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The type of stimuli triggering GnRH secretion has been used to classify mammalian species into two categories: spontaneous or induced ovulators. In the former, ovarian steroids produced by a mature follicle elicit the release of GnRH from the hypothalamus, but in the latter, GnRH secretion requires coital stimulation. However, the mechanism responsible for eliciting the preovulatory LH surge in induced ovulators is still not well understood and seems to vary among species. The main goal of this review is to offer new information regarding the mechanism that regulates coitus-induced ovulation. Analysis of several studies documenting the discovery of β-NGF in seminal plasma and its role in the control of ovulation in the llama and rabbit will be described. We also propose a working hypothesis regarding the sites of action of β-NGF in the llama hypothalamus. Finally, we described the presence of β-NGF in the semen of species categorized as spontaneous ovulators, mainly cattle, and its potential role in ovarian function. The discovery of this seminal molecule and its ovulatory effect in induced ovulators challenges previous concepts about the neuroendocrinology of reflex ovulation and has provided a new opportunity to examine the mechanism(s) involved in the cascade of events leading to ovulation. The presence of the factor in the semen of induced as well as spontaneous ovulators highlights the importance of understanding its signaling pathways and mechanism of action and may have broad implications in mammalian fertility.

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.

Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), release of GnRH from the hypothalamus is triggered when, in the absence of progesterone, systemic estradiol concentrations exceed a threshold. In induced ovulators (e.g., rabbits, ferrets, cats, camelids), release of GnRH is contingent upon copulatory stimuli; hence, ovulation is not a regular cyclic event. Since a classic 1970 Peruvian study, dogma has maintained that physical stimulation of the genitalia during copulation is the primary trigger for inducing ovulation in alpacas and llamas. Exciting results of recent studies, however, provide direct evidence for the existence of an ovulation-inducing factor (OIF) in semen, and compel us to re-examine the mechanism of ovulation in both induced and spontaneous ovulators. Ovulation-inducing factor in seminal plasma is a potent stimulant of LH secretion, ovulation and luteal gland development, and acts via a systemic rather than a local route. OIF is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K. It has a molecular mass of 14kDa, and may be part of a larger protein complex or pro-hormone. The effect of OIF is dose-related and evident at physiologically relevant doses (i.e., as little as 1/100th that present in the ejaculate), and is mediated, in whole or in part, at the level of the hypothalamus in vivo. The factor exists in the seminal plasma of every species in which it has been examined thus far, including Bactrian camels, alpacas, llamas, cattle, horses, pigs, and koalas. Seminal plasma OIF does not appear to be a phylogenetic vestige in spontaneous ovulators since it (1) induced ovulation in pre-pubertal mice, (2) altered ovarian follicular wave dynamics in cows, and (3) elicited LH release in vitro from primary pituitary cell cultures of rats, mice, guinea pigs, rabbits, llamas and cows., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate., Methods: Semen collected from male llamas by artificial vagina was centrifuged and the seminal plasma was harvested and stored frozen. Seminal plasma was thawed and loaded onto a Type 1 macro-prep ceramic hydroxylapatite column and elution was carried out using a lineal gradient with 350 mM sodium phosphate. Three protein fractions were identified clearly (Fractions A, B, and C), where a prominent protein band with a mass of 14 kDa was identified in Fraction C. Fraction C was loaded into a sephacryl gel filtration column for further purification using fast protein liquid chromatography (FPLC). Isocratic elution resulted in 2 distinct protein fractions (Fractions C1 and C2). An in vivo bioassay (n=10 to 11 llamas per group) was used to determine the ovarian effect of each fraction involving treatment with saline (negative control), whole seminal plasma (positive control), or seminal plasma Fractions A, B or C2. Ultrasonography was done to detect ovulation and CL formation, and blood samples were taken to measure plasma progesterone and LH concentrations., Results: Ovulation and CL formation was detected in 0/10, 10/11, 0/10, 2/11, and 10/11 llamas treated with saline, whole seminal plasma, Fractions A, B and C2 respectively (P<0.001). A surge in circulating concentrations of LH was detected within 2 hours only in llamas treated with either whole seminal plasma or Fraction C2. Plasma progesterone concentration and CL diameter profiles were greatest (P<0.05) in llamas treated with Fraction C2., Conclusion: Ovulation-inducing factor was isolated from llama seminal plasma as a 14 kDa protein molecule that elicits a preovulatory LH surge followed by ovulation and CL formation in llamas, suggesting an endocrine effect at the level of the hypothalamus (release of GnRH) or the pituitary (gonadotrophs).

Background: Previously, we documented the presence of ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s) in seminal plasma responsible for inducing ovulation., Methods: In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group) were treated i.m. with whole seminal plasma (positive control), phosphate-buffered saline (negative control), or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or < 5 kDa. In Experiment 2, female llamas (n = 7 per group) were given an i.m. dose of seminal plasma treated previously by: 1) enzymatic digestion with proteinase-K, 2) incubation with charcoal-dextran, 3) heating to 65 degrees C, or 4) untreated (control). In Experiment 3, female llamas (n = 10 per group) were given an i.m. dose of pronase-treated or non-treated (control) seminal plasma. In all experiments, llamas were examined by transrectal ultrasonography to detect ovulation and CL formation. Ovulation rate was compared among groups by Fisher's exact test and follicle and CL diameters were compared among groups by analyses of variance or student's t-tests., Results: In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each), but none ovulated in the other groups (P < 0.001). In Experiment 2, ovulations were detected in all llamas in each treatment group; i.e., respective treatments of seminal plasma failed to inactivate the ovulation-inducing factor. In Experiment 3, ovulations were detected in 0/10 llamas given pronase-treated seminal plasma and in 9/10 controls (P < 0.01)., Conclusions: We conclude that ovulation-inducing factor (OIF) in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

We have recently reported the presence of an ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas-species characterized as induced ovulators. The study was designed to test the hypothesis that the seminal plasma of bulls will induce ovulation in llamas, and to compare the ovulation-inducing effect of seminal plasma of conspecific versus hetero-specific males. The seminal plasma of alpacas, a closely related induced ovulator (Lama pacos), and cattle, a distantly related ruminant species (Bos taurus) considered to be spontaneous ovulators, were compared with that of the llama (Lama glama). Ovulation and maximum corpus luteum diameter were compared by ultrasonography among female llamas (n=19 per group) treated intramuscularly with 2 mL of phosphate buffered saline (PBS, negative control) and those treated with 2 mL of seminal plasma of bulls, alpacas, or llamas (conspecific control). The diameter of the preovulatory follicle did not differ among groups at the time of treatment. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P<0.001). The proportion of females that ovulated was lower (P<0.01) in bull seminal plasma group compared to the groups treated with alpaca or llama seminal plasma (100%). A corpus luteum was detected on Day 8 (Day 0=treatment) in all llamas in which ovulation was detected earlier (Day 2) by ultrasonography. The diameter of the CL did not differ among groups. Results document the presence of an ovulation-inducing factor in the seminal plasma of B. taurus. The interspecies effects of seminal plasma on ovulation and luteal development provide rationale for the hypothesis that OIF is conserved among both spontaneous and induced ovulating species.

The effect of the timing of mating on ovarian response in llamas was evaluated using 20 adult llamas weighing 90-120 kg which had been in oestrus for 5 days and were treated with 20 mg pFSH every 12 h for the following 5 days (total dose: 200 mg of FSH-NIH-P1). They were randomly allocated to Group A (N = 10) and mated immediately at the end of pFSH treatment or to Group B (n = 10) and mated 36 h after the end of pFSH treatment. Llamas of both groups were given hCG (750 iu, i.m.) immediately after mating. A second mating was allowed 12 h later. Ova and embryos were recovered by non-surgical uterine flushing 7 days after the first mating. Ovarian response was immediately evaluated afterwards via laparoscopy. The mean ovulation rate of 4.5 corpora lutea for Group A was significantly lower (P < 0.01) than the mean of 13.8 observed for Group B. The total ovarian response (number of corpora lutea + follicles > 10 mm) was also significantly higher (P < 0.01) in Group B than in Group A. Twenty-seven ova were recovered in each group, corresponding to 60% and 20% (P < 0.01) of the corpora lutea observed in Groups A and B, respectively; however, no significant difference (P > 0.05) in fertilisation rate was observed. The results show that pFSH induces superovulation in llamas treated during oestrus and that a 36-h interval between the end of FSH treatment and mating increases ovulation rate and the total ovarian response but does not affect the number of ova/embryos recovered.