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1.
Theriogenology ; 216: 177-184, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38185017

ABSTRACT

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.


Subject(s)
Camelids, New World , Semen , Pregnancy , Male , Female , Animals , Semen/physiology , Camelids, New World/physiology , Nerve Growth Factor/metabolism , Prospective Studies , Retrospective Studies , Fertility , Spermatozoa/metabolism , Sperm Motility
2.
Domest Anim Endocrinol ; 77: 106645, 2021 10.
Article in English | MEDLINE | ID: mdl-34186420

ABSTRACT

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.


Subject(s)
Corpus Luteum , Nerve Growth Factor , Animals , Cattle , Corpus Luteum/physiology , Estradiol , Estrus Synchronization/methods , Female , Luteinizing Hormone , Male , Nerve Growth Factor/pharmacology , Ovulation , Progesterone
3.
Theriogenology ; 169: 29-35, 2021 Jul 15.
Article in English | MEDLINE | ID: mdl-33932649

ABSTRACT

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.


Subject(s)
Nerve Growth Factor , Ovary/physiology , Semen , Animals , Camelids, New World , Cats , Female , Male , Ovulation , Progesterone
4.
Anim Reprod Sci ; 185: 109-117, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28869109

ABSTRACT

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.


Subject(s)
Animal Husbandry , Mastitis, Bovine/etiology , Animals , Bacterial Infections/veterinary , Cattle , Female , Milk/microbiology , Postpartum Period , Pregnancy , Reproduction , Risk Factors
5.
Theriogenology ; 103: 69-75, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28779611

ABSTRACT

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.


Subject(s)
Camelids, New World/physiology , Corpus Luteum/blood supply , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Nerve Growth Factor/pharmacology , Semen/chemistry , Animals , Corpus Luteum/drug effects , Cytochrome P-450 Enzyme System/genetics , Female , Gonadotropin-Releasing Hormone/pharmacology , Male , Phosphoproteins/genetics , Phosphoproteins/metabolism , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Semen/metabolism
6.
Reprod Domest Anim ; 52(4): 625-631, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28332278

ABSTRACT

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.


Subject(s)
Camelids, New World/physiology , Copulation/physiology , Corpus Luteum/drug effects , Gonadotropin-Releasing Hormone/administration & dosage , Nerve Growth Factor/administration & dosage , Animals , Drug Administration Routes/veterinary , Female , Luteinizing Hormone/blood , Male , Ovary/drug effects , Ovulation/drug effects , Progesterone/blood , Semen , Ultrasonography
7.
Reprod Domest Anim ; 51 Suppl 2: 4-17, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27762054

ABSTRACT

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.


Subject(s)
Nerve Growth Factor/analysis , Ovulation/physiology , Semen/chemistry , Animals , Camelids, New World , Camelus , Corpus Luteum/drug effects , Dose-Response Relationship, Drug , Female , Humans , Luteinizing Hormone/metabolism , Male , Nerve Growth Factor/administration & dosage , Ovary/drug effects , Ovary/metabolism , Ovulation/drug effects , Pregnancy , Reproduction/physiology , Species Specificity
8.
Anim Reprod Sci ; 170: 157-69, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27236376

ABSTRACT

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.


Subject(s)
Cattle/physiology , Glucose/metabolism , Granulosa Cells/drug effects , Ovarian Follicle/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/physiology , 3-Hydroxysteroid Dehydrogenases/genetics , 3-Hydroxysteroid Dehydrogenases/metabolism , 3-O-Methylglucose/metabolism , Animals , Deoxyglucose/metabolism , Female , Follicle Stimulating Hormone/metabolism , Gene Expression Regulation/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Insulin-Like Growth Factor I/metabolism , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Protein Subunits , Radioactive Tracers , Receptors, FSH/genetics , Receptors, FSH/metabolism , Time Factors
9.
Theriogenology ; 84(7): 1096-102, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26164804

ABSTRACT

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.


Subject(s)
Camelids, New World/physiology , Luteinizing Hormone/blood , Nerve Growth Factor/administration & dosage , Ovulation/drug effects , Semen/chemistry , Administration, Intravenous/veterinary , Animals , Corpus Luteum/anatomy & histology , Corpus Luteum/blood supply , Dose-Response Relationship, Drug , Female , Male , Muscles/drug effects , Ovary/diagnostic imaging , Progesterone/blood , Ultrasonography , Uterus/drug effects
10.
Anim Reprod Sci ; 149(3-4): 345-52, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25176642

ABSTRACT

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.


Subject(s)
Camelids, New World/physiology , Corpus Luteum/drug effects , Nerve Growth Factor/pharmacology , Ovulation/drug effects , Ovulation/physiology , Animals , Drug Administration Schedule , Female , Nerve Growth Factor/administration & dosage
11.
Anim Reprod Sci ; 148(3-4): 221-7, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24950997

ABSTRACT

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.


Subject(s)
Camelids, New World/physiology , Corpus Luteum/drug effects , Nerve Growth Factor/isolation & purification , Nerve Growth Factor/pharmacology , Ovarian Follicle/cytology , Semen/chemistry , Animals , Cell Size , Corpus Luteum/diagnostic imaging , Corpus Luteum/physiology , Female , Follicular Phase , Gonadotropin-Releasing Hormone/pharmacology , Male , Ovarian Follicle/diagnostic imaging , Ovulation/drug effects , Ovulation Induction/methods , Ovulation Induction/veterinary , Ultrasonography , Up-Regulation/drug effects
12.
Theriogenology ; 81(8): 1101-1107.e1, 2014 May.
Article in English | MEDLINE | ID: mdl-24582374

ABSTRACT

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.


Subject(s)
Camelids, New World , Corpus Luteum/drug effects , Nerve Growth Factors/administration & dosage , Ovarian Follicle/blood supply , Ovulation/drug effects , Semen/chemistry , Animals , Corpus Luteum/physiology , Female , Gonadotropin-Releasing Hormone/administration & dosage , Injections, Intramuscular/veterinary , Luteinizing Hormone/blood , Male , Nerve Growth Factors/isolation & purification , Ovarian Follicle/diagnostic imaging , Ovarian Follicle/physiology , Progesterone/blood , Semen/physiology , Ultrasonography
13.
Anim Reprod Sci ; 143(1-4): 72-8, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24231049

ABSTRACT

The effect of different ethylene glycol concentrations, times of exposure and vitrification procedure on viability, cleavage and blastocyst rate of in vitro matured alpaca oocytes chemically activated after vitrification was analyzed. In Experiment 1, oocytes were incubated for 12-15 min with different concentrations of ethylene glycol (EG) in the equilibration solution (ES) followed by chemical activation and in vitro cultured for 8 days to determine oocyte viability, cleavage and blastocyst rates. In Experiment 2, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to vitrification solutions containing 25, 35 or 45% of EG for 30s, vitrified and stored at -196°C. In Experiment 3, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to the vitrification solution containing 35% of EG for 15, 30 or 45s, vitrified and stored at -196°C. For Experiments 2 and 3, non-vitrified and vitrified oocytes were activated and cultured in vitro. In Experiment 1, oocyte viability was lowest at concentrations of 6 or 8%, intermediate at 2 or 4% and highest at 0% of EG. Oocyte viability and cleavage rate were affected by EG concentration, time of exposure in the vitrification solution or vitrification procedure in Experiment 2 and 3. Alpaca oocytes were viable after vitrification, given that oocyte viability, cleavage and blastocyst rate were affected by the vitrification procedure, EG concentration and time of exposure in the equilibration and vitrification solutions.


Subject(s)
Camelids, New World , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Ethylene Glycol/pharmacology , Oocytes , Vitrification , Animals , Blastocyst/cytology , Blastocyst/drug effects , Cell Survival/drug effects , Cells, Cultured , Cleavage Stage, Ovum/cytology , Cleavage Stage, Ovum/drug effects , Cryopreservation/veterinary , Dose-Response Relationship, Drug , Female , In Vitro Oocyte Maturation Techniques/veterinary , Time Factors
14.
Anim Reprod Sci ; 138(3-4): 252-60, 2013 May.
Article in English | MEDLINE | ID: mdl-23562451

ABSTRACT

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.


Subject(s)
Animal Nutritional Physiological Phenomena , Caloric Restriction , Camelids, New World/metabolism , Hormones/blood , Ovary/physiology , Animals , Body Constitution/physiology , Caloric Restriction/adverse effects , Caloric Restriction/veterinary , Camelids, New World/blood , Camelids, New World/physiology , Female , Gonadotropin-Releasing Hormone/pharmacology , Leptin/blood , Luteinizing Hormone/blood , Ovary/metabolism , Ovulation/blood , Ovulation/metabolism , Ovulation/physiology , Ovulation Induction/methods , Ovulation Induction/veterinary , Progesterone/blood , Weight Loss/physiology
15.
Anim Reprod Sci ; 133(1-2): 117-22, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22770553

ABSTRACT

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. We hypothesize that OIF stimulates LH release from gonadotroph cells in the anterior pituitary gland. Four experiments were done to determine if purified OIF isolated from llama seminal plasma stimulates LH secretion in pituitary cells using tissue from an induced ovulator (llama) and spontaneous ovulator (cattle). Anterior pituitary cells were cultured in vitro for two days, and on the third day, wells were incubated for 2 h with media containing no treatment (control), GnRH or OIF. Concentrations of LH in the culture medium were measured using radioimmunoassay and compared among groups by analysis of variance. In all experiments, GnRH and OIF treatments induced more LH secretion than untreated controls (P<0.05). A dose-related effect was evident in the llama pituitary cell cultures in that mean LH concentrations were greater (P<0.05) in wells treated with a higher dose of OIF (5.41 ± 0.28 ng/mL) compared to wells treated with a lower dose (2.70 ± 0.50 ng/mL), both of which were higher (P<0.05) than in wells with no treatment (0.87 ± 0.18 ng/mL). Although OIF stimulated LH release in bovine cell cultures, a dose-related effect was not detected. We conclude that OIF stimulates LH secretion from pituitary gonadotrophs in vitro.


Subject(s)
Gonadotrophs/drug effects , Luteinizing Hormone/metabolism , Pituitary Gland/drug effects , Seminal Plasma Proteins/pharmacology , Animals , Camelids, New World , Cattle , Cells, Cultured , Dose-Response Relationship, Drug , Female , Fertility Agents, Female/pharmacology , Gonadotrophs/metabolism , Ovulation Induction , Pituitary Gland/metabolism , Up-Regulation/drug effects
16.
Theriogenology ; 78(5): 1030-9, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22763069

ABSTRACT

Two experiments were designed to determine the effect of purified ovulation inducing factor (OIF) on ovarian function in cattle. In Experiment 1, prepubertal heifers (n = 11 per group) were treated on Day 5 (Day 0 = day of follicular wave emergence) of the follicular wave with an intramuscular dose of saline (1 mL), GnRH (100 µg), or purified OIF (1 mg/100 kg body weight). Ovulation occurred in 9/11 heifers treated with GnRH, and 1/11 heifers in each of the OIF- and saline-treated groups (P < 0.05). Compared to saline-treated controls, OIF treatment was associated with a smaller dominant follicle diameter (P < 0.01), a rise in plasma FSH concentration (P < 0.1), and earlier emergence of the next follicular wave (P < 0.05). In Experiment 2, sexually mature heifers were given either GnRH or purified OIF on Days 3, 6 or 9 of the first follicular wave (i.e., early growing, early static, or late static phase of the dominant follicle; n = 5 per group per day), or were untreated (n = 10). In heifers treated with OIF on Day 6, the dominant follicle diameter profile tended to be smaller than in controls, and was associated with a rise (P < 0.05) in plasma FSH concentrations. A similar rise in FSH was detected after OIF treatment on Day 9. Compared to untreated controls, treatment with OIF and GnRH was associated with a larger CL diameter (Days 3 and 6 groups; P < 0.05) and a greater concentration of plasma progesterone (Days 6 and 9 groups; P < 0.05). Treatment with purified OIF did not induce ovulation in heifers, but hastened new follicular wave emergence in prepubertal heifers, influenced follicular dynamics in a phase-specific manner in mature heifers, and was luteotrophic.


Subject(s)
Camelids, New World/metabolism , Cattle/physiology , Ovary/drug effects , Animals , Cattle/blood , Female , Gonadotropin-Releasing Hormone , Luteinizing Hormone/metabolism , Sexual Maturation , Species Specificity
17.
Theriogenology ; 77(9): 1873-82, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22401833

ABSTRACT

This study was designed to: 1) characterize the effect of ovulation-inducing factor (OIF) on pituitary LH secretion in ovariectomized (OVX) llamas; and 2) determine the effect of OIF on LH secretion in OVX llamas pretreated with estradiol-17ß (E-17ß) or estradiol benzoate (EB). In Experiment 1, intact and OVX llamas (n = 5 or 6 per group) were assigned to a two by two factorial design: 1) Intact llamas treated with 1 mL of phosphate buffered saline (PBS); 2) Intact llamas treated with 1 mg of purified OIF; 3) OVX llamas treated with 1 mL of PBS; or 4) OVX llamas treated with 1 mg of purified OIF. In Experiment 2, intact and OVX llamas (n = 5 or 6 per group) were randomly assigned to the following groups: 1) Intact llamas treated with 1 mg of purified OIF; 2) OVX llamas treated with 1.0 mL of PBS; 3) OVX llamas treated with 1.0 mg of purified OIF; 4) OVX llamas primed with E-17ß, followed by 1.0 mg of purified OIF. Experiment 3 was similar as described for Experiment 2, except that priming was done with EB. In Experiment 1, animal category by treatment and animal category by treatment by time interactions tended (P = 0.08) to affect LH concentration. The effect of OIF on LH released was partly restored (P < 0.05), to the values observed for the intact OIF-treated females, when OVX llamas were primed with E-17ß or BE (Experiments 2 and 3). We concluded that peripheral estradiol concentrations in llamas partially modulates the effect of OIF on pituitary LH secretion; however, other ovarian factor(s) could also participate in this modulatory action.


Subject(s)
Camelids, New World/physiology , Estradiol/metabolism , Luteinizing Hormone/metabolism , Ovary/physiology , Pituitary Gland/metabolism , Seminal Plasma Proteins/pharmacology , Animals , Female , Male , Ovariectomy/veterinary , Ovulation Induction , Semen/chemistry
18.
Theriogenology ; 77(9): 1802-10, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22365705

ABSTRACT

Gonadotrophin releasing hormone (GnRH) is commonly used in llamas to induce ovulation; however, the consequence of reduced doses of GnRH on luteinizing hormone (LH) release, ovulatory response, and subsequent corpus luteum (CL) development and function have apparently not been investigated. Hence, we examined the effect of gradual reduction of gonadorelin acetate (GnRH) dosage on pituitary LH release, ovulatory response, CL development, and plasma progesterone concentrations in llamas. Non-pregnant, non-lactating adult llamas were examined once daily by transrectal ultrasonography, and those with a follicle ≥8 mm in diameter that had grown for three consecutive days were randomly assigned to receive 50 (GnRH50, n = 23), 25 (GnRH25, n = 29), 12.5 (GnRH12.5, n = 29), or 6.25 µg (GnRH6.25, n = 29) of GnRH, or 0.5 mL of PBS (Control group, n = 16) im. In a subset (7 or 8 animals/group), intense blood sampling was done to measure LH concentrations. All females were examined by ultrasonography every 12 h from treatment (Day 0) to Day 2 to determinate ovulation, and thereafter on alternate days until Day 16 to evaluate CL development (9-13 animals/group). Also, blood samples for progesterone determination were taken (9 or 10 animals/group) on alternate days from Days 0-16. Ovulatory response (%) was highest (P < 0.05) in the GnRH50 (82.6), intermediate in the GnRH25 (72.3) and GnRH12.5 (75.9) groups, and lowest in the GnRH6.25 group (48.3). No ovulations were detected in the Control group. Mean peak LH concentrations (ng/mL) were highest (P < 0.05) for GnRH50 (6.2), intermediate for GnRH25 (4.4) and GnRH12.5 (2.9), and lowest for GnRH6.25 (2.2) groups. In addition, based on regression analysis, llamas with an LH peak <4 ng/mL were less likely to ovulate. Llamas given 50 µg of GnRH released more (P < 0.05) pituitary LH and had an LH surge of longer duration than those given 25, 12.5, or 6.25 µg. However, in those that ovulated, neither GnRH treatment nor treatment by time interaction affected (P > 0.05) CL diameter or plasma progesterone concentrations. In summary, reducing the dose of GnRH gradually decreased the magnitude of the preovulatory LH surge and ovulatory response; however, subsequent CL development and plasma progesterone concentrations were not affected.


Subject(s)
Camelids, New World/physiology , Corpus Luteum/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Luteinizing Hormone/metabolism , Ovulation/drug effects , Pituitary Gland/drug effects , Animals , Corpus Luteum/physiology , Female , Gonadotropin-Releasing Hormone/administration & dosage , Ovulation/physiology , Pituitary Gland/metabolism
19.
Anim Reprod Sci ; 129(1-2): 1-6, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22030337

ABSTRACT

This study was conducted to determine the use of repeated transvaginal ultrasound-guided cumulus oocyte complex (COC) aspiration on COC recovery rate, in vitro embryo production (IVP) and subsequent pregnancy rates in Holstein Friesian (HF) and Aberdeen Angus (AA) cows (Experiment 1), and in pregnant and non-pregnant Holstein Friesian cows (Experiment 2). Cycling, non-pregnant HF (n=17) and AA (n=32) cows with 40-70 days postpartum, between 3 and 5 years of age were used in the Experiment 1. All cows were submitted to repeated transvaginal ultrasound-guided COC aspiration twice a week for 5-7 weeks. Cumulus ooctye complexes (COC) were in vitro matured, fertilized and cultured for 8 days. An overall of 100 and 350 embryos from HF and AA cows respectively were cryopreserved using a conventional slow freezing (Experiment 1). A total of 81 and 285 frozen-thawed embryos from HF and AA cows respectively were transferred to recipient cows. Pregnancy diagnosis was performed at 60 and 150 days of gestation using transrectal ultrasonography. In Experiment 2, cycling non-pregnant (n=9) and pregnant (n=8) HF cows were submitted to repeated ultrasound-guided COC aspiration and COC were in vitro matured, fertilized and cultured as in Experiment 1, except that embryos were cryopreserved but not thawed and transferred as described for Experiment 1. The results of this study indicate that COC recovery rate and blastocyts production are affected by the breed of the donor cow. The quality of blastocyts produced from both breed did not differ in terms of pregnancy and calving rates (Experiment 1). The physiologic state of pregnancy did not affect COC recovery rate and blastocysts production per donor/session (Experiment 2). The use of ultrasound-guided COC aspiration and IVP could be a powerful technique to improve the genetic of beef and dairy cattle managed under pasture-based conditions management in the southern Chile.


Subject(s)
Cattle/physiology , Fertilization in Vitro/veterinary , Oocyte Retrieval/veterinary , Oocytes/physiology , Ovarian Follicle/diagnostic imaging , Ovarian Follicle/physiology , Animals , Embryo Transfer/methods , Embryo Transfer/veterinary , Female , Fertilization in Vitro/methods , Lactation , Oocyte Retrieval/methods , Pregnancy , Ultrasonography/methods , Ultrasonography/veterinary
20.
Anim Reprod Sci ; 127(3-4): 213-21, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21899964

ABSTRACT

The objectives of this study were (1) to determine the effect of rabbit seminal plasma on LH secretion and ovulation using the llama animal model as an in vivo ovulation bioassay and (2) to determine the effect of llama or rabbit seminal plasma on ovulation induction in the rabbit model. In Experiment 1, llamas with a growing follicle ≥8mm in diameter were assigned randomly to one of three groups (n=5 per group) and given an intramuscular dose of 1mL of: (a) llama seminal plasma, (b) rabbit seminal plasma, or (c) phosphate buffered saline (PBS; negative control). Blood samples for LH measurement were taken every 15 min from 1.5 h before to 8 h after treatment (Day 0: starting of treatment). Llamas were examined by ultrasonography every 12h from treatment to ovulation, and then every other day until Day 16 after treatment to evaluate corpus luteum (CL) development. Blood samples for progesterone measurement were taken every other day from Day 0 to Day 16. Ovulation was detected in 4 of 5, 5 of 5, and 0 of 0 llamas treated with llama or rabbit seminal plasma and PBS, respectively (P<0.001). After treatment, plasma LH concentration increased and decreased (P<0.01) in the llama and rabbit seminal plasma group but not in the PBS-treated group. No differences were observed on CL development (P≥0.3) and progesterone secretion (P>0.05) between both seminal plasma treated groups. In Experiment 2, receptive female rabbits (n=5-7 per group) were given an intramuscular dose of: (a) 0.5, (b) 1.0 and (c) 2.0mL of either rabbit or llama seminal plasma, (d) 0.5mL PBS (negative control), or (e) 25µg of gonadoreline acetate (GnRH; positive control). Does were submitted to laparotomy 24-36 h after treatment to determine the ovulatory response and the presence of antral and hemorrhagic anovulatory follicles. Ovulation sites (7.0±0.6) were only detected in GnRH-treated does (P<0.01). There was an increase (P<0.01), in the total number of follicles (antral plus hemorraghic follicles) in those females treated with 1mL of rabbit seminal plasma and there was a tendency (P=0.08) for more hemorrhagic anovulatory follicles in does treated with 1.0 and 2.0mL of either rabbit or llama seminal plasma. Results document the presence of OIF in the seminal plasma of rabbits. The differential ovulatory response between species, however, requires further investigation.


Subject(s)
Hormones/isolation & purification , Hormones/pharmacology , Ovulation/drug effects , Semen/chemistry , Animals , Anovulation/pathology , Camelids, New World/metabolism , Efficiency , Female , Hormones/metabolism , Male , Ovulation/physiology , Ovulation Induction/methods , Ovulation Induction/veterinary , Rabbits/metabolism , Rabbits/physiology , Semen/metabolism , Semen/physiology , Sperm Retrieval
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