Hormone Profiles of Mares Affected by the Mare Reproductive Loss Syndrome

While searching for the cause of the Mare Reproductive Loss syndrome (MRLS), we postulated that 1 of 3 tissues in 40–120 D pregnant mares was the likely primary target of the noxious factor that caused early abortions: The corpora lutea (CL), the endometrium or the fetus and/or its membranes. At this stage of gestation, progesterone (P4) is solely produced by luteal tissue, eCG by endometrial cups in the endometrium and oestrogens by the feto‐placental unit. We determined whether concentrations of P4, eCG and/or total conjugated oestrogens (CE) would indicate which tissue was targeted during the MRLS. P4, eCG and CE were measured in single serum samples collected from 216 mares, 60–110 D after ovulation during the 2001 MRLS outbreak. All mares had previously been confirmed pregnant by ultrasonography. The following data was obtained from each mare: Interval from ovulation, pregnancy status and normalcy of fetal fluids at the time of sampling, and pregnancy status 3 weeks after sampling and at term. There were no meaningful differences in hormone concentrations between pregnant mares that had normal and excessively echogenic fetal fluids at the time of sampling. CE were lower (p < 0.05) in mares that aborted after sample collection than in mares the carried to term. In 8 mares from which multiple samples were obtained, CE consistently decreased prior to any decreases in P4 or eCG. Arguments are presented that lead to the hypothesis that the fetal trophoblast was the primary target of the MRLS agent.     

The Effect of Manual Removal of Placenta Immediately after Foaling on Subsequent Fertility Parameters in the Mare

Retained placenta is considered to be a common problem in postpartum mares. The incidence varies from 6% to 54% depending on the breed, with higher incidence in heavy draught mares than in light-weight mares. Retained placenta has been linked to lower postpartum oxytocin concentration, impaired uterine involution, and dystocia. The objective of this study was to assess the effect of early manual removal of placenta immediately postpartum on subsequent fertility parameters (development of free intrauterine fluid, inflammatory status of endometrium, and on pregnancy rates) and to compare them with mares with spontaneous expulsion of placenta. A total of 29 mares, mainly Irish draught, were closely monitored during foaling by closed circuit television and allocated to two groups: (1) mares that expelled the placenta spontaneously within 3 hours of foaling; and (2) mares that were cleansed manually immediately after foal delivery. All mares were examined and scanned 5 and 9 days postpartum, and free intrauterine fluid was recorded; endometrial swabs were taken 9 days postpartum for endometrial cytology and culture. None of the fertility parameters analyzed showed statistical difference between groups 1 and 2. Therefore, it can be concluded that early manual removal of placenta has no detrimental effects on subsequent fertility of mares and, therefore, can be recommended when a veterinarian attends a foaling.     

The preliminary studies on protein profile in retained and not retained foetal membranes in heavy draft mares

Protein profile of the placenta expresses its function and maintenance. Any alterations can be reflected in qualitative and quantitative changes in this profile. The aim of the present study was the evaluation of protein profile in the placenta of mares suffering from the retention of foetal membranes (FMR) by two separation methods and the comparison with physiologically released tissues. Placentas from 14 healthy, heavy draft mares were collected immediately after the expulsion of newborn. Tissues after homogenization and staining with fluorescent dyes were subjected to electrophoretic as well as chromatographic separation. Electrophoretic gels were statistically analysed for the presence and abundance of examined proteins, while some proteins were identified in chromatographic fractions. Out of 248 spots detected in endometrium, 38 differed significantly between FMR and control animals, while in allantochorion, respective values reached 241 and 27 spots (p < .05). Among identified proteins that expressed higher abundance in endometrium of FMR mares than control animals were prostaglandin reductase, dehydrogenase/reductase SDR family, and placental growth factor. These proteins are involved in regulation of parturition. Additionally, the following proteins responsible for physiological activity of a cell—guanine methyl transferase, aspartyl/asparaginyl beta‐hydroxylase and GTP‐binding protein, were identified. These proteins expressed higher abundance in allantochorion of FMR mares than in controls. This preliminary study confirmed the disturbances in protein pattern between foetal membranes in FMR and healthy mares. Further qualitative and quantitative experiments are necessary to deepen our knowledge on the mechanisms of the retention of foetal membranes in mares.     

Retained fetal membranes in the mare: A retrospective study

A retrospective study of 3456 deliveries was conducted from the records of four Standardbred broodmare farms where mares were bred by artificial insemination and maintained under close veterinary supervision. Retained fetal membranes (RFM) were observed in 10.6% of the deliveries. Retained fetal membranes occurred more frequently (p < 0.05) after dystocia and in mares which had RFM the previous year. Retained fetal membranes after normal foaling had no significant effect on the reproductive performance (pregnancy rate, pregnancy loss rate, or foaling rate), nor on the general health of the mares, regardless of the duration of RFM (3 to 144 hours). Postfoaling laminitis was not observed. Oxytocin therapy of mares with RFM starting at two hours postpartum significantly reduced the incidence of RFM ≥ 8 hours. Mares with RFM which had received intrauterine antimicrobials between foaling and first breeding had a foaling rate similar to mares with RFM which had not received intrauterine therapy.     

Histopathologic effects of dimethyl sulfoxide on equine endometrium

Endometrial fibrosis is a major cause of infertility in broodmares. Because of the proven anti-inflammatory effects of dimethyl sulfoxide (DMSO) and its influence on collagen, the effect of DMSO on the endometrium was investigated in mares. Solutions of DMSO (25%, 50%, or 75%) were infused into the uterus of clinically normal mares. Examination of serially obtained biopsy specimens revealed epithelial ulceration and stromal inflammation that were proportional to the DMSO concentration infused, but vasodilatation was not observed. In all mares, the endometrium had returned to normal by day 21 after DMSO infusion.     

Physiological and pathological expression of intermediate filaments in the equine endometrium

The aim of this study was to investigate the expression of the intermediate filaments cytokeratin, vimentin and desmin in the equine endometrium by immunohistological techniques. For this purpose, endometrial biopsies of 151 mares were examined to determine physiological cycle patterns and changes resulting from endometriosis. During the physiological cycle epithelial cells and mesenchymal cells express cytokeratin and vimentin, respectively, whilst desmin and vimentin were coexpressed by the smooth muscle cells. Epithelial coexpression of cytokeratin and vimentin was seen in numerous fibrotic glands and in the uterine glands of three mares with pathologically inactive endometria. Three different staining patterns (basal, perinuclear, diffuse) of vimentin were associated with typical morphological alterations of the affected epithelia. In addition, in 14 cases a stromal coexpression of vimentin and desmin was found, indicating an atypical stromal differentiation in inactive endometria of older mares, barren for several years.     

Equine placenta – A clinician's perspective. Part 1: Normal placenta – Physiology and evaluation

A complex organ, the equine placenta is responsible for fetal nourishment, protection from external and internal insults, and the production and/or metabolisation of various hormones. The endometrial cups are unique structures of the equine placenta that are responsible for producing an essential hormone for equine pregnancy, equine chorionic gonadotropin. Since mares have epitheliochorial placentae, with 6 layers of tissue separating maternal from fetal circulation, almost the entire surface of the chorioallantois must be attached to the maternal endometrium in order to support a developing fetus adequately. The only avillous areas of the normal chorioallantois are: the cervical star, sites of the endometrial cups, areas facing oviductal papillae, folds overlying major allantoic vessels, and fetal foot PADs (placental areas of degeneration). There are characteristic differences between the gravid and the nongravid horn of the chorioallantois. Allantoic vesicles, allantoic pouches, hippomanes, amniotic plaques and yolk sac remnants are normal features of the equine placenta. The clinician should thoroughly examine the entire placenta immediately after its expulsion. The most important aspect of this evaluation is to check for completeness of the chorioallantois, along with identifying any pathological lesions on the fetal membranes or the umbilical cord.     

The role of inflammation and matrix metalloproteinases in equine endometriosis

Equine endometriosis is a multifactorial disease considered to be a major cause of equine infertility. The purpose of this study was to evaluate the reliability of histomorphological grading for biopsy-like samples compared to entire uterine wall samples, to examine the association between the degree of endometriosis with animal age, and to investigate the role of inflammation in endometriosis and the expression of different matrix metalloproteinases in equine endometrium. Histomorphological lesions in 35 uterine samples were examined while comparing biopsy-like samples and entire-wall samples. Seventeen uterine samples were stained with antibodies against MMP-2, MMP-9, MMP-14, and TIMP-2. The morphologic evaluation results of the biopsy-like tissue and entire-wall samples were significantly correlated. Endometriosis in older mares (>12 years of age) was more severe than in young mares (2~4 years of age), confirming the positive correlation between animal age and disease severity, while inflammation was poorly related to the degree of endometriosis. MMP-2 and MMP-14 were detected in stromal cells, while MMP-9 and TIMP-2 were both found in stromal and glandular epithelial cells. There were no significant differences in MMPs expression between the two groups (young vs. old mares). Additional studies on the activity of MMPs could further define the role of these enzymes in equine endometriosis.     

Release of immunoreactive arachidonate metabolites by equine endometrium in vitro

The ability of equine endometrium to release prostaglandin (PG) F, PGE2, and leukotriene (LT) B4 was studied in vitro, using endometrial tissue from diestrous mares. Because of the high cross-reactivity of the PGF antiserum with PGF1 alpha and with PGF2 alpha, results were quoted as total immunoreactive PGF. Significant concentrations of these arachidonate metabolites were released into tissue culture medium between 1 and 24 hours of incubation. Significantly higher concentrations of PGE, but not of PGE2 or LTB4, were released from endometria of mares with chronic endometritis than from genitally normal mares. Prostaglandin F was released only in low concentrations from the endometrium of a mare with pyometra, but concentrations of PGE2 and LTB4 were similar to those of genitally normal mares.     

Common Peripartum Problems in the Mare

Reproductive problems in the perinatal period can adversely affect a mare's future fertility or, worse, may be life threatening to her or her foal. A number of abnormalities can occur, including uterine torsion, uterine artery or uterine hemorrhage, retained fetal membranes, metritis, rectovaginal injuries, and necrotic vaginitis. Because hemorrhage, retained placenta, metritis, and necrotic vaginitis can happen after a normal delivery or after dystocia, all mares should be monitored closely in the first days after parturition for signs of lethargy, depression, colic, sore feet, or anorexia. Methods for diagnosing, managing, and treating these conditions and the possible complications are discussed.     

Histological Changes in the Uterus during Postpartum in the Mare

An histological study of the postpartum period in 29 mares was carried out. Uterine biopsies were taken daily during the first 10 days postpartum in a total of 87 samples. At day 0, equine endometrium was characterized in the surface by the presence of regularly ordered microcaruncles; the stratum spongiosum was oedematous and contained distended and scarce glands. Degenerative changes in microcaruncles and endometrial glands were present on day 1 postpartum. The epithelium of the microcaruncles from 2 to 5 days postpartum showed cytoplasmic vacuolization, karyorrhexis and an inflammatory reaction with neutrophils and phagocitic cells. On day 7 postpartum, the histology of the endometrium was similar to the normal proestrus with cuboidal luminal epithelium and an oedematous stromal tissue. The changes are usually completed within days 9 and 10 postpartum with the histologically typical appearance of estrus in mares.     

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.     

Disposition of desfuroylceftiofur acetamide in serum,placental tissue,fetal fluids,and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis

Macpherson, M. L., Giguère, S., Hatzel, J. N., Pozor, M., Benson, S., Diaw, M., Sanchez, L. C., Vickroy, T. W., Tell, L., Wetzlich, S., Sims, J. Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis. J. vet. Pharmacol. Therap.  36 , 59–67. The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, IM, q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high‐performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40 ± 0.40 μg/mL and 1.06 ± 0.29 μg/mL, respectively. Concentration of DCA in colostrum was 1.51 ± 0.60 μg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 μg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.     

Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies

Pregnant goats were inoculated intravenously or in uterine arteries with Brucella abortus, and tissues from the uterus and placenta were examined by electron microscopy. Identification of B. abortus in placentae was with antibody-coated colloidal gold. B. abortus was first seen in phagosomes of erythrophagocytic trophoblasts and in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Subsequently, trophoblast necrosis and ulceration of chorioallantoic membranes were present. Coincidently, B. abortus was present in the lumen of placental capillaries. In late stages of infection, placental vasculitis was present, and placentomal trophoblasts were separated from maternal syncytial epithelium. In lesions with vasculitis, large numbers of B. abortus were in connective tissue of chorionic villi. Within the placentome, trophoblasts that lined chorionic villi contained no intracellular bacteria and were separated from B. abortus by intact basement membranes. These results suggest that bacteremic B. abortus is endocytosed by erythrophagocytic trophoblasts and that B. abortus replicates in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Replication of brucellae in trophoblastic rough endoplasmic reticulum is unique; we believe that B. abortus may utilize endoplasmic reticulum for synthesis and glycosylation of bacterial membrane proteins or that B. abortus catabolizes trophoblast secretory proteins.     

Factors Affecting the Incidence of Postpartum Fever in Heavy Draft Mares

Postpartum metritis is a common problem in many animal species. In equine medicine, sequelae of postpartum metritis vary from delay in uterine involution to development of systemic acute metritis (SAM), toxemia, and laminitis. Heavy draft mares potentially have higher risk of suffering from SAM than light breed mares. Postpartum fever (PF) mostly appears as a sign of SAM. The purpose of this study is to analyze the factors affecting the incidence of PF in 158 foalings from 62 heavy draft mares by multiple logistic regressions. The objective variable was the incidence of PF, and the explanatory variables were year foaled, month foaled, age of mare, breed of mare (crossbred heavy draft horse vs. Percheron), gestation length, foaling difficulty (easy delivery vs. dystocia), retained fetal membranes, stillbirth, and sex of offspring. Twenty-four out of 158 mares were diagnosed with PF. Stepwise regression analysis showed that breed of dams and foaling difficulty were significant risk factors for PF. Percheron mares had higher risk than crossbred mares (odds ratio: 3.4). Dystocia had higher risk than easy delivery (odds ratio: 3.3). Percheron mares had higher incidence of PF than crossbred mares even in the cases of easy delivery. Risks for damage and contamination of birth canal will be increased especially if the fetal delivery needs assistance. The results of this study suggest that it is necessary to consider the foaling difficulty and the breed of dams for earlier detection of PF in peripartum heavy draft mares.     

Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding‐induced endometritis and the effect of a specific iNOS inhibitor in vitro

Emerging research suggests that the nitric oxide system may play a role in persistent breeding‐induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose‐dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activity in vitro. Six susceptible and six resistant mares were selected based on preset criteria and the results of an intrauterine challenge with killed spermatozoa during oestrus. Endometrial biopsy samples were collected 24 hr post‐challenge and cultured at 37°C for 24 hr in L‐arginine supplemented minimum essential medium with or without a specific iNOS inhibitor (1,400 W dihydrochloride, 1 mM). The medium and the cultured endometrial tissue were collected after 24 hr of culture and assayed for NO and total protein, respectively. Total NO content of the medium, normalized to endometrial tissue wet weight or total protein, was used as a measure of endometrial NOS activity. Non‐parametric tests were applied for statistical analysis. Susceptible mares had significantly greater endometrial NOS activity than resistant mares. The iNOS inhibitor treatment significantly reduced NOS activity in endometrial samples derived from susceptible and resistant mares. These findings provide a basis for in vivo testing of specific iNOS inhibitors as preventative or therapeutic options for PBIE in mares.     

Survival rate and short-term fertility rate associated with the use of fetotomy for resolution of dystocia in mares: 72 cases (1991-2005)

OBJECTIVE: To determine survival rate, complications, and short-term fertility rate after fetotomy in mares. DESIGN: Retrospective study. ANIMALS: 72 mares with severe dystocia. PROCEDURES: Records from 1991 to 2005 were searched for mares with dystocia in which a fetotomy was performed. Data relating to presentation and position of foals; survival rate, complications, and short-term fertility rate in mares; and 45-day pregnancy rate in mares bred 2 to 3 months after fetotomy were recorded. RESULTS: Anterior fetal presentation was detected for 54 of 72 (75%) mares, posterior presentation was detected for 13 (18.1%), and transverse presentation was detected for 5 (6.9%). One fetus in anterior presentation was hydrocephalic. Survival rate after fetotomy was 95.8%. Complications included retained fetal membranes (5.5%), laminitis (6.9%), vaginal and cervical lacerations (2.8%), and delayed uterine involution (2.8%). Mares bred 2 to 3 months after fetotomy had good short-term fertility, with a mean pregnancy rate of 79.4% at 45 days after breeding. CONCLUSIONS AND CLINICAL RELEVANCE: The survival rate was high, compared with rates reported after cesarean section, and short-term fertility rate was similar to those reported for mares that had a controlled vaginal delivery or cesarean section. Fetotomy performed by a skilled veterinarian on a nonviable fetus should be considered as a means of quick and safe correction of dystocia that does not necessarily impair short-term fertility in affected mares.     

Microvascular development and growth of uterine tissue during the estrous cycle in mares

OBJECTIVE: To document uterine growth and microvascular development in the endometrium of uteri with differing degrees of fibrosis as well as uterine growth throughout the estrous cycle of mares. ANIMALS: 30 mares. PROCEDURE: Uterine tissue was obtained during the breeding season from a slaughter facility. Stage of estrous cycle of the mares was assessed on the basis of ovarian structures and plasma progesterone concentrations. Endometrium was characterized by use of light microscopy, and blood vessel walls were marked by histochemical techniques. Microvascular development was evaluated by a computerized image analysis system. Growth of uterine tissue was based on cellular content of DNA and RNA, RNA:DNA, and protein:DNA. RESULTS: Significant differences in vascular density were not observed in the endometrium of uteri obtained from mares euthanatized during the follicular or luteal phase of the estrous cycle, regardless of whether endometrial classification of degree of fibrosis was considered. There was a 3-fold increase in amount of DNA and RNA of endometrial cells in the follicular phase when compared to myometrium. Hypertrophy of endometrial tissue during the luteal phase was reflected by a significant increase in cell protein content and protein:DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Endometrial growth of vascular tissues during the estrous cycle may be coordinated with development of nonvascular tissue. Estrogen and progesterone may play a role in regulation of uterine growth and angiogenesis.     

Fetal maceration and retention of fetal bones in a mare

A 19-year-old Quarter Horse mare was evaluated because of bloody vaginal discharge that was apparent immediately following breeding. On transrectal ultrasonography, it was evident that the uterus was filled with fluid containing echogenic particles; linear hyperechoic structures were also visible. Endoscopy was performed, which revealed a number of bones adhered to the cranial wall and floor of the right uterine horn as well as purulent fluid in both uterine horns. Bacterial endometritis and fetal maceration were diagnosed. The mare was treated with antibiotics, and the fetal bones were manually removed from the uterus. Fetal maceration with intrauterine retention of bones is rare in mares. Use of hysteroscopy supplements ultrasonography in the diagnosis of uncommon conditions of the uterus. Macerated bones may be adhered to the endometrium, thereby requiring manual removal.