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.

Effect of cat seminal plasma and purified llama ovulation-inducing factor (ß-NGF) on ovarian function in queens.

The present study aimed to determine the effect of cat seminal plasma and purified llama ovulation-inducing factor (ß-NGF) on ovarian activity in queens. Queens (n = 6) were used for all the treatments in a crossover design with an interval time between treatments of three interestrus intervals. Forty-eight hours after the detection of an estrus vaginal cytology, queens were given cat seminal plasma (subcutaneous or intramuscular), purified llama ovulation-inducing factor (15 or 35 µg), hCG (75 UI), saline, or were mated with a male. A total of 192 estrous cycles were observed. Estrus length and serum estradiol concentration were 6 ± 1 days (range 2-10 d) and 38 pg/mL (range 10-75 pg/mL), respectively. Queens mated and given hCG showed higher serum progesterone concentration and longer interestrus interval (47 ± 5 d) than that of controls (10 ± 3 d). Sixty-seven percent of queens (4/6) treated with subcutaneous cat seminal plasma, and 17% of those treated with purified llama ß-NGF showed high serum progesterone concentrations along with prolonged interestrus. However, intramuscular administration of cat seminal plasma produced interestrus intervals similar to controls (15 ± 5 d) and basal serum progesterone concentration (<0.50 ng/mL). This study demonstrates that the subcutaneous administration of cat seminal plasma induced ovulation in queens. Therefore, molecules present in cat seminal plasma, contribute to the induction of ovulation in queens. Identifying those molecules will improve the knowledge of queen's reproductive physiology. Also, it could offer a physiologic alternative to induce ovulation in queens when reproductive biotechnologies are used.

The association between subclinical mastitis around calving and reproductive performance in grazing dairy cows.

The objective of this study was to evaluate the effect of subclinical mastitis (SCM) on calving-to-first-service interval (CFS), calving-to-conception interval (CC), and on the number of services per conception (S/C) in grazing Holstein and Normande cows. Primiparous (n=43) and multiparous (n=165) cows were selected from five dairy herds. Two composite milk samples were aseptically collected from each cow at drying-off, and then every week during the first postpartum month. One sample was used for somatic cell count (SCC), and the other one for bacteriological analysis. Cows were followed up to 300 d after calving. Non-parametric and parametric survival models, and negative binomial regression were used to assess the association between SCM, evaluated by SCC and milk culture, and reproductive indices. Staphylococcus aureus, CNS, and Streptococcus uberis were the most frequent isolated pathogens. Subclinical mastitis in the first month of lactation was not associated with CFS; however, the CC interval was longer in cows with SCM compared to healthy cows, the former also had a higher number of S/C.

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.

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.

Ovulation-inducing factor (OIF/NGF) in seminal plasma: a review and update.

The ovulation-inducing effect of seminal plasma was first reported in Bactrian camels over 30 years ago, and the entity responsible was dubbed 'ovulation-inducing factor' (OIF). More recent studies, primarily in llamas and alpacas, characterized the biological and chemical properties of OIF and ultimately identified it as ßNGF. This recent discovery has allowed a convergence of knowledge previously separated by discipline and by mechanism; that is, neurobiology and reproductive biology, and autocrine/paracrine vs endocrine. To preserve this link, we have referred to the seminal factor as OIF/NGF. As a highly conserved protein, the implications of discoveries related to OIF/NGF in reproductive tissues extend beyond the camelid species, and results of recent studies show that the presence and function of OIF/NGF in seminal plasma are conserved among species considered to be induced ovulators as well as those considered to be spontaneous ovulators. The abundance of OIF/NGF in seminal plasma and the effects of seminal plasma on ovarian function strongly support the idea of an endocrine mode of action (i.e. systemic distribution with distant target tissues). This review is intended to provide an update on the progress in our understanding of the nature of OIF/NGF in seminal plasma and its effects on reproductive function in the female, including the effects of dose and route of administration, evidence for ovarian effects in other species, tissue sources of OIF/NGF and early findings related to the mechanism of action of OIF.

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and ß-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and ß-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3ß-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

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.

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.