner et al., 2006). GnRH interacts with high-affinity receptors on gonadotroph cells of the anterior pituitary. The GnRH receptors interact on ligand binding and leads sequentially to activation of different phospholipases and release of free Ca++ and lipid-derived molecules as second messengers. Activation of plasma membrane Ca++ channels, mobilization of Ca++ from intracellular stores, and activation of calmodulin-stimulated cell responses play an important role in GnRH action. In addition, the activation of phospholipase-C (PL- C) is an early response, followed by those of phospholipase A-2 (PLA-2) and phospholipase-D (PLD). Generation of second messengers inositol-1, 4, 5-

6 triphosphate and diacyl-glycerol (DAG) mobilizes intracellular pools of Ca++ and activates protein kinase C (PKC). Both Ca++-dependent conventional and Ca++ independent novel PK isoforms are activated during this process. Arachidonic acid (AA), liberated by activated PLA-2, also participates in PKC activation. Crosstalk between Ca++, AA, and selected lipoxygenase products (e.g. leukotriene C4), and the different PKC isoforms might generate compartmentalized signal transduction cascades on GnRH stimulation of the gonadotropin-producing cells. These include the mitogen-activated protein kinase (MAPK) cascade. Activation of c-jun and c-fos by GnRH stimulation can participate in transcriptional regulation through formation of the transcription factor AP-1. At least partly dissimilar signal transduction systems mediate the GnRH-stimulated acute secretion of gonadotropins and the more prolonged stimulation of new gonadotropin synthesis (Stojilkovic et al., 1994; Stojilkovic and Catt, 1995; Bremner, et al., 2006; Bousfield and Dias, 2011).

Gonadotropin synthesis occurs through the classic process of ribosomal formation of peptide chains, followed by post-translational modifications in the endoplasmic reticulum and Golgi apparatus. These modifications include cleavage of segments of the amino terminus of both α and β subunits and the subsequent addition of carbohydrate moieties to form the mature gonadotropin molecules in the secretory granules. After synthesis and storage in granules, gonadotropins are available for release almost immediately after GnRH stimulation. After exocytosis, the gonadotropins diffuse rapidly into the nearby capillaries and appear in the venous effluent of the pituitary (Bremner, et al., 2006; Bousfield and Dias, 2011).

Measurement of FSH is required for clinical and diagnostic reasons. The definition of “FSH”, its measurable property i.e. biological activity and the corresponding measurement process must be related to the purpose for which the measurement is required. One of the most important requirements for the measurement of FSH is to determine the potency of therapeutic products (Wang, 1988). Steelman and Pohley (1953) reported increased ovarian weight and follicle size in immature or hypophysectomized rats by using hCG and test samples containing an unknown quantity of FSH.

Sairam et al. (1994) studied the isolation and characterization of distinct bioactive forms of LH from male buffalo pituitaries: differences localized to their subunits and suggested that differences in biological potencies could be due to

7 variation in terminal glycosylation and/or differences in branches of this subunit which is known to be important for signal transduction. Experiments in buffaloes proved that the use of gonadotropins in single or in combination prove better results than any other therapy (Samad et al., 1996).

Rensisa and Gatiusb (2007) stated that poor estrus expression, delayed puberty and prolonged intercalving interval compromise the reproductive efficiency of female buffaloes. These limitations are exacerbated during the hot season, when fertility decreases dramatically. Pregnancy rates decrease further because difficulties in detecting estrus. To improve reproductive efficiency, several protocols of estrus and ovulation synchronization have been developed. However, it has recently emerged that a more precise manipulation of follicular development may be needed to achieve better synchrony of ovulation and improve fertility. Researchers have therefore turned their attention to evaluating programs in which hormones such as GnRH, FSH, LH, eCG, hCG, prostaglandins, progesterone and estradiol are administered.

Normal reproductive function requires the precise temporal and quantitative regulation of hormone secretion at all levels of the hypothalamic–pituitary–gonadal axis. The hypothalamus contains gonadotropin-releasing hormone (GnRH) neurons which secrete pulsatile GnRH into the hypophyseal portal blood system through which it is transported to the anterior pituitary gland. GnRH binds to its receptor on gonadotroph cells, stimulating the biosynthesis and secretion of the gonadotropins, (LH) and follicle-stimulating hormone (FSH). LH and FSH travel through the peripheral circulation, acting at the gonads to stimulate gametogenesis (i.e., the development of mature eggs and sperm) and steroidogenesis (i.e., synthesis of the gonadal hormones‒estrogen, progesterone, and ). In the majority of physiologic conditions, the gonadal steroids feedback at the hypothalamus and pituitary to decrease GnRH and gonadotropin secretion (Ehlers and Halvorson, 2013).

Gonadotropin receptors are embedded in the surface of the target cell membranes and coupled to the G-protein system. Signals triggered by binding to the receptor are relayed within the cells by the cyclic AMP second messenger system. Gonadotropins are released under the control of gonadotropin-releasing hormone (GnRH) from the arcuate nucleus and preoptic area of the hypothalamus. The gonads viz. testes and ovaries are the primary target organs for LH and FSH. The

8 gonadotropins affect multiple cell types and elicit multiple responses from the target organs. As a simplified generalization, LH stimulates the Leydig cells of the testes and the theca cells of the ovaries to produce testosterone (and indirectly estradiol), whereas FSH stimulates the spermatogenic tissue of the testes and the granulosa cells of ovarian follicles (Brown, 1994; Dungan et al., 2006; Franceschini, et al., 2006; Campbell, 2009). Developments in biotechnology have led to recognition that there is no single molecule that can be uniquely defined as FSH, and that FSH can induce a range of biological activities (Rose et al., 2000).

Besides GnRH, the secretion of gonadotropins from adenohypophysis is also regulated by activin and inhibins. Activin is a dimer composed of two identical or very similar beta subunits. Inhibin is also a dimer wherein the first component is a beta subunit similar or identical to the beta subunit in activin. However, in contrast to activin, the second component of the inhibin dimer is a more distantly-related alpha subunit. The activin and inhibin protein complexes are both dimeric in structure, and, in each complex, the two monomers are linked to one another by a single disulfide bond. In addition, both complexes are derived from the same family of related genes and proteins but differ in their subunit composition (Xu and Hall, 2006).

Activin is produced in the gonads, pituitary gland, placenta, and other organs. In the ovarian follicle, activin increases FSH binding and FSH-induced aromatization. It participates in synthesis, enhancing LH action in the ovary and testis. In the male, activin enhances spermatogenesis (Chen et al., 2006; Toulis et al., 2010).

In both females and males, inhibin inhibits FSH production and GnRH release from the hypothalamus. However, the overall mechanism differs between the sexes. In females inhibin is produced in the gonads, pituitary gland, placenta and other organs. In female, FSH stimulates the secretion of inhibin from the granulosa cells of the ovarian follicles in the ovaries. In turn, inhibin suppresses FSH. Inhibin secretion is diminished by GnRH, and enhanced by insulin-like growth factor-1 (IGF-1). In males, it is secreted from the sertoli cells, located in the seminiferous tubules inside the testis. Androgens stimulate inhibin production; this protein may also help to locally regulate spermatogenesis. Activins interact with two types of cell surface trans-membrane receptors (Chen et al., 2006).

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The hypothalamic–pituitary–gonadal axis (HPG axis) refers to the interactions between the hypothalamus, pituitary gland, and gonads. This axis is a critical part in the development and regulation of a number of the body systems, such as the reproductive and immune systems. Any change in the hormones produced by each gland, has various widespread and local effects on the body.

The hypothalamus is located in the brain and secretes GnRH. GnRH travels down the anterior portion of the pituitary via the hypophyseal portal system and binds to receptors on the secretory cells of the adenohypophysis. In response to GnRH stimulation these cells produce LH and FSH, which are secreted into the blood stream.

These two hormones play an important role in reproduction. In females, FSH and LH act primarily to activate the ovaries to produce estrogen and inhibin and to regulate the estrous cycle. Estrogen forms a negative feedback loop by inhibiting the production of GnRH in the hypothalamus. Inhibin acts to inhibit activin, which is a peripherally produced hormone that positively stimulates GnRH producing cells. In males, LH stimulates the interstitial cells located in the testes to produce testosterone, and FSH plays a role in initial stages of spermatogenesis (Shepard et al., 2009).

Cattle are polyestrous animals and display estrous behavior approximately every 21 days. The estrous cycle is regulated by the hormones of the hypothalamus (GnRH), the anterior pituitary (FSH and LH), the ovaries (P4; E2 and inhibins) and the uterus (Prostaglandin F2 alpha; PGF2α). These hormones function through a system of positive and negative feedback to govern the estrous cycle of cattle. GnRH was first isolated from the hypothalamus of pigs and is a decapeptide. Its control of the estrous cycle is mediated via its actions on the anterior pituitary which regulates the secretion of the gonadotrophs, LH and FSH4 (Crowe and Mullen, 2011).

One of the most important functions of the HPG axis is to regulate reproduction by controlling ovarian cycles. In females, the positive feedback loop between estrogen and luteinizing hormone helps to prepare the follicle in the ovary for ovulation and the uterus for implantation. When the egg is released, the corpus luteum in the ovary begins to produce progesterone to inhibit the hypothalamus and the anterior pituitary thus stopping the estrogen-LH positive feedback loop. If conception occurs, the placenta produces progesterone; therefore the female cannot

10 ovulate again. If conception does not occur, decreasing secretion of progesterone will allow the hypothalamus to restart secretion of GnRH. The activation of the HPG axis in both males and females during puberty also causes delayed puberty, maturation and anestrus (Weinberg et al., 2008; Sower et al., 2009).

In males, FSH stimulates testicular cells to release androgen-binding protein, which promotes testosterone binding. LH binds to the interstitial cells, causing them to secrete testosterone. Testosterone is required for normal spermatogenesis and inhibits the hypothalamus. Inhibin is produced by the Sertoli cells, which also through inactivating activin inhibits the hypothalamus. After puberty these hormone levels remain relatively constant (Millar et al., 2004; Charlton, 2008).

Baker et al. (2000) investigated the effects of insulin-like growth factor I (IGF-I) and insulin on the function of Coho salmon gonadotrophs in vitro. Dispersed pituitary cells from immature Coho salmon (Oncorhynchus kisutch) were incubated with IGF-I for 1, 3, 7, or 10 days, then incubated with salmon GnRH for an additional 24 h. Medium FSH content before and after GnRH treatment and intracellular FSH content after GnRH treatment were measured. Incubation of pituitary cells with IGF-I for 7 or 10 days increased GnRH-stimulated FSH release and remaining cell content, but did not affect basal release. To examine the specificity of the effects of IGF-I, these workers compared FSH release and cell content of FSH and LH after 10-day incubation with a range of concentrations of IGF-I or insulin. Incubation with physiological concentrations of IGF-I resulted in significantly higher GnRH- stimulated FSH release and remaining cell content of FSH and LH. It was suggested that elevation of gonadotropin levels by IGF-I might be one mechanism by which somatic growth and nutrition promote pubertal development in salmon.

Spicer et al. (1993) determined the effects of insulin, insulin-like growth factor I (IGF-1), testosterone and FSH on proliferation, production of progesterone and production of estradiol and existence of IGF-I mRNA and production of IGF-I in granulosa cells. Cells were collected from small and large follicles from cattle and cultured for 3 or 4 days. They observed that insulin and IGF-I increased cell numbers compared to controls. Moreover, the insulin like growth factor II (IGF-II) and insulin at larger doses did not affect the production of estradiol by cells from small and large follicles. Their research findings were in accordance with the hypothesis that insulin

11 and IGF-I might exert their effect locally on bovine ovarian function and these effects were influenced by dose and size of follicle.

Steroid hormones have a profound influence on the secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These effects can occur as a result of steroid hormones modifying the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, or a direct effect of steroid hormones on gonadotropin secreting cells in the anterior pituitary gland. Utilizing cultured pituitary cells from anestrous ewes, estradiol induced a dose- dependent increase in secretion of LH, but resulted in a dose-dependent decrease in the secretion of FSH (Nett et al., 2002).

Spicer et al. (2002) determined the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P>0.05) on estradiol production by granulosa cells from both small (1–5 mm) and large (≥8 mm) follicles. However, in the presence of ≥3 ng/mL of insulin, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED50) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7–14 ng/mL in the presence of FSH. Similarly, in the presence of ≥3 ng/mL of IGF-I, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the ED50 of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5–6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and was not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin- stimulated estradiol production by 52% on day 1 and 19% on day 2 of treatment. Insulin, IGF-I and FSH also increased (P<0.05) granulosa cell numbers and progesterone production but their maximal effects were less (i.e., <4-fold increase)

12 than their effects on estradiol production. In conclusion, insulin and IGF-I synergize with FSH to directly regulate ovarian follicular function in cattle, particularly granulosa cell aromatase activity.

High-performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. HPLC typically utilizes different types of stationary phases (i.e. sorbents) contained in columns, a pump that moves the mobile phase and sample components through the column, and a detector capable of providing characteristic retention times. The detector may also provide additional information related to the analyte. Analyte retention time varies depending on the strength of its interactions with the stationary phase, the composition and flow rate of mobile phase used, and on the column dimensions. HPLC is a form of liquid chromatography that utilizes small size columns, and higher mobile phase pressures compared to ordinary liquid chromatography (Lloyd et al., 2006; Xiang et al., 2006).

With HPLC, a pump (rather than gravity) provides the high pressure required to move the mobile phase and sample components through the densely packed column. The increased density arises from the use of smaller sorbent particles. Such particles are capable of providing better separation on columns of shorter length when compared to ordinary column chromatography (Lindsay and Kealey, 1987; Snyder et al., 2009).

Enzyme-linked immunosorbent assay also called ELISA, is a commonly used technique for the determination of concentration of different hormones. In between the washes only the ligand and its specific binding counterparts remain specifically bound or immunosorbed by antigen-antibody interactions to the solid phase. The ELISA has been used as a diagnostic tool in medicine, as well as a quality-control check in various industries. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate (Lequin, 2005).

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Narasimhan and Anderson (1981) described a process for growing in vitro normal human and animal pituitary cells for producing large amounts of pituitary hormones i.e. human growth hormone, human ACTH, LH, FSH, PRL or LTH and TSH and also corresponding pituitary hormones of other animal species. Cell cultures were dispersed in amino acid rich nutrient medium supplemented with liver extract, insulin along with other peripheral hormones, antibacterial and antifungal agents and incubated under open-aeration cell growth conditions. The resulting culture was serially sub-cultured several times until the optimum desired cell growth level was achieved and hormones were extracted using conventional techniques. They demonstrated the use of Estradiol monobenzoate for the production of FSH and Medroxyprogesterone for the production of LH. They stated that large amount of pituitary hormones can be obtained in short period of time by using these compounds.

Freshney (1991) reported that pituitary cells isolated from mouse continued to produce pituitary hormones for several subcultures. However, it was indicated that the cell cultures from various animal species may vary in their hormone production.

Kristine and Stone (1992) stated that FSH plays a central role in steroidogenesis and gametogenesis. They concluded that in-vitro assays provide the tool to measure FSH levels in diverse physiologic and pharmacologic states. 'The observed differences in bio and immuno FSH and LH secretion suggest that separate structural entities are recognized by the bioassays versus the immunoassays, and that Radio immunoassay (R1A) does not consistently provide a good estimate of bioactive gonadotropin levels. They observed increases in FSH in GnRH agonist-treated patients with prostate cancer and testosterone-treated normal men, and decreases in the GnRH antagonist treatment in both men and women. Chromatofocusing analysis of serum from GnRH antagonist-treated patients demonstrated a naturally occurring circulating “anti-hormone," and provides the basis to elucidate the role of hormone antagonists in various physiologic, pharmacologic, and pathophysiologic states. Further characterization and eventual purification of these FSH isoforms could aid the understanding of the glycosylation and action of FSH, as well as provide clinical approaches for "anti-hormone" therapies. In addition to GnRH antagonists and testosterone, the observed FSH antagonist isoforms may provide prototypes tor the design of contraceptives.

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Turgeon et al. (1996) stated that pituitary gonadotrophs have the capability to regulate synthesis and secretion of gonadotropins in response to a GnRH signal, and reported 15 fold higher secretion of LH in L beta T2 gonadotrophs. The cell line was generated in the presence of estradiol and dexamethasone. They further reported that the gonadotrophs respond to glucocorticoid treatment with a reversible dampening of proliferation.

The LH secretion from the pituitary is in a pulsatile manner in domestic ruminants; frequency and amplitude of pulsatile pattern varies with the changes in the stages of estrous cycle (Moudgal et al., 1991). The synergistic effect of estradiol and inhibin controls the secretion of FSH from pituitary gland and this secretion of FSH is not pulsatile (Campbell et al., 2004). In humans, cattle and sheep the development of antral follicle greater than 2-4mm diameter is fully gonadotropic dependent. The selection and development of antral follicle is controlled by LH and FSH (Hillier, 2001; Campbell et al., 1996; Gong et al., 1996).

Hormone secretion from the pituitary gland occurs in an episodic or rhythmic manner. This is regulated by the biological clock in the suprachiasmatic nucleus of the hypothalamus. The release of hormones by the anterior pituitary is regulated by feedback control. There is a short feedback loop from the anterior pituitary to the hypothalamus and a long feedback loop of the ultimate hormone on the CNS, hypothalamus or anterior pituitary (Squires, 2003).

The ability of the ovary to produce a dominant follicle which, ovulates a fertilizable egg, is under the control of the endocrine system, most notably by the hormones FSH and LH. Anything that interferes directly or indirectly with the normal action of the gonadotropins can be expected to produce a condition leading to apoptosis and infertility. Research in the past decade has established the concept that FSH and LH signal transduction can be modulated by proteins with growth factor activity (Erickson, 2008).

Surinder et al. (1985) reported addition of insulin and progesterone in medium 199 for ovine pituitary cell culture and stated that the effect of progesterone was maintained up to 20 days and FSH production was augmented with the presence of Insulin in general. They further stated that there was no long term maintenance of the

P4 response unless insulin is present.

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Armstrong et al. (1989) described the role of insulin like growth factor binding protein (IGFBP) in the regulation of IGF bioactivity. These workers measured the expression of IGFBP-2 and -4 messenger’s RNA in small gonadotropin sensitive follicles through in situ hybridization. It was observed that in healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expressions was limited to granulosa and theca tissue, respectively. They observed deficiency of IGFBP-2 mRNA expression in greater gonadotropin dependent follicles. They analyzed the regulation of IGFBP-2 and -4 mRNA expressions in granulosa and theca cells using a serum free cell culture system. It was found that FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, while LH initiated IGFBP-4 mRNA expression in theca cells. Their results provided clue for the presence of various roles for IGFBP-2 and -4 in the developing follicle.

Armstrong et al. (2002) conducted research on heifers by assigning them to two groups with either low or high levels of energy intake and less or increased concentrations of dietary cell-culture protein. They observed the effect of diets on the concentrations of insulin in the plasma, IGF and urea on follicular growth and early embryo development. These dietary doses induced alterations in the ovarian IGF system and elevated the sensitivity of follicles to FSH. They concluded that different mechanisms were involved in mediating the effects of dietary energy and protein on the functions of ovary.

Kawakami et al. (2002) after working in vitro culture of rat lactotrophs, concluded that there is a novel interaction between estrogen and growth factor (insulin) in the regulation of proliferation in estrogen responsive cells.

Pazos et al. (2004) evaluated both the effect of IGF-I on gonadotropin LH- beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The reported that exposure of pituitary cells to recombinant human IGF-I (rhIGF-I; 2 µg/ml) for 72 h markedly stimulated basal LH and FSH release.

Hampton et al. (2004) investigated the importance of gonadotropin in the selection of dominant follicle in cows treated with GnRH agonist. They used Holstein cows and divided them into two groups (control and GnRHa-treated) before treatment. The group treated with GnRH exhibited follicular arrest at <5mm. They

16 further treated cows with FSH for 96 hrs following hourly pulses of luteinizing hormone along with FSH in the last 48 hrs of infusion. They removed the ovaries, counted the follicles and measured their sizes. Furthermore they collected follicular fluid from larger follicles >10 mm. They reported that the number of larger follicles remained the same for GnRH/FSH+LH and GnRH/FSH-treated cows but were lower for the control group. Moreover, androstenedione and estradiol-17β concentrations were highest in the GnRH/FSH+LH group, lower in the control group and least for GnRHa/FSH-treated cows. From their study, they inferred that FSH enhanced the bovine follicular growth beyond 10 mm size and in addition, LH improved the cytochrome P450 17α-hydroxylase expression and androgen production.

According to Dierich et al. (1998), pituitary gonadotropins FSH and LH stimulated the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) were localized in Sertoli cells and ovarian granulosa cells and were coupled to activation of the adenyl cyclase and other signaling pathways. Activation of FSH-Rs was considered essential for folliculogenesis in the female and spermatogenesis in the male. They have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males were fertile but displayed small testes and partial spermatogenic failure. Thus, although FSH signaling was not essential for initiating spermatogenesis, it appeared to be required for adequate viability and motility of the spermatozoa. FSH-R-deficient females displayed thin small uterine horns, small ovaries and were sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes was only moderately altered in FSH-R-mice, drastic sex-specific changes were observed in the levels of various hormones. The anterior lobe of the pituitary gland in females was enlarged and revealed a larger number of FSH and thyroid stimulating hormone (TSH) positive cells. The phenotype of FSH-R-mice was reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Kawakami et al. (1999) compared the effects of 10 nM testosterone (T), 0.1 nM estrogen (E), and 10 nM dihydrotestosterone (DHT) on the LH response to hourly pulses of GnRH as well as the GnRH receptor (GnRH-R) and LH subunit messenger RNA (mRNA) levels in dispersed pituitary cells from intact male monkeys and rats. T suppressed (P<0.01) and E increased (PO.05) GnRH stimulated LH secretion by rat pituitary cells. With monkey pituitary cells, on the other hand, there was non-

17 significant effect of either T or DHT on GnRH stimulated LH secretion. In E-treated monkey cells, a period of initial enhancement (P<0.05) was followed by significant suppression (P<0.05) of LH secretion. GnRH-R mRNA was unchanged by T or E in either rat or monkey cells. T suppressed LHβ (P≤ 0.01) and α-subunit (P≤ 0.01) mRNAs, whereas E increased-subunit (P≤ 0.01), but did not alter LHB mRNA levels in rat cells. In monkey cells, however, neither T nor E affected LHβ or α-subunit mRNA levels significantly. These workers identified different regulatory mechanisms by which testicular steroid hormones controlled LH secretion by the pituitary in male primates and rodents. These workers suggested that the primary site of androgen negative feedback in the male primate is to restrain GnRH pulsatile secretion, whereas in the male rat T also decreases gonadotropin synthesis and secretion by directly affecting the pituitary. E suppresses GnRH-stimulated LH secretion in the primate pituitary, but amplifies the action of GnRH in the rat. Moreover, the action of T to suppress LH secretion and subunit mRNA in male rats is not through decreased GnRH-R gene expression in nonhuman primates. FS mRNA was highly expressed in all fibroblast strains with similar expression regardless of donor species (human or monkey), donor age (neonate or adult), or the organ from which the fibroblast strains were established (skin or pituitary, genital or non-genital skin). Moreover, the band density corresponding to FS-288 was <5-10% of the value for FS-315 in skin fibroblasts as in all other tissues examined. Fibroblast FSH mRNA and protein production were biphasically regulated by dexamethasone. Low concentrations (0.01 and 0.1 nM) increased whereas higher concentrations (>1.0nM) suppressed FSH expression. On the other hand, androgens, activin and pituitary adenyl cyclase- activating peptide (PACAP 38) had no effect. Data established cultured skin fibroblasts as a model to study FS gene expression in humans, and support a role for follistatin in the normal immune response and in the anti-inflammatory actions of glucocorticoids.

Ortmann et al. (1999) conducted experiment on rat pituitary gonadotrophs, demonstrated the interaction of ovarian steroids with GnRH and gonadotropin secretions and concluded that estradiol, but not progesterone, acts as a modulator of adenyl cyclase gonadotrophs. The stimulatory effect of estradiol was thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory

18 effect of estradiol on PACAP stimulated adenyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.

Weil et al. (1999) studied the short term effect of IGF-1 on GTH-I, GTH-II and GH production. It was found that IGF-I did not affect basal GTH-I and GTH-II secretion, although it always inhibited basal GH. Moreover in absence of IGF-I, GTH-I and GTH-II, cells were responsive to GnRH yet these did not observe response for GH cells. They stated that action of IGF-I on the sensitivity to GnRH was different for GTH and GH cells. It was suggested that the action of IGF-I on GTH cell sensitivity to GnRH, was not related to its mitogenic effect.

Denniston et al. (2001) reported that neuropeptide-Y (NPY) receptors present on the anterior pituitary gland did not attenuate GnRH-induced release of LH from anterior pituitary cell cultures. They also reported that cells treated with GnRH alone or GnRH±NPY secreted significantly (P<0.05) higher amount of LH than control (412.1±11.8, 396.2±11.8, 352.8±11.8 ng/ml) media. Moreover there was non- significant (P>0.05) difference in the mean secretion of LH in the cell culture medium of pituitaries collected from Hereford heifers which were in follicular or luteal phase or ovariectomized (417.9103.8, 390.0103.8, 353.3103.8 ng/mL). It was concluded that any effect of NPY on reproduction might be limited to the hypothalamus.

Akhtar and Sajjad-ur-Rahman (2005) compared the organ culture and suspension culture developed from adenohypophysis of adult buffaloes and reported that stimulus with GnRH resulted in higher secretion of FSH (3.89±0.64 mIU/ml) from the suspension culture than 3.08±0.50 mlU/ml from organ culture. The reverse was true for secretion of LH from suspension culture and organ culture, being 3.31±0.63 vs 3.75±0.50 in ILJ/ml, respectively. Culture filtrates were injected to pre- pubertal female rabbits for five days, which resulted in greater activation of GF on the ovaries. It was concluded that buffalo adenohypophysis could be successfully used to develop its in vitro culture and activation of GF on the ovaries of pre-pubertal female rabbits.

Kawakami et al. (2001) have stated that primary pituitary cell cultures are an important tool for understanding pituitary hormone gene expression. Pituitary cell cultures from non-human adult male primate’s pituitary secretory cells were noted to be rapidly overgrown by epithelioid cells with the morphological,

19 immunocytochemical, and proliferative characteristics of folliculostellate cells. Using competitive RT-PCR assays, follistatin mRNA levels were found to increase 4-fold as folliculostellate cells proliferated with time in culture, whereas FSH-mRNA and FSH secretion were suppressed. Follistatin gene expression was stimulated by activin-A and pituitary adcnylate cyclase-activating polypeptide but not by [D-Trp6]-GnRH ethylamide. Testosterone (T) also increased follistatin mRNA levels and follistatin protein secretion. FSH-mRNA was stimulated by [D-Trp6]-GnRH ethylamide and activin but was suppressed by T. The reciprocal relationship between follistatin and FSH-mRNA levels as folliculostellate cells proliferate with time in culture implies a role for folliculostellate cells in the follistatin-activin system in primates. The actions of GnRH and T on follistatin and FSH-mRNA levels in these cultures were opposite to effects observed in pituitary cultures from rats and identify species differences in the control of FSH production that may be folliculostellate cell-related.

Winter and Moore (2007) stated that there is increasing evidence for communication among pituitary cells. Hormone-producing pituitary cells may communicate with each other and with folliculostellate cells. The latter cells surround pituitary hormone-producing cells and are connected by tight junctions to form a network that allows for their coordinated function. Folliculostellate cells are targets of cytokines, peptides, and steroid hormones, and produce growth factors and cytokines, including follistatin, the dynamic regulator of follicle-stimulating hormone (FSH) production that binds activin, and limits activin signaling. Pituitary adenylate cyclase- activating peptide (PACAP) and its receptor are found in folliculostellate cells in which they stimulate transcription of the follistatin gene through cyclic adenosine monophosphate/protein kinase A (PKA) signaling. When PACAP increases, follistatin levels increase, and FSH-beta mRNA is reduced. PACAP also activates gonadotrophs to stimulate transcription of the gonadotropin alpha-subunit gene and lengthen the LH-beta mRNA, presumably to prolong it half-life, and increases responsiveness to GnRH. Accordingly, PACAP differentially regulates FSH and LH, and may prove to be a key player in reproduction through a novel paracrine mechanism.

Chaudhary and Muralidhar (2007) purified and characterized cLH with respect to its size and subunit nature using HPLC. And reported that final product was found to be over 90 fold purified as compared to the starting pituitary extract, and the yield

20 of the final purified LH was found to be 65.3 mg/kilogram of wet pituitary glands. They further reported that purified hormone was capable of stimulating weight increase in the seminal vesicles in immature male rats, with a biopotency equivalent to the 2200 I.U. of hCG per mg of purified cLH. The FSH content of the purified cLH was found to be less than 0.0165% as indicated by in vivo Steelman-Pohley assay.

Hormones play a vital role in regulating the estrous cycle and producing follicles and finally ova. So, reproductive efficiency in terms of maturity, calving interval and estrus regulation is affected by the hormonal profile of animal. At the onset of estrus, LH concentration in blood may be >50 ng/ml, which decreases within a day and LH remains <3 ng/ml in luteal phase and the peak value for LH is achieved after 14.8 hours of estradiol peak concentration (Batra and Panday, 1982). The peak values (57-65 ng/ml) of FSH have been obtained at the beginning of the estrous cycle which were lower (10-17 ng/ml) in the luteal phase (Razdan et al., 1982). The estimated duration of LH surge in buffalo is 7-12 h (Kanai and Shimizu, 1984). On the other hand, receptors for LH are found on the theca interna cells at the tertiary stage and maintained up to the pre-ovulatory stage of development (Richards et al., 1995). LH receptors are also located on the granulosa cells of antral follicles (Garverick et al., 2002).

Looper et al. (2003) determined the effect of steroid on regulation of the synthesis and secretion of gonadotropins by using induced ovulatory cows. The cows were ovariectomized and received intra-vaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intra-vaginal insert (C) for 7 days. Mean serum concentrations of LH and FSH were not significantly influenced by steroid treatments. However, pituitary concentrations of FSH were less in E2- treated cows than in sham-treated cows. The number of GnRH-R was increased in cows treated with E2 but P4 treatment did not influence the number of GnRH-R. Abundance of mRNA for GnRH-R, common-subunit, and FSHß were not affected by treatments. Pituitary concentrations of LH were greater (P<0.05) and concentrations of FSH were less in pro-estrous cows than in ovariectomized, anovulatory cows treated with or without steroids. Abundance of mRNA for GnRH-R, common subunit, LHß and FSHß were similar for proestrus and anovulatory cows. It was concluded that treatment of nutritionally induced anovulatory cows with progesterone and estradiol might cause pulsatile secretion of LH.

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Noseir (2003) studied ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves and reported a significant (P<0.05) increase in the maximum diameter of an-ovulatory follicle was observed during 3-wave inter-ovulatory intervals. Wave 1 was first detected on Day 0, while wave 2 was detected, on average, on Day 8.7 & 7.2 in 2-wave & 3-wave inter- ovulatory intervals, respectively. Wave 3 was detected, on average, on Day 15 in 3- wave inter-ovulatory intervals. The ovulatory follicle showed a significant (P<0.05) increase in diameter compared with the an-ovulatory dominant follicle, in the 2-wave and 3-wave inter-ovulatory intervals. A concomitant significant increase in estradiol concentration and decrease in progesterone concentration accompanied this increase in the diameter of the ovulatory follicle.

According to Gomez et al. (2004) administration of a combination of PMSG and hCG in high doses induced the ovarian hyperstimulation syndrome (OHSS) in rats. hCG has a longer half-life than LH and a greater biological activity, expressed in a higher incidence of complications such as OHSS. Similarly, FSH may also be related to the ovulatory changes within the follicle as there is a simultaneous surge in spontaneous cycles. They compared the capacity of hCG, FSH and LH to induce ovulation and simultaneously prevent OHSS in the animal model. Immature female rats were treated with PMSG for 4 days, and ovulation was triggered with saline, hCG, FSH or LH. They concluded that FSH and hCG, as well as a six fold increase in LH, displayed similar biological activities, including increased vascular permeability (VP) due to excessive vascular endothelial growth factor (VEGF) expression. The use of lower doses of LH produced similar rates of ovulation, while preventing the undesired changes in permeability. These experiments encouraged clinicians to determine the optimal dose of LH to be employed in women in order to trigger ovulation and, at the same time, avoid the risk of OHSS.

Campbell et al. (2004) have reported that regulation of ovarian activity was an integrated process encompassing both extraovarian signals and intrafollicular factors. Initiation of primordial follicle growth and the early stages of folliculogenesis may occur without gonadotropins, but FSH may affect the rate of pre-antral follicle growth. Antral follicle development from 1 to 4 mm in sheep and cattle is completely gonadotropin dependent. These recruited follicles express a range of mRNA encoding

22 steroidogenic enzymes, gonadotropin receptors, and local regulatory factors and their receptors. As follicles continue to mature, there is a transfer of dependency from FSH.

Bergfelt et al. (1994) studied the effect of FSH administration to neutralize the follicular inhibitory effect of follicular fluid. The day-to-day changes in diameter of the largest and second-largest follicles were profiled and compared among treatment groups for days 0-9. They stated that maximum diameter of follicles was observed at day 9, but was smaller in heifers treated with follicular fluid as compared to heifers treated with follicular fluid plus FSH or saline. Moreover, they reported that the number of largest or second largest follicles were similar for treatment with FSH, saline and follicular fluid plus FSH. Whereas the heifers treated with follicular fluid alone exhibited reduced follicle profile. In addition, they stated that heifers treated with follicular fluid exhibited smaller diameter of follicle on days 4-9 and 2-5 days as compared to follicular fluid plus FSH or saline. They concluded that the induction of follicular inhibition by the treatment with follicular fluid was overridden by the exogenous FSH. However, from the available information it is derived that the low FSH concentrations associated with follicle deviation are below the minimal requirements of the smaller or subordinate follicles but are needed for continued growth of the largest or dominant follicle in cattle (Bergfelt et al., 2000).

The effects of various doses of FSH on superovulation in Kamphaeng Saen beef cattle were studied by Nilchuen et al. (2011), who used two different concentrations (200 and 250 mg) of FSH. They synchronized estrus in heifers and cows by injecting 500 µg cloprostenol. They injected FSH twice daily in all animals under study in decreasing doses for four days starting from ninth day of standing estrus. They injected 500 µg cloprostenol on 3rd day of FSH treatment. They inseminated the animals thrice at an interval of 12 hours and treated the animals with 10 µg (GnRH) at the time of 1st insemination. They concluded that the results of 200mg and 250mg FSH were non-significantly (P>0.05) different in terms of percentages of total ova, transferable embryos and number of CL in either heifers or cows.

Hsu and Hammond (1987) studied the effects of FSH, LH and estradiol on the discharge of immunoreactive IGF-I (iIGF). They measured iIGF-I by reversed phase chromatography. They reported that under optimal culture conditions iIGF-I was readily measureable and responsive to various hormonal treatments. The iIGF-I

23 production was 3.12±0.31, 1.78±0.2, 1.58±0.21, 1.33±0.12 and 1.26±0.12 times higher than the control value with E2±FSH, LH±FSH, FSH, LH and E2 treatment of porcine granulosa cells following in vitro culture. It was concluded that gonadal iIGF- I secretion could be activated due to the principal hormones engaged in trophic regulation of the ovary.

Jarrell et al. (1988) stated that there was a need for a biomarker to evaluate numbers of oocytes in any ovary exposed to various exogenous toxicants. After conducting experiments on rats, they concluded that the serum FSH appeared as a biomarker to measure the numbers or size of antral-follicles rather than a measure of the total number of oocytes alone.

Akhtar and Rahman (2005) developed cell culture from adenohypophysis of adult buffaloes and reported significantly (P<0.05) higher mean concentration (3.89±0.64 mIU/mL) for FSH in the culture filtrate treated with GnRH at 100 µL/culture flask than those of control (1.93±0.36 mIU/mL) and culture treated with GnRH at 50 µL (2.59±0.33 mIU/mL). The same trend was found for concentration of LH at both levels of stimulation. They also injected cell free media to pre-pubertal female rabbits up to five days and reported greater activation of Graffian’s follicles (13.66±1.53 vs 5.66±1.53) in the ovaries of treated rabbits than in the controls. It was concluded that buffalo adenohypophysis could be used to develop its in vitro culture.

Hitendra et al. (2004) described a method to isolate FSH from buffalo pituitary glands. They measured buFSH activity by heterologous radioimmunoassay (sensitivity 0.2 ng oFSH/mL). They successfully obtained a biologically active buFSH enriched preparation with 42% recovery. They isolated buFSH as a heterodimer of 30 kDa molecular size, with a 21 kDa presumptive α-subunit by SDS-PAGE, after purification. They evaluated their preparation in terms of biological and physicochemical properties. They also raised a high titer antiserum to buFSH in rabbit. Their developed buFSH and buFSH specific polyclonal anti-sera, had diagnostic and therapeutic applications for improvement of reproductive health of water buffaloes.

Chand et al. (2005) described an improved and cost effective method to isolate FSH from buffalo pituitary glands. The buFSH activity was monitored throughout by a highly sensitive heterologous radioimmunoassay (sensitivity 0.2 ng of FSH/mL) and

24 the in vivo biological activity of the final preparation was also established. A biologically active buFSH-enriched preparation with a moderate recovery (42%) was obtained. The yield of the final buFSH-enriched preparation was 26.5 mg/kg of buffalo pituitary gland. The preparation was characterized in terms of biological and some of its physicochemical properties.

FSH is produced by the pituitary gonadotroph to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. The results of one study indicated that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotroph (Thackray et al., 2006).

The use of gonadotropins in cases of anestrus and to advance the early onset of early puberty in young females is one of the tools to reduce the long calving interval in cows and enhance calf crop and milk production. The super ovulation technology is greatly dependent upon the use of follicular stimulating hormone up to 40mg and pregnant Mare’s Serum Gonadotropins (PMSG or eCG) 1000 IU to 3000 IU in various animal species (Morrow, 1986).

FSH plays major role in the growth and differentiation of GFs. FSH enhances the growth of follicles to pre-ovulatory size in ewes which were made hypogonadotropic by treatment with GnRH analogues for longer period of time. FSH and PMSG are used in cattle and buffaloes for the induction of superovulation (Lindsell et al., 1986; Karaivanov, 1986). The response of superovulation was changed by the variation in the season, age, ovarian status and the stage of estrous cycle when the hormones were administered parenterally (Rahil et al., 1989).

Chappel and Howles (1991) stated that LH was necessary for the production of steroid hormones. During their clinical experiments, they found that increase in serum LH above resting levels might lead to elevated production of androgen and might negatively influence post ovulatory events like conception and implantation. They supported the contention that successful ovulation induction techniques should

25 include the applications of techniques which increase the serum levels of FSH but not LH.

Mahmood et al. (1991) compared the efficacy of PMSG and pFSH with respect to embryos recovered and number of corpora lutea (CL) produced in Pashmina goats. They stated that there was significant (P<0.02) difference in terms of super ovulatory responses. Furthermore, they reported that the rate of embryo recovered was significantly (P<0.001) greater for pFSH as compared to PMSG. Moreover, the rate of embryos recovered and number of resultant CLs was improved in older goats (4 to 6 years) than goats of 1.5 to 3 years age.

Canseco et al. (1992) explored the effects of prostaglandin and FSH on superovulation in Holstein cows. They used 25 mg of GF2α and 50 mg FSH to check their effects on superovulation. They compared single and double injection of PGF2α after FSH administration and proved that split PGF2α injections resulted in better expression of estrus (79% Vs 87%) estrus as compared to single injection. In addition, they stated that using split injections resulted in less time to estrus (55.7 hrs) than a single PGF2α injection (57.4 hrs). The order of occurrence of estrus with different doses of FSH was 35 mg (48 hrs), 40 mg (59 hrs), 37 mg (62 hrs) and 30 mg (56 hrs). According to them, the number of unfertilized ova was greater by using 35 mg FSH as compared to other doses used. Further, they reported that the number of embryos remained unaffected by FSH and PGF2α protocols. They concluded that the FSH and PGF2α affected the time to estrus and estrus expression but they did not affect the embryo recovery rate and quality of the embryos.

Takedom et al. (1995) explored super-ovulatory response induced by single subcutaneous (SC) FSH injection mixed with polyvinylpyrolidone (PVP) in cows. Holstein heifers were given a single injection of pFSH (30mg, sc) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. They administered single injection of PGF2α ≈750 µg in all animals after 48 hours beginning of pFSH treatment. They inseminated the animals twice and embryos were recovered non-surgically. They examined the ovaries by trans-rectal ultrasonography on day 7th or 8th of estrus and plasma concentrations of FSH progesterone and estradiol-17B were determined by radio immunoassays. They stated that the number

26 of transferable embryos recovered and number of CL in group 2 and 3 were similar to that of number of embryos recovered and numbers of CL present in group 4. However, in group 1, one cow out of 5 ovulated single oocyte. They concluded that a single injection of pFSH, mixed with PVP, was sufficient for the induction of superovulation, preserving high FSH plasma level to permit the recovery of sufficient number of embryos for transplantation.

Kelly et al. (1997) worked on the effects of type of FSH used and the method of administration on endocrine pattern, ovulatory response and follicular growth. In first experiment, they compared Follitropin and Pluset given either as a single or multiple injections (8 injections for Follitropin and 10 injections for Pluset). They induced estrus with PGF2α analogue and recovered the embryos after seven days. Their experiment showed better (P<0.05) results for Pluset as compared with Follitropin in terms of ovulation rate and number of degenerated or unfertilized embryos. They stated that multiple injections increased (P<0.05) transferable and freezable embryos significantly and also improved embryo recovery rate. In the next two experiments, they super-ovulated the animals with Pluset and Follitropin by administering as either a single or multiple injections and collected the blood samples for analysis of E2 concentrations. They counted the medium and large size follicles by scanning the ovaries after 72 hours after the first injection of FSH. Moreover, they reported that a single injection of Pluset resulted in higher concentrations of E2 when compared with a single injection of Follitropin at18, 36-48 and 84-96 hours after FSH injection. In addition, they proved similar results for E2 concentrations when multiple injections of Follitropin or Pluset were administered and number of follicles was greater for single Pluset injection than single Follitropin injection on 3rd day after FSH injection. Furthermore, they mentioned that multiple injections of Follitropin or Pluset gave similar results with respect to medium and large follicles throughout the experiment. They concluded that multiple ovulations could be achieved by Pluset or

Follitrpin injections and E2 concentrations were different with single injection of either Pluset or Follitropin.

Hampton et al. (2004) investigated the importance of gonadotropin for the selection of dominant follicle in cows treated with GnRH agonist. They used Holstein cows and divided them into two groups (control and GnRHa-treated) before treatment. The group treated with GnRH exhibited follicular arrest at <5 mm. They

27 further treated cows with FSH for 96 h following hourly pulses of luteinizing hormone along with FSH in the last 48 h of infusion. They removed the ovaries, counted the follicles and measured their sizes. Furthermore they collected follicular fluid from larger follicles >10 mm. They reported that the number of larger follicles remained the same for GnRH/FSH+LH and GnRH/FSH-treated cows but were lower for the control group. Moreover, androstenedione and estradiol-17β concentrations were highest for GnRH/FSH+LH, lower for control and least for GnRHa/FSH-treated cows. From their study, they inferred that FSH enhanced the bovine follicular growth beyond 10 mm size and in addition, LH improved the cytochrome P450 17α- hydroxylase expression and androgen production.

Nayan and Bhardwaj (2013) studied the expression and hormonal regulation of PAPP-A mRNAs in the Indian water buffalo ovary, and reported stage-specific expression of PAPP-A in the whole follicles and postovulatory structures, corpus luteum and corpus albicans. After determination of cDNA sequence of buffalo they further reported that PAPP-A sequences showed more than 90 % homology with reported sequences of cattle, sheep, pig, and human.

Yoshimura et al. (1994) investigated the effects of gonadotropin hormones on follicular growth, oocyte maturation, ovulation, and production of IGF-I in the ovaries of perfused rabbit. They found that ovulation was not observed in any ovary perfused with GH at various concentrations. However, the addition of GH to the perfusate enhanced the follicle diameter in a dose dependent manner. They determined the effect of GH on the concentration of tissue, and found it stimulated the tissue concentration of IGF-I in perfused rabbit ovaries. During their experiment, the exposure to GH stimulated follicular growth, oocyte maturation, and production of IGF-I, indicating that ovary was actually a site of action and reception of GH. They found that GH increased the effects of gonadotropins and suggested GH being involved in the amplification of gonadotropin actions.

Antonio et al. (1999) have stated that FSH is essential for reproductive function since it stimulates the development of ovarian follicles and spermatogenesis. FSH has been widely used in the treatment of female infertility; the use of exogenous FSH also offers considerable potential for the treatment of male infertility.

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Palta and Madan (1996) investigated the hypophyseal response with respect to induction of FSH and LH by GnRH administration in cyclic buffaloes (Bubalus bubalus). They injected two groups of buffaloes with saline or GnRH at 1.0 µg/kg body weight on 14th day of estrous cycle. They measured the plasma FSH and LH levels one hour before and 6 hours after the administration of saline or GnRH. They conducted their study during two seasons (winter and summer) and reported that there was no difference in the LH concentrations induced by GnRH during the two seasons. In addition, they stated that during winter shorter (P<0.05) time was required to attain LH peak moreover, 39% higher (P<0.05) peak area was observed during winter as compared to summer. Moreover, they stated that no effect of season was observed on FSH peak level induced by GnRH (FSH peak area and time required to attain peak FSH was same for both the seasons). They concluded that summer season negatively affected the release of LH induced by GnRH in buffaloes.

After studying the variations in the peripheral levels of FSH, LH, estradiol- 17β and progesterone in Murrah buffalo synchronized and oestrus-induced, Singh et al. (2001) reported that peak LH and FSH levels during oestrus were 38.40 ± 9.21 and 24.04 ± 4.75 ng/ml, respectively and estradiol-17β and progesterone were 19.50 ± 5.51 pg/ml and 0.61 ± 0.25 ng/ml, respectively.

Gonadotropin therapy is now recognized as an essential component in the routine management of infertility; as a result of intensive research FSH has been derived from pituitary extracts, purified preparations from animals, human cadavers and human urine, eventually involving the use of cell culture and recombinant DNA technology (Lunenfeld, 2004).

According to Farnworth (1995) three major types of vacuole that possibly mediate gonadotrophin secretion have been identified in gonadotrophs: the small, dense core secretory granule that is rich in LH; a larger, more diffuse granule that is rich in FSH; and the much smaller, synaptic vesicle-like vacuole that contains no identifiable gonadotrophin. Which of these subserves the tonically regulated, GnRH- independent pathway for secretion of FSH without LH has not yet been determined. It is unlikely to be the small granule, because of its preponderance of LH. It may be a form of synaptic vesicle-like vacuole, which is immunocytochemically 'silent' with respect to FSH, or the FSH-rich larger form of secretory granule, for which a specialized, constitutive-like secretory function has not yet been assigned.

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The two gonadotropins, FSH and LH are pivotal regulators of the development and maintenance of normal fertility by maintaining testicular and ovarian endocrine function and gametogenesis. Too low gonadotropin secretion i.e. hypogonadotrophic gonadism, is a common cause of infertility. There are certain physiological and pathophysiological conditions where gonadotropin secretions or their action are either transiently or chronically elevated; such as during pregnancy, in pituitary tumors, polycystic ovarian syndrome, activating mutations, perimenopause and menopause. These situations can be either the primary or secondary cause of infertility and gonadal pathologies in both sexes (Rulli and Huhtaniemi, 2005).

Gustavo et al. (2001) investigated the effects of IGF-I and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS). They observed that IGF-I initiated maturation of equine oocytes with maximum rate of maturation of the nucleus and it had a positive effect on the maturation rate of the nucleus of equine oocytes. Moreover FCS did not enhance the maturation of nucleus. They stated that both IGF and FSH are compulsory for the maximum production of IGFBP-2. Their research findings indicated that production of IGFBP in the developing ovine ovarian follicle was based on both cell types as well as on follicle size and was regulated by IGF-I and gonadotropins.

FSH, being the central hormone of mammalian reproduction, is necessary for gonadal development and maturation at puberty and for gamete production during the fertile phase of life. FSH regulates folliculogenesis and steroidogenesis by binding to cell surface receptors on the ovary. The later stages of follicular growth and development are primarily controlled by gonadotropins. Although early follicular development can progress independently of gonadotropins, follicles are highly dependent on gonadotropins to successfully achieve ovulatory size (Minj et al., 2007).

Hashizume et al. (2002) examined the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17β (E2) and IGF-I was characterized. They reported that GnRH(100 nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36 h after addition of IGF-I (250 ng/ml), with a maximum at 12 h (48.4 ng/ml media versus 35.4 ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500 ng/ml increased the release by

30

18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2 ng/ml media). E2 (10 nM), IGF-I (250 ng/ml) and combined treatment of E2 plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E2 alone was 37.3% greater than with IGF-I alone (39.0 ng/ml media versus 28.4 ng/ml media) (P<0.05). When E2 and IGF-I were added together (45.6 ng/ml media), the release of LH was significantly greater than with either E2 alone or IGF-I alone (P<0.05). E2 (10 nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250 ng/ml) failed to increase GnRH binding. These results show that IGF-I enhance GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interact with E2 to increase the response to GnRH.

Stormshak and Bishop (2008) have stated that estrogens acting via membrane receptors suppress LH secretion by gonadotrophs and stimulate rapid increase in uterine blood flow. Progesterone acting via a membrane receptor has been shown to inhibit binding of oxytocin to oxytocin receptors in isolated endometrial plasma membranes and stimulate capacitation of spermatozoa. It was suggested that progesterone and estrogens can act non-genomically to alter target cell responses in domestic animals.

The theca cells of the follicle stimulated by luteinizing hormone (LH) synthesize androstenediol (Δ4-A), androstenediol (Δ5-A-diol) and the granulosa cells under follicle-stimulating hormone control aromatize them to estrogens (Longcope, 2008).

According to Biffoni et al. (1994) since most of human menopausal gonadotropin preparations contained significant amount of non-hormonal urine derived proteins, it was suggested that these contaminating proteins were responsible for various allergic reactions. While evaluating the specific 1gE, these workers reported passive cutaneous anaphylaxis in rats and severe allergic reactions in guinea pigs. They also stated that elimination of contaminating proteins significantly reduced the allerginicity of urine derived hFSH preparations.

Kumar et al. (2000) investigated chronic systematic toxicity of immunization with gonadotropins releasing hormone, conjugated to Tetanus toxoid (GnRH-TT) in male rats and rabbits. They reported that haematological and serum chemistry

31 parameters of immunized rats and rabbits were not affected. Most of the changes in serum chemistry of immunized castrated rats were due to withdrawal of androgenic support. The weight of testes, epididymides and accessory sex glands were lower in all immunized animals. Weight of liver, kidney and heart were decreased. Immunization with GnRH-TT had an anti-fertility effect in the male rats. Fertility was restored following cessation of immunization and decline in anti-GnRH antibody titers.

Battaglia et al. (2000) observed that general hypersensitivity- like allergic reactions to intramuscular injections of highly purified urinary follicle stimulating hormone (uFSH-HP) were successfully managed by using intramuscular recombinant FSH (rFSH) for in vitro fertilization in women. To avoid allergic reaction to the protein component from the urine derived FSH preparations, the use of rFSH was suggested in those patients who presented local and/or general hypersensitivity like allergic reactions.

Blood act as a pathological reflector of the status of exposed animals to toxicant and other conditions (Olafedehan et al., 2010). As reported by Isaac et al. (2013) animals with good blood composition are likely to show good performance. Laboratory tests on the blood are vital tools that help detect any deviation from normal in the animal or human body (Ogunbajo et al., 2009). The examination of blood gives the opportunity to investigate the presence of several metabolites and other constituents in the body of animals and it plays a vital role in the physiological, nutrition and pathological status of an organism (Aderemi, 2004; Doyle, 2006). According to Olafedehan et al. (2010) examining blood for their constituents can provide important information for the diagnosis and prognosis of diseases in animals. Blood constituents change in relation to the physiological conditions of health (Togun et al., 2007). These changes are of value in assessing response of animals to various physiological situations (Khan and Zafar, 2005). According to Afolabi et al. (2010), changes in haematological parameters are often used to determine various status of the body and to determine stresses due to environmental, nutritional and/or pathological factors (Etim et al., 2014).

Yakubu et al. (2008) studies the effect of stocking densities on rabbits and reported that rabbits housed at 10 and 14.3/m2 were superior to those at higher densities in packed cell volume, haemoglobin concentration; red blood cell count and

32 mean corpuscular volume, although differences were not statistically significant, moreover, no definite trend was observed in mean corpuscular haemoglobin or mean corpuscular haemoglobin concentration. White blood cell counts; neutrophil, lymphocyte, monocyte and eosonophil values were also not significantly different among rabbits under the varying stocking densities. However, the neutrophil/lymphocyte ratio in blood of rabbits stocked at 20 and 25 rabbits/m2 were more stressed. These results indicate that increasing the density beyond 14.3 rabbits/m2 elicited some negative effects on live performance of weaner rabbits in the savanna zone of a tropical environment.

Umer et al. (2009) stated that no toxicity was observed when extracts were drawn from cultures of buffalo adenohypophysis when injected into pre-pubertal female rabbits.

The influence of FSH on superovulation under descending or ascending plasma progesterone level in Murrah buffaloes was analyzed by Ahmad (2011). He divided the animals into two groups. Group-I buffaloes all having CLs were treated with 75 IU of FSH in descending order for 3 days (twice daily) after ovulation. Group-II buffaloes without having a CL were treated with PGF2α on day 11 post- ovulation. He gave FSH treatment 24 hours post-aspiration and continued till 3 days. He examined the sizes of small, medium and large follicles on 1st, 3rd and 5th day in all groups. He collected blood samples and measured the progesterone (P4) and estradiol concentrations by radioimmunoassay. The results of his study showed that the P4 concentration at day 1 was 0.75 ng/ml and was increased to 2.5 ng/ml at day 5, for CL group, whereas P4 was <0.75 ng/ml for the non-CL group. He stated that non-CL group exhibited higher values for E2 on the 3rd and 5th day. Moreover, he reported that the number of follicles for non-CL group improved significantly compared to CL- group (22.8±2.0vs11.6±2.0). He concluded that FSH treatment improved E2 secretion and the superovulation rate. The superovulation response may be improved by controlling the number of follicles in the absence of CLs.

While studying the effects of FSH extracted from in vitro cultured anterior pituitary cells of male buffalo calves on body and testes weight, serum FSH and total cholesterol and hematological variables in male rabbits Naveed et al. (2014) concluded that in vitro cultured cells of adenohypophysis from male buffalo calves showed FSH activity. This FSH increased testes size, serum FSH, total cholesterol

33 and blood platelets counts and decreased MCV in rabbits. However, it had no effect on weight gain, RBC counts, WBC counts, PCV and MCH.

Terzano et al. (2012) stated that different phases of reproductive cycle are regulated by several sequential events and interactions between hypothalamic releasing hormones, hormones secreted from the pituitary and sex steroids secreted by the ovary. Lack of integration or synchronization or endocrine imbalances at any phase of the sequence may result in reproductive failure.

While studying the biometry of ovaries and follicular count in cycling and non-cycling Nagpuri buffaloes (Bubalus bubalis) Razzaque et al. (2008) reported the average length, width and thickness of ovaries to be 2.21 ± 0.13 cm, 1.39 ± 0.03 cm and 1.49 ± 0.04 cm, respectively, in cycling buffaloes as compared to 2.45 ± 0.07cm, 1.52 ± 0.04 cm and 1.68 ± 0.05 cm, respectively, in non-cycling buffaloes.

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EXPERIMENT-1 CULTURE OF BUFFALO ADENOHYPOPHYSIS AND MEASUREMENT OF GONADOTROPINS ______

MATERIALS AND METHODS

Collection of fetal calf serum

New-born calves were obtained from the Livestock Experimental Station, Department of Livestock Management University of Agriculture Faisalabad. Blood was drained hygienically into the sterilized glass tubes and kept at room temperature for six hours to clot. Thereafter, serum was separated and centrifuged thrice @ 10000 rpm for 10 minutes each time, so that cellular parts were completely removed. Serum was then inactivated by keeping it at 56°C in a water bath for 30 minutes. Inactivated, serum was preserved in the deep freezer at -20°C and was used in the cell culture medium when required.

Preparation of solutions and culture media

The research work was done in the tissue culture laboratory, Department of Theriogenology, University of Agriculture Faisalabad. Pre-sterilized glassware and equipment were used in the experiment. Each experimental procedure was performed hygienically under the laminar air flow system to avoid contamination of the solutions, culture media and in cell cultures (Narasimhan and Anderson, 1981; Nuzzolo and Velucci, 1983; Miyamoto et al., 1999). The detail of preparations of different solutions and culture media is given below:

i. Phosphate buffered saline (PBS)

To prepare 1000 ml of PBS each time sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic (Na2HPO4 2H2O) and potassium phosphate monobasic (KH2PO4) were used as per requirement of the experiment (Table 1). All ingredients were measured separately using electric balance, and mixed gradually in the tri-distilled water to make volume up to 1 liter. The solution was sterilized by autoclaving at 121°C for 20 minutes and was kept in a refrigerator to protect from any bacterial contamination (Dulbecco and Vogt, 1954).

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Table 1: Amount of salts used to prepare 1000 ml of PBS (pH 7.4)

Salt Amount of salt (mg/L) Sodium chloride (NaCl) 8.00 Potassium chloride (KCl) 200 Sodium Phosphate dibasic 2160 (Na2HPO4-7H2O) Potassium phosphate monobasic 200 (KH2PO4)

ii. Medium-199 (M3769-Sigma) and RPMI-1640 (R6504-Sigma) The following ingredients were added into Medium-199 or RPMI-1640 to prepare 1000 ml of each medium:

i. Sodium bicarbonate (NaHCO3) 2.2 g (Sigma) ii. Benzyl penicillin Na 500,000 IU iii. Streptomycin sulphate 500 mg iv. Kanamycin 0.5 g/L v. Amphoteracin 250 µg vi. Tri distilled and de-mineralized water up to 1000ml

One pack of powdered Medium-199 or RPMI-1640 weighing 10.0 g was dissolved in 900 ml of tri-distilled water by keeping the flask in laminar flow cabinet at room temperature. Sodium bicarbonate (2.2g) was dissolved in 29.3 ml of tri- distilled water and poured into the flask. A pre-sterilized magnetic capsule was placed in the flask. The entire solution was stirred by keeping the flask over the magnetic stirrer. Benzyl penicillin-Na (500,000 IU), Streptomycin sulphate (500 mg), Kanamycin (500 mg) and Amphotericin solution measuring 10 ml (250 µg) was added one by one to the medium while stirring. Tri-distilled water was added to the solution to make final volume of 1000 ml. The pH of the medium was adjusted at 6.9- 7.2 by using 1N HCL or 1N NaOH. The medium was filtered using a cellulose membrane with a porosity of 0.22µ and was kept in refrigerator for further use.

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iii. Medium RPMI-1640 (Sigma-R6504) for culturing pituitary cells to produce FSH i. Medium RPMI-1640 100 ml Lexavit 1.0 ml Insulin 1.5 IU Diethyl-stillbesterol 0.5 mg ii. Medium RPMI-1640 100 ml Lexavit 1.0 ml Insulin 1.5 IU Diethyl-stillbesterol 1.0 mg

iv. Medium RPMI-1640 (Sigma- R6504) for culturing pituitary cells to produce LH iii. Medium RPMI-1640 100 ml Lexavit 1.0 ml Insulin 1.5 IU Medroxy-progesterone 2.5 mg iv. Medium RPMI-1640 100 ml Lexavit 1.0 ml Insulin 1.5 IU Medroxy-progesterone 5.0 mg

To achieve optimum growth and proliferation of pituitary gonadotrophs to secrete FSH, the Lexavit 1.0 ml/100 ml; insulin 1.5 IU and diethyl-stillbesterol 0.5 mg or 1.0 mg were added. After adding diethyl-stillbesterol, the medium was thoroughly stirred by placing the flask containing the medium on the magnetic stirrer for at least 10 minutes to make a homogenous solution.

Similarly, to achieve optimum growth and proliferation of pituitary gonadotrophs to secrete LH, Lexavit 1.0 ml/100 ml; insulin 1.5 IU and medroxy- progesterone 2.5 mg or 5.0 mg were added. After adding medroxy-progesterone, the medium was thoroughly stirred by placing the flask containing the medium on the magnetic stirrer for at least 10 minutes to make homogenous solution (Narasimhan and Anderson, 1981).

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v. Trypsin solution (0.05 %)

To make 0.05% Trypsin solution, 50 mg of purified Trypsin powder (Sigma) was dissolved in tri-distilled water to make a final volume of 100 ml. The solution was filtered using a cellulose membrane with porosity of 0.22 µ and kept in refrigerator for further use (Hafez, 2000).

Selection of buffaloes for collection of pituitary gland

Thirty young, healthy and cyclic buffaloes each with a clinically normal reproductive tract were selected from the animals brought to the local abattoir for slaughtering over a period of three months. Before slaughtering, these buffaloes were examined through rectal palpation to ascertain their stages of estrous cycle. Immediately after slaughter, ovaries were observed for the presence of a regressing corpus luteum (CL) and a soft developing Graafian follicle (GF), to confirm that these animals were in pro-estrus.

Collection of buffalo pituitaries

Shortly after slaughter of each selected buffalo, its skull was opened to expose brain and the pituitary gland was excised carefully marking the anterior and posterior lobe. Each pituitary gland was rinsed with PBS to remove blood. The gland was kept in a thermal jug at 37°C in Medium-199 and shifted to the laboratory within 2 hours after the slaughter of buffalo. The adhering tissue to the gland was aseptically trimmed off and the following observations for each gland were recorded with the help of Vernier Calipers:

i. Length (cm)

ii. Width (cm)

iii. Thickness (cm)

Weight of each pituitary gland was recorded in grams using an electrical balance, while volume was assessed by placing the gland in a measured quantity of PBS using a graduated cylinder.

Preparation of tissue fragments

Each gland was rinsed thrice in 70% alcohol to sterilize the surface. Then each gland was rinsed 4 times in PBS and placed in a Petri plate containing Trypsin

38 solution at 37°C. Each gland was incised to remove the posterior part under magnifying lens with the help of a fine surgical blade. The anterior part (adenohypophysis) was sliced and cut in <0.1 mm tissue fragments (Nuzzolo and Velucci, 1983).

Trypsinization and preparation of cell suspension

The fragments of adenohypophysis were transferred to a conical flask containing freshly prepared 0.05% Trypsin solution. The volume of Trypsin solution used was approximately 25 times the volume of the tissues. The flask was placed on an electric shaker @ 500 rpm for 10 min at room temperature to achieve dissociation of cells. Thereafter, the suspension was transferred to a centrifugal tube and centrifuged @ 6000 rpm for 10 min. The supernatant was removed and cells were re- suspended in the fresh solution of Trypsin. The procedure was repeated for 10 times until mono-dispersed cells were obtained using 100µm sterilized stainless steel strainer. Cell suspensions were washed thrice by using Medium-199 to remove Trypsin solution and then centrifuged again, as described above.

Washed cells were re-suspended in Medium-199 to achieve a final concentration of 6x106/ml. For this purpose, a drop of re-suspended cells from each adenohypophysis was mixed with the same volume of 0.4% (w/v) Trypan Blue Stain (Sigma: 108K 2349) for 5 minutes to stain the dead cells. An amount of 10µL from each mixture was removed and placed on the scale of Neubauer hemocytometer, covered with a cover-slip and was allowed to spread over the scale. The scale was examined under light microscope (200X). The cells stained as blue were considered dead. Live cell counts were used to estimate their concentration in the suspension and required amount of Medium-199 was added to achieve a concentration of 6x106 cells per ml (Nuzzolo and Velucci, 1983).

Culture of pituitary cells

An amount of 60 ml fetal calf serum (FCS) was added to 540 ml of Medium RPMI-1640 to achieve 10% concentration FCS and thoroughly mixed. From this mixture, 30 ml was poured in each of 20 tissue culture flasks. These flasks were inoculated with 10 µL cell suspension of buffalo adenohypophysis and placed in a carbon dioxide (CO2) incubator in a mixture of 10% CO2 and 90% filtered air at 38°C. After six hours of incubation, cells in each culture flask were stimulated with

39 gonadotropin releasing hormone (GnRH; Lecirelin, Fatro-Italy) at the dose rate of 0.125 µg per flask. The culture flasks were further incubated for 18 hours and examined for viable gonadotrophs using Trypan blue stain (Nuzzolo and Velucci, 1983). The Medium in the culture flasks (having viable cells stuck at their base) were carefully replaced with treatment Medium RPMI-1640 containing two different concentrations of diethyl-stillbesterol and medroxy-progesterone (Table 2). Among 20 flasks, 16 were used for different treatments, while four culture flasks were kept as a control (Free from treatment).

Table 2: Schedule of treatment to pituitary cells in the culture Medium RPMI-1640

Control Diethyl-stillbesterol Medroxy-progesterone

4 4 4 4 4 0.5 mg/100 ml 1.0 mg/100ml 2.5 mg/100ml 5.0 mg/100ml

The culture flasks were further incubated at 38°C for 72 hours in the CO2 incubator along with 10% CO2 and 90% filtered air. At 72 hours of incubation, the culture medium was withdrawn from each flask and was centrifuged @ 6000 rpm for 10 minutes to remove the cellular part. The supernatants were passed through 0.22 µm syringe filter and were preserved as culture extract in Eppendorf sampling cups at - 20°C in a freezer.

Viability of pituitary cells

Every 24 hours during incubation, 10L culture medium was taken from each flask and placed in a separate test tube, equal amount of Trypan Blue Stain was added. These solutions were kept for five minutes, and examined microscopically for viability of cells in the culture medium. Cells stained with Trypan Blue were considered dead. Culture flask having no viable cells was discarded.

Sterility of extract drawn from cell culture of buffalo adenohypophysis

The culture tubes containing Thioglycolate Broth or PPLO Broth were inoculated with extract drawn from cultures of buffalo adenohypophysis.

40

Measurement of FSH and LH concentration by ELISA

The preserved culture extracts were analyzed for FSH and LH activity by using enzyme-linked immunosorbent assay (ELISA) test kit (BioCheck, Inc.).

Principle of enzyme-linked immunosorbent assay (ELISA)

The assay system utilizes mouse monoclonal anti-FSH for solid phase (micro titer wells) immobilization, and sheep anti-alpha FSH in the antibody-enzyme (horseradish peroxidase) conjugate solution (Musick and Erik, 2002).

Reagents and materials provided with kit

• Antibody-coated micro titer wells, 96-well plate • Reference standard, 0.8ml (0, 1.0, 2.5, 5, 10, 25 ng/ml for calibration of ELISA reader) • Enzyme conjugate reagent, 12ml • TMB color reagent, 12ml • Stop solution (2N HC1), 6ml

Assay procedure

• Anti-FSH coated micro titer wells were placed in the holder. • An amount of 100 µL of each standard and sample was dispensed in the coated micro titer wells. • Enzyme conjugate reagent was added (100µl) into each well and mixed thoroughly for 30 seconds. • The micro titer wells were incubated at 37°C for 3 hours. • Incubated mixtures were flicked into a waste container. • Micro titer wells were rinsed and flicked 5 times with de-ionized water. • The micro titer wells were rinsed five times with diluted wash buffer. • The micro titer wells were struck sharply onto absorbent paper to remove all residual water droplets. • TMB reagent (100 µl) was dispensed into each well, and was gently mixed for 10 seconds. • The micro titer wells were incubated at room temperature for 20 minutes, in the dark. • The stop solution (100µl) was added to each well to stop the reaction.

41

• After gently mixing for 30 seconds, the blue color was completely changed to yellow. • The samples were processed as described above and concentration of FSH and LH were recorded at optical density of 450nm by placing the micro titer wells in the ELISA reader (Stat Fax, 303).

Measurement of hormone concentration by HPLC

Cell culture extracts of buffalo adenohypophysis containing variable concentrations of FSH and LH were prepared specifically for processing by high pressure liquid chromatography (HPLC). For this purpose Gradient HPLC CSW32- Chromatography Station was used. Each sample was prepared such that at least 50µL was injected into the injector. The technique described by Lee et al. (2007) was followed with some modification. Specification of each part of the HPLC used is given below:

i. Degasser unit

The mobile phase (Di-distilled water and ethanol 80:20) was first of all passed through a degasser (DGU-12A, Schimadzu) for the removal of any air bubbles present in the mobile phase.

ii. Flow Controller Valve

After being degassed, the mobile phase was passed through the Flow Controller Valve (FCV-10AL) which mixed both mobile phases in the given ratio by the system controller of HPLC.

iii. HPLC Pump

LC-10AT (Schimadzu) pump was used to maintain and perform the analysis at a specific flow rate.

iv. System Controller

All the commands to follow the method, parameters/conditions were assigned to the System Controller (SCL-10A, Schimadzu). This controller maintained the digital equilibrium of the given commands. Gradient programming for the analysis of amino acids was also assigned to it in the form of a file and was run during injection of each sample.

42

v. Column Oven

Column Oven (CTO-10A, Schimadzu) was used to perform the analysis in the column at a specific temperature. Column was placed inside this oven/incubator. The temperature range of this oven was 25-60°C.

vi. Column

It is the device of HPLC in which actual separation and analysis of sample mixture takes place. A non-polar or reversed phase C-18, Octadecyl Silicate (ODS) column was used for the separation of hormone from the cell culture extract.

vii. Detector

Four detectors were attached with the HPLC i.e. spectral or UV/visible, fluorescent, refractive index and conductivity detector. For this analysis, spectral detector (SPD-10AV, Schimadzu) was used in UV mode at a wavelength of 328 nm.

viii. Integrator

An integrator card was installed in the computer attached with HPLC. This card converted the absorbance recorded by the detector into digital signals in the form of peaks observed on the chromatogram being displayed at monitor of the computer.

ix. Software

Software named CSW32 was installed and used to observe the presence of different analytes being separated from the sample mixtures. This software displayed the integrated signals in the form of peaks on the chromatogram. This software was able to control both isocratic as well as gradient mode of HPLC.

x. HPLC conditions

The analysis of amino acids from the cell culture extract was performed by machine using gradient mode of HPLC at 30°C on reversed phase C-18 Octadecyl Silicate (ODS) column having 15cm length, 4.6 mm diameter and particle size of 5µm. The flow rate was set to 0.7ml/min and absorbance was noted at 328 nm using UV/visible detector. Di-distilled water was used as mobile phase and for auto-mixing of samples. The results of samples were expressed in retention time and peak areas of curve. Results of samples were compared with standard peak curve for FSH and LH

43

(Figure 1) and necessary calculations were made for estimations of hormone concentrations.

Statistical analysis

Mean values (±SE) for concentrations of FSH and LH in treatment and control extracts were computed. In order to study the magnitude of variation among different groups, the data were subjected to analysis of variance, under completely randomized design (Steel et al., 1997) in General Linear Model Procedure by Minitab Statistical Software Computer Package (Anonymous, 1991). Duncan's Multiple Range Test was applied for multiple mean comparisons where necessary (Duncan, 1955).

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RESULTS

CULTURE OF BUFFALO ADENOHYPOPHYSIS AND MEASUREMENT OF GONADOTROPINS ______

Biometry of buffalo pituitaries

The mean (±SE) length, width and thickness of the pituitary glands collected from selected buffaloes were 0.81±0.04, 0.71±0.02 and 0.50±0.02 cm, respectively. The ranges for length, width and thickness of the pituitary glands were 0.74 to 0.97, 0.67 to 0.74 and 0.47 to 0.52 cm, respectively. The mean (±SE) weight of the pituitaries recorded was 0.44±0.07g and range was 0.35 to 0.61g.

Viability of pituitary gonadotrophs Smear prepared from cell culture of buffalo adenohypophysis after 24 hours of incubation showed variable sized viable and dead cells (Plate 1). Smear prepared from cell culture of buffalo adenohypophysis after 48 hours of incubation (Plate 2) showed viable and enlarged mono-nucleated and poly-nucleated pituitary cells.

Sterility of hormones drawn from cell culture

The culture tubes containing Thioglycolate Broth and PPLO Broth inoculated with 10µL of hormone-containing extract from cultures of buffalo adenohypophysis, showed no growth after 48 hours of incubation at 37°C. These culture tubes also showed no turbidity.

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Live cell

Live cell

Dead cell

Plate 1: Photomicrograph showing variable sized viable and dead gonadotrophs in a smear prepared from cell culture of buffalo adenohypophysis after 24 hours of incubation (320 X).

Mono-nucleated

Poly-nucleated

Mono-nucleated

Plate 2: Photomicrograph showing viable and enlarged mono-nucleated and poly- nucleated pituitary cells in a smear prepared from cell culture of buffalo adenohypophysis after 48 hours of incubation (320 X).

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FSH and LH concentrations

The preserved samples of extract drawn from cell cultures of adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2), medroxy-progesterone 2.5 mg (T1), 5.0 mg (T2) and untreated cultures were analyzed separately for the presence of FSH and LH activity, using ELISA test kit (BioCheck, USA) and the results obtained are presented in Table 3. The mean (±SE) activity of FSH (2.33±0.54 ng/ml) for culture treated with 1.0 mg diethyl-stillbesterol was significantly (P<0.05) higher than 0.09±0.07 ng/ml found for untreated cultures (control). However, no difference was found between T1 and T2 (0.21±0.18 Vs 2.33±0.54 ng/ml) cultures treated with diethyl-stillbesterol 0.5 mg and 1.0 mg. Similarly, the mean (±SE) activity of LH in the cultures treated with medroxy-progesterone 5.0 mg showed significantly (P<0.05) higher values (241.8±142.9 ng/ml) than 0.05±0.03 ng/ml found for control. Moreover, non-significant difference was recorded between cultures treated with medroxy-progesterone 2.5 and 5.0 mg, values being 75.58±36.45 Vs 241.8±142.9 ng/ml, respectively.

The commercially available FSH and LH standards were processed first using gradient HPLC (CSW32-Chromatography Station) and UV-visible detector. Thereafter, preserved samples of extract drawn from cell cultures of adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2), medroxy-progesterone 2.5 mg (T1), 5.0 mg (T2) and untreated cultures were processed.

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Table 3: Mean (±SE) activity of FSH and LH in the extract drawn from culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2) or progesterone 2.5 mg (T1), 5.0 mg (T2); and in un- treated culture (control) determined by ELISA.

ELISA

T1 T2 Control

FSH 0.21 ±0.18a 2.33±0.54a 0.09 ±0.07b (ng/ml) LH 75.58±36.45a 241.8±142.9a 0.05±0.03b (ng/ml)

Values with different letters within a row differ significantly from one another (P<0.05)

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Figure 1 represents chromatogram of hormone activity in the form of peak-1 for FSH and peak-2 for LH standard at retention time of 1.827±0.4 and 4.120±0.4 (min), respectively. Peak area and height are prominent. The standards of FSH and LH were estimated to be equivalent to 0.463±0.08 and 1.321±0.08 IU (Table 4). Figure 2 represents chromatogram of extract drawn from in vitro culture of buffalo adenohypophysis kept free of treatment as control. No peak area or height was evident, hence no similar hormonal activity was observed as that of standard used.

Figure 3 and 4 represent chromatograms of extract drawn from in vitro culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2). Peak area and height are also presented. Peaks of FSH are prominent in the chromatograms. Moreover, other peaks of variable heights are also present indicating presence of some other hormones, or like factors having similar activity.

Figure 5 and 6 represent chromatograms of extract drawn from in vitro culture of buffalo adenohypophysis treated with medroxy-progesterone 2.5 mg (T1) and 5.0 mg (T2). Peak area and height of LH are prominent in the chromatograms. As in FSH, other peaks of variable heights are also present indicating presence of some other hormones, or like factors having similar activity.

The data for FSH and LH activity in the extract drawn from in vitro culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2), medroxy-progesterone 2.5 mg (T1) and 5.0 mg (T2) were computed and means are presented in Table 4. The mean (±SE) activity of FSH was estimated to be 0.093±0.081 and 0.213±0.048 IU/50µL, for cell cultures treated with diethyl- stillbesterol 0.5 mg (T1) and 1.0 mg (T2), respectively. Difference between standard and treated groups (T1 and T2) was significant (P<0.05), moreover, between T1 and T2 was also significant (P<0.05). Similarly, the extracts drawn from cell cultures treated with medroxy-progesterone 2.5 mg (T1) and 5.0 mg (T2) having LH like activity was estimated to be 0.117±0.057 and 0.431±0.045 IU/50µL, respectively. Difference between standard and treated groups (T1 and T2) was significant (P<0.05), moreover, between T1 and T2 was also significant (P<0.05). From the results of present experiment, it is concluded that insulin along with Lexavit enhances the GnRH-stimulated release of FSH and LH and significantly (P<0.05) interacts with diethyl-stillbesterol and medroxy-progesterone in vitro culture of buffalo adenohypophysis.

49

Figure 1: Chromatogram derived from commercially available standard gonadotropins by gradient HPLC/UV-visible detector, showing hormone activity in the form of peak-1 for FSH and peak-2 for LH at mean retention time of 1.827±0.4 and 4.120±0.4 (min) and peak area of 34.8 and 65.2%, respectively.

Table 4: Mean (±SE) activity of FSH and LH in the extract drawn from in vitro culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2) or progesterone 2.5 mg (T1), 5.0 mg (T2); and of standards of FSH and LH determined by HPLC.

Standard T1 T2

FSH a c b 0.463±0.081 0.093±0.081 0.213±0.048 (IU)

LH a c b 1.321±0.081 0.117±0.057 0.431±0.045 (IU)

Values with different letters within a row differ significantly from one another (P<0.05)

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Figure 2: Chromatogram of extract drawn from cell culture of buffalo adenohypophysis kept free from any treatment (Control) by gradient HPLC/UV-visible detector, showing low hormone activity in the form of a line for FSH and LH at retention time of 2.087 to 3.853 (min). Peak area for FSH and LH were 5.8 and 9.4%, respectively.

Figures 3: Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol (0.5mg/100ml of Medium-1640). Peaks-1 represents low FSH activity at 1.947 (min) and peak area was 23.1%. Other peaks of variable heights are also present, indicating presence of some other alike hormones/factors having similar activity.

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Figures 4: Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol (1.0mg/100ml of Medium-1640). Peaks-2 represents FSH at 2.093 (min) and peak-3 represents LH at 3.967 (min) and peak area was 20.1 and 5.5%, respectively. Other peaks of variable heights are also present, indicating presence of some other alike hormones/factors having similar activity.

Figures 5: Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with medroxy-progesterone 2.5 mg (T1). Peaks- 2 represents LH at retention time 3.847 (min) and peak area is 18.0%. Other peaks of variable heights are also present, indicating presence of some other alike hormones/factors having similar activity.

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Figures 6: Chromatograms of extract drawn from cell culture of buffalo adenohypophysis treated with medroxy-progesterone 5 mg (T2). Peaks-2 represents FSH and LH at 2.04 and 3.133 (min). Peak area for FSH and LH were 11.2 and 71.642%, respectively, indicating high activity of LH. Other peaks of variable heights are also present, indicating presence of some other hormones or factors having similar activity.

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Estimation of hormones from in vitro culture

From the results of HPLC analysis, mean (±SE) activity of FSH (0.093±0.081 and 0.213±0.048 IU/50µL) and LH (0.117±0.057 and 0.431±0.045 IU/50µL) showed in the in vitro culture of buffalo adenohypophysis treated with low and high doses of diethyl-stillbesterol 0.5 mg (T1) and 1.0 mg (T2) and medroxy-progesterone 2.5 mg (T1) and 5.0 mg (T2) was further estimated to be FSH being 20, 46 IU/ml and LH being 4.489 and 132.85 IU/ml against the standards of FSH and LH, respectively, (Table 5). The amount of FSH and LH secreted in the culture medium were pooled separately and means were computed, being 33.0 and 68.67 IU/ml for FSH and LH, respectively, (Table 6).

Table 5: Estimated amount of hormones separated from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2) or progesterone 2.5 mg (T1), 5.0 mg (T2), compared with standards of FSH and LH.

T1 T2 Standard

FSH 20.0 IU/ml 46.0 IU/ml 100 IU/ml

LH 4.489 IU/ml 132.85 IU/ml 1500 IU/ml

Table 6: Mean concentration of in vitro produced FSH and LH from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol 0.5 mg (T1), 1.0 mg (T2); medroxy-progesterone 2.5 mg (T1), 5.0 mg (T2) along with insulin.

FSH LH

T-1 20 IU/ml 4.489 IU/ml

T-2 46 IU/ml 132.85 IU/ml

Mean 33 IU/ml 68.67U/ml

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DISCUSSION ESTIMATION OF GONADOTROPINS ______

The present experiment was planned to enhance in vitro secretions of gonadotropin (FSH and LH) from cell culture of buffalo adenohypophysis through treatment with estrogen and progesterone along with Insulin. Cell cultures were developed from adenohypophysis collected from adult, healthy and cyclic buffaloes in their pro-estrus stage. These buffaloes were confirmed to be cyclic by observing their ovaries after slaughter for the presence of regressing CL and developing GF. Biometrical observations were recorded for length, width, thickness and weight of the whole pituitary before removing the posterior part.

The mean (±SE) length, width and thickness (0.81±0.04, 0.71±0.02 and 0.50±0.02 cm) of pituitaries collected from selected buffaloes were higher than the previously reported biometrical observations of buffalo pituitaries i.e. 0.79±0.03, 0.69±0.04, 0.56±0.04 cm, respectively. Similarly, mean (±SE) weight (0.44±0.07 g) of the pituitaries was also higher than previous reports (0.26±0.03g), where the pituitaries were collected during met-estrus stage (Akhtar et al., 2012). The higher mean biometrical values of pituitaries recorded during the present study might be due to the fact that all pituitaries were collected from cyclic buffaloes which were in their pro-estrus stage. FSH and LH are synthesized within cells during pro-estrus and they remain packaged within secretory vesicles until released (Hadley, 1992).

Cell cultures were developed in medium RPMI-1640 enriched with fetal calf serum and gonadotrophs were stimulated with GnRH. This was because the pituitary gonadotrophs have the capability to regulate synthesis and secretion of gonadotropins in response to GnRH signals (Turgeon et al., 1996). It is established that GnRH receptors are present in the pituitaries of several species i.e. rat (Naor et al., 1980), human (Wormald et al., 1985), rabbit (Limonta et al., 1986), mouse (Pal et al., 1992), fish (Weil et al., 1992; Peter et al., 1992; Schulz et al., 1993) and in mice (Horn et al., 1991) as specific binding sites with high affinity for GnRH and its potent agonist or antagonist analogs. Hyde et al. (1982) stated that among the rat pituitary cell types, GnRH binding sites are located exclusively in gonadotrophs. Moreover, GnRH binds to bi-hormonal gonadotrophs (expressing LH and FSH), as well as to mono-hormonal

55 cells expressing only LH or FSH (Childs, 1984; Naor and Childs, 1986). In general, two labelled components were reported in rat, rabbit, mouse, sheep and cow pituitaries, both of which were displaced by unlabelled GnRH agonists and antagonists (Iwashita and Catt, 1985).

In the present experiment, cell culture medium was replaced with medium containing insulin and Lexavit as growth promoting factors for pituitary gonadotrophs. Researchers performed experiments on in vitro primary cells culture of pituitary gonadotrophs have suggested that insulin promotes GnRH responses (Adashi et al., 1981, Buggs et al., 2006, Hashizume et al., 2002, Rose et al., 2004, Soldani et al., 1994, Xia et al., 2001). Navratil et al. (2009) and Thackray et al. (2010) also reported that insulin and GnRH interact at the level of pituitary cells, and gonadotroph responses are modified by their interaction. Pituitary adenylate cyclase-activating polypeptide (PACAP) mediates interactions of ovarian steroids with GnRH stimulated cultured pituitary cells and polypeptide increases the sensitivity of pituitary gonadotrophs to GnRH (Ortmann et al., 1999). It was also stated that presumably PACAP actions are mediated via type-I receptors which have been shown to be coupled to adenylate cyclase and phospholipase. PACAP might act as a hypophysiotropic factor and influence several functions of gonadotrophs, including exocytosis of LH and FSH (Culler and Paschall, 1991; Perrin, et al., 1993; Schomerus, et al., 1994; Tsujii, et al., 1994).

Cultured gonadotrophs were treated with estrogen and progesterone as stimulatory agent to increase synthesis and secretion of gonadotropins. It is established that GnRH is the primary regulator of LH and FSH and ovarian steroids serve as the most important modulators (Ortmann et al., 1999) which exert the cyclic variations of gonadotropin release. Thackray et al. (2010) also stated that gonadal steroids and peptides modulate LH and FSH levels via feedback to the anterior pituitary, as well as to the hypothalamus.

In the present study, extract drawn from cell culture of buffalo adenohypophysis treated with diethyl-stillbesterol @ 1.0 mg resulted in higher mean activity of FSH (2.33±0.54 ng/ml) compared with cultures treated with 0.5 mg of diethyl-stillbesterol and untreated culture (control) 0.21±0.18 ng/ml and 0.09±0.07 ng/ml, respectively. The viable pituitary gonadotrophs responded in increased secretion of FSH and LH like activity, when cell culture medium RPMI-1640 was

56 replaced with medium containing insulin and Lexavit as growth promoting factors in the gonadotrophs. Results of the present study are in line with those of Turgeon et al. (1996) and Baker et al. (2000), who stated that pituitary gonadotrophs have the capability to regulate synthesis and secretion of gonadotropins in response to GnRH signals. They further reported 15 fold higher secretion of LH by gonadotrophs in the presence of estradiol and dexamethasone. Similarly, Ortmann et al. (1999) stated that estradiol treatment alone enhance basal as well as GnRH stimulated LH secretion. After studying interactions of ovarian steroids with PACAP and GnRH in anterior pituitary cells, these workers reported that LH release was facilitated by additional short-term progesterone treatment and long-term treatment with estradiol and progesterone led to reduce LH responses to GnRH and PACAP. Lesoon and Mahesh (1992) reported that treating anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the GnRH stimulated FSH release from 0.58 to 0.36ng/ml and GnRH-induced LH release was reduced from 0.54 to 0.19ng/ml. They also reported that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. However, they stated that estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone.

The pituitary gonadotrophs treated with medroxy-progesterone 5.0 mg showed significantly (P<0.05) higher mean activity (241.8±142.9 ng/ml) of LH in their extract than that for cultures treated with medroxy-progesterone 2.5 mg and control, being 75.58±36.45 and 0.05±0.03 ng/ml, respectively. Significant (P<0.05) difference found between 5.0 mg medroxy-progesterone and control group might be due to responsiveness of gonadotrophs to various levels of hormone treatment. Pituitary isolated cells continue to produce pituitary hormones for several sub-cultures and cell cultures from various animal species may vary in their hormone production (Freshney, 1991).

The standard preparations of FSH having 100 IU/ml and LH 1500 IU/ml were processed through HPLC. The results showed estimates of 0.463 IU/50µL for FSH and 1.321 IU/50µL for LH. Extracts drawn from cell culture of buffalo adenohypophysis treated with high and low concentrations of diethyl-stillbesterol or medroxy-progesterone showed estimates of FSH activity as 0.213±0.048 Vs

57

0.093±0.081 IU/50µL and LH activity as 0.431±0.045 Vs 0.117±0.057 IU/50µL, respectively. Gonadotrophs treated with higher concentration of diethyl-stillbesterol or medroxy-progesterone secreted significantly (P<0.05) higher amount of FSH and LH, compared to the lower amount.

Extract drawn from cultured buffalo adenohypophysis treated with low and high (T1 and T2) doses of estrogen or progesterone for secretions of FSH and LH were pooled separately and means were calculated according to the standards of FSH and LH. Mean activity for FSH was calculated to be 33.0 IU/ml and for LH it was 68.67 IU/ml. Results of the present study revealed that treating cell culture of buffalo adenohypophysis with estrogen and progesterone resulted in higher mean values (33.0 and 68.67 IU/ml for FSH and LH) than obtained in previous experiments, being FSH≈ 3.89±0.64 mIU/ml & LH≈ 3.75 mIU/ml, (Akhtar and Rehman, 2005), 2.42±0.01 mIU/ml (Umer et al., 2009) and 2.78 mIU/ml (Akhtar and Rehman, 2009). However, in previous experiments, insulin and Lexavit were not added in the cell culture medium (as growth promoting factors) and no stimulating treatment like estrogen and progesterone was provided to the gonadotropin producing cells.

Present study demonstrate that insulin enhances the GnRH- stimulated release of LH and FSH in cultured buffalo adenohypophysis and insulin interacts with estrogen and progesterone to increase the response to GnRH. The rate of increase in FSH (57.14% and 96.14% with low and high dose of estrogen) and LH (99.93 and 999.98% with low and high dose of progesterone) secretions was higher than reported by Hashizume et al. (2002), who reported 51.6% increase in LH secretion from GnRH bound gonadotrophs treated with estrogen (E2). They demonstrated the role of IGF-I in the release of LH from bovine pituitary cells treated with estrogen and progesterone. They also reported that E2 and IGF-I together or alone significantly enhance the release of LH. The results of present study are also in line with those of Batra and Miller (1985) who demonstrated the effect of insulin while studying progesterone (P4) regulation of FSH production in dispersed ovine pituitary cell culture. They reported that FSH secretion in culture media was decreased by 30-70% in the presence of progesterone. But when insulin was included in the culture medium, the effect of progesterone was maintained for up to 20 days. Thus FSH production, in general, was augmented with insulin; however, there was no long term maintenance of the P4 response unless insulin was present. They also established that

58

FSH production is a phenomenon that is regulated similarly by both estradiol and P4. However, Whitley et al. (1995) reported that GnRH-stimulated release of LH was unaltered by IGF-I treatment in pig pituitary cells. Experiments performed in vitro using primary pituitary cells or gonadotroph cell lines have suggested that insulin promotes GnRH responses (Adashi et al., 1981, Ortmann et al., 1999; Hashizume et al., 2002; Rose et al., 2004; Buggs et al., 2006 and Navratil et al., 2009).

Binding of the hypothalamic GnRH to the pituitary GnRH receptors (GnRHR) stimulates the synthesis and secretion of gonadotropin hormones i.e. LH and FSH (Clayton and Catt, 1981, Gharib et al., 1990 and Ortmann et al., 1999). In the absence of GnRH input to the pituitary gland, gonadotropin production and consequently gonadal function in mammals ceases (Mason et al., 1986a, Mason et al., 1986b). Therefore, it is claimed that the increased secretion of FSH and LH itself is the result of GnRH stimulus and also binding factor for steroidal hormones i.e. estrogen and progestrone. Narasimhan and Anderson (1981) described a process for growing in vitro normal human and animal pituitary cells for producing large amounts of pituitary hormones including FSH and LH. They stated that large amount of pituitary hormones can be obtained in short period of time by using GnRH as primary stimulus to gonadotrophs for increasing FSH and LH secretions. They demonstrated the use of estradiol monobenzoate for the production of FSH and medroxyprogesterone for the production of LH. Similarly, during the present experiment synthetic estrogen was used successfully to stimulate gonadotrophs for the secretion of FSH. Narasimhan and Anderson (1981) also demonstrated supplementation of medium with liver extract, insulin along with antibacterial and antifungal agents. In the present experiment, antibacterial (Benzyl penicillin Na, Streptomycin sulphate and Kanamycin) and antifungal agent (Amphoteracin) were added in the medium to control possible contamination.

Results of present experiment support the findings of Baker et al. (2000), who investigated the effects of insulin on the function of Coho salmon (Oncorhynchus kisutch) gonadotrophs in vitro and stated that supra physiological concentrations of insulin were required to produce more moderate effects on gonadotropin levels. They also reported significantly (P<0.05) higher cellular content of LH after incubation of pituitary cells with insulin than that found in medium without insulin. Soldani et al. (1994) reported insulin’s capability of potentiating anterior pituitary LH release from

59 dispersed rat anterior pituitary cells in vitro. Kanematsu et al. (1991), Soldani et al. (1994); Xia et al. (2001) and Hironori et al. (2006) also reported that addition of IGF- I to rat pituitary cell cultures increased basal secretions of FSH and LH, and GnRH- induced LH secretion. Moreover, Pazos et al. (2004) demonstrated the effect of insulin in male rats and stated that IGF-I stimulated basal release of FSH and LH with increased expression of glycoprotein (GP), however, FSHα was higher than FSHβ and LHβ subunit genes in their study.

Figure 2-5 indicated some of the similarities and variations in the peak height and areas of chromatograms. These similarities might exist due the tonic or basal fashion in the secretions of LH and FSH in individuals. Similarly, Hafez (1987) stated that tonic levels of LH and FSH are controlled by negative feedback from the gonads. The tonic level of LH is not stationary, but shows oscillations about every hour. Therefore, it seems that such mechanism may exist in the present study. The natural FSH and LH are controlled by LH-RH. However, their molecules are structurally different; hence the variation and similarities might be due to differences in gonadotropic and steroidal hormones of individual buffaloes from which pituitaries were collected. Moreover, difference in the amount of hormone secreted might be due to the level of sensitivity of gonadotrophs to stimulus (Hafez, 1987).

In vivo pre-ovulatory surge of LH is initiated by an increase in the circulating estrogen concentrations, which have a positive effect on the hypothalamus inducing release of LH-RH that results in the pre-ovulatory surge of LH and FSH. A complexity of hormone releasing mechanism might exist in in vitro pituitary cultures. This might be due to antagonistic analogues which appear to bind at the receptor sites on the pituitary cells but do not induce the release of one of the LH or FSH and block the action of stimulus. The effects of E2 and P4 on in vitro culture of pituitary gonadotrophs are different from those observed in vivo by Looper et al. (2003). They reported that frequency of LH pulses of ovariectomized, nutritionally induced anovulatory cows was increased (P<0.05) by treatment with E2 and amplitude of LH pulses was greater (P<0.05) in cows treated with E2 or P4 than in cows treated with

E2P4 or sham-treated. Concentrations of LH in the pituitary gland of exsanguinated pro-estrous cows were not affected by steroid treatments; however, pituitary concentrations of FSH were less (P<0.1) in E2 cows than in sham-treated cows.

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The results of present study are supported by findings of Hadley (1992), who stated that each of the pituitary hormones is localized to particular cell type (gonadotrophs) in human, rat quail and frog. Nevertheless, sub populations of gonadotrophs contain only one of the gonadotropins. This may account for the nonparallel release of gonadotropins in response to certain stimuli. Similar trend of hormone level in the extract drawn from various cell culture of buffalo adenohypophysis might exist. Thackray et al. (2010) stated that primary pituitary cell culture is frequently employed to study hormonal effects on gonadotropin synthesis. Whereas, Fallest and Schwartz (1991) stated that there are several caveats to these models that are worth mentioning. First, the endocrine milieu at the time of harvest can affect the results, so caution must be used when interpreting data from animals in different stages of the estrous cycle. Second, gonadotrophs only comprise 5–15% of the cells in the anterior pituitary (Ooi et al., 2004). Four other types of secretory cells including thyrotropes, somatotropes, lactotropes and corticotropes are also present, as well as folliculostellate cells. Many hormones secreted from these cells, such as prolactin and oxytocin from lactotropes, have paracrine effects on gonadotroph cells. Folliculostellate cells have been reported to rapidly overgrow than other pituitary cells in culture (Kawakami et al., 2002) and can affect the experimental outcome since they produce paracrine factors such as follistatin and PACAP (Winters and Moore, 2007 and 2011).

Hormones obtained were preserved at various temperatures; however, due to long short fall of electricity in the country, hormones preserved were equalized at room temperature. Therefore, data obtained was not compare able in respect of various storage temperatures.

Based on the results of present experiment it is concluded that:

1. Diethyl-stillbesterol and medroxy-progesterone enhance the GnRH- stimulated release of FSH and LH from in vitro culture of buffalo adenohypophysis, respectively. 2. Treating in vitro culture of buffalo adenohypophysis with higher dose of estrogen resulted in higher amount of FSH secretion. However, non- significant difference was found in respect of FSH and LH secretion when gonadotrophs were treated with low and high doses of estrogen and progesterone.

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EXPERIMENT-2

EFFECT OF IN VITRO PRODUCED GONADOTROPINS (FSH/LH) ON THE HEALTH AND REPRODUCTIVE PARAMETERS OF FEMALE RABBITS ______

MATERIALS AND METHODS

Experimental rabbits and treatments

Twenty four pre-pubertal female rabbits, aged 10-12 weeks of mixed breed were purchased from local market, for the experiment. These rabbits were kept in the Department of Theriogenology University of Agriculture Faisalabad, under naturally prevailing climatic conditions and were offered lush green fodder ad libitum, and were allowed to acclimatize for one week before the start of experiment. These rabbits were randomly divided into six experimental groups’ viz., A, B, C, D, E and F with four rabbits in each group (Njidda and Isidahomen, 2010). The body weight of these rabbits was measured by electric balance.

Experimental rabbits of groups A and B were injected with sufficient amount of culture extract of buffalo adenohypophysis having FSH activity≈4.0 and 40.0 IU in equally divided dose twice daily at an interval of 12 hours for five days. Similarly, rabbits in groups C and D were injected with sufficient amount of culture extract of buffalo adenohypophysis having LH activity≈8.5 and 85 IU in equally divided dose twice daily at an interval of 12 hours for five days. Rabbits in group E and F were kept as placebo/control-1 (C-1) and control-2 (C-2), respectively, and were injected culture free medium RPM1-1640 in an equal volume, following the same protocol as for treatment of groups A, B, C and D (Rose et al., 2000).

Physical examination of experimental rabbits

Experimental rabbits were thoroughly observed daily for local irritation/redness at the site of injections and for the presence of any hypersensitivity or toxicity sign developed by the injections of in vitro produced FSH and LH from buffalo adenohypophysis.

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Collection of Blood

About 2.0 ml blood was collected from each rabbit through jugular puncture using a sterilized syringe before treatment (day 0) and on day 1, 3 and 6 during treatment with in vitro produced FSH and LH from buffalo adenohypophysis. Each blood sample was divided in to two parts. About 1.5 ml blood was kept in a test tube without anticoagulant and was allowed to stand at room temperature for the separation of serum. The remaining 0.5 ml blood was placed in a heparinized test tube and following hematological observations were recorded for each experimental rabbit as described by Benjamin (1979) and Rahman et al. (2005):

 Total RBC count  Total WBC count  PCV  Haemoglobin (Hb) concentration

The following biochemical constituents were determined in the serum collected at day 0 and 6 using commercially available FSH and LH ELISA kits (BioCheck, USA). Processed sample was observed by ELISA reader (Biotek ELX- 808, USA).

 Serum FSH

 Serum LH

Necropsy findings

On day sixth of the experiment just after collection of the last blood sample, each experimental rabbit was sacrificed. The abdomen was incised; diaphragmatic muscles, peritoneum, liver, kidney, spleen, stomach, urinary bladder, ovaries and uterus, were observed for gross pathological lesions caused by in vitro produced FSH and LH administration.

Ovarian examination

Numbers of observable soft developing GF present on the ovaries was recorded, using magnifying glass. The ovaries of the experimental females were separated and their length, width were recorded with the help of Vernier Calipers. Weight of each ovary was recorded in milligrams using an electrical balance. Volume of each ovary was calculated by dropping the ovary in the measured quantity of PBS

63 kept in a graduated glass cylinder. From the final measurement, previously measured observation was subtracted and the difference was recorded as volume of the ovary (Steelman and Pohley, 1953; Uberoi and Meyer, 1967, Umer et al., 2009).

Histological examination

Ovaries liver, kidney and ovaries were removed. Tissue samples of approximately 10x10x5 mm size were cut from the proximal, distal and middle portions of each organ and placed in buffered formalin solution for fixation. The tissues were then dehydrated, infiltrated, embedded in paraffin blocks, sectioned at 5 micron thickness and stained with haematoxylin and eosin using the standard staining procedure (Humason, 1972).

Statistical analysis

Mean values (±SEM) for various variables were computed. To study the magnitude of variation in these variables among different groups, the data were subjected to analysis of variance considering the completely randomized design (Steel et al., 1997). Least significant difference (LSD) test was applied for multiple mean comparisons where necessary.

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RESULTS AND DISCUSSION

EFFECT OF IN VITRO PRODUCED GONADOTROPINS (FSH/LH) ON FEMALE RABBITS ______

Physical expression of female rabbits

Experimental rabbits were treated with in vitro produced gonadotropins from buffalo adenohypophysis having FSH activity≈4.0, 40.0 IU; LH activity≈8.5 and 85 IU and culture free medium RPMI-1640≈0.125 and 1.25 ml twice daily for five days. They were closely observed during the experimental period. There was no treatment- related mortality in any group in this study. No hypersensitivity or toxic signs were expressed physically by any of the experimental rabbits up to 24 hours after the last injection. There was no sign of swelling at the site of injection. These results are in line with those of Kumar et al. (2000), who reported no hypersensitivity in the rabbits while studying chronic systemic toxicity of immunization with gonadotropin in rats and rabbits. Umer et al. (2009) also reported no sign of toxicity when extract drawn from culture of buffalo adenohypophysis was injected to pre-pubertal female rabbits for five days. During blood sampling there was no effect on the general behavior (aggressiveness or lethargy) in treated rabbits. In early clinical trials with Leuprolide (having activity like gonadotropins) doses as high as 20 mg/day for up to two years caused no adverse effects (Anonymous, 2012).

Effect of FSH and LH on body weight

Data regarding mean (±SE) body weight of pre-pubertal female rabbits used in the experiment are presented in Table 7. Before treatment, mean (±SE) weights of experimental rabbits assigned to groups A, B, C, D, E and F were 722±12.25, 698±15.81, 711±12.25, 708±8.09, 695±10.44 and 721±8.09g, respectively. After treatment of experimental rabbits with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5, 85.0 IU; placebo (C-1 and C-2), their mean (±SE) weights were 712±12.25, 706±12.25, 696±15.81, 717±8.09, 705±8.09 and 696±10.44g, respectively. Statistical analysis revealed non-significant differences among treatments, between treatments and control and between control groups. It indicated that in vitro produced FSH and LH from buffalo adenohypophysis did not affect the body weight of pre-pubertal female rabbits.

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The results of present study are in close agreement with the findings of Jeuniewic et al. (1978), who reported non-significant effect of FSH on body weight of patients. Polyamines play an essential role in tissue growth and differentiation, in body weight increment, in brain organization, and in the molecular mechanisms of hormonal action, intracellular signaling, and cell-to-cell communication (Thyssen et al., 1997). The non-significant change in weight during the experiment might be due to short period of treatment and such changes may require longer period of experimental study. It is therefore, concluded that treating rabbits with in vitro produced pituitary gonadotropins for short period did not affect their body weight.

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Table 7: Mean (±SE) body weight of experimental rabbits before and after treatment with in vitro produced FSH, LH and placebo.

Body weight (g) Treatment Group Before treatment After treatment

A 722±12.25 712±12.25 FSH (4.0)

(IU) B 698±15.81 706±12.25 (40.0)

C 711±12.25 696±15.81 LH (8.5)

(IU) D 708±08.09 717±08.09 (85)

Control-1 E 695±10.44 705±08.09

Control-2 F 721±08.09 696±10.44

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Effect of in vitro produced FSH and LH on hematological variables

Erythrocyte (RBC) count

Data for mean (±SE) RBC count in the blood samples collected from pre- pubertal female rabbits before and after treatment with in vitro produced FSH≈4.0 and 40 IU; LH≈8.5 and 85.0 IU; placebo/control-1 and control-2 at day 0, 1, 3 and 6 are presented in Table 8.

Mean (±SE) RBC counts in the blood of female rabbits before treatment on day-0 were 5.56±0.19, 6.26±0.19, 5.52±0.19, 5.72±0.19, 5.93±0.24 and 5.97±0.24xl06/µL for groups A, B, C. D, E and F, respectively. After treatment of rabbits with in vitro produced FSH≈4.0 and 40 IU; LH≈8.5 and 85.0 IU; placebo/control-1 and control-2 at day-6, mean RBC counts for rabbits in groups A, B, C. D. E and F were 5.76±0.19, 5.98±0.19, 5.62±0.19, 5.54±0.19, 5.93±0.24 and 6.00±0.24xl06/µL, respectively. Significantly (P<0.008) higher mean RBC count (6.26±0.19xl06/µL) was recorded for rabbits in group B compared with 5.52±0.16xl06/µL recorded for rabbits in group C at day-1 of the experiment. However, when experimental rabbits were treated with in vitro produced FSM, LH and placebo, statistical analysis of the data revealed non-significant difference among groups during and after treatment in respect of RBC count in their blood. Similarly, non-significant change was found in respect of mean RBC count being 5.83±0.08, 5.83±0.08, 5.91±0.08 and 5.81±0.08 xl06/µL at day 0, 1, 3 and 6, respectively. Whereas, when rabbits in all groups were compared with each other, rabbits in group E (C-1) showed highest mean RBC count (6.05±0.13 xl06/µL) compared with 5.64±0.11, 6.01±0.10, 5.60±0.08, 5.73±0.08, 6.05±0.13 and 5.95±0.10 xl06/µL recorded for groups A, B, C, D and F, respectively, however, difference among groups after treatment was also non-significant.

The RBC count ranged from 5.1 - 7.8 xl06/µL and mean count was 5.81±0.11 xl06/µL at day-0 before treatment in the blood of experimental rabbits. After treating the experimental rabbits, RBC count ranged from 5.2 to 7.8 x l06/µL and mean was 5.82±0.04 xl06/µL at day-6. Statistical analysis revealed non-significant differences among control and treated groups of experimental rabbits in respect of RBC count.

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Table 8: Mean (±SE) red blood cell count (x106/µL) in the blood of pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Days Group Treatment Group mean D-0 D-1 D-3 D-6

A 5.56±0.19 5.60±0.19 5.64±0.19 5.76±0.19 5.64±0.19 FSH (4.0)

(IU) B 6.26±0.19 6.20±0.19 5.96±0.19 5.98±0.19 6.01±0.19 (40.0)

C 5.52±0.19 5.54±0.19 5.70±0.19 5.62±0.19 5.56±0.19 LH (8.5)

(IU) D 5.72±0.19 5.74±0.19 5.92±0.19 5.54±0.19 5.73±0.19 (85)

Control-1 E 5.93±0.24 6.07±0.24 6.27±0.24 5.93±0.24 6.05±0.12

Control-2 F 5.97±0.24 5.83±0.24 6.00±0.24 6.00±0.24 5.95±0.12

Overall mean 5.83±0.08 5.83±0.08 5.91±0.08 5.81±0.08 5.82±0.04

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The results of present study are in partial agreement with Kumar et el. (2000), who reported non-significantly difference in RBC count in rabbit blood while studying the chronic toxicity and reversibility of anti-fertility effects of immunization against GnRH, values being 6.10±0.20xl06/µL Vs 5.94±0.07xl06/µL before and after completion of the experiment, respectively. However, they reported significant (P<0.01) difference among rats assigned to control, immunized and castrated groups, values being 7.87±0.11, 7.40±0.08 and 7.29±0.14xl06/µL, respectively, after treatment with medium containing gonadotropins hormone like properties.

During the present experiment, mean RBC counts of 5.83±0.08, 5.83±0.08, 5.91±0.08 and 5.81±0.08xl06/µL were recorded at day 0, 1. 3 and 6, respectively, overall mean was 5.82±0.04xl06/µL and range of RBC count was 5.2 to 7.8 x l06/µL; all values were within reference range reported in the literature (Fowler, 1978; Hewitt et al., 1989; Yakubu et al., 2008; Barnes et al., 2008; Black et al., 2009). However, in a previous experiment, Umer et al. (2009) reported significantly (P<0.01) decreased number of RBC (5.57±0.18xl06/µL Vs 6.43±0.28xl06/µL) compared with control when pre-pubertal female rabbits were treated with culture extract of buffalo adenohypophysis containing FSH≈2.78mIU. Hewitt et al. (1989) reported RBC count range from 5.2 to 16.5xl06/µL and mean 6.0±0.6xl06/µL in clinically healthy rabbits. Similarly, Fowler (1978) and Barnes et al. (2008) reported mean RBC count of 7.75xl06/µL and 6.2xl06/µL. Fowler (1978) also reported RBC range from 6.45 to 9.02xl06/µL in rabbits. Olayemi and Nottidge (2007) reported mean RBC count of 8.38±2.30xl06/µL in young and 6.23±2.02xl06/µL in adult New Zealand rabbits. Present results are also in close agreement with those of Yakubu et al. (2008) who reported range of RBC 4.79 to 6.74xl06/µL in healthy rabbits kept at various stocking density. Moreover, they also reported non-significant differences among various groups. The results of present study are also in close range reported earlier in literature (Mitruka and Rawnsley, 1977; Jain, 1986; Kerr, 1989; Akhtar and Rahman, 2005; Burnett et al., 2006; Olayemi and Nottidge, 2007). However, Cetin et al. (2009) reported higher mean RBC count (8.05±0.51xl06/µL) in Angora male rabbits.

No toxic effect or decrease in RBC count was observed in the experimental rabbits. RBC's are involved in the transport of oxygen and carbon dioxide in the body (Isaac et al., 2013). Thus, a reduced RBC count decreases the level of oxygen that would be carried to the tissues. Such changes in haematological parameters are often

70 used to determine various status of the body and to determine stresses or pathological factors (Etim et al., 2014). From the results of present investigations it is concluded that injecting in vitro produced FSH≈4.0, 40 1U; LH≈8.5, 85.0 IU and placebo materials did not result in stress, therefore non-significant effect on RBC count was observed.

White blood cells (WBC) count

Table 9 represents mean (±SE) values for white blood cell (WBC) count in the blood samples collected at day 0, 1, 3 and 6 from pre-pubertal female rabbits treated with in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU and placebo/C-1and C-2.

Results of the present experiment showed that mean (±SE) WBC count in the blood of female rabbits before treatment on day-0 were 7.76±0.30, 7.64±0.30, 7.78±0.30, 7.44±0.30, 7.83±0.39 and 7.53±0.39xl03/µL for groups A, B, C, D, E and F, respectively, and mean was 7.66±0.14xl03/µL. On day-6 of the experiment, WBC counts in female rabbits in groups A, B, C, D, E and F treated with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5, 85.0 IU; placebo/control-1 and control-2 were 7.38±0.30, 7.38±0.30. 7.70±0.30, 7.64±0.30, 7.47±0.39 and 7.67±0.39xl03/µL, respectively, and mean was 7.54±0.14 x l03/µL. Statistical analysis revealed non-significant difference among groups of rabbits in respect of mean WBC count at day-1 and day-6 of the experiment. Group wise mean WBC counts after the completion of treatment were 7.60±0.15, 7.51±0.15, 7.83±0.15, 7.58±0.I5, 7.77±0.19 and 7.63±0.19xl03/µL for groups A, B, C, D, E and F, respectively; statistical analysis showed non-significant difference among groups. Similarly, non-significant difference was found in the mean WBC count (7.66±0.I4, 7.67±0.14, 7.73±0.14, and 7.54±0.14xl03/µL) at day 0, 1, 3 and 6, respectively. Moreover, statistical analysis also revealed non-significant difference among groups in respect of WBC count in their blood, when experimental rabbits were treated with in vitro produced FSH, LH and placebo. The WBC count in the experimental rabbits ranged from 5.8 to 8.6xl03/µL with a mean count of 7.64±0.06 x l03/µL in the blood of experimental rabbits and coefficient of variation was 8.09.

The results of present study are in close agreement with those of Kumar et al. (2000) who reported non-significantly difference in WBC count of rabbit blood while studying the chronic toxicity and reversibility of anti-fertility effects of immunization

71 against GnRH, values being 7.07±0.43 Vs 7.40±0.27xl03/µL in control and immunized rabbits, respectively. They immunized the experimental rabbits with medium containing gonadotropins hormone like activity.

During the present experiment range of WBC (5.8 to 8.6xl03/µL) and mean count (7.64±0.06 x l03/µL) in the blood of experimental rabbits was in close range of reference values reported in the literature (Mitruka and Rawnsley, 1977; Fowler, 1978; Hewitt et al, 1989; Jain, 1986; Kerr, 1989; Akhtar and Rahman, 2005; Olayemi and Nottidge, 2007; Owen et al., 2008; Yakubu et al., 2008; Barnes et al., 2008; Black et al., 2009; Njidda and Isidahomen, 2010). Whereas, Burnett et al. (2006) reported 8.30±0.70 and 8.80±0.50xl03/µL WBC count in juveniles and adult rabbits. They also reported that WBC count was affected significantly (P<0.05) by breed. However, Umer et al. (2009) reported significantly (P<0.01) increased number of WBC count after treating pre-pubertal female rabbits with culture extract of buffalo adenohypophysis having FSH activity≈2.78mIU (10.06±0.11xl03/µL Vs 9.72±0.12xl03/µL). Cetin et al. (2009) reported significantly (P<0.05) decreased WBC count during pregnancy in rabbits compared with non-pregnant, however, no pregnant rabbit was included in the present study.

Range of WBC count (5.8 to 8.6xl03/µL) in the blood of experimental rabbits recorded during the present study is in close agreement with Hillyer (1994) and Anonymous (2009) who reported 5-13 xl03/µL and 4.5-11 xl03/µL WBC's in healthy young rabbits. These results indicated that the rabbits remained healthy because decrease in number of WBC below the normal range is an indication of allergic conditions, anaphylactic shock and certain parasitism, while elevated values (leucocytosis) indicate the existence of a recent infection, usually with bacteria (Ahamefule et al., 2006). The major functions of the white blood cell and its differentials are to fight infections, defend the body by phagocytosis against invasion by foreign organisms and to produce or at least transport and distribute antibodies in immune response (Soetan et al., 2013). From the results of the present investigations it is concluded that injecting in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU and placebo materials did not result in causing stress on rabbits, therefore non-significant effect on WBC count was observed.

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Table 9: Mean (±SE) white blood cell count (x103/µL) in the blood of pre- pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Days Group Treatment Group mean D-0 D-1 D-3 D-6

A 7.76±0.30 7.66±0.30 7.58±0.30 7.38±0.30 7.60±0.15 FSH (4.0)

(IU) B 7.64±0.30 7.46±0.30 7.54±0.30 7.38±0.30 7.51±0.15 (40.0)

C 7.78±0.30 7.88±0.30 7.94±0.30 7.70±0.30 7.83±0.15 LH (8.5)

(IU) D 7.44±0.30 7.64±0.30 7.60±0.30 7.64±0.30 7.58±0.15 (85)

Control-1 E 7.83±0.39 7.53±0.39 8.23±0.39 7.47±0.39 7.77±0.19

Control-2 F 7.53±0.39 7.87±0.39 7.47±0.39 7.67±0.39 7.63±0.19

Overall mean 7.66±0.14 7.67±0.14 7.73±0.14 7.54±0.14 7.64±0.06

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Haemoglobin (Hb) concentration

Data for mean (±SE) Hb concentration in blood samples collected from pre- pubertal female rabbits before and after treatment with in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU; placebo/control-1 and control-2 at day 0, 1. 3 and 6 are presented in Table 10.

Mean (±SE) Hb concentrations in the blood of female rabbits before treatment on day-0 were 11.32±0.18, 11.12±0.18, 10.60±0.18, 10.72±0.18, 10.60±0.23 and 10.73±0.23g/dL for groups A, B, C, D, E and F, respectively. After treatment of rabbits with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5, 85.0 IU; placebo/control-1 and control-2, at day-6 the mean Hb concentrations of rabbits in groups A, B, C, D, E and F were 10.70±0.18, 10.66±0.18, 10.36±0.18, 10.86±0.18, 10.40±0.23 and 10.60±0.23g/dL, respectively. Statistical analysis revealed significantly (P<0.005) higher mean Hb concentration (11.32±0.18 and 11.12±0.18g/dL) for groups A and B than 10.60±0.18, 10.72±0.18, 10.60±0.23 and 10.73±0.23g/dL found for groups C, D, E and F, respectively, at day-1 of the experiment. After treatment with in vitro produced FSH, LH and placebo, statistical analysis of the data revealed non- significant difference among groups during and after treatment in respect of Hb concentration in their blood. Moreover, non-significant difference was found in respect of overall mean Hb concentration being 10.85±0.08, 10.67±0.08, 10.62±0.08 and 10.60±0.08g/dL, at day 0, 1, 3 and 6, respectively.

During the study, Hb concentration in the blood of experimental rabbits ranged from 10.0 to 12.2g/dL with a mean of 11.32±0.30g/dL on day-0 before the start of treatment. After treating the experimental rabbits, Hb concentration in their blood ranged from 10.5 to 10.8g/dL with a mean concentration of 10.70±0.04g/dL recorded at day-6. Statistical analysis revealed non- significant differences among control and treated groups of experimental rabbits in respect of Hb concentration. The results of present study are in agreement with those of Kumar et al. (2000) who concluded that active immunization against GnRH-TT showed no systemic toxic effects in male rats and rabbits, they also reported Hb concentration being 13.14±0.41 and 12.97 ± 0.14g/dL in control and immunized rabbits.

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Table 10: Mean (±SE) haemoglobin concentration (g/dL) in the blood of pre- pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Days Group Treatment Group mean D-0 D-1 D-3 D-6

A 11.32±0.18a 10.74±0.18b 10.50±0.18b 10.70±0.18b 10.82±0.09b FSH (4.0)

(IU) B 11.12±0.18a 11.04±0.18b 10.74±0.18b 10.66±0.18b 10.89±0.09b (40.0)

C 10.60±0.18b 10.60±0.18b 10.82±0.18b 10.36±0.18b 10.60±0.09b LH (8.5)

(IU) D 10.72±0.18b 10.66±0.18b 10.84±0.18b 10.86±0.18b 10.77±0.09b (85)

Control-1 E 10.60±0.23b 10.40±0.23b 10.40±0.23b 10.40±0.23b 10.45±0.11b

Control-2 F 10.73±0.23b 10.60±0.23b 10.40±0.23b 10.60±0.23b 10.58±0.11b

Overall mean 10.85±0.08 10.67±0.08 10.62±0.08 10.60±0.08 10.71±0.04

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Burnett et al. (2006) stated that maturity significantly (P<0.05) affect Hb concentration, being higher (127.7±2.8 Vs 117.5 ±3.7 g/L) in adult than juvenile rabbits. They further reported that male and crossbreed rabbits had higher Hb than females. Umer et al. (2009) also reported Hb concentration ranging from 10.0 to 12.2g/dL in pre-pubertal female rabbits after treating them with culture extract of buffalo adenohypophysis having FSH activity≈2.78mIU. The Hb concentration recorded during our experiment is in accordance with reference values reported in the literature (Mitruka and Rawnsley, 1977; Fowler, 1978; Jain, 1986; Hewitt et al., 1989; Kerr, 1989; Kumar et al., 2000; Akhtar and Rahman, 2005; Burnett et al., 2006; Olayemi and Nottidge, 2007; Owen et al., 2008; Yakubu et al., 2008; Barnes et al., 2008; Cetin et al., 2009 and Black et al., 2009; Njidda and Isidahomen, 2010). From the results of present experiment it is concluded that injecting in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5, 85.0 IU and placebo materials had no effect on Hb concentration in pre-pubertal female rabbits.

Packed cell volume (PCV)

Table 11 represents mean (±SE) values for PCV (%) in the blood samples collected at day 0, 1, 3 and 6 from pre-pubertal female rabbits treated with in vitro produced FSH≈4.0, 40 IU; LH≈8.5. 85.0 IU and placebo/control-1 and controI-2.

Results of present experiment showed that mean (±SE) PCV values in the blood of female rabbits before treatment on day-0 were 34.80±0.81, 34.00±0.81, 34.60±0.81, 34.80±0.81, 31.67±1.05 and 34.67±1.05% for groups A, B, C, D, E and F, respectively, and mean was 34.09±0.37%. Non-significant difference was observed among groups in respect of PCV. At day-6 values for PCV in the blood of female rabbits treated with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5, 85.0 IU; placebo/control-1 and control-2 were 32.80±0.81, 32.40±0.81, 34.00±0.81, 33.40±0.81, 33.00±1.05 and 33.33±1.05% in groups A, B, C, D, E and F, respectively, and mean was 33.16±0.37%. Statistical analysis revealed non- significantly difference among groups in respect of PCV at day-1 and day-6 of the experiment. Group wise mean PCV after the completion of treatment was 34.05±0.40, 33.50±0.50, 33.75±0.41, 34.55±0.41, 33.67±0.52 and 34.17±0.52% for groups A, B, C, D, E and F, respectively, the difference was non-significant. Similarly, non- significant difference was found in the overall mean PCV 34.09±0.37, 33.93±0.37,

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Table 11: Mean (±SE) packed cell volume (%) in the blood of pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Days Group Treatment Group mean D-0 D-1 D-3 D-6

A 34.80±0.81 34.00±0.81 34.60±0.81 32.80±0.81 34.05±0.40 FSH (4.0)

(IU) B 34.00±0.81 33.20±0.81 34.40±0.81 32.40±0.81 33.50±0.50 (40.0)

C 34.60±0.81 33.20±0.81 33.20±0.81 34.00±0.81 33.75±0.41 LH (8.5)

(IU) D 34.80±0.81 34.20±0.81 35.80±0.81 33.40±0.81 34.55±0.41 (85)

Control-1 E 31.67±1.05 34.33±1.05 35.67±1.05 33.00±0.15 33.67±0.52

Control-2 F 34.67±1.05 34.67±1.05 34.00±1.05 33.33±1.05 34.17±0.52

Overall mean 34.09±0.37 33.93±0.37 34.61±0.37 33.16±0.37 33.95±0.18

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34.61±0.37 and 33.16±0.37% at day 0, 1, 3 and 6, respectively. Moreover, statistical analysis also revealed non-significant difference among groups in respect of PCV when experimental rabbits were treated with in vitro produced FSH, LH and placebo. During the experiment PCV (%) in the blood of rabbits ranged from 30.0 to 38.0 with a mean of 33.95±0.18%.

The results of present study are in agreement with those of Kumar et al. (2000) who reported non-significantly difference in PCV of rabbit while studying the chronic toxicity and reversibility of anti-fertility effects of immunization against GnRH, values being 38.24±1.25 and 37.65±0.48% in control and immunized rabbits, respectively. They immunized the experimental rabbits with medium containing hormone having activity like gonadotropins. However, Umer et al. (2009) reported significant (P<0.01) decrease in PCV from 36.69±0.60 to 31.63±0.66% after treating pre-pubertal female rabbits with culture extract of buffalo adenohypophysis having FSH activity. That may be due to physiological stress of using crude culture extract of buffalo adenohypophysis.

During the present experiment, PCV ranged from 30.0 to 38.0% with a mean of 33.95±0.18%. Packed cell volume percentages found during the present study are in close range as reported in the literature (Mitruka and Rawnsley, 1977; Fowler, 1978; Hewitt et al., 1989; Jain, 1986; Kerr, 1989; Akhtar and Rahman, 2005; Ahamefule et al., 2006; Olayemi and Nottidge, 2007; Ewuola and Egbunik, 2008; Owen et al., 2008; Yakubu et al., 2008; Barnes et al., 2008 and Black et al., 2009; Umer et al., 2009; Njidda and Isidahomen, 2010). Yakubu et al. (2008) also reported non-significant differences in PCV being 34.5, 32.9, 28.4 and 28.2% in healthy rabbits kept at various stocking density. Fowler (1978) reported similar values for PCV being 42 to 55% and mean 48%, in healthy New Zealand White rabbits. Moreover, Olayemi and Nottidge (2007) reported mean PCV 30.13±6.10% in healthy young and New Zealand White rabbits. From the results of present investigations it is concluded that injecting in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU and placebo materials did not affect PCV.

Effect of in vitro produced FSH and LH on ovarian biometry

Data for mean (±SE) values for length, width, weight and volume of the ovaries collected from pre-pubertal female rabbits treated with in vitro produced

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FSH≈4.0, 40 IU (group A and B); LH≈8.5, 85.0 IU (group C and D); placebo/control- 1 and control-2 (group E and F) are presented in Table 12, 13, 14 and 15, respectively.

Length and width of rabbit ovaries

Mean (±SE) length of left and right ovary collected from pre-pubertal female rabbits assigned to groups A, B, C, D, E and F was 0.73±0.01, 0.71±0.01, 0.60±0.01, 0.57±0.01, 0.6±0.01 and 0.59±0.01cm, respectively (Table 12). Statistical analysis revealed significantly (P<0.001) higher mean length (0.73±0.01 and 0.71±0.01cm) of ovaries collected from rabbits treated with in vitro produced FSH≈4.0 and 40 IU (A. B) than 0.60±0.01, 0.57±0.01, 0.61±0.01 and 0.59±0.01cm, recorded for groups C, D, E and F treated with LH≈8.5, 85.0 IU; placebo (C-1 and C-2), respectively. Data also showed that mean length of right and left ovary collected from rabbits treated with FSH≈4.0 IU and 40 IU was significantly (P<0.001) higher than other groups, however, difference between left and right ovary was non-significant. Moreover, difference between right and left ovary was also non-significant among all groups. Overall mean length for left and right ovary was 0.64±0.01cm, and ranged from 0.54 to 0.78 cm.

Table 13 represents mean (±SE) width of left and right ovary collected from pre-pubertal female rabbits assigned to groups A, B, C. D, E and F. Statistical analysis showed that mean width of the ovaries collected from rabbits in groups A and B treated with in vitro produced FSH≈4.0 and 40 IU was significantly (P<0.001) higher (0.25±0.00 and 0.24±0.00 cm) than ovaries collected from rabbits in groups C, D, E and F (0.22±0.00, 0.22±0.00; 0.23±0.00 and 0.23±0.00 cm) treated with LH≈8.5, 85.0 IU; placebo/control-1 and control-2, respectively. However, difference between left and right ovary was non-significant in respect of their width among all group. Mean width of ovaries was 0.23±0.00 cm, and range was 0.21 to 0.26 cm.

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Table 12: Mean (±SE) ovarian length (cm) of pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Ovarian length (cm) Treatment Group L R Mean

A 0.72±0.01a 0.74±0.01a 0.73±0.01a FSH (4.0)

(IU) B 0.71±0.01a 0.71±0.01a 0.71±0.01a (40.0)

C 0.59±0.01b 0.60±0.01b 0.60±0.01b LH (8.5)

(IU) D 0.57±0.01b 0.57±0.01b 0.57±0.01b (85)

Control-1 E 0.60±0.01b 0.60±0.01b 0.60±0.01b

Control-2 F 0.60±0.01b 0.59±0.01b 0.59±0.01b

Overall mean 0.63±0.01 0.64±0.01 0.64±0.01

Values with different letters in a column differ significantly (P<0.01)

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Table 13: Mean (±SE) ovarian width (cm) of pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Ovarian width (cm) Treatment Group L R Mean

A 0.25±0.00a 0.25±0.00a 0.25±0.00a (4.0) FSH B 0.24±0.00a 0.24±0.00a 0.24±0.00a (40.0)

C 0.22±0.00b 0.22±0.00b 0.22±0.00b (8.5) LH D 0.22±0.00b 0.23±0.00b 0.22±0.00b (85)

Control-1 E 0.23±0.00b 0.23±0.00b 0.23±0.00b

Control-2 F 0.23±0.00b 0.23±0.00b 0.23±0.00b

Overall mean 0.23±0.00 0.23±0.00 0.23±0.00

Values with different letters in a column differ significantly (P<0.01)

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Roth et al. (2002) and Mcdonald et al. (2006) reported significant increase in width of ovaries after FSH treatment. Present experiment also showed significantly (P<0.001) higher mean length (0.73±0.01 and 0.71±0.0 cm) and width (0.25±0.00 and 0.24±0.00 cm) of the ovaries collected from rabbits treated with in vitro produced FSH≈4.0 and 40 IU (group A, B) compared with mean length (0.60±0.01, 0.57±0.01, 0.61±0.01 and 0.59±0.01 cm) and width (0.22±0.00, 0.22±0.00, 0.23±0.00 and 0.23±0.00 cm) of ovaries collected from groups C, D, E and F treated with LH≈8.5, 85.0 IU; placebo/control-1 and control-2, respectively. Non-significant difference was observed in rabbits treated with LH≈8.5, 85.0 IU; placebo/control-1 and control-2. This might be due to the luteinizing effect of LH or lack of sufficient amount of hormone in placebo material by which the rabbits were treated.

Weight and volume of rabbit ovaries Mean (±SE) weight of left and right ovary collected from pre-pubertal female rabbits assigned to groups A, B, C, D, E and F are presented in Table 14. Results of the study showed significantly (P<0.001) higher mean weight (26.11±0.61 and 26.01±0.61 mg) for ovaries collected from rabbits of groups A and B treated with in vitro produced FSH (4.0 and 40 IU) than weight of ovaries (16.17±0.61, 16.06±0.61, 15.90±0.79 and 15.67±0.79 mg) collected from rabbits in groups C, D, E and F treated with LH≈8.5, 85.0 IU; placebo/control-1 and control-2, respectively. However, difference between left and right ovary in respect of weight was non- significant in all groups. Mean weight for both ovaries was 19.86±0.73 mg and range was 14.20 to 30.22 mg.

The mean (±SE) volume for left and right ovaries collected from pre-pubertal female experimental rabbits assigned to groups A, B, C, D, E and F after injecting in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU; placebo/control-1 and eontrol-2 were 0.29±0.01, 0.30±0.01, 0.17±0.01, 0.17±0.01, 0.15±0.01, 0.15±0.01 x ml3 respectively, and overall mean was 0.21±0.01 x ml3 (Table 15). Statistical analysis revealed that volume of ovaries collected from rabbits in groups A and B treated with in vitro produced FSH≈4.0 and 40 IU was significantly (P<0.01) higher than that for rabbits in groups C, D, E and F treated with LH≈8.5, 85.0 IU or placebo/C-1 and C-2, respectively. It shows that use of in vitro produced FSH≈4.0 and 40 IU in pre-pubertal female rabbits resulted in significantly higher mean length, width, weight and volume of their ovaries. The results of present

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Table 14: Mean (±SE) weight (mg) of ovaries collected from pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Ovarian weight (mg) Treatment Group L R Mean

A 25.63±0.86a 26.57±0.86a 26.11±0.61a FSH (4.0)

(IU) B 25.99±0.86a 26.04±0.86a 26.11±0.61a (40.0)

C 16.07±0.86b 16.26±0.86b 16.17±0.61b LH (8.5)

(IU) D 16.10±0.86b 16.02±0.86b 16.06±0.61b (85)

Control-1 E 16.25±0.11b 15.55±0.11b 15.90±0.79b

Control-2 F 15.35±0.11b 15.98±0.11b 15.67±0.79b

Overall mean 15.23±0.39 19.41±0.39 19.86±0.73

Values with different letters in a column differ significantly (P<0.01)

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Table 15: Mean (±SE) volume (ml3) of ovaries collected from pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

Ovarian volume (ml3) Treatment Group L R Mean

A 0.28±0.01a 0.30±0.01a 0.29±0.01a FSH (4.0)

(IU) B 0.30±0.01a 0.30±0.01a 0.30±0.01a (40.0)

C 0.17±0.01b 0.17±0.01b 0.17±0.01b LH (8.5)

(IU) D 0.16±0.01b 0.17±0.01b 0.17±0.01b (85)

Control-1 E 0.16±0.01b 0.15±0.01b 0.15±0.01b

Control-2 F 0.14±0.01b 0.15±0.01b 0.15±0.01b

Overall mean 0.20±0.00 0.20±0.00 0.21±0.01

Values with different letters in a column differ significantly (P<0.01)

84 study are in line with those of Steelman and Pohley (1953) and Brown and Wells (1966) who reported FSH-induced increase in ovarian weight in immature or hypophysectomized rodents.

Treatment with bovine FSH confirmed its intrinsic property. Similarly, Jacobs (1978) reported increased ovarian weight, after administration of FSH to 24-25 day old Sprague-Dawley immature female rat daily for 3 days. The higher ovarian weight response may potentially provide a plausible qualitative method for detection of FSH. Armstrong et al. (1989) also reported an increased ovarian activity in pre-pubertal (28-30 days old) female rats by injecting preparation having FSH activity. The results of present investigations revealed significantly (P<0.01) higher mean weight 26.11±0.61 and 26.01±0.61 mg of ovaries collected from rabbits treated with in vitro produced FSH ≈4.0 and 40 IU for five days compared with 16.17±0.61, 16.06±0.61, 15.90±0.79 and 15.67±0.79 mg, found for rabbits treated with LH≈8.5. 85.0 IU; placebo control-1 and control-2, respectively. These results are in agreement with those of Mannaerts et al. (1991 and 1994) who reported increased ovarian weight after injecting various amount of FSH in rats. They further stated that when FSH was supplemented with 0.2 to 5.0 IU of hCG, ovarian weight was augmented in a dose- dependent fashion. Umer et al. (2009) also reported significantly (P<0.01) increased weight and volume of rabbit ovaries after treating them with culture extract of buffalo adenohypophysis having FSH activity.

From the results of present experiment it is concluded that treating rabbits with in vitro produced FSH≈4.0 and 40 IU resulted in significantly (P<0.01) higher mean ovarian weight of experimental rabbits. Moreover, non-significant difference found between right and left ovary in respect of their weight might be due to their equal receptivity to the extrinsic hormone action. It is also concluded that non- significant difference between rabbits treated with FSH≈4.0 or 40 IU might be due to the optimum response to both doses of the hormone (Grimek et al., 1976).

Number of Graafian follicles (GF)

Table 16 represents mean (±SE) number of Graafian's follicles (GF) on left and right ovary collected from pre-pubertal female rabbits assigned to groups A, B, C, D, E and F treated with in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU; placebo/C-1 and C-2, respectively.

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Table 16: Mean (±SE) number of Graafian follicles on the ovaries of pre-pubertal rabbits treated with in vitro produced FSH, LH and placebo.

No. of Graafian follicles Treatment Group Left ovary Right ovary Mean

A 9.00±0.43a 9.40±0.43a 9.20±0.31a FSH (4.0)

(IU) B 8.80±0.43a 8.00±0.43a 8.40±0.31a (40.0)

C 0.80±0.43b 0.60±0.43b 0.70±0.30b LH (8.5)

(IU) D 0.40±0.43b 0.40±0.43b 0.40±0.43b (85)

Control-1 E 0.67±0.51b 0.67±0.51b 0.67±0.39b

Control-2 F 0.33±0.55b 0.00±0.55b 0.17±0.39b

Overall mean 3.33±0.19 3.18±0.19 3.69±0.58

Values with different letters in a column differ significantly (P<0.01)

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Mean (±SE) number as 9.20±0.31 and 8.40±0.31 of developing GF present on the ovaries of rabbits in groups A and B treated with in vitro produced FSH≈4.0 and 40 IU, respectively, was significantly (P<0.001) higher than found on the ovaries of rabbits in groups C, D, E and F being 0.70±0.30, 0.40±0.30, 0.67±0.39 and 0.17±0.39, respectively, treated with LH≈8.5, 85.0 IU; placebo/control-1 and control-2, respectively. However, difference among groups C, D, E and F was non-significant in respect of mean number of developing GF. Moreover, difference between left and right ovary was also non-significant among all groups in respect of number of developing GF on them. Mean number of developing GF on left and right ovary of experimental rabbits was 3.33±0.19 Vs 3.18±0.19 and overall mean was 3.69±0.58. Number of GF on the ovaries ranged from 0.00 to 10.00.

Plate 3 represents plain ovaries of a rabbit treated with placebo. Plate 4 represents ovaries of a rabbit treated with in vitro produced FSH≈4.0 IU, showing multiple number of developing Graafian follicles. Macro-photograph (Plate 5) of a rabbit treated with in vitro produced FSH≈40 IU showed ovaries containing multiple numbers of GFs.

The results of present study are in line with those of Steelman and Pohley (1953), Brown and Wells (1966) and Armstrong et al. (1989) those reported significantly increased follicle number in immature or hypophysectomized rodents or increased ovulation in rabbits after FSH injections. Mannaerts et al. (1994) also recorded higher number of antral follicles in a dose-dependent manner, and a gradual shift of small antral follicles to large pre-ovulatory follicles, after treating immature hypophysectomized (hypox) rats twice daily for four days with a total dose either of 2.5 to 40 IU FSH. Results of present study are also in agreement with the findings of Roth et al. (2002) that treatment of cows with FSH resulted in higher mean number of follicles than in untreated cows, while studying the effects of treatment with FSH or bovine somatotropin on the quality of oocytes aspirated during autumn from previously heat-stressed cows. Similar results were found by Balasch et al. (2001). Mcdonald et al. (2006) also reported that exogenous administration of either pFSH or PMSG to hairy nosed Wombats can induce follicular growth and oocyte maturation. It is concluded that the results of present study might be due to effects of FSH resulting in increased ovarian activity. Presence of total number of antral follicle (GF) may be considered more

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Plate 3: Ovaries of a rabbit in control group showing plane surface without Graafian follicles (encircled).

Plate 4: Ovaries of a rabbit treated with FSH showing multiple growing Graafian follicles (arrows).

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Plate 5: Ovaries of rabbit in group B treated with FSH showing multiple Graafian follicles (arrows).

89 sensitive parameter in predicting the ovarian response to gonadotropin treatment for IVF than the total ovarian volume (Yewade et al., 2009).

Serum FSH and LH concentrations in experimental rabbits

Serum samples collected from pre-pubertal female rabbit were processed using HPLC. Data regarding mean (±SE) serum FSH and LH concentrations for pre- pubertal female rabbits assigned to groups A, B, C, D, E and F before and after treatment with in vitro produced FSH≈4.0, 40 IU, LH≈8.5, 85.0 IU; placebo/C-1 and C-2 at day 0 and 6 are presented in Table 17.

The mean (±SE) serum FSH concentrations in pre-pubertal female rabbits assigned to groups A, B, C, D, E and F before treatment at day-0 were 4.38±1.94, 3.54±1.94, 4.53±1.94, 3.13±1.94, 3.61±2.45 and 4.09±2.45 IU/ml. Serum FSH level in rabbits of groups A, B, C and D were significantly (P<0.001) increased to 422.41±7.63, 462.70±7.63, 308.40±7.63 and 273.15±7.63 IU/ml after five days by treating them with in vitro produced FSH≈4.0, ≈40 IU, LH≈8.5 and 85.0 IU, respectively. However, non-significant change was observed in rabbits treated with placebo material as C-1 and C-2 being 3.61±2.45 and 4.09±2.45 IU/ml Vs 3.71±2.45 and 9.18±2.45 IU/ml.

The mean (±SE) serum LH concentrations in the blood of pre-pubertal female rabbits assigned to groups A, B, C, D, E and F at day-0 were 3.85±1.13, 4.12±1.13, 4.27±1.13, 3.58±1.31, 3.48±1.32 and 3.67±1.32 IU/ml, respectively. After five days of treatment with in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU; placebo/control-1 and control-2 at day-6 the serum LH concentrations were 22.85±3.13, 41.12±3.13, 34.27±3.13, 43.58±3.31, 4.48±2.52 and 5.67±2.52 IU/ml. Statistical analysis revealed significantly (P<0.01) higher mean LH after five days of treatment with in vitro produced FSH≈4.0, 40 IU; LH≈8.5, 85.0 IU/ml.

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Table 17: Mean (±SE) serum FSH and LH (mIU) in the experimental rabbits before and after treatment with in vitro produced FSH.

FSH LH Treatment Group Day-0 Day-6 Day-0 Day-6

A 4.38±1.94a 422.41±7.63b 3.85±1.13a 22.85±3.13b FSH (4.0)

(IU) B 3.54±1.94a 462.70±7.63b 4.12±1.13a 41.12±3.13b (40.0)

C 4.53±1.94a 308.40±7.63b 4.27±1.13a 34.27±3.13b LH (8.5)

(IU) D 3.13±1.94a 273.15±7.63b 3.58±1.13a 43.58±3.13b (85)

Control-1 E 3.61±2.45a 3.71±2.45a 3.48±1.32a 4.48±2.52a

Control-2 F 4.09±2.45a 9.18±2.45a 3.67±1.32a 5.67±2.52a

Values with different letters in a row for each hormone differ significantly (P<0.01)

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From the results of the present study it is concluded that treating rabbits with in vitro produced FSH≈4.0, 40 IU; LH≈8.5 and 85.0 IU resulted in higher mean FSH and LH in their blood. These results are in accordance with those of Akbar et al. (1974) who reported serum FSH being 66±8 ng/ml during follicular or luteal phase and in estrus cow 78±8 ng/ml and significant increase in FSH coinciding with LH around estrus after injection of 250 µg of GnRII. They also reported non-significant change from luteal phase to estrus without treatment. While, studying the effect of rabbit doe-litter separation on 24-hour changes of LH, FSH and prolactin release in female and male suckling pups, Cano et al. (2005) reported mean values for FSH being 66.1±13.6, 89.9±22.6 ng/ml and for LH being 6.13±1.37, 6.97±1.57 ng/mL in control and isolated females pups of rabbits. Though, the measuring units are different, however, the ratio between FSH and LH is very close as found during the present investigations.

Necropsy findings

The gonadotropins are cleared from the blood by both the kidney and the liver. Small amounts are also bound to the gonads, but this accounts for little of the hormone clearance. About 80 to 90% of the gonadotropins are metabolized in the hepatic and renal parenchyma. The half-life of FSH i.e. 4 to 5 hours is much longer than that of LH being 30 to 60 minutes; this is due partly to the higher sialic acid content of FSH, which impairs clearance, particularly by the liver, and partly to the high proportion of sulfated carbohydrate termini in LH, which accelerates its hepatic clearance (Bremner et al., 2006). Therefore, liver and kidney were examined microscopically. No gross pathological lesion was observed in the liver, kidney, spleen, stomach, urinary bladder, ovaries, uterus, diaphragm and peritoneum of pre- pubertal female experimental rabbits after the injections of in vitro produced gonadotropins from buffalo adenohypophysis. Plate 6 represent photomicrograph of an ovary collected from a rabbit in group A, treated with in vitro produced FSH. Multiple numbers of Graafian's follicles are prominent, while another photomicrograph (Plate 7) of a ovary collected from a control rabbit shows small non-developing GF.

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Plate 6: Photomicrograph of an ovary collected from a rabbit treated with in vitro produced FSH, showing multiple growing Graafian follicles.

Plate 7: Photomicrograph of an ovary collected from a rabbit treated with placebo (control) showing small non-developing GF.

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Photomicrograph (Plate 8) of a liver collected from a rabbit treated with in vitro produced FSH shows normal parenchyma and normal appearance of cellular mass, no pathological lesion is seen. Kidney of a rabbit treated with high amount of gonadotropin is shown in Plate 9 with normal muscular and excretory tissue.

These results are in agreement with those of Reza et al. (2001) who also reported no pathological change in the liver while studying ovarian hyper-stimulation syndrome followed by administration of human exogenous gonadotropins in rabbits. However, they reported congestion in the uterus, ovaries having multiple corpus luteum after ovulation, congestion on the lungs and hemorrhages present on the kidneys, whereas no such changes were observed in any of experimental rabbit during the present study. Kumar et al. (2000) concluded that immunization with GnRH had no systemic toxicological effects in the adult male rats and rabbits. They also reported no pathological changes in the immunized animals.

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Plate 8: Photomicrograph of liver collected from a rabbit treated with in vitro produced FSH showing normal parenchyma.

Plate 9: Photomicrograph of kidney collected from a rabbit treated with in vitro produced FSH showing normal muscular and secretory tissue.

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The following conclusions can be drawn from results of second experiment of this study:

1. In vitro produced FSH and LH from buffalo adenohypophysis when administered in ten time high dose to pre-pubertal female rabbits did not effect on most of the hematological parameters in rabbits, viz., RBC, WBC count; PCV and MCH. 2. Although the in vitro produced FSH and LH increased the platelet count and decreased MCV compared to placebo treated rabbits (control group), the values of both these parameters were within normal ranges for rabbits 3. The significantly (P<0.001) increased ovarian size and activation of numerous small GF's are good indicator for the presence of hormone activity in the purified extract of in vitro culture of buffalo adenohypophysis. 4. No mortality occurred during the experiment indicate the safety of hormones produced.

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EXPERIMENT-3

EFFECT OF IN VITRO PRODUCED FSH ON SERUM FSH, LH, ESTROGEN AND PROGESTERONE CONCENTRATIONS AND ON OVARIAN BIOMETRY IN BUFFALOES ______

MATERIALS AND METHODS

Experimental animals and treatments

For this experiment nine healthy and acyclic buffaloes about eight years of age with history of normal parturition and clinically normal reproductive tract were used. These buffaloes were maintained under the naturally prevailing climatic conditions at Department of Theriogenology, University of Agriculture Faisalabad. These buffaloes were palpated rectally, none of them showed GF or CL on their ovaries; approximate size of ovaries was recorded. The experimental buffaloes were offered good quality green fodder @ 10% body and clean drinking water ad. libitum. They were randomly divided into three groups A, B and C, with three buffaloes in each group.

The experimental buffaloes of group A were treated with in vitro produced FSH≈500 IU daily in two divided doses given s/c at an interval of 12 hours for five days. Buffaloes in group B were treated with Folltropin-V≈5mg, twice daily at an interval of 12 hours for five days and buffaloes in group C were treated with culture medium RPM1-1640 as placebo/control. After the last treatment on day-6 and day-9, these buffaloes were further palpated rectally and approximate size of ovaries was recorded, moreover, presence of functional structure was also recorded.

Determination of serum FSH, LH, estrogen and progesterone concentrations in buffaloes About 10.0 ml blood was drawn from each experimental buffalo daily before injecting treatment material. Thereafter serum was separated from each sample and stored at -20°C until analyzed for the concentration of FSH, LH, estrogen and progesterone. The serum samples were processed using bovine FSH and LH, ELISA kits (Endocrine Technologies Inc. USA). For estrogen and progesterone, serum samples were processed using commercially available EL1SA kits (Biocheck Inc, USA). Hormone concentrations were determined as per instruction of the manufacturer. Processed samples were observed by placing the micro titer well plates

97 in the ELISA reader (Biotek ELX-808, USA), FSH and LH concentration of experimental buffaloes were recorded at optical density of 450nM. Following assay procedure was adopted:

Assay procedure

• Anti-FSH, LH, estrogen or progesterone coated micro titer wells were placed in the holder. • An amount of 100 µL of each standard and sample was dispensed in the coated micro titer wells. • Enzyme conjugate reagent was added (100µl) into each well and was mixed thoroughly by shaking for 30 seconds. • The micro titer wells were incubated at 37°C for 3 hours. • Incubated mixtures were dumped into a waste container. • Micro titer wells were rinsed and dumped 5 times with wash buffer. • The micro titer wells were struck sharply onto an absorbent paper and residual water droplets were removed. • TMB reagent (100 µl) was dispensed into each well, and was gently mixed for 10 seconds. • The micro titer wells were incubated at room temperature for 20 minutes, in the dark. • A volume of 100µl of stop solution was added to each well to stop the reaction for measurement of estradiol-17β and progesterone, whereas a volume of 50µl of stop solution was added to each well to stop the reaction for measurement of FSH and LH. • After gently mixing for 30 seconds, the blue color was completely changed to yellow. • The samples were processed as described above and concentration of FSH, LH, estrogen and progesterone were recorded at optical density of 450nM by placing the micro titer wells in the ELISA reader (Biotek ELX-808, USA).

Statistical analysis

Mean values (±SEM) for various variables were computed. To study the magnitude of variation in these variables among different groups, the data were subjected to analysis of variance considering the completely randomized design (Steel

98 et al., 1997). Least significant difference (LSD) test was applied for multiple mean comparisons where necessary.

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RESULTS

______

Serum FSH and LH concentration in buffaloes treated with in vitro produced FSH The mean (±SE) concentration of serum FSH i.e. 41.6±0.9 and 42.1±0.9 ng/ml found on day one before treatment was significantly (P<0.01) raised to 98.4±0.9 and 93.1±0.9 ng/ml on day 6th of the experiment in buffaloes treated with Folltropin-V and buffaloes treated with in vitro produced FSH from buffalo adenohypophysis, respectively (Table 18). Use of Folltropin-V resulted in significantly (P<0.01) high mean serum FSH concentration 98.4±0.9ng/ml than that of 93.1±0.9ng/ml recorded in buffaloes treated with in vitro produced FSH. Buffaloes treated with Folltropin-V and in vitro produced FSH from buffalo adenohypophysis showed significantly (P<0.01) raised serum FSH concentration than those buffaloes treated with placebo/control. Non-significant change was observed in serum FSH concentration of buffaloes kept as control during the experiment. Treating buffaloes with in vitro produced FSH and Folltropin-V resulted in increased serum FSH day to day. However, no such change in serum FSH concentration was observed in control group of buffaloes.

The overall mean (±SE) serum FSH concentration for experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo was 65.5±0.3, 67.7±0.3 and 46.3±0.3 ng/ml, respectively. Treating buffaloes with Folltropin-V resulted in significantly (P<0.01) higher mean serum FSH concentration (67.7±0.3 ng/ml) than 65.5±0.3 and 46.3±0.3 ng/ml recorded for buffaloes treated with in vitro produced FSH and placebo. Moreover, difference between buffaloes treated with in vitro produced FSH and placebo was also significant (P<0.01) in respect of serum FSH concentration (65.5±0.3 Vs 46.3±0.3ng/ml). When overall mean for serum FSH concentration was computed day by day the results showed linear increase from day 1 to 6th i.e. 41.6±0.5, 45.3±0.5, 54.7±0.5, 64.5±0.5, 72.2±0.5 and 80.2±0.5 ng/ml, respectively.

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Table 18: Mean (±SE) concentration of serum FSH (ng/ml) in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Days of experiment Group Mean 1 2 3 4 5 6

In vitro produced 42.1±0.9 45.1±0.9a 57.6±0.9b 71.3±0.9b 84.0±0.9a 93.1±0.9b 65.5±0.3b FSH

Follitropin- 41.6±0.9 46.4±0.9a 59.8±0.9a 74.5±0.9a 85.3±0.9a 98.4±0.9a 67.7±0.3a V

Control 41.1±0.9 44.5±0.9a 46.5±0.9c 47.7±0.9c 48.5±0.9b 49.2±0.9c 46.3±0.3c

Overall 41.6±0.5 45.3±0.5 54.7±0.5 64.5±0.5 72.2±0.5 80.2±0.5 59.8±0.3 Mean

Values having different letters within the same column differ significantly (P<0.01).

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The mean (±SE) serum LH concentrations recorded on day one before treatments were 4.6±0.3, 3.7±0.3 and 4.6±0.3 ng/ml in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and control, respectively (Table 19). On day 6th of the experiment, serum LH concentration in buffaloes treated with in vitro produced FSH, Folltropin-V and control were raised to 29.1±0.3, 34.9±0.3 and 6.9±0.3 ng/ml, respectively. Buffaloes treated with Folltropin-V showed significantly (P<0.01) higher (34.9±0.3 ng/ml) mean serum LH concentration than 29.1±0.3 and 6.9±0.3 ng/ml in buffaloes treated with in vitro produced FSH and placebo/control, respectively. Difference between buffaloes treated with in vitro produced FSH and placebo/control was also significant (P<0.01) being 29.1±0.3 Vs 6.9±0.3 ng/ml in respect of serum LH concentration.

Serum LH concentration in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo was not changed significantly up to day 5th of treatment. However, a sharp and significant (P<0.01) increase in serum LH concentration was recorded on day 6th of the experiment (Table 19). The overall mean (±SE) serum LH concentrations for experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo were 8.1±0.1, 9.2±0.1 and 4.6±0.1 ng/ml, respectively. Use of Folltropin-V also resulted in significantly (P<0.01) high overall mean serum LH concentration, values of 9.2±0.1 Vs 8.1±0.1 and 4.6±0.1 ng/ml were recorded for buffaloes treated with in vitro produced FSH and placebo/control. However, no difference was found in mean serum LH concentration of buffaloes kept as placebo/control up to day 6 of the experiment. When overall mean for LH concentration was computed on day by day, the results showed non-significant difference up to day 5, however, significantly (P<0.01) higher mean serum LH was recorded on day 6 of the experiment being 4.3±0.2, 4.1±0.2, 3.3±0.2, 4.4±0.2, 3.9±0.2 and 23.6±0.2 ng/ml, for six days, respectively.

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Table 19: Mean (±SE) concentration of serum LH (ng/ml) in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Days of experiment Group Mean 1 2 3 4 5 6

In vitro produced 4.6±0.3 3.5±0.3 3.2±0.3 4.5±0.3 3.7±0.3 29.1±0.3b 8.1±0.1b FSH

Follitropin- 3.7±0.3 4.3±0.3 3.3±0.3 4.4±0.3 4.4±0.3 34.9±0.3a 9.2±0.1a V

Control 4.6±0.3 4.5±0.3 3.4±0.3 4.3±0.3 3.8±0.3 6.9±0.3c 4.6±0.1c

Overall 4.3±0.2 4.1±0.2 3.3±0.2 4.4±0.2 3.9±0.2 23.6±0.2 7.3±0.1 Mean

Values having different letters within the same column differ significantly (P<0.01).

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Effect of in vitro produced FSH/LH on serum estrogen and progesterone concentrations in buffaloes

Serum samples collected from experimental buffaloes were processed using commercially available estrogen and progesterone EL1SA kits (Biocheck Inc, USA) and processed samples were observed by ELISA reader (Biotek ELX-808, USA). Data are presented in Tables 20 and 21.

The mean (±SE) serum estrogen concentration found on day one before treatment was 110.4±0.8, 110.4±0.8 and 110.2±0.8 pg/ml in experimental buffaloes assigned to groups FSH, Folltropin-V and control, respectively (Table 20). On day 6th of the experiment, serum estrogen concentration in buffaloes treated with in vitro produced FSH and Folltropin-V was significantly (P<0.01) raised to 173.0±0.8 and 179±0.8 pg/ml, respectively, whereas no difference was recorded in buffaloes treated with placebo from day 1 to 6 being 110.2±0.8 Vs 112.1±0.8 pg/ml in respect of serum estrogen concentration. No change was recorded in estrogen concentration in the blood of experimental buffaloes up to day 4 by any of the treatment, however, a significant (P<0.01) increase was recorded on day 5 leading to day 6 in buffaloes treated with in vitro produced FSH and Folltropin-V.

When overall mean (±SE) serum estrogen concentration for experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo was compared, it was noted that buffaloes treated with in vitro produced FSH and Folltropin-V showed significantly (P<0.01) higher (123.5±0.5, 125.9±0.5 pg/ml) estrogen concentration than 110.7±0.5 pg/ml found in buffaloes treated with control, however, difference between experimental buffaloes treated with in vitro produced FSH and Folltropin-V was non-significant.

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Table 20: Mean (±SE) serum estrogen concentration (pg/ml) in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Days Group Mean 1 2 3 4 5 6

In vitro produced 110.4±0.8 110.6±0.8 110.7±0.8 110.7±0.8 125.8±0.8b 173.0±0.8b 123.5±0.5 FSH

Follitropin- 110.4±0.8 111.3±0.8 111.4±0.8 111.4±0.8 132.0±0.8a 179.1±0.8a 125.9±0.5 V

Control 110.2±0.8 110.3±0.8 110.4±0.8 110.4±0.8 110.7±0.8c 112.1±0.8c 110.7±0.5c

Overall 110.3±0.4 110.8±0.4 110.8±0.4 110.9±0.4 122.8±0.4 154.7±0.4 120.0±0.5 Mean

Values having different letters within the same column differ significantly (P<0.01).

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The mean (±SE) serum progesterone concentrations in experimental buffaloes assigned to group for treatment with in vitro produced FSH, Folltropin-V and control were 5.8±0.1, 5.8±0.1 and 5.7±0.1 ng/ml, respectively, on day one before treatment (Table 21). On day 6th after 24 hours of last treatment the mean serum progesterone concentration was 1.7±0.1, 1.4±0.1 and 4.5±0.1 ng/ml for buffaloes treated with in vitro produced FSH, Folltropin-V and placebo, respectively. Statistical analysis revealed significantly (P<0.01) decreased concentration of progesterone in the blood of buffaloes treated with in vitro produced FSH, Folltropin-V and control. However, difference between buffaloes treated with in vitro produced FSH and Folltropin-V was non-significant.

Comparison of overall mean (±SE) serum progesterone concentration (4.0±0.08, 3.7±0.08 and 4.8±0.08 ng/ml) for experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo, respectively, revealed significant (P<0.01) difference among three groups. However, buffaloes treated with Folltropin- V showed lowest and control group showed highest serum progesterone concentration. The trend of decrease in the serum progesterone concentration was slow in control group than those treated with in vitro produced FSF and Folltropin-V.

Correlation of serum estrogen concentration was positive with LH and FSH being r=0.3 and r=0.716, respectively, whereas, serum estrogen was negatively correlated with progesterone r=-0.697. Serum progesterone was negatively correlated with LH and FSH being r=-0.206 and r=0.326. Data for correlation is presented in Table 22.

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Table 21: Mean (±SE) serum progesterone concentration (ng/ml) in experimental buffaloes treated with in vitro produced FSH, Folltropin-V and control.

Days Group Mean 1 2 3 4 5 6

In vitro produced 5.8±0.1 5.4±0.1a 4.6±0.1a 3.8±0.1b 2.6±0.1b 1.7±0.1b 4.0±0.08b FSH

Follitropin- 5.8±0.1 5.0±0.1b 4.1±0.1b 3.3±0.1c 2.7±0.1b 1.4±0.1b 3.7±0.08c V

Control 5.7±0.1 4.9±0.1b 4.7±0.1a 4.6±0.1a 4.6±0.1a 4.5±0.1a 4.8±0.08a

Overall 5.8±0.1 5.1±0.1 4.4±0.1 3.9±0.1 3.3±0.1 2.5±0.1 4.16±0.08 Mean

Values having different letters within the same column differ significantly (P<0.01).

Table 22: Correlation between serum LH; FSH; estrogen and progesterone.

Serum LH Serum estrogen Serum progesterone

Serum estrogen 0.300*

Serum progesterone -0.206NS -0.697**

Serum FSH 0.342* 0.716** -0.326*

NS = Non-significant

* = Significant (P<0.05)

** = Significant (P<0.01)

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Effect of in vitro produced FSH on ovarian biometry of buffaloes

Length and width of buffalo ovaries

Data for mean (±SE) ovarian length and width of experimental buffaloes treated with in vitro produced FSH, Folltropin-V and placebo are presented in Table 23.

Analysis of variance under GLM revealed significantly (P<0.01) higher mean length (2.2±0.1 Vs 2.5±0.1cm and 2.0±0.1 Vs 2.3±0.1cm) for ovaries of experimental buffaloes after treating them with in vitro produced FSH and Folltropin-V, respectively. Statistical analysis also revealed significantly (P<0.01) higher mean width (1.7±0.1 Vs 2.1±0.1cm and 1.6±0.1 Vs 2.2±0.1cm) of ovaries of the experimental buffaloes treated with in vitro produced FSH and Folltropin-V, respectively. Whereas, no difference was found in the mean length and width of ovaries in buffaloes treated with placebo.

Graafian follicles

Ovaries were rectally examined for the development and presence of functional structure i.e. GF and CL. It was observed that there was numerous small GF on the ovaries of buffaloes treated with in vitro produced FSH and Folltropin-V. One of the buffalo treated with Folltropin-V showed a GF < 1.1 cm in size on one of her ovary at the end of treatment. Other GF found were less than 0.5 cm in size. In control group of buffaloes no prominent GF was observed on their ovaries. No active corpus luteum could be detected on the ovaries of any animal.

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Table 23: Group wise mean ovarian length (cm) and width (cm) of experimental buffaloes before and after treatment with in vitro produced FSH, Follitropin-V and control.

Group

In vitro produced Follitropin-V Control Mean FSH

Before 2.2±0.1b 2.0±0.1b 2.0±0.1 2.2±0.0 treatment Length After 2.5±0.1a 2.3±0.1a 2.0±0.1 2.3±0.0 treatment

Mean 2.4±0.1 2.1±0.1 2.0±0.1 2.2±0.0

Before 1.7±0.1b 1.6±0.1b 1.6±0.1 1.7±0.0 treatment Width After 2.1±0.1a 2.2±0.1a 1.6±0.1 2.0±0.0 treatment

Mean 1.9±0.1 1.9±0.1 1.6±0.1 1.8±0.1

Values with different letters within the same column for each parameter differ significantly (P<0.01).

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DISCUSSION Effect of in vitro produced FSH on serum FSH, LH, estrogen and progesterone concentrations and on ovarian biometry in buffaloes ______Estrogen and progesterone are considered to act synergistically in their effects on sensitive tissues; moreover, these hormones also act antagonistically. The activity of gonadotropins is usually not considered species specific; there is a wide variation in potency, depending upon the animal in which they are used. Moreover, they are capable of producing antibodies and immunological response. However, the antibodies or anti-hormones that appear may be species specific, but the hormones themselves are not (Zarrow et al., 1964).

GnRH is a peptide hormone produced by specific neurons in the hypothalamus. It drives the pulsatile release of FSH and LH from anterior pituitary. In vivo it acts as neurotransmitter and neuromodulator during the estrous cycle very close to estrus when the level of estrogen is maximal; estrogen sensitizes the pituitary cells binding to GnRH. Moreover, when the level of estrogen is maximal, the positive feedback on GnRH neurons in the surge center of hypothalamus initiates a massive and transient release of GnRH, which dramatically increases LH release from the pituitary, while negative feedback by estrogen reduces FSH release (Squires, 2003). However, during the present experiment, higher amount of estrogen resulted in increased FSH secretion from in vitro culture of gonadotrophs. This might be due to the effects of estrogen or volume of gonadotrophs receptors sensitive to estrogen.

The mean (±SE) concentration of serum FSH (42.1±0.9 and 41.6±0.9 ng/ml) found on day one before treatment was significantly (P<0.01) raised to 93.1±0.9 and 98.4±0.9 ng/ml on day 6th of the experiment in buffaloes treated with in vitro produced FSH and Folltropin-V. The results of present study are in close agreement with those of Schams et al. (1977) who reported that average values for FSH in a healthy cow remained above 30.0 ng/ml during estrous cycle and rose too little above 100 ng/ml during pro-estrus, just before LH peak. Similarly, Singh et al. (2001) reported peak FSH and LH levels during oestrus 38.40 ± 9.21 and 24.04 ± 4.75 ng/ml in synchronized Murrah buffaloes induced to oestrus. Results of the present study are also in close agreement with those of Terzano et al. (2012) who stated that peak value of FSH is averaged as 70-80 ng/ml in buffalo after stimulation with GnRH and this

110 peak begins at each follicular wave. Moreover, difference between buffaloes treated with in vitro produced FSH and Folltropin-V was significant (P<0.01) being 93.1±0.9 Vs 98.4±0.9 ng/ml. The difference in serum FSH may be due to higher purity of commercially available Folltropin-V than that of in vitro produced FSH from buffalo adenohypophysis which was not yet optimized in respect of purification.

The mean (±SE) concentrations of serum LH (4.6±0.3 and 3.7±0.3 ng/ml) found on day one before treatment were significantly (P<0.01) raised to 29.1±0.3 and 34.9±0.3 ng/ml on day 6th of the experiment in buffaloes treated with in vitro produced FSH and Folltropin-V. Terzano et al. (2012) also reported similar values of LH around 0.72-3.0 ng/ml during a major part of the estrous cycle and peak values being 20-40 ng/ml on the day of estrus. During the present study, use of Folltropin-V resulted in significantly (P<0.01) high mean serum LH concentration (34.9±0.3 Vs 29.1±0.3 ng/ml). The results obtained might be due to variation in FSH:LH ratio or higher purity of Folltropin-V resulting in higher estrogen level (179.1±0.8 Vs 173.0±0.8 ng/ml) in Folltropin-V treated buffalo than that of treated with in vitro produced FSH. Statistical analysis of data revealed positive correlation of serum estrogen with FSH and LH. Non-significant change was observed in the serum LH concentration of buffaloes kept as control. Whereas buffaloes treated with in vitro produced FSH and Folltropin-V showed significantly (P<0.01) raised serum LH concentration than those of control. It is obvious that low serum LH concentration in control group of buffaloes was due to lack of treatment. Avenell et al. (1985) also reported similar concentration of serum LH 5.0 ng/ml in Swamp buffaloes recorded at stages during the estrous cycle other than at estrus. In cyclic animals, different physiological states may be due to the frequency of GnRH released from the hypothalamus and changes in the ratio of LH to FSH may occur. Furthermore, the ability to induce follicle growth and ovulation is dependent on the pattern and frequency of GnRH pulses and the concomitant effect on differential secretion of LH and FSH (Crowe and Mullen, 2011).

FSH is required for normal reproductive functions in all mammals and its extrinsic use stimulates maturation of GF in the females of all species (Richards and Hedin, 1988). The maturing GF also produces increasing amount of estradiol (Robert et al., 1994; Morrow, 1986; Balasch et al., 2001; Driancourt, 2001). In cycling animals, peripheral progesterone concentration is minimum (0.1 ng/ml) on the day of

111 estrus and rises to peak level (1.6-3.6ng/ml) on day 13 to 15 of the cycle, during luteal phase (Ahmed et al., 1977; Bachalaus, 1979). Statistical analysis of the data revealed increasing trend in serum estrogen and decreasing trend in serum progesterone of experimental buffaloes treated with Folltropin-V and in vitro produced FSH from buffalo adenohypophysis. Similarly, a concomitant significant (P<0.01) increase in estradiol concentration and decrease in progesterone concentration was reported by Noseir (2003). The mean (±SE) concentration of serum progesterone (5.8±0.1 and 5.8±0.1 ng/ml) found on day one before treatment was significantly (P<0.01) decreased to 1.4±0.1 and 1.7±0.1 ng/ml on day 6th of experiment in buffaloes treated with Folltropin-V and in vitro produced FSH, respectively. Use of Folltropin-V resulted in significantly (P<0.01) low mean serum progesterone concentration (1.4±0.1 Vs 1.7±0.1 ng/ml). These results are in line with previously reported information in literature. Most of the workers indicated that peripheral plasma progesterone profile in buffalo is very similar to that of cattle (Wetteman et al., 1972; Kanai and Shimizu, 1984; Shah and Mehta, 1992; Kamboj and Prakash, 1993; Mondal et al., 2001; Mondal and Prakash, 2002a; Mondal and Prakash, 2002b, Mondal and Prakash, 2002c; Mondal et al., 2003). Buffaloes treated with Folltropin-V and in vitro produced FSH showed significantly (P<0.01) higher serum estrogen concentration than control due to the extrinsic use of FSH. The higher mean (±SE) serum estrogen concentration (110.3±0.4 - 154.4±0.4 pg/ml) and serum progesterone concentration (2.5±0.1 - 5.8±0.1 ng/ml) found during present study in experimental buffaloes compared with previously reported values might be due to variation in breed or animals having different reproductive status.

Significant (P<0.05) difference in ovarian size of experimental buffaloes after treatment with Folltropin-V and in vitro produced FSH indicated positive effect of treatment materials having biological activity like FSH. Moreover, increased size of ovaries in the treated buffaloes was also due to the presence of numerous small GFs, though the size of GFs was not greater than 0.5 cm, whereas, in untreated/control group of buffaloes no GF was observed and no change was recorded in length and width of ovaries in these animals. Present results might be due to the a-cyclic status of experimental buffaloes. Razzaque et al. (2008) also reported similar findings in Nagpuri buffaloes, with ovarian length and width being 2.45±0.07 and 1.52±0.04 cm, respectively. Previous studies have indicated that buffaloes have lower reproductive

112 efficiency and buffalo ovaries bear lower follicular population (Madan, 1990 and Totey et al., 1991). Our results are also in line with those of Patel et al. (2009), who stated that buffaloes (Bubalus bubalis) in general are known to be very poor responders to superovulation protocols in comparison to cattle. While working for superovulation in Toda buffaloes using Folltropin-V. Patel et al. (2009) also reported significant increase in the length and width of ovaries and average size of left and right ovary being 24.67±2.35 and 26.11±1.71 mm. They further reported delayed ovulation and a lower number of recruited follicles during superovulation.

There were numerous small GFs on the ovaries of buffaloes treated with in vitro produced FSH from buffalo adenohypophysis and Folltropin-V compared to control. In buffaloes treated with Folltropin-V, one GF reached up to 1.1cm. However, it did not attain ovulatory capability, whereas, GFs gain the ovulatory capability when they reach at least 10.25±1.28 mm (Rohilla et al., 2005).

From the results of the present experiment it appears that higher values of FSH may not be sufficient to improve the follicular growth, which might be due to the limited sensitization or priming of receptors found on the follicles. This is why an optimum dose of FSH must be administered for reasonable follicular response. The number of injections and method of administration are also important for the higher ovulation rates (Singh and Madan, 1998). The in vitro produced FSH resulted in comparatively higher estrogen level as compared to control which might be due to numerous small sized GFs and estrogen secretion from theca interna cells, but the values were lower than Folltropin-V treated buffaloes. Similarly, the progesterone level was lower for buffaloes treated with in vitro produced FSH than those of control. Statistical analysis of data also revealed negative correlation of estrogen with FSH and LH, values being r=-0.326 and r=-0.206, respectively. Hormones are regulated by negative and positive feedback mechanism in sexually active animals (Hafez, 1987), and very few studies on superovulation have combined data from follicular observations, embryo recovery results and hormonal profiles of FSH and estrogen throughout the super ovulatory period.

The results of the present study are in accordance with those of Demoustier et al. (1988), who reported that serum FSH level was increased soon after the administration of FSH and Folltropin-V, attained peak level after 3 hours, and then decreased uniformly. Moreover, FSH level after 12 hours post-injection could not be

113 detected. We found that subcutaneous injection of FSH resulted in the slower release in to the blood stream and this route was found to be more efficient in maintaining reasonable level of FSH in the blood for longer period. The method and schedule of hormone inoculation in buffalo adopted during the present experiment was similar to that of Roberts and Echternkamp (1993), who also used injection of FSH twice daily for five days and reported increased serum FSH level required for ovulation. The differences between in vitro produced FSH and Folltropin-V in respect of high mean serum FSH and LH level in experimental buffaloes might be due to the varying level of tissue/cell receptivity and receptor sensitization (Zarrow et al., 1964 and Squires, 2003).

Kelly et al. (1997) reported an increased number of small follicles in heifers responding adequately to superovulation predominantly on the second day of pFSH treatment. This could be related to the greater number of medium sized follicles and supported the hypothesis that administration of gonadotropins increased the number of antral follicles and decreased the number of atretic follicles, especially at the size of antral follicles and antrum formation (Monniaux et al., 1984). The animals treated with single injection of FSH did not result in satisfactory growth of medium sized follicles. On the other hand, treating twice daily at an interval of 12 hours for 5 days resulted in the growth of small sized follicles, those may reflect higher amount of peripheral estrogen from numerous small sized GF. Our results are supported by Bevers and Dieleman (1987), who stated that the synthesis of E2 from pre-ovulatory follicles was reflected in the blood estrogen level which increased slowly till the LH surge occurred.

Variation in mean range of FSH might be due to the fact that FSH and LH are influenced by the same regulatory hormones (e.g. GnRH and estradiol); there are a number of instances when their patterns of secretion diverge. One such example is seen during the follicular phase of the estrous cycle, when secretion of LH increases while secretion of FSH decreases. Moreover, preventing the interaction of GnRH with receptors in the anterior pituitary gland by hypothalamic-pituitary disconnection, immune-neutralization, or a GnRH antagonist essentially abolishes secretion of LH but has little effect on the basal secretion of FSH. Although GnRH stimulates secretion of FSH, the magnitude of the response is normally only a fraction of that observed for LH. One possible reason for this is that the second messenger system(s)

114 responsible for inducing secretion of FSH may be different from that responsible for stimulating secretion of LH (Kile and Nett, 1994). Moreover, the discrepancies might be due to differences in biological potencies in terminal unit of the hormone (Sairam et al., 1994) or the fact that intra-ovarian regulation of follicular development is controlled by many factors like GH, insulin, and IGF-I and hormones like FSH and LH may be species-specific (Nayan et al., 2013). In conclusion, the use of in vitro produced FSH≈500 IU and Folltropin-V resulted in higher serum FSH, estrogen, greater ovarian size (length, and width) and numerous small sized GF in buffaloes.

115

CONCLUSIONS

Based on the results of the present study, it is concluded that:

1. Diethyl-stillbesterol and medroxy-progesterone enhances the GnRH- stimulated release of FSH and LH from in vitro culture of buffalo adenohypophysis, respectively, and also interact with insulin and Lexavit.

2. Treating in vitro culture of buffalo adenohypophysis with higher dose of estrogen resulted in higher amount of FSH secretion, however, non-significant (P>0.05) difference was found in respect of low and high doses of estrogen as treatment, similarly, higher concentration of progesterone resulted in significantly (P<0.05) higher amount of LH secretion. 3. In vitro produced FSH and LH from buffalo adenohypophysis when administered in 10 time high dose to pre-pubertal female rabbits did not effect on most of the hematological parameters in rabbits, viz., RBC, WBC count; PCV and MCH. However, the in vitro produced FSH and LH increased the platelet count and decreased MCV compared to placebo treated rabbits (control group), the values of both these parameters were within normal ranges for rabbits. The significantly (P<0.001) increased ovarian size and activation of numerous small GFs are good indicators for the presence of hormone activity in the purified extract of in vitro culture of buffalo adenohypophysis. No mortality occurred during the experiment, indicating the safety of hormones produced. 4. The in vitro produced FSH showed activity in dairy buffaloes by significantly (P<0.01) increased serum FSH, LH, estrogen, ovarian size, and decreased serum progesterone concentration. 5. Quality and quantity of hormone used might not be sufficient to develop the GFs to reach at optimum size suitable for ovulation.

116

SUGGESTIONS

It is suggested that in future in vitro produced FSH and LH may be concentrated using prep-HPLC system so that more purified form could be prepared for commercialization of the product.

117

SUMMARY ______

In this project, the first experiment was planned to enhance the secretion of gonadotropins i.e. FSH and LH by treating in vitro culture of buffalo adenohypophysis with estrogen (0.5, 1.0 mg/100ml) and progesterone (2.5, 5.0 mg/100ml). Insulin and liver extract were also added in the culture medium as growth promoting factors. The amount of FSH and LH produced were estimated by ELISA and HPLC techniques. Results showed significant (P<0.05) increase in the secretion of FSH and LH in estrogen and progesterone treated cultures compared with control. The FSH and LH obtained were estimated to be 33.0 IU/ml and 68.67 IU/ml, respectively, in proportionate to standard of FSH (100.0 IU/ml and LH 1500.0 IU/ml). Experiment was repeated and sufficient amount of FSH and LH was obtained. Repeated procedure proved that the technique can be used for further production of FSH and LH.

The second and the third experiment were planned to study bioactivity of the FSH and LH obtained through in vitro culture by injecting them in pre-pubertal female rabbits and dairy buffaloes. In the second experiment, four groups of pre- pubertal female rabbits were treated with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5 and 85 IU (s/c), twice daily in divided doses at an interval of 12 hours for five days. Similarly, one group of rabbits was treated with placebo as control-1 and one was untreated kept as control-2. In the third experiment, nine healthy but acyclic dairy buffaloes were divided into three groups viz., A, B and C. Buffaloes in group A were treated with in vitro produced FSH≈500 IU (s/c) daily in divided doses at an interval of 12 hours for five days. Buffaloes in group B were treated with Folltropin-V≈5mg (s/c), twice daily and buffaloes in group C were treated with placebo/control.

There was no treatment-related mortality in any group no hypersensitivity or toxic signs were seen in any of the experimental rabbit/buffalo. None of the experimental rabbit showed change in general behavior, aggressiveness/lethargy and body weight. The in vitro produced FSH≈4.0, 40 IU, LH≈8.5 and 85 IU did not affect body weight and most of the blood parameters i.e. RBC and WBC count, PCV and hemoglobin (Hb) concentration of female rabbits. Moreover, FSH≈40 IU resulted (P<0.05) in increased ovarian activity showing developing GFs along with (P<0.05)

118 increased mean length, width, weight and volume of ovaries compared with those treated with FSH≈4 IU, LH≈8.5, 85.0 IU and controls. Treating rabbits with in vitro produced FSH≈40 IU, LH≈85.0 IU also resulted in significantly (P<0.001) increased serum FSH (260-470 Vs 4-6 mlU/ml) and serum LH (28-48 Vs 4-5 mlU/ml), compared with rabbits of control group.

Treating buffaloes with in vitro produced FSH and Folltropin-V resulted in significantly (P<0.01) high serum FSH, LH and estrogen concentration at day 5 to 6, whereas progesterone concentration was decreased (P<0.01). Day to day increase in serum FSH was also significant (P<0.01). Increase in serum LH was observed at day 5 to 6. Though the size of ovaries was increased showing numerous small GF’s, however, no one attained ovulatory capability. These, hormones obtained from the culture materials showed bioactivity in the experimental animals without any toxicity.

119

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Appendix-I

Equipment Column Oven: CTO-10A (Schimadzu) ELISA reader (Biotek ELX-808, USA) Flow Controller Valve: FCV-10AL (Schimadzu) HPLC degasser: DGU-12A (Schimadzu) HPLC spectral detector: SPD-10AV (Schimadzu) LC-10AT: (Schimadzu) Neubauer hemocytometer: Octadecyl Silicate: ODS Software CSW32 Syringe Filter: (Z26034-7 Sigma) System Controller: SCL-10A (Schimadzu)

ELISA Kits FSH and LH ELISA kits (Endocrine Technologies Inc. USA)

Medium RPMI-1640 (R6504-Sigma) Medium-199 (M3769-Sigma)

Chemicals, Hormones and Reagents

Amphotericin: (Sigma-Aldrich) Benzyl penicillin-Na: (Pfizer, Pakistan) Diethyl-stillbesterol (Star, Pakistan) GnRH: Lecirelin: (Fatro, Italy) Hydrochloric acid (HCL): (Emerk, Germany) Kanamycin (500 mg): (Spic, China) Lexavit: ( Medroxy-progesterone: (Pfizer, Pakistan) Potassium chloride (KCl): (Sigma-Aldrich) Potassium phosphate monobasic (KH2PO4): (Sigma-Aldrich) Sodium bicarbonate (NaHCO3): (Sigma-Aldrich) Sodium chloride (NaCl): (Sigma-Aldrich) Sodium hydroxide (NaOH): (Sigma-Aldrich) Sodium Phosphate dibasic (Na2HPO4-7H2O) : (Sigma-Aldrich) Streptomycin Sulphate: (Spic, China) Tri distilled and de-mineralized water: (Lab Deptt. of Theriogenology, UAF) Trypsin powder: (Sigma-Aldrich) Trypan Blue Stain (Sigma: 108K 2349)

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