Effects of estradiol on uterine perfusion in anesthetized cyclic mares affected with uterine vascular elastosis.

Uterine vascular elastosis in mares is characterized by degeneration of uterine vasculature through thickening of the elastin layers. Factors commonly associated with this degeneration include age, parity, and chronic uterine endometritis. Affected mares have also been shown to exhibit decreases in uterine blood flow and perfusion of the uterus. Due to the increased thickness of the elastin layers, we hypothesize that vasodilatation of the uterine vasculature is also impaired. To test the functionality of these vessels, we evaluated the vasodilatory effects of estradiol on the uterine vascular bed in mares with normal vasculature and mares with severe elastosis. Both groups were tested in estrus and diestrus. Fluorescent microspheres were used to determine basal blood perfusion, followed by the intravenous administration of 1.0 µg/kg of 17ß-estradiol. After 90 min, perfusion was measured once again to determine the vascular response to estradiol. Control mares in estrus displayed a significant increase in total uterine blood flow after the administration of estradiol when compared to baseline levels. No other group had a significant increase in total blood flow and perfusion after estradiol administration. The administration of estradiol in control mares induced regional increases in perfusion in the uterine horns and uterine body during estrus and only in the uterine horns during diestrus. Mares affected by elastosis exhibited no regional differences in perfusion levels post-estradiol administration. The difference in the vasodilatory response induced by estradiol between reproductively healthy mares and mares affected with elastosis indicates that the functionality of the affected vessels is compromised.

Effects of vascular elastosis on uterine blood flow and perfusion in anesthetized mares.

In the uterus of the mare, data obtained using transrectal Doppler ultrasonography indicate that uterine blood flow (UBF) is dynamic and changes throughout the estrous cycle. Degenerative lesions in the uterus are associated with subfertility and infertility. Among these lesions, vascular elastosis has been reported in aged, multiparous, and infertile mares. Angiosis of the uterine vasculature could potentially compromise UBF. The objectives of this experiment are to determine levels of UBF and perfusion of reproductively healthy mares and compare them to levels of subfertile/infertile mares affected by uterine vascular elastosis. Twenty mares were classified on the basis of degree of vascular degeneration and stage of cycle. A fluorescent microsphere technique was used to measure reproductive organ perfusion, where microspheres were injected into the left ventricle of the heart and became trapped in capillary beds in proportion to blood flow and tissue perfusion. The reproductive tract was removed, sectioned, and the fluorescent intensity evaluated to measure blood flow and perfusion. Additionally, full-thickness samples of the uterine wall were examined postmortem to further assess the degree of vascular degeneration in all layers of uterine wall. The mean value of uterine perfusion for the control mares during estrus (n = 5) was higher (P < 0.01) than that during diestrus (n = 5); 17.6 and 11.9 mL/min/100g, respectively. For the subfertile/infertile mares, the mean value of tissue perfusion was not different (P > 0.05) during estrus (n = 5) and diestrus (n = 5); 5.9 and 7.2 mL/min/100g, respectively. Uterine perfusion in subfertile/infertile mares affected by elastosis was lower than that of control mares during both estrus (P < 0.01) and diestrus (P < 0.01). The differences in baseline levels of perfusion between the control and elastosis groups indicate that elastosis of the uterine vasculature is associated with decreased uterine perfusion during both phases of the estrous cycle. In the uterus, a compromise in UBF could have implications in endometrial glandular development, postbreeding endometritis, uterine clearance, development of the conceptus, and overall fertility.

The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro.

The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5% CO2. The COCs were stained with 10 µg/mL propidium iodide and 10 µg/mL fluorescein isothiocyanate-labeled Lens culinaris agglutinin. Chromatin configuration and distribution of cortical granules were assessed via confocal microscopy. Compared to control, COCs incubated with eGH had: more oocytes that reached metaphase II (35/72, 48.6% vs. 60/89, 67.4%, respectively; P=0.02); greater concentrations of testosterone (0.21 ± 0.04 vs. 0.06 ± 0.01 ng/mL; P=0.01), progesterone (0.05 ± 0.01 vs. 0.02 ± 0.00 ng/mL; P=0.04), and oestradiol (76.80 ± 14.26 vs. 39.58 ± 8.87 pg/mL; P=0.05) in the culture medium, but no significant differences in concentration of androstenedione. Based on Real Time RT-PCR analyses, expression of the eGH-R gene was greater in cumulus cells and COCs at the start than at the end of in vitro maturation. Positive immunostaining for eGH-R was present in cumulus cells, the oocytes and granulosa cells. In conclusion, addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation, perhaps mediated by the presence of eGH-R in ovarian follicles.

Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes.

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus-oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 µg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

Uterine vascular degeneration is present throughout the uterine wall of multiparous mares. Colinearity between elastosis, endometrial grade, age and parity.

Vascular degeneration is present in endometrial vessels of multiparous aged mares. The lesions associated with vascular degeneration consist of enlargement, duplication and splitting of the membrana elastica interna and perivascular deposits of elastin. However, there are no similar data available for deep myometrial vessels and the vascular layer. The objectives of the present study were to characterize the status of vasculature in full-thickness uterine necropsy samples and to correlate these findings to endometrial grade, age, and parity. Elastosis was present in myometrial vessels, as well as in large arteries and veins located between the circular and longitudinal myometrial layers. Vascular degeneration was associated with number of foals (P < 0.001) and endometrial grade (P < 0.05), but not with mare age (P > 0.05). Endometrial grade was associated with age (P < 0.001) and vascular grade (P < 0.05), but not with number of foals (P > 0.05). The presence of elastosis in the myometrial vessels was related to problems associated with chronic uterine infection (CUI) and delayed uterine clearance (DUC) of infertile mares. Uterine contractility was impaired in mares affected by CUI and/or DUC and could be related to a lack of myometrial blood flow. Additionally, degeneration of large vessels in the vascular layer may indicate a general compromise in uterine blood flow and fertility. The main conclusions were the presence of vascular elastosis in large deep myometrial vessels as well as in endometrial vessels, and that the factor with the strongest association with vascular degeneration was number of foals (P < 0.001), followed by endometrial grade (P < 0.05), but no association with mare age.

Biological and clinical significance of anti-Müllerian hormone determination in blood serum of the mare.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor ß superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologous enzyme immunoassay for determination of serum AMH in the horse, and to determine concentrations of AMH in the blood of mares during the estrous cycle, pregnancy, and in mares with granulosa-cell tumors. Mares with normal estrous cycles (n = 6) and pregnant mares (n = 6) had blood samples collected throughout one interovulatory period and monthly throughout gestation, respectively. Mares diagnosed with GCT had blood samples taken before (n = 11) and after ovariectomy (n = 5). Tumors were sectioned and fixed for immunohistochemistry and snap frozen for immunoblot analyses and RT-qPCR. In normal cyclic mares and in pregnant mares, there was no effect of cycle stage or month of gestation on serum AMH concentrations. In GCT mares, serum concentrations of AMH (1901.4 ± 1144.6 ng/mL) were higher than those in cyclic (0.96 ± 0.08 ng/mL) or pregnant (0.72 ± 0.05 ng/mL) mares and decreased after tumor removal. Both AMH and AMH receptor (AMHR2) immunolabeling and expression were detected by immunohistochemistry in the tumor and cyst fluid obtained from mares with GCTs. Therefore, we concluded that AMH was a useful biomarker for detection of granulosa-cell tumors in mares.

Expression of anti-Müllerian hormone (AMH) in equine granulosa-cell tumors and in normal equine ovaries.

Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is expressed by granulosa cells in females of many mammalian species, and circulating AMH concentrations have been used to monitor granulosa-cell tumors (GCT) in women. The objective was to characterize expression of AMH in equine GCT, and in normal equine ovaries, based upon immunohistochemistry (IHC), using a polyclonal primary antibody directed against human AMH. Equine GCT (n=27) and normal equine ovaries (n=10) were examined by IHC. In addition, sera from four mares with GCT were characterized for AMH bioactivity, based upon suppression of Müllerian duct development in the fetal rat. Immunolabeling with alpha-AMH was localized to granulosa cells in equine GCT, as well as within antral follicles in normal ovaries. Expression of AMH first appeared in granulosa cells of small growing follicles and was most intense in small antral follicles; large antral or atretic follicles had reduced immunolabeling. Omission of the primary antibody or incubation of the primary antibody with the corresponding blocking peptide eliminated immunolabeling of granulosa cells in GCT and in normal antral follicles, confirming the specificity of the immunolabel. Sera from mares with GCT had increased AMH bioactivity compared to control sera. In conclusion, AMH was strongly expressed by granulosa cells in equine GCT and in normal antral follicles. Therefore, anti-Müllerian hormone may be a useful biomarker for detection of GCT in the horse.

The diagnosis and treatment of endometritis in the mare: yesterday and today.

Diagnosis and treatment of endometritis in the mare has been controversial and mostly empirical. The lack or inability of researchers to establish or develop a model that can serve as a standard or control makes this area of equine reproduction difficult to address scientifically. However, major advances have been made, particularly with the demonstration of the importance of uterine contractility in the elimination of bacteria, fluid, and inflammatory products from the uterus after breeding. This review provides a historical perspective of what has been done, and where we are now, in the approach to the diagnosis and therapy of endometritis in the mare.

Expression of anti-Müllerian hormone (AMH) in the equine testis.

Anti-Müllerian hormone (AMH) induces regression of Müllerian ducts during male fetal development; in the human male, it is expressed in Sertoli cells during fetal development (and through puberty). The objective was to characterize expression of AMH in the fetal, neonatal, prepubertal, and adult equine testis, as well as in equine cryptorchid testes, in select testicular neoplasms, and in intersex gonads, based upon immunohistochemistry (IHC). Testes were removed from equine fetuses at 5.5, 10, and 11 months of gestation, at 12 months of age, and from adult stallions. In addition, cryptorchid testes, testis tumors (teratomas, seminomas, Sertoli cell tumors), and male intersex gonads were examined by IHC for expression of AMH using a goat polyclonal primary antibody (alpha-AMH) directed against a C-terminal peptide antigen from human AMH. Immunolabeling with alpha-AMH was localized to Sertoli cells within the developing seminiferous tubules of fetal, neonatal and prepubertal equine testes, with no expression detected in Sertoli cells from normal adult equine testes. Furthermore, expression was detected in cryptorchid testes (in animals up to 3-4 years of age) and in Sertoli cell tumors and male intersex gonads. In conclusion, AMH was strongly expressed by Sertoli cells in fetal, neonatal and prepubertal equine testes, but not in normal adult testes. That AMH was expressed in cryptorchid testes may provide a useful biomarker for detection of cryptorchid testes, as well as for immunohistochemical characterization of testicular tumors and intersex gonads in the horse.

Uterus unicornis in two mares.

Two American Paint Horses, a 3-year-old nulliparous mare and a 7-year-old primiparous mare, presented for recent infertility and a pre-breeding examination, respectively. Examination of the internal reproductive tract of both mares using transrectal palpation and ultrasonography revealed the presence of the cervix, uterine body, left uterine horn and bilateral ovaries. The right uterine horn could neither be palpated nor imaged. The clinical diagnosis of uterus unicornis in one mare was confirmed at necropsy, which revealed combined aplasia of the right uterine horn and oviduct.

Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production.

The present 2-year study investigated the feasibility of using porcine zona pellucidae (pZP) as antigen for immunocontraception in American black bears. Sows, 3-6 years of age, were administered either two doses of 250 microg pZP with Freund's adjuvant (n = 10) or adjuvant alone (n = 5), one in April and one in May, and were kept away from the boars until June. Serum samples were collected before injections and before denning (November). The presence of sows with cubs at side was observed during premature emergence from denning. First-year results indicated that anti-pZP antibody titres in vaccinated sows were 2.5-9.0-fold (range) higher compared with non-vaccinated sows and that the vaccinated sows were threefold less likely to become pregnant (P = 0.167). Control and vaccinated bears produced 1.6 and 0.2 cubs per sow, respectively (P = 0.06). The second-year study investigated the feasibility of using pZP sequestered in a controlled-release pellet and a water-soluble adjuvant (QS-21) to avoid regulatory problems associated with Freund's adjuvant. Sows in the treatment group (n = 22) were administered a single dose of an emulsion of 250 microg pZP and 150 microg QS-21 plus a pellet containing 70-90 microg pZP for delayed release as booster dose. Control sows (n = 5) received the QS-21 adjuvant in pellet alone. Serum samples were collected before inoculations (April) and before denning (November). Seven cubs were born to the five control sows, but none was born to the 22 vaccinated sows (P < 0.001). Anti-pZP antibody mean absorbance ratios in control sows remained at background levels, whereas vaccinated sows had ratios fourfold higher than controls. Two-dimensional polyacrylamide gel electrophoresis and immunohistochemical localisation confirmed immunoreactivity of sera from inoculated bears. We conclude that cub production in the American black bear can be effectively limited with either two injections of 250 microg pZP or a single inoculation of partially purified pZP sequestered in controlled-release pellets.

Effect of porcine zonae pellucidae immunisation on ovarian follicular development and endocrine function in domestic ewes (Ovis aries).

Domestic ewes (Ovis aries) were immunised with porcine zonae pellucidae (pZP) or pZP conjugated to keyhole limpet haemocyanin (KLH) in adjuvant(s) to examine the feasibility of the species to serve as a model for further development of pZP-based vaccines in ungulates. Two immunisation groups were employed, with a third group receiving only adjuvant (n = 5 per group). Early in the study, oestrous activity was monitored by the use of a vasectomised ram fitted with a marking harness. Eventually, ewes were exposed to an intact ram for breeding. In addition, weekly serum and every-other-day faecal samples were collected to measure pZP antibodies and progesterone metabolite concentrations respectively. At the conclusion of the study, fecundity was established, and ovarian tissue was examined. Ewes immunised against pZP : KLH with adjuvant produced minimal antibody absorbance levels, displayed normal oestrous cycles, became pregnant upon introduction of the intact ram and exhibited normal ovarian histopathology. Ewes immunised against pZP with adjuvant produced high antibody absorbance levels, were acyclic following primary immunisation and were infertile. Examination of the ovarian tissue revealed atrophic changes that included: (1) the absence of growing follicles; (2) significant reduction in the number of primordial follicles; and (3) the presence of abnormal granulosa cell clusters lacking oocytes. Antisera displayed immunoreactivity to the major components of pZP, and immunohistochemical labelling of ovarian tissue showed specificity to the ZP. These data are the first generated in an ungulate species showing deleterious effects of pZP immunisation on folliculogenesis and oestrous cyclicity.

Persistence of anti-zonae pellucidae antibodies following a single inoculation of porcine zonae pellucidae in the domestic equine.

In this study of equids, we investigated the antibody response and the effect on the estrous cycle following a single inoculation of porcine zonae pellucidae (pZP) employing controlled-release methodology. We also investigated the use of two different water-soluble adjuvants as an alternative to oil-based adjuvants. Twenty-seven domestic mares were inoculated with various formulations of pZP and adjuvant. We showed that the anti-pZP antibodies generated as a result of the inoculations persisted for at least 43 weeks (length of the study). Of the various formulations used in the study, pZP and QS-21 water-soluble adjuvant, administered in combination with an emulsified preparation of pZP and Freund's Complete Adjuvant generated a significantly (P < 0.05) higher titer of anti-pZP antibodies when compared with other formulations employing the water-soluble adjuvant, Carbopol. Hormone analyses for cyclicity indicated a high incidence and extended duration of persistent corpora lutea among the treated mares. The positive control group of mares receiving two standard inoculations of pZP and Freund's Complete and Incomplete Adjuvants, as well as the placebo group of mares injected with QS-21 only, also exhibited high incidences of persistent corpora lutea. However, all mares eventually returned to normal cyclicity. The basis for the high incidence and extended duration of persistent corpora lutea was unexplained. The results demonstrate for the first time the persistent generation of anti-pZP antibodies following a single inoculation of pZP incorporated into a controlled-released preparation in the horse. This study further suggests that a single inoculation of pZP sequestered in a controlled-release lactide-glycolide polymer may serve as an alternative to traditional two-inoculation protocols for contraception investigations in the equine.

Effect of GnRH conjugated to pokeweed antiviral protein on reproductive function in adult male dogs.

This study evaluated the effect of a GnRH analogue conjugated to the cytotoxin, pokeweed antiviral protein (PAP), on reproductive function in adult, male dogs. Four dogs received 0.0042 mg GnRH-PAP kg(-1) hourly for 36 h, and four other dogs received 0.1 mg GnRH-PAP kg(-1) as one bolus injection daily for three consecutive days. One dog received a single bolus (0.1 mg x kg(-1)). Three adult male dogs received GnRH without the PAP conjugate, as controls. Twenty-five weeks after the initial treatment, all treated dogs received 0.1 mg GnRH-PAP kg(-1) as a single administration, whereas dogs in the control group received 0.0045 mg kg(-1) of the GnRH analogue. Serum concentrations of testosterone and LH were determined by radioimmunoassay, and testis size was measured for 9 months after treatment. Stimulation tests (5 microg GnRH kg(-1)) were used to evaluate LH release (-15, 0, 30, 60, 90, 120 min), which was assessed by measuring area under the curve. Serum testosterone concentrations were significantly lower (P<0.05) after treatment in the bolus and hourly groups than in the control group. Testosterone concentrations fell to less than 50 pg x ml(-1) in three of four dogs in the bolus group and one of four dogs in the hourly group by week 8-9 after treatment. Basal LH was lower (P<0.05) in the bolus and hourly groups than in the control group between weeks 0 and 33 after treatment. Treatment with GnRH-PAP reduced (P<0.05) LH release after GnRH stimulation in the bolus and hourly groups compared with the control group. Testis volume was lower (P<0.05) in all treated versus control dogs. In conclusion, administration of the conjugate GnRH-PAP at a 25 week interval resulted in a major disruption of reproductive parameters in male dogs; this effect was maintained for 11-12 weeks after a second injection of GnRH-PAP.

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods.

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.

Field testing of immunocontraception on white-tailed deer (Odocoileus virginianus) on Fire Island National Seashore, New York, USA.

Application of contraception for the control of suburban populations of white-tailed deer (Odocoileus virginianus) has been much debated, but few data are available on field applications and even fewer on population effects. Between 1993 and 1997, 74-164 individually known female deer living on Fire Island, New York, USA, were treated remotely with an initial shot of 65 microg porcine zona pellucida (PZP) in Freund's complete adjuvant followed by booster injections of 65 microg PZP in Freund's incomplete adjuvant. Starting in 1996, progressively increasing numbers of deer were treated with vaccinating/marking darts. Estimates of population density and composition, using distance sampling methods, began in 1995 in selected portions of the study area. Between 1993 and 1997, fawning rates among individually known, treated adult females decreased by 78.9% from pretreatment rates. Population density in the most heavily treated area increased by 11% per year from 1995 to March 1998 and then decreased at 23% per year to October 2000. In 1999-2000 surveys, fawns comprised 13-14% of the total population in the most heavily treated area, versus 16-33% in nearby untreated areas. These results show that PZP can be delivered effectively to sufficient deer to affect population density and composition in some environments, but that technical and logistical improvements are needed before contraception can be used widely to manage suburban deer populations.

Porcine zona pellucida (PZP) immunocontraception of wild horses (Equus caballus) in Nevada: a 10 year study.

Porcine zona pellucida (PZP) immunocontraception was investigated for possible use in free-roaming wild horses in the western USA. A protocol of two injections (3-4 weeks apart) of vaccine lasting 1 year was first used and a single-injection controlled-release vaccine of 1 year duration was developed and tested in the field. Studies of a presumptive vaccine of 2 year duration were initiated. The parameters of anti-PZP antibody titre response, pregnancy testing and offspring production were used, and PZP vaccine was found to provide up to 94% infertility in free-roaming wild mares. In addition, a single-injection PZP vaccine of 1 year duration and containing a controlled-release component of PZP in a polymer matrix can provide infertility equivalent to the two-injection PZP vaccine. All the PZP vaccine preparations tested were associated with a return to normal fertility within 1 year. During the course of these studies, attention was given to practical aspects of management application of PZP contraception. Preparation of the controlled-release portion of the vaccine in pellets, which fit into the needle of a dart or syringe, has simplified vaccine handling and permitted long-term storage of the controlled-release component. Vaccine delivery is now performed using a jabstick on captured mares restrained in a field stock chute during routine horse gathers. Provision of a vaccine-training programme has maximized personnel safety during vaccine preparation and use.

Equine oocyte maturation with epidermal growth factor.

Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum). Each group was divided further into 3 treatments with tyrphostin A-47, a specific tyrosine kinase inhibitor, at 0, 10(-4) and 10(-6) mmol/l. Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period. Immunohistochemical detection of EGF-receptor (EGFR) was performed using a streptoavidin-biotin method. The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively. Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs. 26.9% in controls, respectively). The specific-tyrosine kinase inhibitor A-47 was effective in suppressing EGF-effect on EGF-cultured oocytes; no significant differences were observed in EMS-supplemented oocytes when cultured with A-47. EGF-receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells. This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A-47, suggests a physiological role for EGF in the regulation of equine oocyte maturation. The results should help successful development of assisted reproductive technology in the horse.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.