A preliminary study of the efficacy of fluphenazine as a treatment for fescue toxicosis in gravid pony mares

This study was designed to determine the efficacy of a single injection of the long-acting D₂-dopamine receptor antagonist, fluphenazine deconoate, on the clinical symptoms and plasma prolactin concentrations of mares grazing endophyte-infected tall fescue. Twelve mares were maintained on an 80% endophyte-infected tall fescue pasture. Group T (n=6) received 25 mg of fluphenazine deconoate i.m. on day 320 of gestation, while group C (n=6) served as untreated controls. Daily plasma samples were obtained and analyzed for prolactin using a homologous equine radioimmunassay. The prolactin data were then grouped according to week after treatment (day 320 to 327=Week 1; day 328 to 335=Week 2) and to week prior to parturition (day 0 to −7=Week −1; day −8 to −14=Week −2). A single injection of fluphenazine had no effect on experiment-wide plasma prolactin concentrations or on prolactin concentrations relative to week of treatment or parturition. However, a treatment by time period interaction was observed. Plasma prolactin concentrations were higher (p<.05) in treated mares (35.6±29.1 ng/ml) on week 2 compared to control mares (14.9±14.2 ng/ml). In addition, there was a trend (p=.09) for fluphenazine treated mares (68.1±64.2 ng/ml) to exhibit higher prolactin concentrations one week prior to parturition when compared to control animals (27.8±27.4 ng/ml). Peak prolactin was higher (p<.05) in fluphenazine treated mares and in mares which foaled normally. Gestation lengths were shorter (p<.05) in group T (330±2.9 days) compared to group C (341±3.2 days). It appeared that treated mares exhibited fewer clinical signs of fescue toxicosis than mares in group C. Four out of six of the untreated mares exhibited at least one of the clinical signs of fescue toxicosis (agalactia, stillbirth, thickened placenta, retained placenta) while only one treated mare showed obvious clinical symptoms. These data indicate that a single injection of a long-acting dopamine receptor antagonist may be beneficial in reducing the effects of fescue toxicosis in pregnant mares grazing endophyte-infected tall fescue pastures. However, additional research is needed to determine the most effective dosage and administration times.     

Inhibin activity in the mare and stallion

An overnight double antibody RIA, employing a rabbit antiserum raised to bovine 31 kDa inhibin (rAs-#1989, NICHD) and purified bovine 31 kDa inhibin (bINH-I-90/1, NICHD) as trace and standard, was validated to measure immunoreactive inhibin (iINH) concentrations in equine peripheral plasma, follicular fluid (FF), ovarian vein (OV) plasma, testicular tissue extracts (TTE) and testicular vein (TV) plasma. The dynamic relationship of iINH and follicle stimulating hormone (FSH) was investigated during the estrous cycle of the mare and the annual reproductive cycle of the stallion.In the RIA, parallel dose-response curves were observed between the bovine inhibin standard and serial dilutions of equine FF, OV, TTE, TV and plasma. The average recovery of a known amount of purified bovine inhibin added to gelding plasma was approximately 100%. In the inhibin bioassay, serial dilution of equine FF and TTE were observed to be parallel to the bovine inhibin standard. A five-fold difference (p<0.05) between jugular and gonadal vein plasma iINH concentrations was observed in the mare and an eight-fold difference (p<0.05) was observed in the stallion. Plasma levels of iINH in ovariectomized mares or geldings were undetectable in the RIA.Concentrations of FSH, estradiol and iINH changed significantly in the mare during the estrous cycle (p<0.05). Immunoreactive inhibin levels were highest (0.54 ± 0.06 ng/ml) on the day of ovulation, declined rapidly following ovulation and reached a nadir (0.21 ± 0.03 ng/ml) on day 7 post-ovulation. Plasma iINH and estradiol concentrations followed a similar profile and were found to be positively correlated (r=0.7064; p<0.01), whereas iINH and FSH levels demonstrated an inverse relationship (r=−0.7359, p<0.01) throughout the estrous cycle. Concentrations of FSH were also inversely related (−0.8498, p<0.01) with estradiol during the cycle. In the stallion, plasma iINH and FSH levels changed significantly during the year (p<0.05). The iINH profile reflected seasonal changes in testicular activity, with highest concentrations in late spring (3.37 ± 0.44 ng/ml) and lowest concentrations in the fall (2.21 ± 0.33 ng/ml). Plasma concentrations of iINH were positively correlated (r=0.7691, p<0.01) with FSH concentrations throughout the year.In conclusion, a specific and sensitive RIA for iINH has been validated for plasma and biological fluids in the horse. Furthermore, the gonads appear to be the source of bioactive and immunoreactive inhibin as observed in other species. The dynamic relationship between iINH and FSH that is present in both the mare and stallion suggests that iINH may be a useful marker of gonadal activity in this species.     

Investigations on the Endometrial Response to Intrauterine Administration of N‐Acetylcysteine in Oestrous Mares

In mares, mating‐induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N‐acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki‐67), lectins and periodic acid‐Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC‐ and C‐mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC‐treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki‐67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC‐treated mares than in C‐mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti‐inflammatory effect on the equine endometrium was observed.     

Pathology of maternal genital tract, placenta, and fetus in equine viral arteritis

Six pregnant mares were given equine viral arteritis virus intravenously. Tissues from genital tracts, placentae, and fetuses were examined by light and electron microscopy to study the mechanism of abortion. Four mares which died with acute disease had diffuse vacuolation of endometrial epithelium and systemic necrotizing vasculitis. Two of these mares had dead fetuses and two had live fetuses; virus was isolated from tissues of one live fetus. Placentae of mares dying from acute disease did not have lesions attributable to infection; virus was isolated from two of these placentae. One of the two mares which recovered from clinical disease aborted a dead fetus eight days after inoculation. The mare had severe necrotizing myometritis and virus was isolated from maternal ovaries and from fetal tissues. The fetus did not have lesions attributable to arteritis virus. These results suggest that although fetal death may occur in utero during acute equine viral arteritis, abortion probably is due to lesions in the uterus of the mare.     

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18–20 h (no sperm added). In Ex oocytes, TCM‐199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10⁶ sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY‐stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.     

马胚胎附植及其分子调控研究进展

胚胎附植是处于活化状态的囊胚滋养层细胞与处于接受态的母体子宫上皮细胞之间逐步建立组织和生理上联系的过程。马（马属动物）在该过程中先后存在侵入性的绒毛膜带（子宫内膜杯）和非侵入性的尿囊绒毛膜（微子叶型上皮绒毛膜胎盘）,是一种集侵入性与非侵入性胚胎滋养层于一体的哺乳动物。多种分子共同调节妊娠母马的胚胎附植过程。作者综述了马胚胎附植特点、过程与相关分子调控的研究进展,对减少母马妊娠早期胚胎丢失与流产具有重要参考价值。     

Clinical presentation,treatment and possible causes of persistent endometrial cups illustrated by two cases

Persistent endometrial cups are relatively rare in the mare and the occurrence in 2 successive pregnancies, as described in this issue, is an intriguing finding. This article describes an additional 2 cases of endometrial cups persisting in excess of 12 months in mares that had experienced pregnancy loss. Both mares demonstrated irregular ovarian activity in the form of repeated formation of haemorrhagic anovulatory follicles. A definitive diagnosis was made by visualisation of cup tissue by hysteroscopy and demonstration of equine chorionic gonadotropin (eCG) in the mares' serum. Biopsy of endometrial cups was made under visual direction and the mares were treated by chemical curettage with kerosene. The clinical presentation, treatment and possible causes of persistent endometrial cups are reviewed and discussed.     

Effects of Neodymium:Yttnum Aluminum Garnet Laser Irradiation on Endometrium and on Endometrial Cysts in Six Mares

Effects of neodymium:yttrium aluminum garnet (Nd:YAG) laser irradiation on equine endometrium were evaluated In vitro and in six mares with endometrial cysts. The Nd:YAG laser was applied to six endometrial sites, in each of five uterine specimens, with power densities of 5659 to 33,954 J/cm². Depth of tissue ablation was measured and graded on histologic sections of the tissue lesions. Power density had a significant effect on the depth of tissue ablation (p < .001). Grade 3 lesions (full-thickness ablation of the endometrium) were created with energy densities of 16,977 to 33,954 J/cm². Six mares had endometrial cysts treated by photoablation. Two of the four mares that were reproductively sound but barren, despite appropriate breeding, produced foals after treatment. One mare remained reproductively unsound after treatment, and another mare that was treated postpartum was bred successfully.     

Influence of Seroreactivity to Leptospira and Reproductive Failures in Recipient Mares of Equine Embryo Transfer Programmes

The aim of this study was to demonstrate that seroreactivity against Leptospira is significantly associated to the reproductive efficiency of recipient mares of an embryo transfer (ET) programme. A serosurvey was conducted from August 2007 to March 2009 in five herds from Rio de Janeiro, Brazil, with high rates of reproductive failure, as early embryonic death (>12%), abortion (>12%) and perinatal death. Detailed information about the losses was obtained from practitioner. A total of 338 recipient mares were tested by microscopic agglutination test, and 226 (66.9%) were seroreactive, mainly against serovars Bratislava and Copenhageni. Seroreactivity could be associated to reproductive failure (p < 0.001), and it was demonstrated that a seroreactive mare is 1.8 times more likely (relative risk – RR) to present reproductive failure than a seronegative one, particularly in relation to early embryonic death (p < 0.0001; RR 8.4) but also to abortions (p < 0.0001; RR 3.5), and to perinatal death (p < 0.05; RR 7.3). Therefore, seroreactivity to Leptospira is associated to reproductive failure in all phases of pregnancy in recipient mares, impairing equine ET programmes.     

Comparison of endometrium before and after repair of third-degree rectovestibular lacerations in mares.

The endometrial response of mares to repair of third-degree rectovestibular lacerations was evaluated. Endometrial biopsy specimens from 8 mares with third-degree rectovestibular laceration were obtained immediately before surgery and from 9 to 15 days after repair. Presurgical endometrial biopsy specimens were classified as category I for 2 mares; category II, attributable to slight endometritis, for 5 mares; and category III, attributable to moderate-to-severe endometritis, for 1 mare. Within 15 days after rectovestibular repair, all endometrial biopsy specimens were classified as category I. Results indicated that mares with third-degree rectovestibular injuries are candidates for breeding by artificial insemination by 2 weeks after repair of the injury.     

Key Factors Affecting Reproductive Success of Thoroughbred Mares and Stallions on a Commercial Stud Farm

To evaluate factors contributing to fertility of thoroughbred mares, data from 3743 oestrous periods of 2385 mares were collected on a large thoroughbred farm in Ireland. Fourteen stallions (mean age 8.3 years; range 4–15 years) had bred 2385 mares (mean age 9.4 years; range 3–24 years). Maiden mares accounted for 12%, mares with a foal at foot for 64%, and barren, slipped or rested mares for 24% of the total. The mean pregnancy rate per cycle was 67.8% (68.6% in year 1 and 66.9% in year 2). Backward stepwise multivariable logistic regression analysis was utilized to develop two models to evaluate mare factors, including mare age, reproductive status, month of foaling, dystocia, month of cover, foal heat, cycle number, treatments, walk‐in status and stallion factors including stallion identity, stallion age, shuttle status, time elapsed between covers and high stallion usage on the per cycle pregnancy rate and pregnancy loss. Old age (p < 0.001) and cover within 20 days post‐partum (p < 0.003) were associated with lowered pregnancy rates. High mare age (p < 0.05) and barren, slipped or rested reproductive status (p = 0.05) increased the likelihood of pregnancy loss. Uterine inflammation or infection, if appropriately treated, did not affect fertility. Only high usage of stallions (used more than 21 times in previous week) was associated with lowered (p = 0.009) pregnancy rates. However, shuttle stallions were more likely to have increased (p = 0.035) pregnancy survival, perhaps reflecting a bias in stallion selection. In conclusion, mare age exerted the greatest influence on fertility; nonetheless, thoroughbreds can be effectively managed to achieve high reproductive performance in a commercial setting.     

Stored of Hsp72/Hsp73 in Germinal Vesicle-stage Mouse Oocytes

Heat-shock proteins (hsps) Hsp72 and Hsp73 are the stored maternal proteins found in mouse oocytes. Both hsps appear in mouse oocytes at germinal vesicle (GV) and metaphase II (M-II)-stages as previously demonstrated by immunoblotting analysis. In this report, we further determined the presences of Hsp72/Hsp73 proteins in mouse embryos at stages of 2-pronucleus, arrested 1-cell, 2-cell, arrested 2-cell, 4-cell, arrested 4-cell, 8-cell to morula and blastocyst. Except for the blastocyst stage, the Hsp72/Hsp73 proteins were detectable in most embryo stages. The concentration of Hsp72/Hsp73 in GV-stage oocytes was higher than that in M-II-stage oocytes, and in any stages of embryos before implantation. A dramatical increase in Hsp72/Hsp73 expression was found at the 2-cell stage. Together with these findings, we speculated that hsps accumulated or stored earlier in the GV-stage mouse oocytes to protect the oocytes against environmental influences acting on ovary, and hsps may be required for zygotic gene activation and provided a protective effect against apoptosis.     

Changes in Serum Concentrations of Prolactin,Progestagens, and Estradiol-17β and Biochemical Parameters During Peripartum in an Agalactic Mare

The purpose of this study was to investigate the changes in serum concentrations of prolactin (PRL), progestagens, and estradiol-17β and biochemical parameters during peripartum in an agalactic mare, as well as to study the periparturient indices of the foal. A 4-year-old Thoroughbred, primiparous, pregnant mare was diagnosed as agalactic from clinical conditions such as absence of observable udder development and weight loss of her foal after parturition. Serum PRL concentrations of the agalactic mare during prepartum tended to be lower than those of the control mares (19.5 ng/mL and 67.0 ± 15.0 ng/mL on the day of parturition, respectively). The progestagens and estradiol-17β concentrations were not markedly different between the agalactic mare and the control mares. Concentrations of γ-glutamyl transferase of the agalactic mare were higher than those of the control mares (P < .05). Although the serum immunoglobulin G concentrations of the foal of the agalactic mare after 24 hours of age were lower than those of the foals of the control mares (P < .05), all periparturient indices regarding the agalactic mare were considered to be within the normal range. In contrast, insufficient udder development and milk production was observed in the agalactic mare through the pre- and postpartum periods. We have ruled out the known causes for agalactia in mares based on other parameters, clinical symptoms, and nutritional causes. Clinical symptoms and PRL parameters in this case report are consistent with the category of agalactia in mares of unexplained causes, and we have determined additional blood parameters associated with agalactia in mares that are consistent with this category.     

山羊早期胚胎发育的初步研究

本实验以黑龙江地方山羊为材料,经FSH超数排卵后,在不同时间屠宰母山羊,并冲洗输卵管及子宫,获取新鲜卵及各发育时间的胚胎。实验中发现山羊的排卵时间为发情开始后约30小时。受精卵的第一次卵裂的发生在排卵24小时以后。2细胞、4细胞、8细胞、16细胞、桑椹及胚泡期胚胎所处的时间分别为排卵后约32～42、48～52、62、72、96小时以及7～8天。16细胞期以前的胚胎移行于输卵管中,桑椹胚及胚泡则移行于子宫中。在每一时间从每只羊中所收集的胚胎基本上处于几个相邻的发育时期。在桑椹胚的动物极有明显的突起,这可能是内细胞群已开始形成的标志。到胚泡期时,胚胎体积增大,透明带变薄。正在孵化或已经孵化的胚泡中,内细胞群外端无滋养层细胞包围。     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Protective factors in mammary gland secretions during the periparturient period in the mare

Changes in mare mammary secretion composition were characterized prior to and after foaling. Eighteen mares were used for collection of mammary secretions from 23 days prepartum through 44 days postpartum. Concentrations of lactose, total protein, IgG and lysozyme (activity assay) were determined. Considerable variabilty was observed among mares. Mean lactose concentrations by mare were lower (P<.05) in the prepartum period compared with the postpartum period. Mean total protein and immunoglobulin G concentrations by mare were higher (P<.05) in the prepartum period compared with the postpartum period. Mean lysozyme activities by mare tended to be lower (P<.08) during the prepartum period compared with the postpartum period. Mammary secretion protein profiles, characterized by SDS-PAGE, were consistent with changes in concentrations of specific proteins measured in the secretions. Dynamic changes occurring in mammary secretions during the transition from prepartum accumulation of colostrum to postpartum production of milk include factors like IgG and lysozyme which have protective roles in the neonatal foal.     

Consequences of Intrauterine Enrofloxacin Infusion on Mare Endometrium

Endometritis is an important cause of infertility in mares. Enrofloxacin is a broad-spectrum antibiotic to which most equine endometritis pathogens are not resistant. The objective of this study was to determine whether enrofloxacin is safe to use as a conventional intrauterine infusion treatment. Nine healthy mares received intrauterine infusions of enrofloxacin (Baytril 100, 100 mg/mL, Bayer Health Care LLC, Animal Health Division) at 2.5 mg/kg daily for 3 days. Ultrasonographic examination and vaginal examinations were performed during the study. Endometrial biopsies were performed before treatment (S0) and 24 hours after the last treatment (S1) to evaluate acute effects. For evaluating chronic effects, biopsies were performed at 14 days (S2) and 60 days posttreatment (S3). Biopsies were graded histologically by the Kenny and Doig category scale. Difference in histological biopsy grade before and after treatment was compared between biopsies by using a repeated-measures one-way analysis of variance. and significant changes in grades were used to assess treatment effects. The vaginal and ultrasonographic examination after intrauterine infusion of enrofloxacin showed that all mares had severe purulent vaginitis and uterine fluid accumulation of ≥2 cm, with ≥1.5-cm thickening of the endometrial wall which persisted in most mares until the end of the study. Histologically, there was acute endometrial ulceration, necrosis, and hemorrhage in biopsy S1 in all mares, categorized as grade III. In biopsy S2, most mares developed fibrosis and inflammation graded as IIb (four of nine mares) or III (four of nine mares). In biopsy S3, fibrosis was extensive and had variable inflammation, graded as IIb (two of nine mares) or III (five of nine mares), with some mares healing to grade IIa (two of nine mares). There was an overall worsening of endometrial biopsy grade from I to III at S3 compared with S0 (P < .001). These results confirm that enrofloxacin is not suitable for conventional intrauterine infusion treatment in mares.