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.

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC 12 ) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.neurotrophins | hypothalamus | fertility | neuroendocrine I n a monograph nearly 50 y ago, Thaddeus Mann summarized the natural properties of seminal plasma as a vehicle for sperm transport, a controller of sperm motility and capacitation, and as a stimulant of uterine contractility (1). Notwithstanding Mann's admonishment to resist the temptation "to assign to every newly discovered chemical constituent of semen a major role in the process of fertilization," recent isolation of a protein factor in seminal plasma (2-4) suggests an additional role of the ejaculateas an inducer of ovulation.The role of the fluid portion of the ejaculate, and the male accessory glands responsible for producing it, has been enigmatic. From an evolutionary perspective, it has been suggested that the male accessory glands likely originated as the machinery for producing a copulatory plug, which has the "chastity effect" of preventing the sperm of other males from entering the female tract, as well as minimizing sperm loss after insemination (5). If this is so, then the persistence of an elaborate accessory gland system in many species in which plug formation does not occur may be viewed as nothing more than an evolutionary vestige.The first reports of an ovulation-inducing factor (OIF) in semen resulted from the observation that ovulation occurred after intravaginal or intramuscula...

BackgroundThe 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.MethodsSemen 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.ResultsOvulation 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.ConclusionOvulation-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).

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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.