C. Aurich

Influence of different semen extenders and seminal plasma on PMN migration and on expression of IL-1β, IL-6, TNF-α and COX-2 mRNA in the equine endometrium

After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oestrous cycle before experiments were started. No differences in volume, pH, specific density or cell count of lavage fluid were found between the treatments. A significantly (p<0.01) lower number of leukocytes in the endometrium was identified in pre-experiment biopsies (68+/-5 leukocytes per field) compared to PBS (154+/-32), SP (175+/-22), SM (193+/-29) and EY treatments (113+/-17). PMN numbers were lower (p<0.01) after infusion of EY (23+/-10) compared to PBS (59+/-21) and SM extender (69+/-21). The number of eosinophils increased after inoculation of SP (p<0.05 vs. PBS, SM and EY). All treatments increased expression of interleukins (IL)-1beta and 6, tumor necrosis factor-alpha (TNF-alpha) and cyclooxgygenase-2 (COX-2) in the endometrium compared to pre-experiment values. Expression of COX-2 mRNA was significantly higher after infusion of SM than after PBS treatment (p<0.04). In conclusion, extender alone as well as seminal plasma and PBS causes an inflammatory endometrial response with the least pronounced response induced by EY-based semen extender.

Effects of season, age, sex, and housing on salivary cortisol concentrations in horses

Analysis of salivary cortisol is increasingly used to assess stress responses in horses. Because spontaneous or experimentally induced increases in cortisol concentrations are often relatively small for stress studies, proper controls are needed. This requires an understanding of the factors affecting salivary cortisol over longer times. In this study, we have analyzed salivary cortisol concentration for 6 mo in horses (n = 94) differing in age, sex, reproductive state, and housing. Salivary cortisol followed a diurnal rhythm with the highest concentrations in the morning and a decrease throughout the day (P < 0.001). This rhythm was disrupted in individual groups on individual days; however, alterations remained within the range of diurnal changes. Comparison between months showed highest cortisol concentrations in December (P < 0.001). Cortisol concentrations increased in breeding stallions during the breeding season (P < 0.001). No differences in salivary cortisol concentrations between nonpregnant mares with and without a corpus luteum existed. In stallions, mean daily salivary cortisol and plasma testosterone concentrations were weakly correlated (r = 0.251, P < 0.01). No differences in salivary cortisol between female and male young horses and no consistent differences between horses of different age existed. Group housing and individual stabling did not affect salivary cortisol. In conclusion, salivary cortisol concentrations in horses follow a diurnal rhythm and are increased in active breeding sires. Time of the day and reproductive state of the horses are thus important for experiments that include analysis of cortisol in saliva.

Gene Expression of ACTH, Glucocorticoid Receptors, 11βHSD Enzymes, LH‐, FSH‐, GH Receptors and Aromatase in Equine Epididymal and Testicular Tissue

Glucocorticoids (GCs) are important mediators of the stress response and have been implicated in the function and regulation of testicular functions in different species. In many tissues, intracellular glucocorticoid activity is controlled by either or both of the two known isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 and 2, which interconvert active and inactive GCs. Little is known about the effects of stress on fertility in the equine species. The main objective of the present study was to investigate the expression of receptors for GCs and adrenocorticotropic hormone [ACTH, melanocortin 2 receptor (MC2R)] as well 11βHSD1 and 11βHSD2 in male equine epididymal and testicular tissue. In addition, expression of aromatase P-450 and receptors for luteinizing hormone (LHR), follicle stimulating hormone (FSHR) and growth hormone (GHR) was studied. Reverse transcriptase PCR and quantitative real-time PCR were performed in tissue from the epididymis (caput and cauda) and testes collected from nine healthy mature stallions (age 4-10 years). mRNA for ACTH and GC receptors as well as 11βHSD1 and -2 were found in epididymal and testicular tissue. Expression of the genes studied was always positive in testicular tissue, while it was inconsistent in epididymal tissue. Quantitative gene expression in relation to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was significantly correlated (R = 0.403, p < 0.001). Quantitative PCR in relation to β-actin revealed significant differences in the gene expression of 11βHSD1, 11βHSD2, LHR, FSHR, MC2R and aromatase between tissue collected from caput epididymidis, cauda epididymidis and testicular parenchyma (p < 0.05). With GAPDH, differences between tissues were significant for 11βHSD1, 11βHSD2 and MC2R (p < 0.05) In addition, high concentrations of mRNA of aromatase and receptors of LH and FSH were found in testicular tissue, while a pronounced expression of GH receptor was present in epididymal tissue. The results support the hypothesis of an interaction between the pituitary-adrenal axis and testicular function in the stallion.

Expression of 11β-Hydroxysteroid Dehydrogenase Type 1 and Glucocorticoid Receptors in Reproductive Tissue of Male Horses at Different Stages of Sexual Maturity

Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β-hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre-pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) were used. Pre-pubertal horses showed higher testicular gene expression of 11βHSD1, 11βHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11βHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)-dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11βHSD enzymes and the oxidative 11βHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.

195 EXPRESSION OF MESENCHYMAL STROMAL CELL (MSC) MARKERS IN THE EQUINE ENDOMETRIUM AND IN VITRO INFLUENCE OF STEROID HORMONES ON ENDOMETRIAL-DERIVED MSC

Mesenchymal stromal cell (MSC) are multipotent precursor cells that have been isolated from many tissues, including endometrium in some species. These cells are necessary for tissue homeostasis, which in the cycling equine endometrium is regulated in part by changes in concentration of steroid hormones. The expression of oestrogen and progesterone receptors during the oestrous cycle has been studied before, but MSC gene expression is not reported as well as the effects of steroid hormones on in vitro proliferation of endometrial MSC. This study was designed to investigate the influence of steroid hormones on endometrial MSC proliferation in vitro and to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, and CD105) in the healthy equine endometrium during the oestrous cycle. Equine endometrial tissue was collected postmortem (n=6) and digested using a dissociation medium and mucin-1-bound magnetic beads were utilised to remove epithelial cells from the resulting single-cell solution. The cells were expanded in culture and, at passage 4, incubated with 3 different concentrations of oestradiol and progesterone for 5 days. For the proliferation analysis the Alamar Blue® assay was used according to manufacturer instructions. Endometrial biopsies, for quantitative RT-PCR analysis, were taken from healthy mares (n=5) on Day 5 and 13 post-ovulation, during oestrus (1 follicle >3.5cm, pronounced uterine oedema), and seasonal anestrous (seasonal anovulation). The ΔCt values were used for statistical analysis using SPSS Statistics 22 (IBM Corp., Armonk, NY). Data for quantitative PCR are presented as gene expression relative to the mean of 18S and GAPDH. No significant differences in proliferation could be detected in the various groups incubated with steroid hormones compared with the controls supplemented with charcoal-stripped fetal bovine serum. Detectable levels of mRNA for all 5 MSC markers analysed were present throughout the oestrous cycle. While the levels of CD73 were consistent, the expression of 3 MSC markers (CD29, CD44, and CD105) was elevated at Day 13. This difference was substantial between Day 13 and oestrus for CD29 (37.6±6.2 and 12.2±3.4; P<0.01) and CD105 (8.3±0.9 and 4.5±0.6; P<0.05), and between Day 5 and 13 for CD29 (7.4±2.3 and 37.6±6.2) and CD44 (12.9±1.8 and 4.1±0.3; P<0.01). In contrast, CD90 expression was higher at oestrus (27.8±3.8) than at Day 5 (6.7±0.9) or 13 (12.0±2.1; P<0.01). Elevated quantities of MSC marker transcripts during late diestrus might be linked to the preparation of the equine endometrium for the proliferation phase associated with oestrus. However, the in vitro proliferation of endometrial-derived MSC is not influenced by the steroid hormones, although gene expression of steroid hormone receptors is present throughout the oestrous cycle of the mare. In summary, this study shows that the equine endometrium expresses MSC markers, and it does so at variable levels throughout the oestrous cycle; however, cell proliferation in vitro is not influenced by steroid hormones. This information will be useful for future studies aiming to derive endometrial MSC from mares.

Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.

120 ANTRAL FOLLICLE COUNT OF 2-YEAR-OLD MARES IS HIGHLY CORRELATED WITH ANTI-MÜLLERIAN HORMONE CONCENTRATIONS AT 24 to 28 WEEKS OF AGE

Success of assisted reproductive techniques, as determined by the response to hormonal treatments and embryo quality, can successfully be predicted by the concentration of anti-Müllerian hormone (AMH) in plasma of several species. Being able to predict ovarian follicular reserve of prepubertal female horses (fillies) would help to select fertile broodmares and reduce costs associated with animal upkeep. The objectives of this work were to (1) assess AMH dynamics in female horses during the first year of life and (2) determine whether AMH concentrations detected in plasma of prepubertal fillies are correlated with AMH concentrations and antral follicle count (AFC) after puberty. Warmblood fillies (n=14) born from February to May of 1 year in the same stud were used. Blood samples for AMH determinations were collected from birth onward every 4 weeks up to the age of 1 year. At 2 years, blood samples were collected and AFC was determined by transrectal ultrasonography. The AMH concentrations were determined by ELISA (AL-115, Ansh Laboratories, Webster, TX, USA). Transrectal ultrasonography was used to determine the AFC, which corresponds to the total number of antral follicles detected with ultrasound. Statistical analysis was done with the SPSS Statistics 24 software (SPSS Inc., Chicago, IL, USA). The AMH was detectable in the plasma of all animals from birth onward. At birth, mean AMH concentration was 4.5±1.2ngmL-1. The AMH concentration increased and peaked between 24 weeks (8.7±4.4ngmL-1) and 28 weeks (6.7±2.1ngmL-1) and subsequently decreased again (52 weeks: 3.9±0.9ngmL-1). Very high variation among individuals during the first year was lost at 2 years of age. The AMH concentration at 2 years was highly correlated with AMH concentration at birth (r=0.62, P<0.05) and with AFC (r=0.78, P<0.001). Also, AMH concentration (r=0.73, P<0.01) and AFC (r=0.6, P<0.05) at 2 years were highly correlated with AMH concentrations at 24 and 28 weeks. Gestational length (337±1 days), parity of the dam (4.6±0.8), and placental weight (6983±352g) did not influence AMH concentrations at any time. Our results demonstrate that AMH is detectable in blood of female horses from birth onward. Despite its high variability between individuals up to 52 weeks, strong correlations were observed during the first 2 years of life. High correlations to AFC at 2 years suggest that determination of AMH in prepubertal female horses helps to predict the ovarian reserve and fertility in postpubertal life.