Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains viii, 71 p. : ill. Includes abstract. Includes bibliographical references (p. 60-71).

Prolactin receptors (PRLr) were detected in anestrous (n=3), winter cycling (n=2), follicular (n=3) and luteal phase (n=3) equine ovaries by IHC. Follicle stages evaluated were primordial, preantral and antral. Receptors were detected in all follicle stages and in CL. PRLr staining was not different (P > 0.05) between primordial and preantral, but was greater (P < 0.001) in antral follicles. Primordial follicles stained weakest in anestrous and follicular phase ovaries, followed by luteal phase ovaries and was most intense in winter cycling. Staining in preantral follicles was weakest in anestrus, followed by follicular phase and highest in winter cycling and luteal phase. Staining was most intense in antral follicles with no difference (P > 0.05) between any of the reproductive states. Oocytes and ovulation fossa also possessed PRLr. In conclusion, concentrations of PRLr are highest in large, antral follicles suggesting a mechanistic role for PRL around the time of ovulation.

It has been reported world wide that over the past fifty years production has dramatically increased in dairy cattle but at the same time fertility rates have steadily declined, particularly in mature cows. Fertility of heifers that were bred for the first time has not been affected. One of the major reasons for such fertility decline in mature cows could be impaired progesterone production. Therefore relationships of parity with reproductive performance, its effect on progesterone concentrations and genes associated with progesterone synthesis were examined in this thesis. In the first experiment, breeding records of 163 Holstein heifers and cows in 1st, 2nd, and 3rd/4th parities were used to compare pregnancy rates among heifers and parity cows and between parity cows. Progesterone levels of heifers, 1st, 2nd, and 3rd/4th parity (10 animals each group) were measured from milk and blood samples. First and second inseminations pregnancy rates were higher in heifers compared to other parity cows. Furthermore 1st parity cows showed higher pregnancy rates than 2nd and 3rd/4th parity cows. However, P₄ levels were not significantly different among animals of different parity. In the second experiment, expression levels of steroidogenic genes – StAR, P450scc, 3-β HSD; apoptotic genes Bax and Bcl-2; and HSP70 in corpus luteum obtained from six heifers and three 2nd/3rd parity lactating cows were compared using RT-PCR. Relative optical density with house keeping gene was obtained for each gene. Analysis of variance revealed that expression levels of steroidogenic and Bax genes are higher (p<0.05) in cows than heifers. HSP70 gene and Bcl-2 gene expressions were not different (P>0.05) between the two groups. This study confirmed a clear relationship between parity and reproductive performance. There was no significance relationship between parity and circulating progesterone levels. Steroidogenic genes expression was higher in lactating cows than heifers and no differences were seen in mRNA levels of Bcl2, and HSP70 genes between heifers and mature cows. Bax mRNA expression was higher in mature cows suggesting that the lifespan of corpus luteum may be compromised in 2nd and 3rd parity cows, resulting in early embryonic mortality and reduced pregnancy rates. / Land and Food Systems, Faculty of / Graduate

The objective of this study was to examine hepatic steroid inactivation and luteal function throughout bovine gestation. In pregnant beef cows, cytochrome P450 3A activity decreased from mid- to late-gestation, while progesterone concentrations tended to increase from mid- to late-gestation. Uridine diphosphate-glucuronosyltransferase activity per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Total corpus luteum (CL) blood perfusion tended to be increased in pregnant vs non-pregnant dairy cows. Hepatic portal blood flow per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Hepatic steroid inactivating enzyme activity, CL blood perfusion, and portal blood flow did not differ between pregnant and non-pregnant beef cows. There was no difference in progesterone concentrations in pregnant vs non-pregnant dairy or beef cows. The current study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.

Fescue toxicosis is a common problem in beef cattle grazing endophyte-infected (E+) tall fescue. Symptoms include decreases in feed intake, weight gain, and reproductive efficiency along with vasoconstriction. The mechanisms by which fescue toxicosis affects the bovine reproductive tract have yet to be discovered. The objective of this study was to determine if the onset of fescue toxicosis conditions would alter the blood perfusion observed in the CL and peripheral concentrations of progesterone in cattle. We hypothesized that during fescue toxicosis, the vasoconstrictive symptoms would lead to a reduction in CL blood perfusion thus decreasing peripheral concentrations of progesterone. Overall, fescue toxicosis was induced as cows fed an E+ treatment diet had greater rectal temperatures and reduced pulse pressure and mean arterial pressure measurements; however, total blood perfusion of the CL and peripheral concentrations of progesterone did not differ in cattle under fescue toxicosis compared to those without.

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Establishment and maintenance of pregnancy is a central concern to the cattle industry, as it strongly impacts efficiency and profitability of beef cow-calf operations. The objective of this study was to determine if nutrient restriction impacts ovulatory follicle size and corpus luteum (CL) development and function of Bos taurus cows enrolled in estrous synchronization. A total of 26 Angus cows were housed in a facility equipped with a Calan gate system for individual animal intake. Cows were stratified by body weight (BW), and randomly assigned one of two nutritional treatments: 1) 100% of nutrient requirements (MTN; n=13) or 2) 70% of nutrient requirements (REST; n=13). Individual daily intakes were measured and adjusted weekly based on BW. Cattle underwent an acclimation period of 14 days and were exposed to nutritional treatments for 30 days prior to estrous synchronization. Body weight was measured daily using an automated scale and a conventional livestock scale at the beginning and end of the experiment. Cows were synchronized using a 7-day CO-synch + CIDR protocol beginning on day -10. Ultrasonography of the ovaries was performed at each event of the estrous synchronization protocol on days -10, -3, 0, 5, and 7. Blood samples were taken on days -10, -3, and daily from day 0 through 7 to observe changes in progesterone (P4). Data were analyzed as repeated measures using the MIXED procedure of SAS. Initial BW tended to differ between treatments (P = 0.07; MTN 597 ± 32 kg, REST 604 ± 32 kg), but MTN had greater final BW (P < 0.001; 687 ± 24 and 556 ± 27 kg, respectively) and greater average daily gain (1.35 ± 0.18 and -0.72 ± 0.21 kg/d, respectively, P < 0.001) than REST. Diameter of the largest follicle was similar (P = 0.851) between treatments at CIDR insertion (12.6 ± 0.6 mm) and CIDR removal (12.9 ± 0.4 mm) but was greater (P < 0.05) for MTN than REST cows at 60 hrs after CIDR removal (14.01 ± 0.6 and 12.37 ± 0.5 mm, respectively). Volume of CL was similar (P > 0.1) at 5 (3211 ± 113 mm3) and 7 (5280.3 ± 212 mm3) days after ovulation. Concentration of P4 did not differ on days -10, -3, or 0-5. However, on days 6 and 7, P4 was greater (P < 0.05) for MTN than REST (2.07 ± 0.15 and 1.65 ± 0.15, and 2.27 ± 0.15 and 1.83 ± 0.15 ng/mL, respectively). In conclusion, nutrient restriction to 70% of maintenance during estrous synchronization negatively affects diameter of the ovulatory follicle and circulating P4, but it did not affect CL volume in multiparous Bos taurus beef cows. / Master of Science / Ensuring successful pregnancy in beef cow-calf operations is crucial for the efficiency and profitability of the cattle industry. This study investigates the effects of nutrient restriction on ovulatory follicle size and corpus luteum (CL) volume in Angus cows undergoing estrous synchronization. A total of 26 cows were subjected to either a maintenance diet meeting 100% of nutrient requirements (MTN) or a diet providing 70% of nutrient requirements (REST). Intakes were updated weekly using computer software. The cows underwent a 30-day nutritional treatment before synchronization of ovulation. Results revealed that cows on the maintenance diet exhibited greater final body weight and average daily gain compared to those on the restricted diet. While estrus expression showed a numerical increase in MTN cows, the impact was not statistically significant. Analysis of ovulatory follicle size demonstrated that MTN cows had larger follicles 60 hours after synchronization compared to REST cows. Surprisingly, corpus luteum volume did not differ between the two groups at 5 and 7 days after ovulation. Additionally, circulating progesterone (P4) levels were affected by nutrient restriction, with notable differences observed on days 6 and 7. In summary, nutrient restriction during ovulation synchronization negatively influenced ovulatory follicle size and P4 levels, but did not affect corpus luteum volume in mature Angus cows. These findings contribute valuable insights for the cattle industry, emphasizing the importance of proper nutrition for optimal reproductive health in beef cows.

Inadequate uterine environment is one of the main causes of pregnancy failure in cattle. Progesterone (P4) supplementation during early diestrus may either induce a receptive uterine status or shorten luteal lifespan and reduce fertility. Regulation that leads to either outcome is currently unclear. In this thesis, four studies were conducted to test the main hypothesis that the pre-implantational conceptus plays a major role on preventing P4-luteolytic effects in Bos indicus beef cows, due the supplementary P4. In a fifth study the importance of the uterine luminal milieu on the establishment of pregnancy was evaluated. P4 was supplemented by injecting 150 mg of long acting injectable P4 (iP4) on 3 days post-ovulation. In the last study, uterine luminal flushings were performed on days 1, 4 and/or 7 post-estrus, aiming to deplete the uterine milieu. In this thesis, we evidenced that uterus played a key role on determining the iP4-luteolytic response. In this sense, first study revealed that higher uterine exposure to estradiol (E2) during pre-ovulatory period prevented P4-luteolytic effect, but did not increase overall pregnancy outcome. Further analysis, revealed that optimal uterine estradiol exposure is required for beneficial effects of iP4. Cows with large preovulatory follicle or with small follicle, but exposed to the exogenous estradiol presented higher pregnancy rates. Next, we demonstrated that iP4 hindered CL formation, but this had a minor impact on iP4-induced luteolysis. About half of iP4 supplemented cows presented early luteolysis, which occurred by day 15 post-ovulation. Role of the embryo to inhibit iP4-induced early luteolysis relied on its capability to establish pregnancy. In the third study, we demonstrated that iP4-inhibition of growth of the first-wave dominant follicle was related to early luteolytic onset, and this was independent of number of waves in the cycle (two vs. three). Three-wave cycles favored embryonic capacity to inhibit early luteolysis. In the fourth study, we failed to demonstrate that P4 supplementation supported embryonic survival. This was despite of the transfer of 5 embryos to each recipient cow to maximize embryonic signaling. In the last study we disturbed the composition of the uterine environment and negatively affected, but did not abolish, embryonic survival. Overall, from the results obtained in the course of this thesis, we conclude that variability in fertility rates after P4 supplementation, are in part attributable to the complexity of uterine function programming by sex steroids, rather than caused by the incidence of early luteolysis. Furthermore, we highlighted that a sub-optimal composition of the uterine environment is a major contributor to embryonic losses in beef cattle. / Um ambiente uterino deficiente é uma das principais causas de falha gestacional em bovinos. A suplementação com progesterona (P4) durante o diestro inicial estimula a receptividade uterina, mas também pode encurtar a fase luteal, prejudicando a fertilidade. Os fatores que levam a um dos dois resultados não são claros. Nesta tese, quatro estudos foram conduzidos para testar a hipótese principal de que o concepto pré-implantacional bloqueia o efeito luteolítico antecipado causado pela P4 suplementar. Em um quinto estudo, avaliou-se a importância do ambiente uterino no estabelecimento da gestação. A suplementação com P4 foi realizada pela administração de 150 mg de P4 injetável (iP4) de longa ação 3 dias após a ovulação. No último estudo, lavados uterinos foram coletados nos dias 1, 4 e/ou 7 após o estro, com o objetivo de depletar o ambiente uterino. Nestes estudos, o útero desempenhou um papel crucial na determinação da resposta luteolítica da iP4. O primeiro experimento demonstrou que a exposição uterina a maiores concentrações de estradiol (E2) durante o período pré-ovulatório bloqueou efeito luteolítico da iP4, mas não resultou em incremento na taxa de prenhez. Análises complementares revelaram que a suplementação de iP4 teve efeito positivo na fertilidade quando houve uma ótima exposição do útero ao E2 pré-ovulatório. Vacas com maiores folículos pré-ovulatórios ou com pequenos folículos, mas suplementadas com estradiol exógeno, apresentaram maiores taxas de prenhez. A exposição sub-ótima ou exagerada ao E2 foi deletéria ao efeito embriotrófico da P4. Em seguida, demonstramos que a iP4 prejudicou o desenvolvimento luteal, porém, esse não foi o principal fator relacionado com a antecipação da luteólise. Aproximadamente metade das vacas suplementadas com iP4 apresentaram luteólise precoce, que ocorreu no dia 15 após a ovulação. O efeito do embrião sobre o processo luteolítico antecipado foi dependente da sua capacidade de estabelecer a gestação. No terceiro estudo, verificamos que a redução do desenvolvimento do folículo dominante da primeira onda esteve associado à ocorrência de luteólise precoce, e isso foi independente do número de ondas no ciclo (dois vs. três). No entanto, ciclos de três ondas favoreceram a capacidade embrionária de inibir a luteólise precoce. No quarto estudo, nós não evidenciamos um efeito embriotófico da iP4 reduzindo a mortalidade embrionária, mesmo quando 5 embriões foram transferidos para cada receptora, com objetivo de maximizar a sinalização embrionária. No último estudo, a composição do ambiente uterino foi alterada por lavagens uterinas e isso afetou negativamente a prenhez, porém não aniquilou as chances de sobrevivência embrionária. A partir dos resultados obtidos nessa tese, concluímos que a variabilidade nas taxas de fertilidade após a suplementação com P4 é, em grande parte, determinada pela complexidade na programação da função uterina pelos hormônios ovarianos, em vez de ser causada pela incidência de luteólise precoce. Além disso, concluímos que uma composição sub-ótima do ambiente uterino é um dos principais contribuintes para as perdas embrionárias em bovinos de corte.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Restricted until May 2007. Bibliography: leaves 97-114.

This study was designed to evaluate the possible existence of masked gonadotropin binding sites in the corpus luteum of the rhesus monkey. Pretreatment of macaque luteal particulates and cells with neuraminidase increased LH binding. In vitro exposure to alcohols also enhanced LH binding to these preparations. Ethanol modulation of LH binding was a time- and temperature-dependent process. The optimal concentration of ethanol for enhancing LH uptake was inversely proportional to the incubation temperature. Longer straight-chain alcohols were more potent than ethanol in increasing LH binding. Ethanol and neuraminidase increased the number of binding sites with no affect on affinity. Moreover, the effects of ethanol and NA were additive. Since alcohols and temperature are modulators of membrane fluidity, we examined the hypothesis that the unmasking of gonadotropin binding sites may be related to changes in the fluid state of the lipid bilayer of the luteal membrane. First, membrane fluidity was estimated from the fluorescence polarization of the membrane probe diphenylhexatriene. Conditions which resulted in enhanced gonadotropin binding (1-8% ethanol, increased temperature), increased the fluidity of luteal membranes. Moreover, changes in gonadotropin binding were highly correlated (r = -0.97) with changes in membrane fluidity under these conditions. Pretreatment of luteal particulates with neuraminidase had no apparent effect on membrane fluidity. Second, gonadotropin receptors were removed from the luteal membrane by detergent solubilization, and the effects of ethanol on soluble receptors were compared to those on receptors associated with the lipid bilayer. Solubilization resulted in the recovery of 50% more gonadotropin binding sites than are available in particulate preparations of the corpus luteum; these sites displayed lower affinity for gonadotropin. Moreover, conditions which increase LH binding to luteal particulates (1-8% ethanol at 25C) decreased LH uptake by soluble receptors. The data suggest that two populations of LH binding sites are masked within the membranes of the monkey corpus luteum. The ability of two markedly different agents, alcohol and neuraminidase, to increase LH binding indicates the diverse mechanisms may modulate the masking/unmasking of gonadotropin receptors in target cell membranes. As such, changes in membrane fluidity may play an important role in this response.