Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

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BackgroundVertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0).ResultsWe hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively.ConclusionsThe functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

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In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal's age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

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During the early luteal phase, low progesterone concentrations delay downregulation of endometrial progesterone receptors. This contributes to impaired histotroph production at Day 14 of pregnancy (Beyer et al. 2019 Theriogenology 125, 236-241). Until the beginning of placentation (i.e. Day 37 of pregnancy), nutritional supply of the equine conceptus depends on histotrophy alone. The aim of the present study was to analyse development of the equine conceptus under reduced plasma progesterone concentrations until shortly before placentation. Fertile Haflinger mares (n=11; 4-11 years old) were examined daily by transrectal ultrasonography, and when in oestrus, they were inseminated every 48h until spontaneous ovulation. Mares were randomly assigned to either the treatment group and received the prostaglandin F2α agonist cloprostenol (PGF2α; 125μg) once daily for 3 days after ovulation (Beyer et al. 2019) or the control group and left untreated. After conceptus collection on Day 34, mares were allowed one oestrous cycle for recovery and subsequently assigned to the opposite treatment, thus serving as their own controls. From Day 10 after ovulation, conceptus development including size, uterine fixation, and heartbeat detection was evaluated daily by transrectal ultrasonography. On Day 34, conceptus and fetal membranes were recovered transcervically. The recovered material was weighed, measured, and stored for further analysis. Conceptuses underwent microcomputed tomography that was evaluated by Amira (Thermo Fisher Scientific). Statistical comparison for differences between control and treatment pregnancies was performed by nonparametric Wilcoxon test or chi-square analysis. The day of first visualisation of the conceptus and of the embryo proper and heartbeat did not differ between treatments. Uterine fixation occurred on Day 19.3±0.5 in conceptuses from PGF2α-treated pregnancies but on Day 16.7±0.4 in controls (P&lt;0.05). At 34 days of gestation, the conceptuses from PGF2α-treated mares were smaller (P&lt;0.05) than control conceptuses when measured with ultrasound callipers in utero (maximal length: PGF2α 17.4±0.3mm, control 19.0±0.4mm). Conceptus weight determined after recovery was less in treated pregnancies (PGF2α 2.5±0.2 g; control 3.5±0.3 g; P&lt;0.05). Microcomputed tomography analysis of selected inner organs showed some differences in development. Seven complete pairs of undestroyed conceptuses were available. Mean numbers of 33.4±3.2 and 18.8±7.5 bronchi were detected in control and PGF2α conceptuses, respectively (P&lt;0.05). Heart volume did not differ, but communication between ventricles was detected in only 1/7 control but 5/7 PGF2α conceptuses (P&lt;0.05). The footpad was present in 6/7 control and 0/7 PGF2α conceptuses (P&lt;0.001). In conclusion, subphysiological progesterone concentration during the early luteal phase delays development of the equine conceptus before placentation. The condition may contribute to early conceptus loss in horses, which occurs in up to 20% of pregnancies.