The purpose of the studies reported in this thesis was to provide further evidence in support of the hypothesis that ovulation-inducing factor (OIF) is a component of seminal plasma which is conserved amongst mammals. Based on studies conducted in vivo, the results indicate that males ejaculate a substance during copulation which is responsible for the ovulatory and luteotrophic effect in female camelids. In our lab we have developed an <i>in vivo</i> llama bioassay to study the presence and biological effects of OIF in seminal plasma from different species.<p> The objective of the first experiment within the first study was to determine if llama seminal plasma would stimulate ovulation in prepubertal mice. Mice were treated with a single 0.1 mL intraperitoneal dose of 1) phosphate-buffered saline (negative control), 2) 5 µg gonadotropin-releasing hormone (GnRH), 3) 5 IU of human chorionic gonadotropin (hCG) or 4) llama seminal plasma. Results indicate that prepubertal mice treated with GnRH, hCG or llama seminal plasma stimulated similar proportions of mice to ovulate, which were all higher than the proportion of mice that ovulated after saline treatment. The number of oocytes observed under a stereomicroscope was also higher in all treatment groups than in mice treated with saline. However, the number of oocytes observed was lower in mice treated with seminal plasma than those treated with GnRH, both of which were similar to the number of oocytes observed in hCG treated mice.<p> In a second part of this study the corollary that OIF is present in the seminal plasma of horses and pigs was examined. Seminal plasma from horses or pigs was administered intramuscularly to female llamas and ovulation was monitored using transrectal ultrasonography. Llamas were treated with an intramuscular dose of 1) phosphate buffered saline (negative control), 2) llama seminal plasma (positive control), 3) equine seminal plasma or 4) porcine seminal plasma. Ovulations were detected in llamas treated with seminal plasma while none were observed in saline-treated llamas. The proportion of llamas that ovulated when treated with equine seminal plasma was higher than llamas treated with saline. The proportion of llamas that ovulated after porcine seminal plasma tended to differ from negative control groups, but did not reach statistical significance. The proportion of llamas that ovulated after equine or porcine seminal plasma treatment was lower than animals treated with llama seminal plasma which indicates that either OIF is not present in equal concentration among mammals, or that OIF is not structurally the same across mammals.<p> The second study was carried out to test the hypothesis that OIF stimulates LH secretion at the level of the anterior pituitary gland. The second objective was to determine if the degree of LH release was related to the dose of OIF treatment. Anterior pituitary cells (2 x 10^6 cells/ well) from either llamas (reflex ovulator) or cattle (spontaneous ovulator) were incubated for 2 hours with either media containing no treatment (control), GnRH or OIF. In all experiments, GnRH and OIF stimulated more LH secretion than control groups. An effect of dose was evident in the llama pituitary cell culture where mean LH concentrations were greater in wells treated with a higher dose of OIF in comparison to wells treated with a lower dose, both of which were higher than in wells with no treatment. Although OIF stimulated LH release in bovine cell cultures, an apparent dose response was not detected. Results indicate that the preovulatory LH surge observed after OIF treatment in camelids may be the result of OIF directly stimulating LH release from gonadotrope cells within the anterior pituitary gland. In conclusion these results illustrate that the presence and the response to OIF is conserved among species that share no relation or common reproductive strategy.

Master of Science / Department of Animal Sciences and Industry / Joann M. Kouba / The objective of these studies was to investigate the efficacy of an intravaginal triptorelin acetate (TA) gel as an ovulation-inducing agent in mares. In Exp 1, 24 mares were stratified by parity and age and randomly assigned to 3 treatment groups receiving either: 5 mL TA gel (500 μg TA; TA5), 10 mL TA gel (1,000 μg TA; TA10), or 5 mL vehicle gel only (CON). Following the appearance of a follicle ≥ 25 mm, blood samples were obtained every 24 h until treatment administration for measurement of luteinizing hormone (LH) concentrations. Once a follicle ≥ 35 mm in diameter was detected, treatment was administered intravaginally. Following treatment, blood samples were collected and ovaries were scanned via transrectal ultrasonography every 12 h until 48 h post-ovulation. Both TA5 and TA10 tended (P = 0.08) to experience a brief surge in LH by 12 h post-treatment. Regarding LH concentrations, there was a significant (P < 0.005) treatment by time interaction. The interval from treatment to ovulation was not different (P > 0.05) between groups, nor was there a difference (P > 0.05) in the percentage of mares ovulating within 48 h of treatment administration. We hypothesized that LH was not staying elevated long enough for ovulation to occur in a greater percentage of mares. Furthermore, more frequent sampling and scanning was needed to get a more robust characterization of the effect of TA on LH and a more accurate timeframe for when ovulation was occurring. Experiment 2 involved the same CON and TA5 treatment groups; however, the TA10 treatment was split into two 5-mL doses of TA gel, administered 24 h apart (two 500-μg doses of TA; TA5x2). Blood collection and ultrasonography occurred every 12 h upon detection of a follicle ≥ 25 mm in diameter. Once a follicle ≥ 35 mm was detected, treatment was administered and ultrasonography and blood collection occurred every 6 h until 48 h post-ovulation. Both TA5 and TA5x2 had a significant increase (P < 0.05) in LH by 6 h post-treatment, which was declining by 12 h post-treatment. The second dose administered to TA5x2 failed to elicit an increase in LH (P > 0.05). Overall, the treatment by time interaction was significant (P < 0.005) in regard to LH and the interval from treatment to ovulation was shorter (P < 0.01) in TA5 and TA5x2 compared with CON. In conclusion, TA gel increased LH concentrations and hastened the interval from treatment to ovulation in mares in Exp. 2, but not Exp. 1, without an advantage in the timing of ovulation noted between the 5 or 10-mL doses, or administration of two 5-mL doses given 24 h apart. The results of these studies suggest that further testing is needed to effectively evaluate the efficacy of TA gel as an ovulation-inducing agent in mares.

No description available.

Proper understanding of ovarian follicular and luteal development is essential to improve and optimally control reproductive function in domestic animals and to unravel causes of infertility in animals and humans. MicroRNAs (miRNAs) are key post-transcriptional gene regulators in multiple processes involving tissue growth and differentiation. The studies in this thesis were carried out to identify and characterise expression profiles of miRNAs and their potential roles during ovarian follicular development in the cow. The first study aimed to identify expression profiles of miRNAs during antral follicle growth. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. An heterologous microarray approach followed by RT-qPCR validation was used to identify and compare miRNA profiles between large (13–16 mm) healthy and small (4–8 mm) follicles. A unique subset of 7 miRNAs (miR-144, miR-202, miR-210, miR-451, miR-652, miR-873 and miR-876) was identified that were enriched in the Large Healthy follicles relative to small follicles and Large Atretic follicles. Further characterisation revealed that many of these miRNAs were highly expressed in the granulosa compartment of the follicle, predominantly in the mural granulosa cells, indicating their potential involvement in regulating granulosa cell function. Expression patterns of miRNAs in the ovarian tissues were generally reflected in the follicular fluid, thus follicular fluid may be used to monitor follicular health. Finally, tissue-wide screening of miRNAs identified miR-202 as a gonad-specific miRNA in the cow. The aim of the second study was to identify potential roles of the miRNAs enriched in Large Healthy follicles. Using in silico approaches, about 1700 bovine genes were identified as the predicted targets of those miRNAs. Putatively targeted signalling pathways are primarily involved in cell survival, proliferation and differentiation. Further, as expected, top predicted gene targets (SPRED1, ATG7, TGFBR2) showed an expression pattern in follicular tissues that was opposite to the patterns of the targeting miRNAs. However, expression patterns of miR-202 or miR-210 during follicular growth could not be recapitulated in gonadotrophin-stimulated cells in vitro and moreover, over-expression or inhibition of miR-202 in these cells did not result in changes in target mRNA levels. The third study investigated the expression profiles of miRNAs during follicle-luteal transition. Microarray analyses identified 9 miRNAs that were upregulated and 14 miRNAs that were downregulated between Large Healthy follicles and early corpus luteum in the cow. Predicted targets of these miRNAs were associated with signalling pathways involved in cell survival, proliferation and differentiation, indicating that these miRNAs might play significant regulatory roles during ovulation and early luteal development. Further, expression of some of these miRNAs and their putative targets during luteinisation in vivo could be recapitulated in cultured granulosa cells providing a system to study the functional roles of these miRNAs during follicle-luteal development. In summary, this is the first study identifying unique subsets of miRNAs associated with follicular development and the follicle-luteal transition in the cow which may be important for female fertility.

The purpose of the studies reported in this thesis was to increase our understanding of the reproductive physiology of South American camelids. Studies were conducted in llamas and alpacas to investigate methods to electively control ovarian follicular dynamics, to determine the effects of hormone preparations or biological factors derived from seminal plasma on ovulation induction, and to evaluate the establishment of superstimulatory protocols to induce a consistent ovarian follicular response for oocyte collection. The first study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas, and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating. In the first experiment, lutenizing hormone (LH) and follicular ablation treatments were most effective for inducing follicular wave synchronization, while estradiol plus progesterone (E/P) treatment was intermediate. In the second experiment, llamas were assigned randomly to Control, (E/P), and LH groups. A single, fixed-time natural mating was permitted 10 to 12 days after treatment. The pregnancy rate was higher (P<0.05) for synchronized llamas (LH and E/P groups combined) than for non-synchronized llamas (Control group). The second study was done to compare the effects of hormonal treatments and natural mating on ovulation induction, interval to ovulation, and luteal development in llamas. No differences were detected among groups (mated, LH, and GnRH) in ovulation rate (80%, 91%, 80%, respectively; P = 0.6), or interval from treatment to ovulation (30.0 ¡À 0.5, 29.3 ¡À 0.6, 29.3 ¡À 0.7 h, respectively; P = 0.9). Similarly, no differences were detected among groups (mated, LH, and GnRH) in maximum corpus luteum (CL) diameter. The third study documents the existence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. In Experiment 1, female alpacas were given alpaca seminal plasma or saline intramuscularly (im) or by intrauterine infusion. Only alpacas that were given seminal plasma im ovulated. In Experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 ¡À 0.7 hours after seminal plasma treatment. In Experiment 3, female llamas were given llama seminal plasma, GnRH, or saline im, and ovulation was detected in 6/6, 5/6, and 0/6 llamas, respectively (P < 0.001). Treatment was followed by a surge (P < 0.01) in plasma LH concentration beginning 15 minutes and 75 minutes after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05), and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). The fourth study describes the presence of an OIF in the seminal plasma of Bos taurus ¨C a species conventionally considered to ovulate spontaneously - contains OIF. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P < 0.001). The ovulation rate was lower (P < 0.01) in bull seminal plasma group compared to that in the groups treated with alpaca or llama seminal plasma (100%). The fifth study was conducted to determine a local versus systemic effect of ovulation-inducing factor in seminal plasma. Ovulation rate in the seminal plasma intramuscular group (93%) was higher (P < 0.01) than seminal plasma intrauterine group (41%), while the seminal plasma intrauterine curettage group was intermediate (67%). The sixth study was done to determine the time required for llama oocyte to reach the maturation stage, and to establish a superstimulatory treatment for oocyte collection. Llama oocytes reached second metaphase as early as 28 h after in vitro culture. The FSH- and eCG-treated groups did not differ (P = 0.85) with respect to the number of follicles ¡Ý6 mm at the time of cumulus-oocyte complex (COC) collection (17.9 ¡À 2.2 vs 17.7 ¡À 2.2), the number of COC collected (10.7 ¡À 2.1 vs 11.2 ¡À 2.3 per llama), or the collection rate per follicle aspirated (71 vs 74%). Finally, in the last study, the effect of two superstimulatory treatments was evaluated on ovarian response and COC collection efficiency and oocyte maturation in alpacas. No difference (P = 0.54) was observed between FSH and eCG- treated alpacas in the number of expanded (11.5 ¡À 2.9 vs 8.8 ¡À 2.8) or compact COC collected with ¡Ý3 layers of cumulus cells (12.5 ¡À 4.3 vs 14.3 ¡À 2.6; P = 0.72). No difference (P = 0.1) was detected between FSH and eCG groups in the number of expanded COC at first metaphase (1.2 ¡À 1.2 vs 1.7 ¡À 0.6) or second metaphase stage (8.5 ¡À 1.9 vs 6.0 ¡À 2.1) respectively. In conclusion, these studies demonstrated that the control of ovarian follicular wave emergence and ovulation induction in llamas will contribute consistently to the establishment of fixed-time natural or artificial insemination as well as recipient synchronization in embryo transfer programs. The discovery of an ovulatory molecule in the semen of this species generates a new area of research regarding the ovulation mechanism in induced ovulators. Characterization of this factor may have important implications in the diagnosis and treatment of male and female infertility. Finally, the superstimulatory treatments and oocyte development studies will establish the baseline for the development of an in vitro embryo production system in llamas and alpacas.

The purpose of the studies reported in this thesis was to increase our understanding of the reproductive physiology of South American camelids. Studies were conducted in llamas and alpacas to investigate methods to electively control ovarian follicular dynamics, to determine the effects of hormone preparations or biological factors derived from seminal plasma on ovulation induction, and to evaluate the establishment of superstimulatory protocols to induce a consistent ovarian follicular response for oocyte collection. The first study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas, and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating. In the first experiment, lutenizing hormone (LH) and follicular ablation treatments were most effective for inducing follicular wave synchronization, while estradiol plus progesterone (E/P) treatment was intermediate. In the second experiment, llamas were assigned randomly to Control, (E/P), and LH groups. A single, fixed-time natural mating was permitted 10 to 12 days after treatment. The pregnancy rate was higher (P<0.05) for synchronized llamas (LH and E/P groups combined) than for non-synchronized llamas (Control group). The second study was done to compare the effects of hormonal treatments and natural mating on ovulation induction, interval to ovulation, and luteal development in llamas. No differences were detected among groups (mated, LH, and GnRH) in ovulation rate (80%, 91%, 80%, respectively; P = 0.6), or interval from treatment to ovulation (30.0 ¡À 0.5, 29.3 ¡À 0.6, 29.3 ¡À 0.7 h, respectively; P = 0.9). Similarly, no differences were detected among groups (mated, LH, and GnRH) in maximum corpus luteum (CL) diameter. The third study documents the existence of an ovulation-inducing factor (OIF) in the seminal plasma of alpacas and llamas. In Experiment 1, female alpacas were given alpaca seminal plasma or saline intramuscularly (im) or by intrauterine infusion. Only alpacas that were given seminal plasma im ovulated. In Experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 ¡À 0.7 hours after seminal plasma treatment. In Experiment 3, female llamas were given llama seminal plasma, GnRH, or saline im, and ovulation was detected in 6/6, 5/6, and 0/6 llamas, respectively (P < 0.001). Treatment was followed by a surge (P < 0.01) in plasma LH concentration beginning 15 minutes and 75 minutes after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05), and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). The fourth study describes the presence of an OIF in the seminal plasma of Bos taurus ¨C a species conventionally considered to ovulate spontaneously - contains OIF. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P < 0.001). The ovulation rate was lower (P < 0.01) in bull seminal plasma group compared to that in the groups treated with alpaca or llama seminal plasma (100%). The fifth study was conducted to determine a local versus systemic effect of ovulation-inducing factor in seminal plasma. Ovulation rate in the seminal plasma intramuscular group (93%) was higher (P < 0.01) than seminal plasma intrauterine group (41%), while the seminal plasma intrauterine curettage group was intermediate (67%). The sixth study was done to determine the time required for llama oocyte to reach the maturation stage, and to establish a superstimulatory treatment for oocyte collection. Llama oocytes reached second metaphase as early as 28 h after in vitro culture. The FSH- and eCG-treated groups did not differ (P = 0.85) with respect to the number of follicles ¡Ý6 mm at the time of cumulus-oocyte complex (COC) collection (17.9 ¡À 2.2 vs 17.7 ¡À 2.2), the number of COC collected (10.7 ¡À 2.1 vs 11.2 ¡À 2.3 per llama), or the collection rate per follicle aspirated (71 vs 74%). Finally, in the last study, the effect of two superstimulatory treatments was evaluated on ovarian response and COC collection efficiency and oocyte maturation in alpacas. No difference (P = 0.54) was observed between FSH and eCG- treated alpacas in the number of expanded (11.5 ¡À 2.9 vs 8.8 ¡À 2.8) or compact COC collected with ¡Ý3 layers of cumulus cells (12.5 ¡À 4.3 vs 14.3 ¡À 2.6; P = 0.72). No difference (P = 0.1) was detected between FSH and eCG groups in the number of expanded COC at first metaphase (1.2 ¡À 1.2 vs 1.7 ¡À 0.6) or second metaphase stage (8.5 ¡À 1.9 vs 6.0 ¡À 2.1) respectively. In conclusion, these studies demonstrated that the control of ovarian follicular wave emergence and ovulation induction in llamas will contribute consistently to the establishment of fixed-time natural or artificial insemination as well as recipient synchronization in embryo transfer programs. The discovery of an ovulatory molecule in the semen of this species generates a new area of research regarding the ovulation mechanism in induced ovulators. Characterization of this factor may have important implications in the diagnosis and treatment of male and female infertility. Finally, the superstimulatory treatments and oocyte development studies will establish the baseline for the development of an in vitro embryo production system in llamas and alpacas.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vi, 56 p. : ill. Includes abstract. Includes bibliographical references (p. 45-56).

Background: Intrauterine Insemination (IUI)is one of the common first-line assisted reproductive technologies (ART) for couples suffering infertility. Controlled ovarian stimulation (COH) with clomiphene citrate (CC) or aromatase inhibitor (like Letrozole) is often used in adjunct to IUI to increase the pregnancy outcome. Both CC and letrozole can be given alone as a single ovulation induction agent or they can be combined with injectable gonadotropin for purpose of superovulation. Study objectives: To systematically review the efficacy and adverse outcomes of letrozole and CC for supervulation in infertility patients undergoing IUI. Method: Systematic review of pertinent randomised controlled trials (RCT) using the bibliographic databases EMBASE, PubMed, Cochrane, Medline (OVID), Academic Search Premier and CINAHL. References of selected articles identified were hand-searched for additional relevant citations. RCTs that have compared the pharmacological performance of CC and letrozole as a single agent or combination with equal dose of gonadotropins were included. Results: Ten published randomized controlled trials were included in this review. The mean age, infertility diagnosis and duration of infertility of the recruited participants were comparable. Pregnancy rate was found to be comparable in clomiphene citrate (CC) group and letrozole (L) group. Higher peak estrogen concentration and greater number of dominant follicles were reported in CC group. Endometrial thickness was found significantly greater in L group. Adverse outcomes of rate of miscarriage, multiple pregnancies, ectopic pregnancies, OHSS and fetal anomalies were not significantly different between the two intervention groups. Conclusion: Letrozole and CC, considered equally patient-friendly agent due to oral route administration. Both agents achieved similar pregnancy rates without any increased risk of adverse events in either group. Letrozole can be used as alternative first-line OI agent to CC in reproductive treatments. Drug selection for patients should be done according to the cost effectiveness, duration of therapy, characteristics and compliance of patients. / published_or_final_version / Public Health / Master / Master of Public Health

No description available.

No description available.