Seminal Plasma Induces Ovulation in Llamas in the Absence of a Copulatory Stimulus: Role of Nerve Growth Factor as an Ovulation-Inducing Factor

Abstract

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.

Mammalian species have been classified as either spontaneous or induced ovulators based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus (1, 2). In induced or reflex ovulators (eg, rabbit, Bactrian camel, llama, alpaca, cat, ferret), neural signals elicited by the act of copulation trigger hypothalamic GnRH secretion, which evokes a preovulatory discharge of LH from the pituitary gland (1). In turn, this surge of LH secretion induces ovulation. Although different stimuli (tactile, olfactory, and visual) have been associated with eliciting/facilitating ovulation in reflex ovulators, the physical stimulation of penile intromission or cervical stimulation has been ascribed a pivotal role in triggering the preovulatory LH surge and subsequent ovulation (1). Mating-induced ovulation has been documented in several species of the Camelidae, such as llamas (3–5), alpacas (3, 4, 6, 7), and Bactrian camels (8). In a classic study in alpacas (6), penile intromission was the main physical stimulus found to induce ovulation. However, the use of vasectomized males did not allow a distinction between ovulation induced by the mechanical stimulation of coitus or that resulting from deposition of seminal plasma in the female genital tract. Subsequent studies in Bactrian camels, alpacas, and llamas (8–12) challenged this earlier concept (5, 6) by showing that ovulation could be induced by intravaginal or intrauterine infusion of seminal plasma in the absence of a male. Ratto et al (10) reported that 41% of llamas (7 of 17) ovulated after intrauterine infusion of 2 mL of seminal plasma. These authors also observed that the ovulatory rate increased to 67% (10 of 15) when curettage of the endometrium was performed prior to seminal plasma deposition to mimic the endometrial erosion resulting from repetitive penile intromission during natural mating (13).

Recent studies in llamas, alpacas (10, 14–16), and Bactrian camels (17–19) have documented the presence of a potent ovulation-inducing factor (OIF) in seminal plasma. The existence of this factor(s) has raised the possibility that ovulation in these species may not depend on the activation of a neural pathway but instead is elicited by semen-derived chemical signals. This OIF has been biochemically and functionally characterized (14, 16, 20–25; for review see reference 26) and has been shown to also have a luteotrophic effect by im or intrauterine infusion in llamas and alpacas (12, 14, 22). A recent study demonstrated that OIF purified from the seminal plasma of llamas has an amino acid sequence identical to that of β-nerve growth factor (NGF) (21).

Two recent studies conducted in llamas demonstrated that β-NGF alone is able to induce ovulation (11, 12). These reports showed that ovulation could be induced in 90% of llamas receiving an intrauterine infusion (IUI) of 5 mL of seminal plasma or 20 mg of purified seminal plasma (sp) β-NGF, the amount contained in a normal ejaculate. In both cases, ovulation was preceded by a preovulatory LH surge. However, these studies demonstrated neither the ability of spβ-NGF to reach the bloodstream upon intrauterine deposition of seminal plasma nor a temporal association of circulating spβ-NGF with LH secretion. The present study was designed to accomplish the following: 1) discern between the physical stimulatory effect of natural mating and a potential chemical stimulatory input provided by seminal plasma on ovulation in llamas and 2) determine whether circulating levels of β-NGF increase after mating or IUI of seminal plasma and whether this change is temporally associated with the preovulatory LH surge.

Materials and Methods

Animals and handling conditions

The study was conducted in the Llama Research Farm at the Universidad Austral de Chile, Valdivia, Chile (39° 38'S, 73° 5′W y 19 m above sea level). Llamas were kept in pasture and provided supplementary hay, feed pellet (crude protein 14%, crude fat 2.5%, crude fiber 12%), and ad libitum water. The procedures were performed according to the standards and conditions set forth by the university bioethics committee.

Semen collection and seminal plasma preparation

Semen was collected in an artificial vagina from five males during the 3 months prior to starting the experiments, as previously described (12). A total of 58 ejaculates were obtained from five males, with an average volume of 5 mL per ejaculate. Ejaculates were centrifuged and the supernatant was decanted to remove spermatozoa. A drop was evaluated microscopically to confirm the absence of cells. Sperm-free seminal plasma was pooled in 5- to 6-mL aliquots and stored at −80ºC.

Urethrostomy

The urethrostomy procedure consisted of opening the urethra to the perianal region to have a male able to mate naturally without intrauterine ejaculation. Urethrostomy was conducted in three 5-year-old male llamas, weighing approximately140 kg. The procedure was performed in the Teaching Veterinary Hospital at the Faculty of Veterinary Science, Universidad Austral de Chile. Animals were anesthetized by im administration of acepromazine (0.1 mg/kg) and atropine (0.04 mg/kg), followed by ketamine (1.0 mg/kg) and xylazine (0.25 mg/kg). The anesthesia was maintained by intranasal administration of a mixture of isoflurane (1.5%) and oxygen. After surgery, animals received antiinflammatory/analgesic therapy (ketoprofen, 4.4 mg/kg every 12 h, by 5 d) and an antibiotic (enrofloxacin 5 mg/kg every 8 h) for 7 days. They were also provided with a urethral catheter to allow normal urination during the first postoperatory week. The animals were monitored daily for signs of inflammation or infection for 1 month after surgery. After 3 months of recovery, they were evaluated for mating behavior. When males presented normal mating behavior, including penile erection, time of copulation (>20 min), penetration and ejaculation from the urethral opening in the perianal region, they were used for the study.

Animal treatments

Mature nonpregnant llamas (n = 30), 4–8 years of age, and weighing 120–140 kg were examined daily by transrectal ultrasonography using an ultrasound scanner (MyLab 30; Esaote) equipped with a 7.5-MHz linear array transducer. Llamas with a growing preovulatory size follicle (>7 mm) were randomly assigned to one of four groups: 1) IUI of 5 ml PBS (n = 6, negative control); 2) IUI of 5 mL of seminal plasma (n = 6); 3) single mating with an urethrostomized male (n = 6); and 4) single mating with an intact male (n = 7).

To carry out the IUI, an insemination pipette was guided through the cervix via transrectal palpation, and 2.5 mL of either PBS or seminal plasma was infused into each uterine horn. The volume of seminal plasma used for the IUI was determined based on the average ejaculatory volume of 5.5 mL from 56 ejaculates collected by an artificial vagina, with a total protein concentration ranging from 15 to 17 mg/mL of seminal plasma (27). Considering that spβ-NGF represents 30% of the total protein present in seminal plasma (26), a dose of 5 mL of seminal plasma should mimic the concentration of spβ-NGF deposited into the female genital tract in a natural mating (12).

To verify the occurrence of ovulation, the ovaries were examined by transrectal ultrasonography, as outlined above, every 12 hours from the time of treatment until ovulation. Ovulation was defined as the sudden disappearance of a large follicle (>7 mm) detected during the previous examination and was confirmed by the presence of a corpus luteum (CL) on day 8 after treatment.

LH analysis

One day before blood sampling, a catheter was inserted into the jugular vein of each female to minimize the effects of stress and handling on LH concentrations. Blood samples were collected into heparinized tubes every 15 minutes for 8 hours, beginning 15 minutes before treatment. The samples were centrifuged at 1500 × g for 10 minutes and the plasma obtained was stored at −20ºC until assayed for LH. Plasma LH concentrations were determined using a double-antibody RIA, as previously described (12, 23, 24). Intra- and interassay coefficients of variation were 4.7% and 7.5%, respectively. The lower limit of detection of the assay, defined as 90% of buffer control, was 0.1 ng/mL.

NGF analysis

Plasma β-NGF levels were measured in blood samples collected during the first 3 hours of sampling. The protein was detected using an enzyme-linked immunoassay (ELISA Duoset, number DY256; R&D Systems, Inc) developed to detect human β-NGF. Samples were processed and measured in duplicate according to the protocol recommended by the manufacturer. The lower limit of detection was 31.3 pg/mL. To determine whether the kit can detect llama β-NGF, we assessed the ability of serial (1:2) plasma dilutions of plasma to generate displacement curves parallel to that of ELISA standard recombinant human β-NGF diluted with blocking buffer (recommended by the manufacturer). The blood samples tested were the blood plasma pooled from four llamas with an expected high level of β-NGF and the standard β-NGF diluted in blood plasma pooled from four llamas with an expected low level of β-NGF. The range of values detected by these serial dilutions ranged from 2000 to 62.5 pg/mL.

Statistical analysis

Nonserial data (ie, follicle size at the time of treatment; CL diameter; basal, peak, and mean plasma β-NGF levels) were compared between groups by a one-way ANOVA. Serial data (plasma LH and β-NGF concentrations) were compared by a one-way ANOVA for repeated measures (Proc-mixed in SAS; Statistical Analysis System Institute Inc) to determine the effects of treatment and day- and treatment-by-day interaction. When main effects or their interaction were significant (P ≤ .05), we used the Tukey's post hoc test for multiple comparisons to assess differences and the Dunnett's test to compare experimental groups with the control group. A two-way ANOVA was used to compare the total output of β-NGF and LH, considering the main factor treatment groups or time and their interaction. A Bonferroni post hoc test was used for multiple comparisons. The ovulation rate was compared among groups by χ² analysis. Values were expressed as mean ± SEM. The displacement curves observed in the β-NGF ELISA kit were analyzed by regression (STATGRAPHICS 17; Statpoint Technologies, Inc).

To determine the association between circulating β-NGF and LH levels, data were analyzed using the bestglm package written by Xu and McLeod (http://www.stats.uwo.ca/faculty/aim) as implemented in R 3.2 (R Core Development Team [28]; http://www.R-project.org). Using these tools we selected the most informative linear regression model relating total LH output measured during a 3-hour sampling period with plasma β-NGF levels attained during the first, second, and third hour of sampling. We also considered the presence or absence of mechanical stimulation (ie, llamas mating with an intact or an urethrostomized male). Bestglm uses the leaps algorithm (29) to efficiently evaluate models based on all possible predictor subsets from an entire input predictor set and ranks each model by its balance of predictive ability and parsimony based on the Bayesian information criterion (30). The per-hour β-NGF levels were summed from 15–75 minutes, 90–135 minutes, and 150–180 minutes. We also calculated the Pearson correlation for total LH output with per-hour β-NGF values to determine whether a significant correlation existed between the β-NGF values and the total LH output at any individual time point included in our regression model.

Results

The diameter of the largest follicle at the time of treatment was similar among groups (Table 1). The ovulatory rate was identical in llamas given an IUI of seminal plasma or mated with an intact male. No ovulations were detected in females given an IUI of PBS or mated with an urethrostomized male (Table 1). The presence of a CL was confirmed on day 8 in all animals that had ovulated.

Table 1.

Ovarian Response in Llamas Given an IUI of PBS or Seminal Plasma or Mated to an Urethrostomized or Intact Male

End Points	Treatment Groups
	PBS (n = 6)	Urethrostomized Male (n = 6)	Seminal Plasma (n = 6)	Intact Male (n = 7)
Follicle size at the time of treatment, mm	9.6 ± 0.8	9.2 ± 0.7	8.8 ± 0.6	10.5 ± 0.7
Ovulation rate, %	0/6 (0)	0/6 (0)	5/6 (83.3)a	6/7 (85.7)a
CL diameter on day 8, mm			13.0 ± 0.4	13.0 ± 0.8

^aP < 0.01 vs group injected with PBS or mated with an urethrostomized male. Values are mean ± SEM.

A significant increase (P < .01) in plasma LH concentration was observed 1 hour after treatment in females mated with an intact male and those receiving an IUI of seminal plasma (Figure 1). Peak values were detected 2.25 and 4.5 hours after treatment, respectively. Importantly, plasma LH levels were significantly higher (P < .01) between 1 hour after treatment and the end of the sampling period in the animals receiving an IUI of seminal plasma or mated to an intact male compared with females given an IUI of PBS or mated to an urethrostomized male. From 1.5 hours after treatment onward, plasma LH levels were higher in females mated with an intact male than in llamas receiving an IUI of seminal plasma (P < .01). Plasma LH did not differ between llamas receiving an IUI of PBS or mated with an urethrostomized male and remained at basal levels in both groups for the entire sampling period (Figure 1).

Figure 1.

Plasma LH concentration (mean ± SEM) in llamas after IUI of PBS (n = 6) or seminal plasma (n = 6) or after mating with an urethrostomized (n = 6) or intact male (n = 7). X Within each group denotes the first significant increase from pretreatment values (P < .001). Y Within each group represents the maximal concentration achieved after treatment (P < .01). Z Within each group represents the first significant (P < .01) decrease from the maximal LH levels achieved after treatment. *, LH levels significantly greater (P < .01) than group administered an IUI of seminal plasma; **, interval during which plasma LH values were significantly higher (P < .001) in the group administered seminal plasma than in the animals mated with an urethrostomized male or administered PBS; ***, interval during which plasma LH levels were higher in the group mated with an intact male than in the group mated with an urethrostomized male or administered PBS (P < .001). No significant differences in LH levels between females mated to a urethrostomized male or subjected to IUI of PBS were detected. Tx, treatment.

Pretreatment plasma β-NGF levels did not differ among groups but peaked 15 minutes after treatment in both females mated with an intact male and females receiving an IUI of seminal plasma (Figure 2A). The β-NGF levels were significantly higher in females mated with an intact male compared with the other three groups between 15 minutes after treatment and the end of the sampling period (Figure 2A). The β-NGF levels were also significantly higher in the group receiving an IUI of seminal plasma than in animals given an IUI of PBS or mated to a urethrostomized male. The ELISA used to measure llama β-NGF was able to reliably estimate a large range of β-NGF concentrations in plasma as evidenced by the parallelism of the dilution curves containing plasma to that of standard β-NGF (Figure 2B; R² = 0.96).

Figure 2.

A, Plasma β-NGF concentration in llamas after IUI of either PBS (n = 5) or seminal plasma (n = 6) or mating with an urethrostomized (n = 5) or intact male (n = 6). *, Times at which β-NGF levels were higher in llamas receiving an IUI of seminal plasma relative to those receiving IUI of PBS or mated to an urethrostomized male (P < .05); **, interval during which plasma β-NGF values were significantly higher (P < .001) in llamas mated with intact male in relation to all other groups. Tx, treatment. B, Validation of a human ELISA β-NGF assay to quantify blood plasma β-NGF levels in llamas. The β-NGF provided in the ELISA kit was serially diluted (1:2) in either block solution (1) or in blood plasma pooled from four llamas expected to have low circulating β-NGF levels (2; because they had low LH levels). In addition, a blood plasma pool from animals expected to have high levels of β-NGF (3; because they had elevated LH values) was serially diluted (1:2) and found to display a curve parallel to that of standard β-NGF.

The integrated changes in the plasma β-NGF and LH levels resulting from the different treatments are displayed in Figure 3. Whereas the β-NGF levels increase sporadically toward the end of the sampling period in animals given an IUI of PBS or mated to an urethrostomized male (Figure 3, upper panels), they increase promptly (within 15 min) in animals either mated to an intact male or receiving an IUI of seminal plasma (Figure 3, lower panels). The β-NGF levels were lower in the latter group than in llamas mated to an intact male, but they were significantly higher between 15 minutes and 3 hours after treatment than pretreatment values and values detected in llamas treated with PBS or mated to a urethrostomized male (Figure 3, lower panels).

Figure 3.

Comparison of plasma LH and β-NGF concentration in llamas after IUI of either PBS (n = 5) or seminal plasma (n = 6) or after mating with an urethrostomized (n = 5) or intact male (n = 6). Interval during which plasma LH and β-NGF concentration differed from pretreatment values (P < .01). *, P < .05, **, P < .01, ***, P < .001) vs pretreatment levels. Tx, treatment.

Individual profiles of circulating β-NGF levels showed that β-NGF levels varied little in animals receiving an IUI of PBS (Figure 4, upper panels). Short-duration elevations of β-NGF levels were detected by the second half of the sampling period in some animals mated with an urethrostomized male (Figure 4, upper middle panels). In contrast, β-NGF levels increased early and strikingly in both females receiving an IUI of seminal plasma and llamas mated with an intact male (Figure 4, lower panels). This increase was more sustained in the latter group (Figure 4, lower panel). Interestingly, llamas receiving an IUI of seminal plasma exhibited a plasma profile of β-NGF levels that appeared to be episodic (Figure 4, lower middle panel).

Figure 4.

Individual profiles of plasma β-NGF in female llamas receiving an IUI of either PBS or seminal plasma or mated to an urethrostomized (urethro) or intact male. The letters A, B, and C followed by a number refer to individual llamas.

The total output of β-NGF and LH per hour observed over 3 hours of sampling increased significantly only in those females given an IUI of seminal plasma or mated with an intact male (Figure 5A). To analyze the potential association that may exist between plasma β-NGF and LH levels, we used the bestglm package described in Materials and Methods and identified three possible models. The top-ranked model predicting LH output based on the β-NGF levels included the Y-intercept (β₀ = 19.6, P = .087) and the total β-NGF levels detected during the first hour of the study (β₁ value = 0.0019, P = .003). The second-ranked model included terms for total β-NGF levels at hours 1 and 3, and the third-ranked model was based on total β-NGF levels at hour 2 alone. The top-ranked model based solely on β-NGF levels at hour 1 was also superior to the intercept-only model using the F test of overall model significance (P = .003, 1 and 18 df). This result is consistent with the significant difference in β-NGF levels at hour 1 observed between the combined data from animals exposed to seminal plasma (receiving an IUI of seminal plasma and mated to intact males) considered as a whole vs the groups not exposed to seminal plasma (P < .001, Wilcoxon rank sum test). Whereas all per-hour β-NGF levels correlated significantly with total LH output, the correlation of hour 1 β-NGF levels with LH totals was most significant (r = 0.62, P = .003, Figure 5B).

Figure 5.

A, Total β-NGF and LH output (mean ± SEM) 1, 2, or 3 hours after treating llamas with an IUI of PBS (n = 5) or seminal plasma (SP; n = 5), or after mating with an urethrostomized male (UM; n = 6) or intact male (IM; n = 6; mean ± SEM). A significant effect of group (P < .001), time (P < .001), and interaction (P < .001) was found for both plasma β-NGF and LH levels. *, P < .05, **, P < .01, ***, P < .001) vs groups receiving either an IUI of PBS or mated with an urethrostomized male. B, Scatterplot of the relationship that exists between plasma β-NGF levels detected during the first hour of treatment and the total LH output measured during the 3-hour study. The solid line represents the best-fit line from a regression analysis plotting total (3 h) plasma LH levels vs the first hour of plasma β-NGF levels.

Discussion

The results of this study demonstrate the following: 1) neither penile intromission nor the physical stimulation of coitus is able to induce an LH surge and trigger ovulation in llamas in the absence of seminal plasma, 2) deposition of seminal plasma into the female genital tract per se is sufficient to elicit an LH surge and ovulation in the absence of mating, and 3) mating with an intact male or IUI of seminal plasma elicits an increase in circulating β-NGF levels that is positively correlated with a preovulatory discharge of LH. By showing an essential role of seminal plasma in eliciting ovulation, our results call into question the long-held belief that llamas are reflex ovulators that require the physical stimulation of coitus to ovulate (5, 6).

Penile intromission, along with other sensory stimuli, is thought to be the main physical stimulus triggering the ovulatory cascade in induced-ovulation species (1, 31). However, studies in alpacas (10) and Bactrian camels (8, 32), two species considered to be reflex ovulators, have demonstrated that cervicovaginal mechanical stimulation alone, in the absence of coitus stimulation, is unable to induce either an LH surge or ovulation. Although vaginal-cervical stimulation with a glass rod induced a significant increase of GnRH and LH concentrations in ovariectomized ferrets, this change is seen only in the presence of a male (33). Furthermore, Ramirez and Soufi (34) observed that although GnRH levels increased in the cerebrospinal fluid of 60% of rabbits subjected to vaginal-cervical stimulation, none of these animals ovulated. Surprisingly, the IUI of saline induced ovulation in 37% of rabbits in the absence of a significant increase of plasma LH levels (35), suggesting that this particular ovulatory response is not centrally mediated. In support of this view, elevated plasma LH levels followed by ovulation were observed only in rabbits artificially inseminated with raw semen (35), suggesting that substances contained in semen are able to activate the hypothalamic-pituitary unit to release LH and elicit ovulation.

Although earlier reports had shown the ability of an IUI of seminal plasma or coitus to induce a preovulatory LH surge followed by ovulation in llamas (12, 26, 36), the present study is the first to specifically compare the ability of natural copulation and seminal plasma to induce ovulation in the absence of each other. Interestingly, our results show that the ovulatory rate was similar in females mated with an intact male and those given an intrauterine infusion of seminal plasma, but plasma LH levels attained were greater after coitus. This observation is consistent with the well-established concept that only a fraction of the preovulatory LH surge is required for a full ovulatory response (37). It also emphasizes the existence of an important physiological difference between the two conditions. During natural mating of llamas, the penis enters the uterine cavity and mechanically disrupts the endometrial mucosa via repeated intromissions that may last up to 40 minutes (13). Loss of endometrial integrity is likely to facilitate the transfer of bioactive substances, such as spβ-NGF, from the semen into the bloodstream. This view is supported by the present results, showing that mating with an intact male resulted in an elevation of circulating β-NGF levels that was greater than that observed after IUI of seminal plasma and was maintained during the 3-hour period. We do not believe that this difference is due to spβ-NGF degradation during the storage of seminal plasma because in our experience spβ-NGF retains its ability to induce ovulation after either several cycles of freezing and thawing or after incubating the seminal plasma at 37ºC for 12 hours (16). In keeping with these observations, a previous report (10) showed that endometrial curettage increased (from 41% to 67%) the ovulatory rate in alpacas subjected to an IUI of seminal plasma. It should also be noted that the pattern and time course of LH release in females mated with an intact male or subjected to an IUI of seminal plasma was similar to that described earlier in the studies giving seminal plasma either im or iv to llamas or alpacas (12, 14, 20, 21) or after natural mating (3, 36).

β-NGF is a major component of seminal plasma in llamas and alpacas (30% of the total protein content). Although present at much lower levels in other mammals, including humans, the semen of most mammals contains β-NGF (26). Our results show that circulating β-NGF levels increase rapidly (within 15 min) in female llamas after mating with an intact male or after IUI of seminal plasma. Although sporadic increases in plasma β-NGF levels were noted in urethrostomized and PBS-treated animals, these changes were not sustained, occurred late during the sampling period, and were not associated with either an increase in LH secretion or ovulation. The increase in circulating β-NGF levels detected in llamas mating to an intact male or given an IUI of seminal plasma was sustained, lasting for the 3 hours' duration of the sampling period. Importantly, the first significant increase was observed 45 minutes earlier than the first significant LH increase, suggesting that both phenomena are causally related. In fact, a detailed statistical analysis of these results demonstrated that the total amount of LH release during the 3-hour sampling period depends largely on the level of β-NGF achieved during the first hour of observation. At this time, β-NGF levels remained at basal values in animals receiving PBS or mated to an urethrostomized male. These results provide for the first time evidence that spβ-NGF contained in seminal plasma not only reaches the bloodstream within minutes after IUI of seminal plasma, but it also attains levels sufficiently elevated to elicit LH release.

Altogether, our findings are consistent with a model in which spβ-NGF is transferred after mating from the uterine cavity to the systemic circulation reaching the hypothalamus to stimulate GnRH secretion as previously proposed (23) or the pituitary to directly stimulate release of LH (38, 39). A recent report using another reflex ovulator, the musk shrew (40) as the animal model, showed that coitus activates kisspeptin neurons in the hypothalamus. These authors concluded that an increased release of kisspeptin is the mechanism by which coitus elicits a preovulatory LH surge and ovulation in this species. It is tempting to speculate that a similar neuroendocrine pathway underlies the ability of spβ-NGF to elicit ovulation in llamas.

Finally, it should be mentioned that administration of seminal plasma from spontaneous ovulators, such as bulls, horses, and pigs, elicits ovulation in llamas (41). Because components of seminal plasma can also hasten the preovulatory surge in cows or ovulation in pigs (42, 43), the study of ovulation inducing molecules contained in semen may represent a new avenue of investigation to define the potential role of these substances in the ovulation mechanism in either induced or spontaneous ovulators.