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BOR - Papers in Press, published online ahead of print May 11, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040097
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BIOLOGY OF REPRODUCTION 73, 452–457 (2005)
DOI: 10.1095/biolreprod.105.040097
© 2005 by the Society for the Study of Reproduction, Inc.

Ovulation-Inducing Factor in the Seminal Plasma of Alpacas and Llamas1

Gregg P. Adams 2, 3, Marcelo H. Ratto 3, Wilfredo Huanca 4, and Jaswant Singh 3

Veterinary Biomedical Sciences,3 Western College of Veterinary Medicine, University of Saskatchewan, Saskatchewan, Canada S7N 5B4 Faculty of Veterinary Medicine,4 San Marcos University, Lima 1, Peru


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies were conducted to document the existence of an ovulation-inducing factor in the seminal plasma of alpacas (experiment 1) and llamas (experiment 2) and to determine if the effect is mediated via the pituitary (experiment 3). In experiment 1, female alpacas (n = 14 per group) were given alpaca seminal plasma or saline intramuscularly or by intrauterine infusion. Only alpacas that were given seminal plasma i.m. ovulated (13/ 14, 93%; P < 0.01). In experiment 2, ovulation was detected in 9/10 (90%) llamas at a mean of 29.3 ± 0.7 h after seminal plasma treatment. Plasma progesterone concentrations were maximal by Day 9 and were at nadir by Day 12 posttreatment. In experiment 3, female llamas were given llama seminal plasma, GnRH, or saline i.m., 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 min and 75 min after treatment with GnRH and seminal plasma, respectively. Plasma LH remained elevated longer in the seminal plasma group (P < 0.05) and had not yet declined to pretreatment levels after 8 h. Compared with the GnRH group, corpus luteum tended to grow longer and to a greater diameter (P = 0.1) and plasma progesterone concentration was twice as high in the seminal plasma group (P < 0.01). Results document the existence of a potent factor in the seminal plasma of alpacas and llamas that elicited a surge in circulating concentrations of LH and induced an ovulatory and luteotropic response.

camelids, corpus luteum function, gonadotropin-releasing hormone, luteinizing hormone, ovary, ovulation, ovulation-inducing factor, semen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovulation in mammals involves pulsatile release of GnRH from the mediobasal nuclei of the hypothalamus into the hypophyseal portal system with subsequent release of LH from the gondotrophs of the anterior pituitary into the systemic circulation [1, 2]. Elevated circulating concentrations of LH elicit a cascade of events within the mature follicle culminating in follicle wall rupture and evacuation of its fluid and cellular contents [3]. 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 [4]. In spontaneously ovulating species (e.g., human, sheep, goats, cattle, horse, pigs), release of GnRH from the hypothalamus is triggered when, in the absence of progesterone, systemic estradiol concentrations exceed a certain threshold [5–9]. As a consequence of regularly occurring luteolysis and development of one or more estrogen-producing follicles, a preovulatory surge in circulating concentrations of LH occurs at regular intervals. In induced ovulators (e.g., rabbits, ferrets, cats, camelids), however, neural signals from copulatory stimulation trigger GnRH secretion from the hypothalamus, followed by the preovulatory release of LH from the pituitary [4]. Similar to spontaneous ovulators, a surge in the circulating concentration of LH appears to be requisite for ovulation in induced ovulators, but its occurrence is contingent on copulatory stimuli; hence, ovulation is not a regular cyclic event.

The phenomenon of induced ovulation has been demonstrated in llamas [10], alpacas [11], and Old World camels [12, 13], but very few studies have been conducted to determine the factors responsible for eliciting ovulation in camelids. In the only study of its kind in New World camelids [14], ovulation induced by natural mating in llamas was associated with a rise in plasma LH concentration beginning within 15 min of mating. In an early, classic study in alpacas [15], the ovulation rate was compared among females that 1) were nonmounted, 2) were mounted only, followed with or without artificial insemination, 3) had interrupted mating, 4) had sterile mating (vasectomized male), followed with or without artificial insemination, 5) had a single uninterrupted mating (intact male), or 6) had multiple uninterrupted matings. It was concluded that mounting accompanied by penile intromission was necessary to stimulate ovulation. From these early studies, the concept that physical stimulation of the genitalia during copulation is the primary trigger for inducing ovulation in alpacas and llamas has become dogma.

This dogma, however, is being challenged by the suggestion that a chemical substance may be present in the semen that mediates the ovulatory cascade. The existence of such a substance was initially reported by investigators in China, who concluded that some factor in the semen was responsible for eliciting ovulation in Bactrian camels, rather than the mechanical stimulation of copulation [16]. Ovulation occurred after intravaginal [16, 17] or intramuscular/ intrauterine [18] administration of Bactrian seminal plasma to female Bactrian camels. In this regard, results of one study in alpacas [19] appear contradictory to the initial alpaca study [15] in that artificial insemination (intravaginal deposition of alpaca semen) was associated with ovulation in 6/10 alpacas and 5/8 llamas. In a more recent report [20], alpaca seminal plasma stimulated LH secretion from primary culture of rat pituitary cells in vitro. The authors suggested that the putative ovulation-inducing factor in seminal plasma had GnRH-like activity but was not GnRH because its biological activity on rat pituitary cells was not suppressed when anti-GnRH antibodies were added to the culture media.

The objective of this study was to document the existence of an ovulation-inducing factor in the seminal plasma of alpacas and llamas. Experiment 1 was designed specifically to document the existence of an ovulation-inducing factor (OIF) in alpacas and to compare the effects of intrauterine versus intramuscular administration. Experiment 2 was designed to determine the existence of OIF in llamas before a controlled study (experiment 3) designed to determine if the effect of OIF is associated with a preovulatory surge in LH. The studies were designed to determine the effects of seminal plasma on ovulation rate, interval to ovulation, and corpus luteum function and to compare the effect of seminal plasma with that of GnRH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1

The study was conducted during January–March at the Quimsachata Research Station in the Department of Puno, Peru (15°S, 71°W, and 4500 m above sea level). Semen was collected from eight male alpacas using an artificial vagina [21] over a period of 2 mo (16 ejaculates per animal). Ejaculates were diluted 1/1 (v/v) with phosphate buffered saline (PBS, Gibco, Grand Island, NY) and centrifuged for 30 min at 1500 x g. The supernatant was decanted from the spermatozoa and a drop was evaluated by microscopy to confirm the absence of cells. If spermatozoa were detected, the sample was centrifuged again in like manner. Sperm-free seminal plasma was stored at –20°C before the experiment. Upon thawing, the diluted seminal plasma samples from all eight males were pooled and kanamycin sulfate (Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 25 µg/ml.

Mature nonlactating female alpacas (n = 70), ≥3 yr of age and weighing an average of 70 kg, were examined daily by transrectal ultrasonography (Aloka SSD 500, Instruments for Science and Medicine Inc., Vancouver, BC, Canada) using a 7.5-MHz linear-array transducer attached to a rigid probe extension [22, 23]. Alpacas were selected (n = 58) when a growing follicle of ≥8 mm diameter was detected (i.e., capable of ovulating 24) and was then assigned randomly to four groups in a 2 x 2 factorial experiment (n = 14 per group), and 1 ml of diluted alpaca seminal plasma was given intramuscularly or by intrauterine infusion or 1 ml of PBS was given intramuscularly or by intrauterine infusion (negative control). Although not part of the original experimental design, an additional group of six mature nonlactating female alpacas from the same herd became available during the experimental period and were treated by intrauterine infusion of 5 mg of LH (Lutropin-V; Bioniche Animal Health Canada Inc., Belleville, ON, Canada) when a growing follicle ≥8 mm was detected by transrectal ultrasonography [24]. Intramuscular injections were given in the semimembranosus muscle using a 20-gauge 40-mm-long needle, and intrauterine infusions were accomplished by passing a pipette through the cervix via transrectal manipulation.

Alpacas were examined daily by transrectal ultrasonography until Day 3 (Day 0 = treatment) to detect ovulation and again on Day 8 to detect the presence of a corpus luteum (CL). Ovulation was defined as the sudden disappearance of a large follicle (≥8 mm) that was detected during the previous examination and was confirmed by subsequent detection of a CL [25].

Experiment 2

The study was conducted from May to August at the University of Saskatchewan, Saskatchewan, Canada (52°N, 106°W and 500 m above sea level) to determine if llama seminal plasma would induce ovulation in llamas, as it did in alpacas (experiment 1), and to provide necessary information for the design of a controlled comparison of the effects of GnRH versus seminal plasma (experiment 3), with adequate numbers for statistical interpretation (i.e., ovulation rate, interval to ovulation, CL and progesterone profiles. Semen was collected from four mature (5- to 7-yr-old) male llamas using an artificial vagina over a period of 2 mo (24 ejaculates per animal). As in experiment 1, samples were diluted 1/1 (v/v) with PBS and centrifuged for 30 min at 1500 x g. Sperm-free seminal plasma was stored at –20°C. Upon thawing, the seminal plasma from all four males was pooled and kanamycin sulfate was added to a final concentration of 25 µg/ml.

Mature nonlactating female llamas (n = 15), ≥4 yr of age and weighing 90–150 kg, were given 5 mg Armour Standard luteinizing hormone (Lutropin-V; Bioniche Animal Health) i.m. to synchronize follicular-wave emergence among animals [24]. Twelve days after LH treatment, llamas with a follicle ≥8 mm in diameter (n = 10) were given 1.5 ml of diluted llama seminal plasma i.m. The ovaries were examined by transrectal ultrasonography every 4 h from the time of seminal plasma treatment until ovulation or 48 h, whichever came first. Ultrasonographic examination was performed once daily thereafter for 15 days to monitor CL growth and regression. As in experiment 1, ovulation was defined as the sudden disappearance of a large follicle (≥8 mm) that was detected during the previous examination. The onset of luteal regression was defined as the first day on which the corpus luteum began a progressive decrease in diameter leading to a minimum on the last day of the observational period. Blood samples were collected into heparinized tubes (Vacutainer Systems; Becton Dickinson, Franklin Lakes, NJ) by jugular venipuncture on Days 0, 3, 6, 9, 12, and 15 (Day 0 = day of seminal plasma treatment). Blood samples were centrifuged at 1700 x g for 25 min and the plasma was stored at –20°C.

Plasma progesterone concentrations were determined using a commercial, double-antibody radioimmunoassay kit (Coat-a-Count total progesterone, DPC; Diagnostic Products Corporation, Los Angeles, CA), as described previously [26]. All samples were analyzed in a single assay. The intraassay coefficients of variations were 1.8%, 4.9%, and 1.8%, respectively, for reference plasma progesterone concentrations of 1.8, 2.9, and 14.6 ng/ml.

Experiment 3

Results of experiment 2 were used to design a study to determine if the ovulatory effects of seminal plasma treatment are associated with pituitary release of LH, by comparison with the effects of GnRH treatment. The study was conducted in the autumn (October–November) after experiment 2 using a different group of llamas from the same herd at the University of Saskatchewan. Mature nonlactating female llamas (n = 25), ≥4 yr of age and weighing 90–150 kg, were given 5 mg Armour Standard LH (Lutropin-V) to synchronize follicular wave emergence among animals, as described in experiment 2. Twelve days after LH administration, llamas with a follicle ≥8 mm diameter were assigned randomly to three groups (n = 6 per group), in which 1.5 ml of llama seminal plasma, 1.5 ml of PBS (negative control group), or 50 µg of GnRH (Cystorelin, Merial Canada Inc., Victoriaville, PC, Canada; positive control group) were given by intramuscular injection. The same pool of llama seminal plasma collected during experiment 2 was used in experiment 3. The ovaries were examined by transrectal ultrasonography once daily from the day of treatment (Day = 0) to Day 15 to detect ovulation and CL development, as described in experiment 1.

Blood samples for measurement of plasma LH concentration were collected in heparinized tubes by jugular venipuncture every 15 min for 8 h starting immediately before treatment (Time 0 = treatment). A jugular catheter (inner and outer diameters of 1.0 and 1.5 mm, respectively) was fixed in place 1 day before frequent blood sampling to minimize the effects of handling stress on plasma LH concentrations. Blood samples were centrifuged at 1700 x g for 25 min and the plasma was stored at –20°C. Plasma LH concentrations were measured using a double-antibody radioimmunoassay [27]. Concentrations of LH are expressed in terms of NIAMDD-oLH-24. The minimum detectable limit of the assay was 0.026 ng. The range of the standard curve was 0.026 ng (80% ligand labeled LH) to 0.19 ng (20% ligand labeled LH). The intra- and interassay coefficients of variation were 6.3% and 6.0%, respectively, for the high reference plasma LH concentration (0.79 ng/ml). The intra- and interassay coefficients of variation were 17% and 15%, respectively, for the low reference plasma LH concentration (0.17 ng/ml).

Blood samples were collected every 2 days from Day 3 (Day 0 = treatment) to Day 17 for measurement of plasma progesterone concentration and assayed as described in experiment 2. All samples were analyzed in a single assay. The intraassay coefficients of variation were 5.8%, 7.6%, and 2.9%, respectively, for reference plasma progesterone concentrations of 1.4, 2.3, and 11.6 ng/ml.

Statistical Analyses

Nonserial data (i.e., follicle size at the time of treatment, maximum CL diameter, day on which the CL was first detected, onset of CL regression) were compared between groups by analyses of variance. For statistical analyses of serial data and preparation of figures, CL diameter, LH, and progesterone data were centralized to the day of treatment (Day 0). Serial data were compared by analysis of variance for repeated measures (Proc-mixed in SAS, Statistical Analysis System Institute Inc., Cary, NC) to determine the effects of treatment, day, and treatment-by-day interaction. When main effects or their interaction were significant (i.e., P ≤ 0.05), means on a given day were compared by the method of least significant difference. Ovulation rates were compared among groups by chi-square analysis.

The experimental protocols were approved by the University of Saskatchewan Committee on Animal Care and Supply under the guidelines of the Canadian Council on Animal Care.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Two alpacas assigned to the intrauterine PBS group and two assigned to the intrauterine seminal plasma group were excluded from the study because their small size precluded transrectal manipulation for intrauterine infusion.

The diameter of the largest follicle at the time of treatment did not differ among groups (P = 0.65). Ovulation was observed only in the group treated by intramuscular administration of seminal plasma (Table 1). Ovulations were detected on Day 1 in six alpacas and on Day 2 in the remaining seven alpacas (Day 0 = day of treatment). A corpus luteum was detected on Day 8 in all 13 ovulatory alpacas. Ovulation and luteal development were not detected in the negative control groups nor in any of the intrauterine treatment groups (Table 1).


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  		TABLE 1. Effect of intramuscular or intrauterine administration of alpaca seminal plasma, PBS, or LH on ovulation and CL formation in alpacas (mean ± SEM; experiment 1)

Experiment 2

The diameter of the largest follicle at the time of treatment was 9.7 ± 0.4 mm (mean ± SEM). Ovulation was detected in 9/10 (90%) llamas after i.m. administration of llama seminal plasma. The mean interval from treatment to ovulation was 29.3 ± 0.7 h; 6 llamas ovulated at 28 h and the remaining 4 ovulated at 32 h. The corpus luteum was first detected on Day 2.3 ± 0.2, it reached a maximum diameter of 11.5 ± 0.5 mm on Day 6.9 ± 0.3, and began to regress on Day 9.7 ± 0.3 (Day 0 = treatment). Plasma progesterone concentrations were elevated by Day 6, were maximal by Day 9, and were at nadir by Day 12 (Fig. 1).



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  		FIG. 1. Corpus luteum diameter and plasma progesterone concentration (mean ± SEM) in llamas (n = 9) after intramuscular administration of llama seminal plasma (experiment 2)

Experiment 3

The diameter of the largest follicle at the time of treatment did not differ among treatment groups and ovulation occurred in all llamas treated with seminal plasma, all but one treated with GnRH, and none treated with PBS (Table 2). During the 8-h period following treatment, plasma LH concentration increased and decreased (P < 0.01) in the GnRH (positive control) and seminal plasma groups but not in the PBS (negative control) group (Fig. 2). Plasma LH concentration in the GnRH group began to increase (P < 0.05) and was higher (P < 0.05) than in the other groups by 15 min after treatment. The first significant increase in LH in the seminal plasma group was detected 1 h after treatment. At 2 h after treatment, plasma LH concentrations were similarly elevated in the GnRH and seminal plasma groups (Fig. 2). Within individuals, maximum plasma LH concentration occurred at 1.4 ± 0.2 and 1.9 ± 0.2 h after GnRH and seminal plasma treatment, respectively (P = 0.06). Plasma LH concentration remained elevated for a longer period in the seminal plasma group than in the GnRH group (P < 0.05) and had not yet declined to pretreatment levels by the end of the sampling period (8 h). In the GnRH group, plasma LH began to decrease (P < 0.05) at 5 h after treatment and was similar to pretreatment levels by 5.5 h after treatment.


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  		TABLE 2. Effect (mean ± SEM) of intramuscular administration of llama seminal plasma, GnRH, or PBS on ovulation and luteal development in llamas (Day 0 = day of treatment; experiment 3)



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  		FIG. 2. Plasma LH concentrations (mean ± SEM) in female llamas after intramuscular treatment with llama seminal plasma, GnRH, or phosphate buffered saline (PBS; experiment 3). abc On a given day, values with no common superscript are different among groups (P < 0.05). x Within group, the first increase from pretreatment (time 0) concentration (P < 0.05). y Within group, the maximum concentration (P < 0.05). Z Within group, the first decrease from maximum concentration (P < 0.05). * Within group, the last value is higher than the pretreatment value (P < 0.05). ** Within group, the last value is not different from the pretreatment value (P = 0.9)

The CL tended to grow for a longer period and to a greater diameter in the seminal plasma group compared with the GnRH group (group-by-day interaction, P = 0.1; Fig. 3). On an individual-animal basis, differences between GnRH and seminal plasma groups in maximum CL diameter and the day of maximum diameter were not significant (Table 2). Plasma progesterone concentrations were highest (P < 0.05) in the seminal plasma group, intermediate (P < 0.05) in the GnRH group, and remained basal in the PBS group. In both seminal plasma and GnRH groups, progesterone concentrations increased sharply to peak values by Day 7 and decreased sharply to nadir by Day 11 (Fig. 4).



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  		FIG. 3. Diameter profile (mean ± SEM) of the corpus luteum in female llamas after intramuscular treatment with llama seminal plasma or GnRH (experiment 3)



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  		FIG. 4. Plasma progesterone concentrations (mean ± SEM) in female llamas after intramuscular treatment with llama seminal plasma, GnRH, or phosphate buffered saline (PBS; experiment 3). abc On a given day, values with no common superscript are different (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Copulation in llamas and alpacas is an unusually protracted event, often lasting 30–50 min [15, 28], and involves constant gutteral humming by the male, clasping and treading of the male's fore- and hindlimbs while mounted on a receptive female in sternal recumbency. While the role of physical, visual, auditory, and olfactory stimuli remains to be elucidated, results document the existence of an OIF in the seminal plasma of alpacas (experiment 1) and llamas (experiment 2) and that the mechanism of action of OIF involves a preovulatory surge in circulating concentrations of LH (experiment 3). These results in alpacas and llamas support earlier observations made in Bactrian camels [16– 18] and challenge the notion that physical stimuli associated with copulation are causative of ovulation in camelid species [15].

One of the most important findings in this study was the potency of the effect of seminal plasma treatment. A relatively conservative dose of seminal plasma was chosen in the present study (0.5–1 ml of raw seminal plasma) based on the reported average volume of the ejaculate in alpacas and llamas (2–3 ml) [29, 30]. Despite the modest dose used in this study, the effects of seminal plasma were profound. Collectively, 28 of 30 (93%) alpacas and llamas given seminal plasma intramuscularly in the present series of experiments ovulated compared with 5 of 6 (83%) given GnRH and 0 of 20 (0%) given PBS. The duration of the surge in plasma concentration of LH was significantly greater after treatment with seminal plasma than with GnRH, and progesterone secretion from subsequent CL was double that of the GnRH group.

Interestingly, the posttreatment surge in LH was later in the seminal plasma group compared with the GnRH group; i.e., the first significant increase occurred 1 h later, the maximum concentration occurred 2 h later, and the first significant decrease occurred 2.5 h later than in the GnRH group. In addition, posttreatment plasma LH concentrations remained elevated above pretreatment levels in the seminal plasma group for at least 8 h (end of sampling period), whereas LH had returned to basal levels by 5.5 h post-GnRH treatment. These observations provide rationale for the hypothesis that OIF and GnRH affect pituitary LH release differently and are different molecules. This is consistent with the observation that the LH-secreting effect of alpaca seminal plasma on rat pituitary cells in vitro was not suppressed when anti-GnRH antibodies were added to the primary culture [20].

While the disparity in amplitude and duration of the LH surge between GnRH- and seminal plasma-treated llamas did not influence the ovulation rate or interval to ovulation, it was associated with a subtle increase in CL diameter profile and a striking increase in plasma progesterone concentration in the seminal plasma group. This observation provides rationale for the hypothesis that the degree of luteogenesis is directly proportional to the duration of the preovulatory LH surge. Results of studies done in primates [31] are consistent with this hypothesis; a prolonged surge of LH (48–50 h) during the periovulatory phase was necessary to achieve normal luteinization of granulosa cells, expression of progesterone receptors, and development of a functional CL. Shorter endogenous LH surges (14 h) elicited by exogenous GnRH given after ovarian stimulation protocols in primates were associated with deleterious effects on luteinization of granulosa cells and CL development and function [31]. Studies conducted in rabbit [32] and rats [33, 34] documented that changes in concentration and duration of gonadotropins during the periovulatory period can influence changes in oocyte maturation, granulosa cell luteinization, and corpus luteum formation.

The ovulation rate and interval to ovulation after seminal plasma treatment in llamas (experiment 2) were similar to those reported previously in llamas after natural mating or hormonal treatment [35]. In the latter study, a direct comparison of natural mating, LH treatment (5 mg Lutropin-V), and GnRH treatment (50 µg Cystorelin) revealed no differences in ovulation rate (80%, 91%, 80%, respectively), interval to ovulation (30.0 ± 0.5, 29.3 ± 0.6, 29.3 ± 0.7 h, respectively), CL diameter profiles, or plasma progesterone profiles. Relatively infrequent sampling in experiment 2 limits the ability to interpret the effects of seminal plasma treatment on subsequent progesterone production.

The difference in the effects of seminal plasma given by intramuscular injection versus intrauterine infusion was unexpected and supports the notion that the effect of seminal plasma involves a systemic rather than a local pathway. None of the alpacas given seminal plasma by intrauterine infusion in the present study ovulated. This result is consistent with the finding that ovulation rate was not increased by artificial insemination of female alpacas that were allowed to be mounted only (i.e., no intromission; 15) but is in contrast with the findings of another study in which ovulation was observed in 6/10 alpacas and 5/8 llamas after intravaginal deposition of alpaca semen [cited in 19]. In three separate studies in Bactrian camels, ovulation was induced by intravaginal or intrauterine infusion of whole semen or seminal plasma in ≥75% of females [16–18]. The reason for the disparity in results is not clear, but differences appear too great to be due to chance alone. It is interesting to note that, in the present study, alpacas given an intrauterine infusion of LH also failed to ovulate. The dose of LH used was the same as that used in previous studies [24, 35], in which ovulation was induced in 27/30 and 10/11 llamas after intramuscular administration, respectively. Differences may be attributed to differential adsorption from the genital mucosa compared with the muscle. In this regard, copulation in alpacas and llamas is prolonged (30–50 min) and ejaculation is intrauterine [36]. A normal sequela of copulation is acute, transient inflammation of the endometrium as a result of repeated abrasion by the penis [36]. Perhaps absorption of OIF in seminal plasma subsequent to natural mating is facilitated by the hyperemia of the excoriated endometrium. Test of this hypothesis might include curettage of the endometrium at the time of intrauterine infusion of seminal plasma.

The categorical distinction between induced and spontaneous ovulators is not as clear as the label implies. Authors of early studies suggested that camels are spontaneous ovulators [37–39] and, even in llamas and alpacas, there are conflicting reports on whether spontaneous as well as induced ovulations can occur [40, 41]. Based on laparotomy or necropsy examinations, the ovulation rate of unmated alpacas (spontaneous ovulation) was reported to be about 5% [15]. In a critical ultrasound study [42], the incidence of spontaneous ovulation was 8% in unmated llamas, and ovulation failure rate was 10% in mated llamas. Rodents appear to occupy an intermediate position between induced and spontaneous ovulators. Ovulation in mice and rats occurs spontaneously, but CL development and function are contingent on mating [43, 44]. The distinction between spontaneous and induced ovulators is further blurred by the finding that ovulation was hastened in gilts by intrauterine application of a pronase-sensitive fraction of boar seminal plasma [45]. Further, the existence of a GnRH-like molecule was detected in studies on human seminal plasma [46]. Although the nature and function of these molecules remains unknown, they may represent an evolutionary vestige of a common ancestry or indeed play a pivotal role in the ovulatory mechanism or a role in gamete interaction [47].

In summary, results clearly document the existence of a potent factor in the seminal plasma of alpacas and llamas that elicited a surge in circulating concentrations of LH and inducing ovulation in more than 90% of animals treated. The presence of a potent ovulation-inducing factor in seminal plasma would seem an evolutionary asset and one on which natural selection pressure would be brought to bear. The existence of OIF in camelids begs the question of its existence and its effect in other species. The discovery of this novel factor may have broad implications on our understanding of ovulation and on diagnosis and treatment of ovulatory perturbations in this and other species.


   ACKNOWLEDGMENTS
 
Animals and animal maintenance in Peru were provided by the Quimsachata Research Station under the sponsorship of San Marcos University of Lima, Peru. We thank Bioniche Animal Health Canada Inc. and Merial Canada Inc. for providing Lutropin and Cystorelin, respectively. We also thank R. Jaiswal, R. Duggavathi, M. Colazo, P. Malhi, D. Saravanan, T. Bousquet, and C. Olazabal for technical assistance in data collection.


   FOOTNOTES
 
1 Supported by grants from the Canadian Llama and Alpaca Association, the Alpaca Research Foundation, and the Inter-American Institute for Cooperation in Agriculture. Back

2 Correspondence: FAX: 306 966 7405; gregg.adams{at}usask.ca Back

Received: 17 January 2004.

First decision: 21 February 2005.

Accepted: 25 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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