A Dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2017 / Gonadotropin-releasing hormone (GnRH) binds to GnRH receptors (GnRHR) in the pituitary and stimulates release of gonadotropins, which control reproduction. It has been proposed that the congenital Glu2.53(90)Lys GnRHR mutation causes infertility by disrupting a salt-bridge important for GnRHR protein expression. To investigate its role in GnRHR function, Glu2.53(90) was mutated to residues that mimic or remove its side-chain properties. Mutant receptors were assessed for inositol phosphate signaling and radioligand binding. Receptors with small or negatively-charged substitutions for Glu2.53(90) exhibited no measurable function. Stabilizing receptor expression by appending a carboxy-terminal tail recovered function of the Glu2.53(90)Lys and Glu2.53(90)Ala GnRHRs, but not the conservative Glu2.53(90)Asp mutant. Receptors with uncharged (Gln) or hydrophobic (Leu, Phe) substitutions that cannot form salt-bridges with Lys3.32(121) were fully functional. Although the positively-charged Arg substitution decreased binding affinity, it preserved GnRHR function, confirming that interaction with the positively-charged Lys3.32(121) is not required. Comparing the GnRHR with structurally-related G protein-coupled receptors revealed that the equivalent residue of rhodopsin, Met2.53(86), interacts with Trp6.48(265). Mutating Trp6.48(280) of the GnRHR to Ala and Arg disrupted GnRH-stimulated function, confirming a role in expression. The Trp6.48(280)Arg GnRHR with an appended carboxy-terminal tail had decreased GnRH binding affinity. The preserved function of mutant receptors with large hydrophobic or positively-charged amino acid substitutions suggests that the size of the Glu2.53(90) is important for stabilizing GnRHR structure. Decreased affinity of mutant receptors with larger (Arg) substitutions for Glu2.53(90) and Trp6.48(280) suggest that both residues make conserved intramolecular interactions that stabilize receptor protein expression and configure the extracellular GnRHR structure. (250 words) / MT2017

In Experiment 1, 12 cyclic mares were assigned randomly to one of two groups (n = 6/group): 1) Control, saline; and 2) GnRH, 100 mu g/h. Between 3 and 6 d after ovulation (Day 0), Alzet osmotic minipumps (Model 2ML1) containing saline or GnRH were placed subcutaneous and connected to a jugular infusion catheter. Five-min samples were collected from the intercavernous sinus (ICS) of 10/12 mares (5/group) during 8 h on Day 4, followed by an additional 6-h intensive sampling period 36 h after induced luteal regression (Day 6). Treatment with GnRH markedly increased (P < 0.01) secretion of LH during both luteal and follicular phases. During the luteal phase, treatment with GnRH eliminated the very large, intermittent secretory episodes of LH characteristic of controls and produced frequent episodes of LH release of short duration. In Experiment 2, 12 anovulatory mares and 3 mares with some residual follicular activity (n = 15) were used during the fall (December 5 to 20) and winter (February 15 to 29) seasons. Mares were assigned randomly to: 1) Control, 2) GnRH-20; continuous infusion of GnRH at 20 mu g/h, or 3) GnRH-100; continuous infusion of GnRH at 100 mu g/h. Treatments were administered subcutaneously for 14 d using Alzet minipumps. Both the 20- and 100-mu g/h treatments increased (P less than 0.01) mean circulating concentrations of LH compared to controls before the winter solstice, but mares did not respond to the GnRH- 20 dose after the winter solstice. GnRH-100 caused a seasonally-independent increase (P less than 0.0001) in follicle size and ovulation frequency compared to controls The equine gonadotrope responded to continuous administration of high-dose GnRH during both ovulatory and anovulatory seasons, but was less responsive late compared to early in the anovulatory season.

Objectives were to determine if low-dose, continuous infusion of GnRH from Fall to Spring, would prevent seasonal anovulation in mares. Twenty Quarter Horse mares, ages 18 mo to 24 yrs, were stratified by age and body condition score and assigned randomly to either a saline control (n = 9) or GnRH (n = 11) treatment group. Treatments were instituted between September 23 and October 9, 2002. Gonadotropinreleasing hormone was delivered in 0.9% physiological saline via Alzet osmotic minipumps (Model 2004) placed sc at the base of the neck, with Silastic sham pumps placed in control mares. Pumps were inserted on day 3 following ovulation or during the follicular phase if ovulation had not occurred. Delivery rate of GnRH was 2.5 ug/h (60 ug/d) for the first 60 d, followed by 5.0 ug/h (120 ug/d) thereafter, with all pumps replaced every 30 d. By December 1, all mares had become anovulatory and remained anovulatory until February. Mean serum concentrations of LH were not affected by treatment in anovulatory mares. In contrast, control mares that exhibited ovulatory cycles after treatment onset had higher (P < 0.05) mean concentrations of LH during all phases of the estrous cycle except diestrus. Mean serum concentrations of FSH were not affected by treatment, but were lower (P < 0.05) from November though January relative to all other months in anovulatory mares. Interovulatory intervals in mares that cycled temporarily did not differ between groups. Ovulatory control mares had slightly larger (P < 0.10) follicles overall than GnRH-treated mares; however, ovulatory follicle diameters for control and GnRH-treated mares did not differ. Ovulatory control mares had higher (P < 0.10) mean concentrations of progesterone during metestrus and late diestrus. In a subgroup of control (n =5) and GnRH-treated (n = 5) mares, total releasable pools of LH in response to 1 mg GnRH did not differ between groups. Ovulation resumed in 3 control and 3 GnRH-treated mares by March 30. Results indicate that continuous infusion of native GnRH at the doses employed herein is not sufficient to maintain ovulatory cycles during the anovulatory season.

Observing cows in estrus and inseminating them at the optimal time are necessary steps for effective reproductive management of a dairy herd. However, increasing herd sizes can lead to reproductive inefficiency resulting in decreased profits on dairy herds. Synchronization of estrus, through pharmacological control, has been used to improve reproductive efficiency. Systematic breeding programs provide an organized approach for administering artificial insemination (AI) at first service. Moreover, reproductive management is based on a methodical approach for the entire herd rather than for the individual cow. Seven-hundred and thirty four Holstein cows from 16 commercial dairy herds were used to conduct this study evaluating three systematic breeding protocols; 14-d PGF2a, timed AI (TAI), and GnRH-PGF2α, in comparison with an untreated control group. Eight herds relied on visual observation as their primary method for detection of estrus, and 8 herds utilized the HeatWatch® (DDx, Inc., Denver, CO) electronic estrus detection system. The average days to first postpartum AI were longer for untreated control cows when compared to the other breeding protocols. First AI conception rates did not differ among control, 14-d PGF2a, or GnRH-PGF2a protocols, but were higher than the TAI protocol. However, first AI pregnancy rates were higher for untreated controls versus hormonally treated cows. Estrus characteristics associated with each protocol were also evaluated and no difference was detected across treatments. An economic analysis determining cost per pregnancy for each protocol when considering drug costs, and pregnancy rates, resulted in the highest cost per pregnancy for TAI followed by GnRH-PGF2a and 14-d PGF2a. These programs should be considered as tools for convenience and efficiency of estrus detection; however, reduced labor costs from less time spent on estrus detection may be offset by the cost of the drug protocols. Cost effectiveness must be calculated on an individual herd basis when deciding whether a systematic breeding program is the appropriate choice. / Master of Science

Orientador: Rita Cássia Menegati Dornelles / Co-orientador: Janete Aparecida Anselmo-Franci / Banca: Maristela de Oliveira Poletini / Banca: Jacqueline Nelisis Zanoni / Resumo: As alterações nos componentes reprodutivos do eixo hipotálamo-hipófise-gônadas em muitas fêmeas de mamíferos determinam a transição gradual de ciclos reprodutivos regulares para ciclos irregulares, com perda de fertilidade. A interação dos neurônios do hormônio liberador de gonadotrofinas (GnRH) e esteróides gonadais representa função chave na neurobiologia do envelhecimento, pois a sobreposição temporal da senescência endócrina e neural está mecanicamente interligada pelas alças de retroalimentação. Estímulos do locus coeruleus (LC) para a área pré-óptica (APO) e eminência mediana são essenciais para a liberação das gonadotrofinas e seus neurônios apresentam receptores para estrógeno e progesterona, sugerindo controle dos esteróides ovarianos. Neste estudo foi avaliado a atividade de células neuronais localizadas em áreas e núcleos envolvidos com o controle de ação dos neurônios GnRH de ratas Wistar no período de transição para a aciclicidade. Para este trabalho foram utilizadas fêmeas Wistar cíclicas (4 meses) e acíclicas (18-20 meses) submetidas à decapitação ou perfusão às 10, 14 e 18 h na fase do diestro. Após serem retirados, os cérebros dos animais decapitados foram congelados e armazenados para posterior determinação do conteúdo de GnRH hipotalâmico e do conteúdo de noradrenalina e dopamina na APO. Os cérebros perfundidos foram cortados seriadamente em secções coronais de 30 μm para a APO e o LC e... / Abstract: Changes in reproductive components of the hypothalamic-pituitary-gonadal axis in many female mammals determine the gradual transition from regular reproductive cycles to irregular cycles, with loss of fertility. The interaction of neurons of gonadotropin-releasing hormone (GnRH) and gonadal steroids represents key role in the neurobiology of aging, because the temporal overlap of endocrine and neural senescence is mechanically interconnected by feedback loops. Stimulation of the locus coeruleus (LC) for the preoptic area (POA) and median eminence are essential for the release of gonadotropins and their neurons have receptors for estrogen and progesterone, suggesting control of ovarian steroids. Therefore, in this study we evaluated the activity of neuronal cells located in areas and nuclei involved in the control of action of GnRH neurons of female rats during the transition to acyclicity. For this study, we used cyclic female (4 months) and acyclic (18-20 months) rats underwent perfusion or decapitation at 10, 14 and 18 h of diestrus day. The brains from decapitated animals, after removed, were frozen and stored for subsequent determination of the hypothalamic GnRH content and the noradrenaline and dopamine content in the POA. The perfused brains were serially cut into coronal sections of 30 μm to POA and LC and subsequently submitted to immunohistochemical labeling for Fos (FRA) and FRA / TH, respectively. For quantitative analysis of the POA were considered plates containing AVPe being the counting of neurons FRA-ir performed from the insertion of the box with... / Mestre

Sexual selection and the maintenance of species diversity in Lake Malawi cichlid fishes are greatly dependent on optical communication, which is influenced by environmental, physiological and endocrinological factors. The diversity in spectral sensitivity of cichlids has been partially attributed to differences in opsin gene expression, with each species preferentially expressing a subset of seven possible genes. Hormones such as gonadotropin releasing hormone (GnRH) can mediate changes in gene expression and the presence of GnRH immunoreactive fibers and GnRH receptors throughout the retinal layers make it an excellent candidate for mediating changes in visual processes. Effects of exogenous GnRH administration on the visual system of zebra cichlids (Metriaclima zebra) via prolonged release cholesterol implants and intubation was investigated using electroretinogram (ERG) recordings, quantitative real-time RT-PCR and in situ hybridization. Three week and ten week sampling periods were used in the intubation study. No obvious differences in spectral sensitivity were evident when looking at a-wave, b-wave and d-wave components of the ERG waveform in any of the treatment groups. A multiple mechanism model was used to describe the cone mechanisms mediating spectral sensitivity and this analysis showed that the activity of cones was shaped by opponent and non opponent cone interactions based on subsets of five opsin genes previously described in cichlids (SWS1, SWS2b, RH2b, RH2aβ, and RH2aα). Although differences in the spectral sensitivity between control and GnRH-treated fish were not evident on a functional level, there were changes in the gonadosomatic index in the intubation group. Quantitative real-time RT-PCR (qRT-PCR) and in situ hybridization demonstrated that treatment with a synthetic GnRH3 analogue using the oral intubation delivery system resulted in statistically significant changes in opsin gene expression in both three week and ten week treatment groups, specifically the upregulation of RH2b and the downregulation of RH2a opsin genes. Moreover, in situ hybridization analysis showed that the pattern of labeling for the RH2a and RH2b riboprobes corroborated the changes in opsin gene expression found in the qRT-PCR data. In contrast, GnRH treatment using the cholesterol implant delivery system did not result in significant changes in spectral sensitivity or opsin gene expression. / Thesis (Master, Biology) -- Queen's University, 2011-01-05 22:57:11.308

Background: Hypothalamic kisspeptin and neurokinin B (NKB) are central regulators of GnRH and thus gonadotropin (LH and FSH) secretion. Men and women with loss-of-function mutations in NKB-kisspeptin pathway show hypogonadotropic pubertal delay with reduced GnRH/LH pulsatility. Studies in patients with defects in NKB signalling suggest that kisspeptin is functionally downstream of NKB, although there are very limited data on the relevance of the NKB pathway in normal men or women, and no hierarchical data on this. The studies described in this thesis have investigated the interaction between these neuropeptides in the control of human reproduction in conditions of varying sex-steroid environment, and in states of fast and slow LH secretion (men, menopause, various stages across the menstrual cycle). Overall hypothesis: Pharmacological blockade of NKB signalling will decrease LH secretion by modulating GnRH/LH pulsatility, indicating the involvement of the NKB pathway in normal human reproductive function. It is also hypothesised that this will not abrogate the stimulatory kisspeptin response, revealing a functional hierarchy whereby NKB signalling is upstream of kisspeptin. Research strategy: A specific neurokinin-3 receptor antagonist (NK3R antagonist, AZD4901) was administered 40 mg twice daily orally for 7 days with and without kisspeptin-10 (KP-10) challenge. Response of reproductive hormones (serum and urinary where applicable) was measured. LH was sampled every 10 minutes for 8 hours to assess LH pulsatility by blinded deconvolution. Results: Role of neurokinin B and kisspeptin in healthy men Six healthy men underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist reduced LH and testosterone secretion, whilst stimulatory LH response to KP-10 was unaffected. LH pulse frequency was unchanged by the NK3R antagonist but basal (nonpulsatile) and pulsatile LH secretion was markedly reduced. Role of neurokinin B and kisspeptin in postmenopausal women Eleven postmenopausal women underwent LH pulsatility study pre-treatment and on day 7 of NK3R antagonist administration with iv KP-10 bolus (0.3 μg/kg) at 6 hours. NK3R antagonist decreased LH secretion. Basal (nonpulsatile) LH secretion also fell and while LH pulse frequency did not change in a group as a whole, it did fall in the 8 of 11 postmenopausal womenwith hot flushes. These women reported a reduction in hot flush frequency (3.4±1.2 vs 1.0± 0.6 flushes/day with NK3Ra, p=0.008) and severity whilst on NK3R antagonist. LH response to KP-10 was minimal and unaffected by the NK3R antagonist. Role of neurokinin B across different phases of menstrual cycle The effect of NK3R antagonist on ovarian function was compared in early follicular (n=13), late follicular (n=6) and luteal phase (n=6) to no treatment control cycle. Early follicular: NK3R antagonist was commenced from cycle day 5-6. The diameter of the leading follicle was smaller than in controls at the end of treatment (9.3±0.4 vs 15.1±0.9 mm, p < 0.0001). Serum estradiol was also reduced and the endometrium was thinner. Although NK3R antagonist had no effect on LH pulse frequency, basal (nonpulsatile) LH secretion was decreased, suggesting that NKB modulates GnRH secretion. After stopping treatment, follicle development resumed and estradiol secretion increased thereby delaying the LH surge in 11/13 women (LH surge cycle day 22±1 vs 15±1, p=0.0006). The delayed LH surge and ovulation were confirmed by a similarly delayed rise in urinary progesterone and prolonged cycle length. NK3R antagonist did not affect luteal function. Late follicular: NK3R antagonist was administered from the emergence of a dominant follicle (≥12mm). Whilst there was an LH surge in all treated cycles, estrogen feedback was perturbed by the NK3R antagonist, as there was increased variation in the timing of LH surge compared to control cycle. NK3R antagonist had no effect on the growth of a dominant follicle and luteal function was unaffected. Luteal: NK3R antagonist was administered from day +2-3 of the disappearance of the dominant follicle. NK3R antagonist reduced the variation in the timing of peak estradiol secretion. Estradiol and progesterone concentrations remained unchanged, suggesting that luteal function was overall unaffected by this treatment. No difference in mean LH was observed, although LH pulsatility was not assessed. Role of neurokinin B and kisspeptin in the mid-cycle LH surge A model of follicular phase (cycle day 9-11) administration of estradiol (200μg/day) to induce LH secretion at 48 hours was used in twenty women, mimicking LH surge. In this model, KP-10 infusion (4μg/kg/hr for 7 hours) enhanced LH secretion, the response of which was directly correlated with estrogen concentration, indicating a role of kisspeptin in estrogen feedback. Pre-treatment with NK3R antagonist decreased LH pulse frequency and whilst the immediate LH response to KP-10 was unaffected, it blunted the duration of this response and abolished the relationship between estradiol and kisspeptin-induced LH secretion. Conclusions: These data indicate the role of NKB-KP pathway in regulating human reproductive function and that this is via the modulation of pulsatile GnRH secretion. Whilst NKB is predominantly proximal to kisspeptin, the hierarchy is more complex than simply linear in the control of human HPG axis. Manipulation of NKB-KP signalling has therapeutic potential in regulating GnRH/LH secretion in wide range of clinical settings, including contraception, sex-steroid dependent disorders and in the treatment of hot flushes.

Made available in DSpace on 2014-06-11T19:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-08-02Bitstream added on 2014-06-13T19:12:30Z : No. of bitstreams: 1 000742177.pdf: 1654502 bytes, checksum: 866898964213e96038c110158bf8a746 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / As alterações nos componentes reprodutivos do eixo hipotálamo-hipófise-gônadas em muitas fêmeas de mamíferos determinam a transição gradual de ciclos reprodutivos regulares para ciclos irregulares, com perda de fertilidade. A interação dos neurônios do hormônio liberador de gonadotrofinas (GnRH) e esteróides gonadais representa função chave na neurobiologia do envelhecimento, pois a sobreposição temporal da senescência endócrina e neural está mecanicamente interligada pelas alças de retroalimentação. Estímulos do locus coeruleus (LC) para a área pré-óptica (APO) e eminência mediana são essenciais para a liberação das gonadotrofinas e seus neurônios apresentam receptores para estrógeno e progesterona, sugerindo controle dos esteróides ovarianos. Neste estudo foi avaliado a atividade de células neuronais localizadas em áreas e núcleos envolvidos com o controle de ação dos neurônios GnRH de ratas Wistar no período de transição para a aciclicidade. Para este trabalho foram utilizadas fêmeas Wistar cíclicas (4 meses) e acíclicas (18-20 meses) submetidas à decapitação ou perfusão às 10, 14 e 18 h na fase do diestro. Após serem retirados, os cérebros dos animais decapitados foram congelados e armazenados para posterior determinação do conteúdo de GnRH hipotalâmico e do conteúdo de noradrenalina e dopamina na APO. Os cérebros perfundidos foram cortados seriadamente em secções coronais de 30 μm para a APO e o LC e... / Changes in reproductive components of the hypothalamic-pituitary-gonadal axis in many female mammals determine the gradual transition from regular reproductive cycles to irregular cycles, with loss of fertility. The interaction of neurons of gonadotropin-releasing hormone (GnRH) and gonadal steroids represents key role in the neurobiology of aging, because the temporal overlap of endocrine and neural senescence is mechanically interconnected by feedback loops. Stimulation of the locus coeruleus (LC) for the preoptic area (POA) and median eminence are essential for the release of gonadotropins and their neurons have receptors for estrogen and progesterone, suggesting control of ovarian steroids. Therefore, in this study we evaluated the activity of neuronal cells located in areas and nuclei involved in the control of action of GnRH neurons of female rats during the transition to acyclicity. For this study, we used cyclic female (4 months) and acyclic (18-20 months) rats underwent perfusion or decapitation at 10, 14 and 18 h of diestrus day. The brains from decapitated animals, after removed, were frozen and stored for subsequent determination of the hypothalamic GnRH content and the noradrenaline and dopamine content in the POA. The perfused brains were serially cut into coronal sections of 30 μm to POA and LC and subsequently submitted to immunohistochemical labeling for Fos (FRA) and FRA / TH, respectively. For quantitative analysis of the POA were considered plates containing AVPe being the counting of neurons FRA-ir performed from the insertion of the box with... / FAPESP: 12/14464-6

The recent decline and disappearance of many amphibians around the world is thought to be the sign of an impending sixth mass extinction that is driven by disease, habitat loss and pollution. Reproductive technologies are now required to establish captive colonies followed by reintroduction into suitable habitats. The AMPHIPLEX method is a hormone mixture that has successfully stimulated spawning in several amphibians. However, its extensive application requires further experimentation and knowledge regarding the basic neuroendocrine control of reproduction in amphibians. The role of the catecholamine neurotransmitter dopamine in the regulation of spawning and gonadotropin synthesis was investigated using multiple time- and dose-related approaches in the field and laboratory. These end points were explored in two distantly-related frog species: the Northern leopard frog (Lithobates pipiens) and the Western clawed frog (Silurana tropicalis). Northern leopard frogs were injected during the natural breeding season with three doses of a gonadotropin-releasing hormone agonist (GnRH-A) (0.1 μg/g , 0.2 μg/g and 0.4 μg/g) alone and in combination with two doses of the selective dopamine receptor D2 antagonist metoclopramide (MET) (5 μg/g and 10 μg/g). Injected animals were allowed to breed in mesocosms in an outdoor field. Time to amplexus and oviposition were assessed, and egg mass release, incidences of amplexus, egg mass weight, total egg numbers and fertilization rates were measured. The results revealed no statistically significant interaction between GnRH-A and MET on amplexus and oviposition. A series of GnRH-A dose-response spawning studies were conducted in the Western clawed frog. The current findings indicate that partial ovulation, male sexual behavior and fertilization can be induced by 4 μg/g of GnRH-A alone and in combination with 10 μg/g of MET. This represents a first step towards understanding basic neuroendocrine reproductive mechanisms in this species. These spawning results were paired with a second end point which explored the molecular mechanisms of gonadotropin synthesis in response to GnRH-A and MET alone and in combination. Pituitary gene expression results in the Northern leopard frog indicate a potentiating action of MET when combined with GnRH-A on the mRNA levels of gonadotropin subunits 36 hours following injection. The postulated mechanisms of action are through the upregulation of gonadotropin-releasing hormone receptor 1 and the downregulation of dopamine receptor D2. Such gene expression pathways were similarly explored in the Western clawed frog, however no significant changes in pituitary gonadotropin and receptor gene expression were present at 12 hours post-injection. The hypothesized inhibitory action of dopamine was supported by pituitary gene expression analysis, but not by spawning outcome. The results from this study provide a fundamental framework for future time- and dose-response investigations to improve current spawning methods in amphibians.

Two studies were conducted to evaluate the effects of 5 or 7 d post-AI GnRH on first service PR, plasma P4, and CL volume in lactating dairy cows synchronized using 72 h Cosynch. All cows were synchronized and randomly assigned to one of three treatment groups: Control – no additional GnRH; 5 d – GnRH 5 d after TAI; 7 d – GnRH 7 d after TAI. In the first study, P4 concentrations were evaluated in samples collected at five separate times and CL volume and number were recorded at 30 d pregnancy examination for Holstein (n = 77) and Jersey (n = 33) cows. GnRH treatment did not affect PR (Control - 47.2%, 5 d GnRH - 40.5%, 7 d GnRH – 44.7%) or P4, but increased TCLV compared to controls (Control – 7.33 cm3, 5 and 7 d GnRH – 10.77 cm3). Incidence of accessory CL increased PR (94.7 vs. 60.6%), P4 (6.95 vs. 5.88 ng/mL), and TCLV (15.51 vs. 6.78 cm3) compared to cows with a spontaneous CL. Cows classified as cycling based on P4 evaluation had significantly higher PR than acyclic cows (54.4 vs. 16.1%). In the second study, Holstein cows (n = 1055) were submitted to the same experimental protocol and evaluated for first service PR. Post-AI GnRH treatment did not significantly affect PR. Primiparous cows (32.8%) tended to have higher PR than multiparous cows (27.6%), but GnRH treatment had no influence on this relationship. In conclusion, GnRH post-AI did not affect PR. Further evaluation of accessory CL incidence is warranted as it significantly affected PR. (Abbreviations: AI – artificial insemination, CL – corpus luteum, PR – conception rate, P4 – progesterone, TCLV – total corpus luteum volume) / Master of Science