Mauricio Silva

Is an ovulation-inducing factor (OIF) present in the seminal plasma of rabbits?

The objectives of this study were (1) to determine the effect of rabbit seminal plasma on LH secretion and ovulation using the llama animal model as an in vivo ovulation bioassay and (2) to determine the effect of llama or rabbit seminal plasma on ovulation induction in the rabbit model. In Experiment 1, llamas with a growing follicle ≥8mm in diameter were assigned randomly to one of three groups (n=5 per group) and given an intramuscular dose of 1mL of: (a) llama seminal plasma, (b) rabbit seminal plasma, or (c) phosphate buffered saline (PBS; negative control). Blood samples for LH measurement were taken every 15 min from 1.5 h before to 8 h after treatment (Day 0: starting of treatment). Llamas were examined by ultrasonography every 12h from treatment to ovulation, and then every other day until Day 16 after treatment to evaluate corpus luteum (CL) development. Blood samples for progesterone measurement were taken every other day from Day 0 to Day 16. Ovulation was detected in 4 of 5, 5 of 5, and 0 of 0 llamas treated with llama or rabbit seminal plasma and PBS, respectively (P<0.001). After treatment, plasma LH concentration increased and decreased (P<0.01) in the llama and rabbit seminal plasma group but not in the PBS-treated group. No differences were observed on CL development (P≥0.3) and progesterone secretion (P>0.05) between both seminal plasma treated groups. In Experiment 2, receptive female rabbits (n=5-7 per group) were given an intramuscular dose of: (a) 0.5, (b) 1.0 and (c) 2.0mL of either rabbit or llama seminal plasma, (d) 0.5mL PBS (negative control), or (e) 25μg of gonadoreline acetate (GnRH; positive control). Does were submitted to laparotomy 24-36 h after treatment to determine the ovulatory response and the presence of antral and hemorrhagic anovulatory follicles. Ovulation sites (7.0±0.6) were only detected in GnRH-treated does (P<0.01). There was an increase (P<0.01), in the total number of follicles (antral plus hemorraghic follicles) in those females treated with 1mL of rabbit seminal plasma and there was a tendency (P=0.08) for more hemorrhagic anovulatory follicles in does treated with 1.0 and 2.0mL of either rabbit or llama seminal plasma. Results document the presence of OIF in the seminal plasma of rabbits. The differential ovulatory response between species, however, requires further investigation.

Cetrorelix suppresses the preovulatory LH surge and ovulation induced by ovulation-inducing factor (OIF) present in llama seminal plasma

BackgroundThe purpose of the study was to determine if the effect of llama OIF on LH secretion is mediated by stimulation of the hypothalamus or pituitary gland.MethodsUsing a 2-by-2 factorial design to examine the effects of OIF vs GnRH with or without a GnRH antagonist, llamas with a growing ovarian follicle greater than or equal to 8 mm were assigned randomly to four groups (n = 7 per group) and a) pre-treated with 1.5 mg of GnRH antagonist (cetrorelix acetate) followed by 1 mg of purified llama OIF, b) pre-treated with 1.5 mg of cetrorelix followed by 50 micrograms of GnRH, c) pre-treated with a placebo (2 ml of saline) followed by 1 mg of purified llama OIF or d) pre-treated with a placebo (2 ml of saline) followed by 50 micrograms of GnRH. Pre-treatment with cetrorelix or saline was given as a single slow intravenous dose 2 hours before intramuscular administration of either GnRH or OIF. Blood samples for LH measurement were taken every 15 minutes from 1.5 hours before to 8 hours after treatment. The ovaries were examined by ultrasonography to detect ovulation and CL formation. Blood samples for progesterone measurement were taken every-other-day from Day 0 (day of treatment) to Day 16.ResultsOvulation rate was not different (P = 0.89) between placebo+GnRH (86%) and placebo+OIF groups (100%); however, no ovulations were detected in llamas pre-treated with cetrorelix. Plasma LH concentrations surged (P < 0.01) after treatment in both placebo+OIF and placebo+GnRH groups, but not in the cetrorelix groups. Maximum plasma LH concentrations and CL diameter profiles did not differ between the placebo-treated groups, but plasma progesterone concentrations were higher (P < 0.05), on days 6, 8 and 12 after treatment, in the OIF- vs GnRH-treated group.ConclusionCetrorelix (GnRH antagonist) inhibited the preovulatory LH surge induced by OIF in llamas suggesting that LH secretion is modulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus.

Ovarian estradiol modulates the stimulatory effect of ovulation-inducing factor (OIF) on pituitary LH secretion in llamas

This study was designed to: 1) characterize the effect of ovulation-inducing factor (OIF) on pituitary LH secretion in ovariectomized (OVX) llamas; and 2) determine the effect of OIF on LH secretion in OVX llamas pretreated with estradiol-17β (E-17β) or estradiol benzoate (EB). In Experiment 1, intact and OVX llamas (n = 5 or 6 per group) were assigned to a two by two factorial design: 1) Intact llamas treated with 1 mL of phosphate buffered saline (PBS); 2) Intact llamas treated with 1 mg of purified OIF; 3) OVX llamas treated with 1 mL of PBS; or 4) OVX llamas treated with 1 mg of purified OIF. In Experiment 2, intact and OVX llamas (n = 5 or 6 per group) were randomly assigned to the following groups: 1) Intact llamas treated with 1 mg of purified OIF; 2) OVX llamas treated with 1.0 mL of PBS; 3) OVX llamas treated with 1.0 mg of purified OIF; 4) OVX llamas primed with E-17β, followed by 1.0 mg of purified OIF. Experiment 3 was similar as described for Experiment 2, except that priming was done with EB. In Experiment 1, animal category by treatment and animal category by treatment by time interactions tended (P = 0.08) to affect LH concentration. The effect of OIF on LH released was partly restored (P < 0.05), to the values observed for the intact OIF-treated females, when OVX llamas were primed with E-17β or BE (Experiments 2 and 3). We concluded that peripheral estradiol concentrations in llamas partially modulates the effect of OIF on pituitary LH secretion; however, other ovarian factor(s) could also participate in this modulatory action.

Seminal plasma induces ovulation in llamas in the absence of a copulatory stimulus: Role of nerve growth factor as an ovulation-inducing factor

Llamas are considered to be reflex ovulators. However, semen from these animals is reported to be rich in ovulation-inducing factor(s), one of which has been identified as nerve growth factor (NGF). These findings suggest that ovulation in llamas may be elicited by chemical signals contained in semen instead of being mediated by neural signals. The present study examines this notion. Llamas displaying a preovulatory follicle were assigned to four groups: group 1 received an intrauterine infusion (IUI) of PBS; group 2 received an IUI of seminal plasma; group 3 was mated to a male whose urethra had been surgically diverted (urethrostomized male); and group 4 was mated to an intact male. Ovulation (detected by ultrasonography) occurred only in llamas mated to an intact male or given an IUI of seminal plasma and was preceded by a surge in plasma LH levels initiated within an hour after coitus or IUI. In both ovulatory groups, circulating β-NGF levels increased within 15 minutes after treatment, reaching values that were greater and more sustained in llamas mated with an intact male. These results demonstrate that llamas can be induced to ovulate by seminal plasma in the absence of copulation and that copulation alone cannot elicit ovulation in the absence of seminal plasma. In addition, our results implicate β-NGF as an important mediator of seminal plasma-induced ovulation in llamas because ovulation does not occur if β-NGF levels do not increase in the bloodstream, a change that occurs promptly after copulation with an intact male or IUI of seminal plasma.

The effect of repeated administrations of llama ovulation-inducing factor (OIF/NGF) during the peri-ovulatory period on corpus luteum development and function in llamas

The objective of the study was to test the hypothesis that repeated administrations of OIF/NGF during the peri-ovulatory period (pre-ovulatory, ovulatory, early post-ovulatory), will enhance the luteotrophic effect in llamas. Female llamas were examined daily by transrectal ultrasonography in B- and Doppler-mode using a scanner equipped with a 7.5-MHz linear-array transducer to monitor ovarian follicle and luteal dynamics. When a growing follicle ≥7mm was detected, llamas were assigned randomly to one of the three groups and given 1mg of purified OIF/NGF im (intramuscular) (a) pre-ovulation (single dose; n=12), (b) pre-ovulation and at the time of ovulation (2 doses, n=10), or (c) pre-ovulation, at the time of ovulation, and 24h after ovulation (3 doses, n=10). The pre-ovulatory follicle diameter at the time of treatment, ovulation rate and the first day of CL detection did not differ (P=0.3) among groups. However, maximum CL diameter was greatest (P=0.003) in llamas in the 2-dose group, and smallest in the 3-dose group. Accordingly, the 2 dose-group had the largest day-to-day profile for CL diameter (P<0.01), area of CL vascularization (<0.01), and plasma progesterone concentration (P=0.01) compared to the other groups. Interestingly, the luteal response to 3-doses of OIF/NGF during the peri-ovulatory period was not different from a single dose. In conclusion, OIF/NGF isolated from llama seminal plasma is luteotrophic and the effect on CL size and function is affected by the number and timing of treatments during the peri-ovulatory period.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of β-nerve growth factor of seminal plasma origin in female llamas

The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of β-nerve growth factor (β-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF-treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that β-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.

Effects of nutritional restriction on metabolic, endocrine, and ovarian function in llamas (Lama glama)

The objectives of the study were to determine the effects of nutritional restriction on ovarian function in llamas. Mature female llamas were assigned randomly to a Control group, fed 100% of maintenance energy requirements (MER) (n=8), or a Restricted group (n=8) fed from 70% to 40% of MER until a body condition score of 2.5 was attained. Blood samples were taken every-other-day to determine plasma concentrations of LH, estradiol, leptin and metabolic markers, and follicular dynamics were monitored daily by ultrasonography for 30 days (Experiment 1). Llamas were then treated with GnRH to compare the ovulatory response and corpus luteus (CL) development between groups (Experiment 2). Blood samples were taken to measure LH, leptin, progesterone and metabolic markers and ovarian structures were assessed as in Experiment 1. Llamas in the Restricted group had lower body mass and body condition scores than those in the Control group (P<0.001). Plasma concentrations of cholesterol, non-esterified fatty acids, triglycerides, and urea were higher in the Restricted group (P<0.05) than in the Control group. The day-to-day diameter profiles of the dominant follicles were smaller (P<0.05) in the Restricted group than in the Control group but plasma estradiol concentration did not differ. The ovulation rate and LH secretion in response to GnRH did not differ. Day-to-day profiles of CL diameter, plasma progesterone and leptin concentrations were smaller (P<0.01) in the Restricted group. In conclusion, nutritional restriction in llamas was associated with suppressed follicle and CL development, and lower plasma concentrations of progesterone and leptin.

Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter

Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as β-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7-10 vs >10mm) on ovulation rate, CL diameter and function in llamas. Llamas (n=32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1mg purified OIF/NGF in the presence of a follicle of 7-10mm in diameter; (b) 50 μg of GnRH in the presence of a follicle of 7-10mm in diameter; (c) 1mg purified OIF/NGF in the presence of a follicle >10mm in diameter; (d) 50 μg of GnRH in the presence of a follicle >10mm in diameter. Llamas were examined by ultrasonography every 12h from treatment to Day 2 (Day 0=treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P<0.001), however plasma progesterone concentration (n=15/per group) was higher (P<0.05) in the OIF/NGF - than that of the GnRH - treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment.

Effect of location and stage of development of dominant follicle on ovulation and embryo survival rate in alpacas

This study was designed to determine the effect of location of the preovulatory dominant follicle and stage of ovarian follicle development on ovulation rate and embryo survival in alpacas. In Experiment 1, mature lactating alpacas were randomly assigned to one of two groups according to the location of the dominant follicle detected by ultrasonography: (a) Right ovary (RO, n=96) or (b) Left ovary (LO, n=108). All females were mated once by an intact adult male. Ovulation rate, CL diameter and embryo survival rate (heartbeat) were assessed by ultrasonography on Days 2 (Day 0=mating), 8 and 30, respectively. Ovulation rate (96.5 and 96.3% for RO and LO group, respectively), corpus luteum (CL) diameter (10.2 and 10.6 mm for RO and LO group, respectively) and pregnancy rate (60.2 and 56.7% for RO and LO group, respectively) did not differ among groups. In Experiment 2, lactating alpacas (n=116) were submitted to ultrasonic-guided follicle ablation to synchronize follicular wave emergence. Afterwards, daily ultrasonography examinations were performed and females were randomly assigned to the following groups according to the growth phase and diameter of the dominant follicle: (a) early growing (5-6 mm, n=27), (b) growing (7-12 mm, n=30); (c) static (7-12 mm, n=30), or (d) regressing phase (12-7 mm, n=29). All alpacas were mated with a proven intact male, except five alpacas from early growing group that rejected the male. Females were examined by ultrasonography on Day 2 (ovulation rate), Day 8 (CL diameter), and Days 15, 20, 25, 30 and 35 (embryo survival by the presence of embryo proper and heartbeat). No differences were detected in ovulation rate among groups (96%, 97%, 100%, and 97%) or in CL size (10.3, 11.7, 11.1, and 11.1 mm, for early growing, growing, early static and regressing, respectively). Although, embryo survival rate at Day 35 after mating was numerically greatest in growing (65.5%), intermediate in early growing (52.4%) and static (53.3%), and least in regressing phase (42.9%), there were no differences among groups. Results suggest that neither location nor stage of development of the dominant follicle has an influence on ovulation and embryo survival rate in alpacas.

Vitrification of in vitro mature alpaca oocyte: effect of ethylene glycol concentration and time of exposure in the equilibration and vitrification solutions.

The effect of different ethylene glycol concentrations, times of exposure and vitrification procedure on viability, cleavage and blastocyst rate of in vitro matured alpaca oocytes chemically activated after vitrification was analyzed. In Experiment 1, oocytes were incubated for 12-15 min with different concentrations of ethylene glycol (EG) in the equilibration solution (ES) followed by chemical activation and in vitro cultured for 8 days to determine oocyte viability, cleavage and blastocyst rates. In Experiment 2, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to vitrification solutions containing 25, 35 or 45% of EG for 30s, vitrified and stored at -196°C. In Experiment 3, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to the vitrification solution containing 35% of EG for 15, 30 or 45s, vitrified and stored at -196°C. For Experiments 2 and 3, non-vitrified and vitrified oocytes were activated and cultured in vitro. In Experiment 1, oocyte viability was lowest at concentrations of 6 or 8%, intermediate at 2 or 4% and highest at 0% of EG. Oocyte viability and cleavage rate were affected by EG concentration, time of exposure in the vitrification solution or vitrification procedure in Experiment 2 and 3. Alpaca oocytes were viable after vitrification, given that oocyte viability, cleavage and blastocyst rate were affected by the vitrification procedure, EG concentration and time of exposure in the equilibration and vitrification solutions.