M.H.T. Troedsson

Susceptibility to persistent breeding-induced endometritis in the mare: Relationship to endometrial biopsy score and age, and variations between seasons

The objectives were to: (1) investigate the associations of age and endometrial biopsy score with uterine fluid retention after insemination; and (2) determine if a strict classification of susceptibility to persistent breeding-induced endometritis (PBIE) based on biopsy score, endometrial cytology, and fluid retention after inseminations, is consistent over subsequent breeding seasons. In Experiment 1, 57 mares were inseminated with 10(9) freeze-killed sperm during estrus and evaluated for uterine fluid retention 48 h and 96 h after insemination. Comparisons were made between fluid retention and biopsy score or age. In Experiment 2, a subset of 14 mares was classified for susceptibility to persistent breeding-induced endometritis in two subsequent breeding seasons. Biopsy score and age were associated with fluid retention (P < 0.001). In addition, age was related to biopsy score (P < 0.001). Of the mares examined for susceptibility, 36% (5 of 14) changed status during subsequent seasons. Three mares changed to a more severe classification (intermediate to susceptible, or resistant to intermediate), whereas two mares changed to a less severe classification (susceptible to intermediate).

Maternal recognition of pregnancy in the horse: a mystery still to be solved

Maternal recognition of pregnancy in the horse is the sum of events leading to maintenance of pregnancy; in a narrow sense, maternal recognition of pregnancy refers to the physiological process by which the lifespan of the corpus luteum is prolonged. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. The presence of the conceptus reduces pulsatile prostaglandin F(2α) secretion by the endometrium during early gestation in the mare, partly attributed to the reduced expression of cyclooxygenase-2. Cyclooxygenase-2 has therefore been suggested as one of the regulators of endometrial prostaglandin F(2α) release modified by the antiluteolytic factor secreted by the conceptus. In addition, altered oxytocin responsiveness has been implicated in the adjustment of prostaglandin release in pregnant mares. While conceptus mobility has proven to be essential for establishment of pregnancy, conceptus-derived oestrogens and prostaglandins, principally prostaglandin E(2), have not been confirmed as the critical antiluteolytic factor. Various ways to induce prolonged luteal function in the non-pregnant mare will be highlighted in the current review, specifically, how they may pertain to the process of maternal recognition of pregnancy. Furthermore, recently published microarray experiments comparing the transcriptome of pregnant and non-pregnant endometria and different stages of conceptus development will be reviewed. Findings include the prevention of conceptus adhesion, the provision of nutrients to the conceptus and the avoidance of immunological rejection, among others.

The interrelationship between anti-Müllerian hormone, ovarian follicular populations and age in mares.

REASONS FOR PERFORMING STUDY Anti-Müllerian hormone (AMH) is a granulosa-cell-derived glycoprotein, which plays an important inhibitory role during folliculogenesis. Concentrations of AMH are highly correlated with antral follicle counts (AFCs) in other species, which in turn are related to follicular reserve. Relatively little is known about AMH and AFC in the mare. OBJECTIVES To determine plasma AMH concentrations and AFCs in mares of different ages, to measure the repeatability of AMH concentrations and AFCs within and across oestrous cycles and to assess the relationship between plasma AMH concentrations and AFCs with regard to mare age and follicle size. STUDY DESIGN An observational study examining the relationship between AMH, AFC and age in 45 mares. METHODS Young (3-8 years), middle-aged (9-18 years) and old mares (19-27 years) were examined by transrectal ultrasonography over 2 or 3 oestrous cycles. Plasma AMH concentrations and AFCs were determined, and antral follicles were classified by size into different groups. RESULTS Plasma AMH concentrations varied widely between mares within similar age groups. Antral follicle counts were significantly lower in old mares than in young and middle-aged mares, and AMH concentrations were significantly lower in old than in middle-aged mares. A positive relationship was detected between AFC and AMH, and this relationship varied by mare age with a strong correlation in older mares (ρ = 0.86; P<0.0001), a moderate correlation in middle-aged mares (ρ = 0.60; P = 0.01) and no correlation in young mares (ρ = 0.40, P<0.4). The AMH concentrations were significantly related to the number of antral follicles between 6 and 20 mm in diameter, and the repeatability of AFCs and AMH concentrations was high within and between oestrous cycles. CONCLUSIONS Our findings indicate that the relationship between AMH and AFC varies across age groups, and concentrations of AMH might be a better reflection of reproductive age than calendar age.

Changes in maternal androgens and oestrogens in mares with experimentally-induced ascending placentitis

REASONS FOR PERFORMING STUDY While advanced stages of ascending placentitis can be diagnosed by transrectal ultrasonography and clinical signs, early stages can be missed. Thus, additional tools could enhance assessment of placental health. OBJECTIVES To characterise peripheral dehydroepiandrosterone sulphate (DHEA-S) and testosterone concentrations in mares carrying normal pregnancies (Study 1) and compare plasma concentrations of DHEA-S, testosterone, oestradiol 17-β (oestradiol) and oestrone sulphate (OES) in mares with or without placentitis (Study 2). STUDY DESIGN Longitudinal cohort study of healthy mares (Study 1) and controlled experiment (Study 2). METHODS In Study 1, mares had serum samples collected from 100 days of gestation to term. In Study 2, pregnant mares (260-280 days gestation) were assigned to a control group or a group with placentitis. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Blood was collected at inoculation/commencement for control mares (day = 0) and daily for 12 days post inoculation (DPI) or until abortion. Steroid concentrations were determined by immunoassays. Concentrations of steroids in Study 2 were also evaluated relative to days from abortion (DFA -8 days to 0). RESULTS In Study 1, DHEA-S peaked by 180 days gestation, while testosterone concentrations were progressively increased from Days 100 to 180 with a plateau until ~240 days and a progressive decline until 290 days of gestation. In Study 2, concentrations of DHEA-S and testosterone were not significantly different between groups. There were significant effects of time (oestradiol P = 0.0008, OES P = 0.01) and time-by-group interactions (oestradiol P<0.001, OES P<0.0001) for oestrogen concentrations. For mares with experimental placentitis, concentrations of oestradiol were significantly reduced at -6, -2, -1 and 0 DFA, while OES concentrations were significantly reduced on the day before abortion (0 DFA). CONCLUSIONS Testosterone and DHEA-S were increased and varied through pregnancy. Oestrogens but not androgens decreased significantly in mares with experimentally-induced ascending placentitis.

Characterization of prostaglandin E2 receptors (EP2, EP4) in the horse oviduct

Biological effects of prostaglandin E2 are mediated via one of four receptors designated EP1, EP2, EP3 and EP4 which are encoded by separate genes. In general, EP1 and EP3 induce smooth muscle contraction whereas EP2 and EP4 induce smooth muscle relaxation. The objective of the current study was to characterize the expression of the genes for PGE2 receptors (EP2 and EP4) in the horse oviduct based upon immunohistochemistry (IHC) and quantitative PCR (qPCR). Oviductal tissue was collected from mares at estrus (n=5), at 5 days post-ovulation (n=4), and from prepubertal mares (n=5). Isthmic and ampullar regions of the oviduct were fixed for IHC or preserved for RNA isolation. Prostaglandin E2 receptors EP2 and EP4 were strongly expressed by the luminal epithelium of both the isthmic and ampullar regions of the horse oviduct with a lesser immuno-expression noted within the smooth muscle in both regions of the oviduct. Based upon qPCR, relative amounts of EP2 or EP4 mRNA did not differ across estrous cycle stage or from prepubertal mares. However, across region and estrous cycle stage, relative amount of EP2 was greater (P<0.05) than EP4, and relative amount of EP2 mRNA was greater (P<0.001) in the ampullar compared with the isthmic oviduct. The relative roles of these receptors in regulating oviduct function in the mare remains to be determined.

Equine Pre-Implantation Conceptuses Express Neuraminidase 2 - A Potential Mechanism for Desialylation of the Equine Capsule

During the second and third week of pregnancy, the equine conceptus is covered by an acellular glycoprotein capsule. This capsule contains glycoproteins resembling those of the mucin family with sialic acid making up a high proportion of the carbohydrate. Coinciding with conceptus fixation, a marked decline in sialic acid content of the capsule occurs, which has been proposed to contribute to cessation of conceptus mobility. Herein, we describe the expression of neuraminidase 2 (NEU2) by pre-implantation stages of equine conceptus development. NEU2 transcript abundance was examined in conceptuses obtained 8, 10, 12, 14 and 16 days after ovulation; highest levels were observed 16 days after ovulation. Transcript abundance observed in endometrial tissue was on average 474-fold lower than in conceptus tissue. Protein expression was localized to trophoblast cells and capsular material. Functionality of NEU2 was shown using an Amplex Red reagent-based assay. NEU2, formerly known as sialidase 2, belongs to a family of enzymes that cleave sialic acid from polysaccharide chains. The expression of NEU2 described herein provides a mechanism by which the conceptus can regulate the sialic acid content of its own capsule. The timely desialylation coinciding with conceptus fixation has been suggested integral for establishment of normal pregnancy.

Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis

Endometritis in horses caused by Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) may be underdiagnosed due to traditional diagnostic methods lacking sensitivity and specificity. We serendipitously identified a bacterial growth medium (bActivate) that appeared capable of inducing growth of dormant S. zooepidemicus, which subsequently allowed detection by standard diagnostics. To assess the effect of bActivate we compared its ability to activate dormant S. zooepidemicus in a group of potentially infected subfertile mares with phosphate-buffered saline (PBS). All mares had to test negative for S. zooepidemicus on a low-volume uterine lavage, be negative on endometrial cytology and without clinical signs of endometritis to be included in the investigation. The mares were instilled with bActivate or PBS in the uterus. Growth of S. zooepidemicus was induced by bActivate in 64% (16/25) and PBS in 8% (1/12) of the mares, respectively (p<0.002). In vitro studies supported that some strains of S. zooepidemicus were able to form persister cells tolerating 32-times of the minimal inhibitory concentration of penicillin compared to normal growing cells. Persister cells had not acquired penicillin resistance, but seemed to tolerate the antimicrobial due to dormancy. This is, to our knowledge, the first description of controlled growth induction of dormant bacteria from a subclinical infection. Moreover we demonstrated how endometritis can origin from a reservoir of dormant bacteria residing within the endometrium, and not only as an ascending infection. Further studies should aim at determining the prevalence of dormant S. zooepidemicus, impact of activation on diagnostic and treatment efficacy, uterine health and mare fertility.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN Observational study. METHODS Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Removal of bacteria from stallion semen by colloid centrifugation.

Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not processed by Single Layer Centrifugation) were cultured and the bacteria identified. In experiment 1, doses of E. coli from 2×10(2) to 2×10(7) colony forming units were added to aliquots of semen. In experiment 2, Taylorella equigenitalis or a mix of E. coli, Klebsiella pneumoniae and Streptococcus equi subsp. zooepidemicus (approximately 7×10(6), 5×10(6), and 6×10(6)cfu, respectively) were added to 15mL aliquots of semen. In experiment 1, more than 90% of the bacteria were removed where loading doses were >×10(4)cfu/mL. In experiment 2, varying proportions of different bacteria were removed, ranging from 68% for naturally occurring Corynebacterium spp. to >97% for added cultured E. coli. Thus, Single Layer Centrifugation can separate spermatozoa from many, but not all bacteria in stallion ejaculates and could be a useful alternative to adding antibiotics to semen extenders to control bacterial contamination. However, further research is needed to determine the effect of small numbers of bacteria on sperm quality.

The expression of interferon-stimulated gene 15 in equine endometrium.

Establishment of pregnancy is critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The up-regulation of the interferon-stimulated protein 15 kDa (ISG15) during pregnancy has been described in various species and has been hypothesized to be part of the molecular repertoire that makes the uterus receptive to conceptus development. In the current study, the expression of ISG15 and enzymes involved in ISG15ylation was examined at the mRNA and protein level in equine endometrium at Day 14 of the luteal phase and at Day 14 and 50 of pregnancy. ISG15 mRNA showed a 2.63-fold higher expression at Day 14 of pregnancy when compared to Day 14 of the cycle, while mRNA abundance at Day 50 of pregnancy was unchanged compared to Day 14 of the cycle. Upon Western blot analysis using anti-ISG15 antibody, several higher molecular weight bands could be observed, representing proteins conjugated to ISG15. No free ISG15 could be detected. The pattern of ISG15 reactive proteins differed from those observed in non-uterine samples. Upon immunohistochemistry, ISG15 reactive proteins located primarily to luminal and glandular epithelial cells, while stromal cells showed weaker staining. In conclusion, the expression of ISG15-conjugated proteins in equine endometrium did not differ between cyclic and pregnant 14 days after ovulation and Day 50 of pregnancy. It is hypothesized that the unique subset of ISG15ylated proteins expressed in endometrial tissue contributes to normal cellular function and that, unlike other species, the modification of ISG15-conjugated proteins is not an active contributor to conceptus-maternal interaction in the mare.