A comparative study of the effects of intramuscular administration of gonadorelin, mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified ß-NGF on luteal development in llamas.

The aim of this study was to compare the effect of the intramuscular administration of 50 µg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta-nerve growth factor from seminal origin (spß-NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 µg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spß-NGF contained in 4 ml of PBS (phosphate-buffered saline) (spß-NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spß-NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spß-NGF influences its luteotrophic effect in llamas.

Predictive characteristics of male fertility in alpacas with special reference to seminal NGF.

Recent studies document the LH-releasing pathway of nerve growth factor (NGF) in male camelids and that the LH response to seminal NGF is associated with elevated plasma testosterone concentration. Results provide rationale for the hypothesis that NGF in semen is associated with male fertility. In Experiment 1, the association between the amount of NGF in the ejaculate and characteristics of the male reproductive system was examined in alpacas. The concentration of NGF was measured by radioimmunoassay in semen samples collected from male alpacas (n = 47) and correlated with sperm morphology and motility, and measurements of the male reproductive anatomy. Most ejaculates had NGF concentrations that, based on previous studies, triggered ovulation in female camelids, however, we only found a positive correlation between NGF concentration with sperm concentration, thread formation and total NGF, and a negative correlation with pH. In Experiment 2, a retrospective analysis was carried out to determine if breeding performance during the previous season was related to recent concentrations of seminal NGF in male alpacas (n = 22). Birth rates tended to be correlated with sperm concentration and total amount of NGF in the ejaculate (P = 0.09). Experiment 3 was a prospective study to determine the relationship between seminal NGF (n = 8 male alpacas) and ovulation and pregnancy rates in a breeding trial. No association was detected between seminal NGF concentration and ovulation rate, pregnancy rate, or LH response in the female. We conclude that among the breeding males used in our study, the abundance of seminal NGF was correlated with sperm concentration and thread formation, however, it was not predictive of male fertility in alpacas. Examination of males not previously selected as breeding stock may be expected to include a broader range of seminal NGF and provide a more comprehensive understanding of the relationship between seminal NGF and male fertility.

Heterologous beta-nerve growth factor (ß-NGF) given at the LH surge enhances luteal function in dairy heifers.

Genetic selection for high yield milk production has led to a decline in dairy cattle's reproductive performance over the last 40 years. Low progesterone (P4) plasma content following ovulation is associated with suboptimal fertility in dairy cattle. Several pieces of evidence indicate that the protein beta-nerve growth factor (ß-NGF) that is present in the male seminal plasma exerts potent ovulatory and luteotrophic effects following systemic administration in camelids but also in other species. In this study, we determine whether systemic administration of purified llama ß-NGF given at the induced preovulatory luteinizing hormone (LH) peak improves corpus luteum (CL) function in dairy heifers subjected to an estradiol (E2) / P4 estrus-synchronization protocol. To achieve this, we first determined plasma E2 and LH hormone profiles to establish the timing of the estradiol benzoate (EB)-induced LH peak in estrus-synchronized heifers. Then, we tested whether the administration of ß-NGF given at the end of this peak affects the CL and its function by analyzing diameter, vascular area, and P4 output. Our results show that, with the estrus-synchronization protocol applied, plasma LH concentrations peaked (P < 0.01) 40-h and 16-h after removal of the bovine intravaginal device (DIB; containing 1.0 g of P4) plus cloprostenol injection and subsequent EB administration, respectively; after peaking, plasma LH concentrations remained stable for the next 8-h to then return to basal levels. Heifers synchronized with this protocol and receiving a dose of 1 mg of ß-NGF at the end of the LH peak (ie, 48-h after DIB removal) did not show significant differences in CL diameter, but these exhibited a greater CL vascular area (P = 0.01) than the observed in vehicle-injected heifers. Furthermore, plasma P4 concentration in ß-NGF-treated heifers was higher (P = 0.001) than those quantified in vehicle-injected heifers. These results support the use of ß-NGF in estrus-synchronization protocols to improve the early luteal function in dairy heifers.

Nerve growth factor from seminal plasma origin (spß-NGF) increases CL vascularization and level of mRNA expression of steroidogenic enzymes during the early stage of Corpus Luteum development in llamas.

The objectives of the study were to determine the effect of seminal plasma ß-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 µg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spß-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1/P450scc and STAR expression by RT-PCR. Ovulation was similar between llamas treated with GnRH or spß-NGF and CL diameter did not differ between GnRH or spß-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P < 0.01) in llamas from the spß-NGF than GnRH-treated group by Day 4 and 8. Plasma progesterone concentration was higher (P < 0.05) in llamas from the spß-NGF compared to females of GnRH group by Day 4 and 8. The proportion of small and large luteal cells did not differ between GnRH or spß-NGF groups by Day 8. CYP11A1/P450scc was upregulated 3 folds at day 4 and 10 by spß-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spß-NGF. In conclusion, the luteotrophic effect of spß-NGF could be related to an increase of vascularization and up regulation of CYP11A1/P450scc and STAR transcripts enhancing progesterone secretion.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of ß-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 ß-nerve growth factor (ß-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 ß-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.

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas.

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 µg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.

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 ß-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.

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.

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.