Juliane Kuhl

The PGF2α agonists luprostiol and d-cloprostenol reliably induce luteolysis in luteal phase mares without evoking clinical side effects or a stress response

In the present study we have evaluated a possible stress reaction in response to two different PGF2α analogs-luprostiol and D-cloprostenol--and their effects on estrous cycle characteristics. In a cross-over-design eight mares received in alternating order either luprostiol (Treatment LUP; 3.75 mg im), D-cloprostenol (Treatment CLO; 22.5μg im) or saline (Treatment CON; NaCl 0.9% 0.5ml im) on day 8 after ovulation. Injection of either LUP or CLO, but not of CON resulted in a significant decline of progesterone concentration in plasma to baseline concentrations within two days (time: p<0.001, treatment: p<0.01, time × treatment: p<0.05). The treatment to ovulation interval was significantly shorter in LUP and CLO than in CON cycles (LUP: 9.4 ± 0.4 d; CLO: 9.4 ± 1.3 d; CON: 16.1 ± 0.8 d; p<0.001). Injection of either LUP or CLO, but not of CON significantly increased salivary cortisol concentration (immediately before injection: CON 1.3 ± 0.2, LUP 1.4 ± 0.3, CLO 1.4 ± 0.3 ng/ml; 60 min after injection: CON 1.0 ± 0.3, LUP 8.0 ± 1.4, CLO 4.2 ± 0.7 ng/ml; time: p<0.01, treatment: p<0.001, time × treatment: p<0.001). Heart rate decreased over time (p<0.05) independent of treatment and no changes in heart rate variability were detected. Injection of the PGF2α analogs CLO and LUP reliably induced luteolysis and apart from a transient increase in salivary cortisol concentration no signs of a physiological stress response or apparent side effects occurred.

Treatment with human chorionic gonadotrophin before ovulation increases progestin concentration in early equine pregnancies.

For prevention of early conceptus loss in the horse, treatment with progestins has become common practice. In cattle, treatment with human chorionic gonadotrophin (hCG) during the early postovulatory phase stimulates endogenous progesterone synthesis, which is an important factor for maintenance of early pregnancy via stimulation of endometrial function and conceptus development. In the present study we have therefore investigated the influence of treatment with hCG either for induction of ovulation or during the early luteal phase on plasma progestin concentrations, size of the corpus luteum and size of the conceptus in early pregnant mares. We hypothesized that hCG treatment stimulates progestin secretion and conceptus development. In Experiment 1, induction of ovulation with hCG (1500 IU i.v.; n=14) significantly increased progestin concentration between days 5 and 15 after ovulation compared to untreated controls (n=28; p<0.05; e.g. day 5 hCG i.v.: 17.2 ± 1.9, control: 13.9 ± 0.8 ng/ml). A significant interaction (p<0.05) of hCG treatment with size of the conceptus between days 30 and 40 of pregnancy was detected. In Experiment 2, treatment of mares with hCG (5000 IU) on day 5 after ovulation (n=12) did neither affect progestin secretion (e.g. day 8 hCG: 15.4 ± 1.6, control: 17.6 ± 1.2 ng/ml) nor luteal tissue area (e.g. day 8 hCG: 9.0 ± 0.7, control: 7.6 ± 1.4 cm(2)) compared to untreated mares (n=9). In conclusion, treatment of mares with hCG for induction of ovulation within 48 h before ovulation but not on day 5 of the luteal phase stimulates progestin secretion and may enhance conceptus development via stimulation of endometrial function during early pregnancy.

Effects of oral supplementation with β-carotene on concentrations of β-carotene, vitamin A and α-tocopherol in plasma, colostrum and milk of mares and plasma of their foals and on fertility in mares

In this study, effects of oral β-carotene supplementation to mares (β-carotene group: 1000 mg/day, n = 15; control group: n = 15) from 2 weeks before foaling until 6 weeks thereafter on concentrations of β-carotene, vitamin A and α-tocopherol in plasma, colostrum and milk and plasma of their foals were determined. In addition, effects on fertility were studied. Beta-carotene concentrations increased in plasma and colostrum of β-carotene-supplemented mares compared to control mares (p < 0.05). In mares of both groups, β-carotene concentrations were higher in colostrum than in milk (p < 0.05). In foals, β-carotene concentrations increased with colostrum uptake and were higher in foals born to supplemented mares (p < 0.05; control group: 0.0003 ± 0.0002 μg/ml on day 0, 0.008 ± 0.0023 μg/ml on day 1; β-carotene group: 0.0005 ± 0.0003 μg/ml on day 0, 0.048 ± 0.018 μg/ml on day 1). Concentrations of vitamin A and α-tocopherol were higher in colostrum than in milk (p < 0.05) but did not differ between groups. Concentration of α-tocopherol in plasma of mares decreased over time and in foals, increased markedly within 4 days after birth. All but one mare (control group) showed oestrus within 2 weeks post-partum. Occurrence of oestrus did not differ between groups. More mares of the control group (7/7 vs. 5/12 in the β-carotene group) became pregnant after being bred in first post-partum oestrus (p < 0.05). In conclusion, β-carotene supplementation to mares increased β-carotene concentrations in plasma, colostrum and milk of mares and plasma of their foals but had no positive effects on fertility.

Sperm Quality during Storage Is Not Affected by the Presence of Antibiotics in EquiPlus Semen Extender but Is Improved by Single Layer Centrifugation

Contamination of semen with bacteria arises during semen collection and handling. This bacterial contamination is typically controlled by adding antibiotics to semen extenders but intensive usage of antibiotics can lead to the development of bacterial resistance and may be detrimental to sperm quality. The objective of this study was to determine the effects of antibiotics in a semen extender on sperm quality and to investigate the effects of removal of bacteria by modified Single Layer Centrifugation (MSLC) through a colloid. Semen was collected from six adult pony stallions (three ejaculates per male). Aliquots of extended semen were used for MSLC with Equicoll, resulting in four treatment groups: control and MSLC in extender with antibiotics (CA and SA, respectively); control and MSLC in extender without antibiotics (CW and SW, respectively). Sperm motility, membrane integrity, mitochondrial membrane potential and chromatin integrity were evaluated daily by computer-assisted sperm analysis (CASA) and flow cytometry. There were no differences in sperm quality between CA and CW, or between SA and SW, although progressive motility was negatively correlated to total bacterial counts at 0 h. However, MSLC groups showed higher mean total motility (P < 0.001), progressive motility (P < 0.05), membrane integrity (P < 0.0001) and mitochondrial membrane potential (P < 0.05), as well as better chromatin integrity (P < 0.05), than controls. Sperm quality remained higher in the MSLC groups than controls throughout storage. These results indicate that sperm quality was not adversely affected by the presence of antibiotics but was improved considerably by MSLC.

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.

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.