Selection of frozen-thawed stallion semen by microfluidic technology.

The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 µl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 µl) for stallion semen. Frozen-thawed semen from 15 stallions that were previously classified as being of 'good fertility' (GF; n = 8; pregnancy rate ≥ 40%) and 'poor fertility' (PF; n = 7; pregnancy rate < 20%), respectively, was used. Each ejaculate was assessed before and after microfluid recovery for kinetics (CASA), membrane integrity (MI) (SYBR14/PI), membrane functionality (MF) (HOS test), acrosome integrity (Spermac Stain Kit), morphology (Spermac stain kit), mitochondrial membrane potential (MMP) (JC-1) and chromatin integrity (aniline blue staining). Sperm concentration was reduced after sperm recovery in both groups, but more markedly in frozen-thawed semen of PF stallions (p < .05). Microfluid recovery increased total motility, MI, MF and MMP. While there was a significant increase in the percentage of progressively motile sperm after sperm microfluid recovery, there was a decrease in DAP, DSL, VAP, VSL, LIN, WOB and ALH (p < .05). A slight increase (p < .05) was detected in beat-cross frequency. The present results suggest that the ZyMot Multi (850 µl) device selects a specific sperm population from any stallion ejaculate with motile sperm and could therefore be a valid tool for in vitro testing with the aim to predict the fertility of frozen-thawed stallion semen.

Transcription-dependent spreading of the Dal80 yeast GATA factor across the body of highly expressed genes.

GATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well defined in vitro, the in vivo selectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites, correlating with nitrogen- and/or Dal80-sensitive gene expression. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies requires active transcription. Consistently, Dal80 co-immunoprecipitated with the initiating and post-initiation forms of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.

Bases of antisense lncRNA-associated regulation of gene expression in fission yeast.

Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene ctt1 revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to ctt1 induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on ctt1 induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast.

Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

Effects of dietary L-arginine supplementation to early pregnant mares on conceptus diameter-Preliminary findings.

The importance of the amino acid L-arginine (ARG) for conceptus growth and litter size has been demonstrated in various species. L-arginine is part of embryo-derived polyamines, a substrate for nitric oxide synthase and stimulates protein synthesis by the embryo. In the present study, we have investigated whether dietary L-arginine supplementation stimulates early conceptus growth in mares. Warmblood mares with singleton pregnancies received either an arginine-supplemented diet (approximately 0.0125% of body weight, n = 12) or a control diet (n = 11) from days 15 to 45 after ovulation. Diameter of the embryonic vesicle (days 14, 17, 20 of pregnancy) and size of the embryo respective foetus (length and maximal diameter, days 25-45 of pregnancy at 5-day intervals) were determined by transrectal ultrasound. At foaling, weight and size of the foal and the placenta were determined. Blood for determination of equine chorionic gonadotrophin (eCG) and progestin concentrations was collected repeatedly. Neither eCG nor progestin concentration in plasma of mares differed between groups at any time. No effects of arginine treatment on diameter of the embryonic vesicle between days 14 and 20 of pregnancy were detected. Diameter of the embryo/foetus on days 40 to 45 of pregnancy strongly tended to be enhanced by arginine supplementation (p = 0.06). Weight and size of neither the foal nor placenta at birth differed between groups. In conclusion, L-arginine supplementation was without negative effects on early equine embryos and may support embryonic growth at the beginning of placentation.

Implication of the estrogen receptors GPER, ESR1, ESR2 in post-testicular maturations of equine spermatozoa.

Estrogen receptors ESR1, ESR2 and GPER are present on mature ejaculated horse spermatozoa, suggesting these cells as putative targets for estrogens. Indeed, spermatozoa are exposed to high level of estrogens during the transit in the male and female genital tracts but their roles are not investigated. So, we evaluated in vitro the role of 17ß-estradiol during post-testicular maturations: regulation of motility, capacitation and acrosome reaction. Moreover according to the pseudo-seasonal breeder status of the stallion, we analyzed the putative seasonal variations in the presence of ESRs in spermatozoa. We showed that ESRs are more present on stallion sperm during the breeding season. We showed that capacitation and acrosome reaction are independent of estradiol action in horse. Estradiol can weakly modulate the motility and this effect is strictly associated with GPER and not with ESR1 and ESR2. The subcellular localization of GPER in the neck on stallion sperm is coherent with this effect. It seems that estrogens are not major regulators of sperm maturations associated to mare genital tract, so they could act during the epididymal maturations.

Stallion spermatozoa: putative target of estrogens; presence of the estrogen receptors ESR1, ESR2 and identification of the estrogen-membrane receptor GPER.

Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysis mainly showed a flagellar localization for both receptors. Immuno-TEM experiments revealed that they are mostly located near the membranes, which are classically associated with rapid, non-genomic, effects. Moreover, we evidenced the expression of the seven transmembrane estradiol binding receptor GPER in colt testis. The protein was also localized at the connecting piece in mature spermatozoa. In conclusion, our results suggest that horse spermatozoa are a target for estrogens, which could act on several receptors either during the epididymal transit and/or in the female genital tract.

Tumor necrosis factor-α is transferred to equine neonates via colostrum but is not associated with their health status.

We followed the hypothesis that equine neonates with reduced transfer of tumor necrosis factor-α (TNFα) are at increased risk of neonatal infection. We investigated TNFα concentrations in colostrum of healthy mares and blood of their neonates in a non-hospitalized population of Warmblood mares where delivery, neonatal adaptation and health was closely monitored by veterinarians. Concentration of TNFα and IgG was determined in colostrum respective milk and in neonatal blood collected immediately after delivery and 18 h thereafter in 97 foals that were assigned to groups failure of passive transfer (FPT; n = 31) and control (CON; n = 66) based on serum IgG concentration at 18 h of age. Foal health was assessed repeatedly during the first 24 h of life. Statistical analysis was done with p < 0.05 indicating significance. There were no significant differences between foal groups FPT and CON regarding age and parity of dams, gestation length (FPT 343 ± 10, CON 340 ± 8 days) and foal sex. Concentrations of TNFα in colostrum at birth and in foals at 18 h varied but did not differ between groups (colostrum FPT 6.1 ± 9.1, CON 9.9 ± 31.5 ng/ml; foal FPT 2.3 ± 5.9, CON 2.4 ± 5.3 ng/ml; n.s.). There was an increase in the mean serum TNFα concentration until 18 h in foals (n.s. between groups). Results of the present study confirm previous findings of TNFα transfer from the mare to the neonate via colostrum but do not suggest that transfer of TNFα via colostrum is important for protection of the neonate against infectious diseases.

In mares resistant to endometrial infection, periovulatory treatment with ecbolic drugs does not influence uterine clearance or luteal development.

We aimed to determine associations between experimentally impaired uterine clearance or treatment with ecbolic drugs on luteal development in estrous mares after insemination. In a crossover design, eight mares were treated with saline (CON), clenbuterol (CLEN), oxytocin (OXY) and carbetocin (CARB) from the day of first insemination until 2 days after ovulation. Between treatments, the mares rested for one cycle. Estrous mares were examined for the presence of free intrauterine fluid by transrectal ultrasound. Endometrial swabs for cytology and bacteriology were collected on days 1 and 14. Blood samples were collected daily before AI until day 14 after ovulation for determination of progesterone and PGF2α metabolites (PGFM). Differences between treatments were compared by a general linear model for repeated measures (SPSS 29). One mare was excluded because of a uterine infection in the control cycle. In all other mares, only minor amounts of free intrauterine fluid were present after insemination and decreased over time (P<0.05) with no treatment x time interaction. There was no effect of treatment on polymorphonucleated cells (PMN) in endometrial cytology after ovulation or PGFM secretion. Progesterone release from day 1-14 as well as pregnancy rate and conceptus size on day 14 was not influenced by treatment. In conclusion, treatment with clenbuterol does not impair uterine clearance in estrous mares resistant to endometritis. Repeated injection of the oxytocin analogue carbetocin during the early postovulatory period is not detrimental to corpus luteum function and can be recommended to enhance uterine clearance.

Protamine 2 and phospholipase C zeta 1 are possible biomarkers for the diagnosis of male subfertility in frozen-thawed stallion semen.

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = -0.495), PRM2/ZPBP (r = -0.645) and CRISP3/ACR (r = -0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.

Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

CONTEXT: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. AIMS: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. METHODS: Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4µg/day for 4weeks followed by 8µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. KEY RESULTS: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. CONCLUSIONS: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. IMPLICATIONS: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Shipping duration and temperature influence the characteristics of cryopreserved horse semen stored in different shipping devices for up to 14 days.

This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ -150 °C) or on dry ice (≤ -78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intact sperm (SYBR14/PI), high mitochondrial membrane potential (HMMP; JC1) and DNA fragmentation. Temperature inside the containers was monitored continuously. Until d7, no changes were observed in TMot, PMot, and membrane-intact spermatozoa. Thereafter, all three parameters decreased in semen stored on dry ice but not in a dry shipper (time p < 0.001, time x shipping device p < 0.001). The HMMP decreased continuously over time in both containers with a more pronounced decrease on dry ice compared to the dry shipper (shipping device p < 0.01, time p < 0.001, time x device p < 0.001). The DNA fragmentation increased on d10-14 on dry ice and d14 in the dry shipper (time p < 0.001, time x device p < 0.01). In conclusion, frozen horse semen can be safely stored for up to 7 days on dry ice. Sperm DNA integrity and HMMP, however, were adversely affected after 14 days in both shipping devices.

Abundance of Anti-Muellerian hormone in cat ovaries and correlation of its plasma concentration with animal age, weight and stage of the estrous cycle.

In female animals of different species, Anti-Müllerian hormone (AMH) is produced by follicular granulosa cells and has been associated with the ovarian follicle pool. Because concentration of AMH in plasma of ovary-intact female cats is apparently more variable than previously assumed, we have analysed AMH concentration in blood of cats (n = 93) presented for routine ovariectomy and assessed ovarian histology and AMH protein expression in the surgically removed ovaries. We hypothesised that AMH is synthesized only in preantral and small antral follicles and that plasma AMH concentration reflects the antral follicle count (AFC). Corpora lutea were detected in 35% of the female cats, whereas plasma progesterone concentration was ≥1 ng/mL in 57% of the cats. Follicular cysts were present in 15 cats (16%). Positive immunostaining for AMH protein was detected in close to all primordial and antral follicles, ovarian cysts, 70% of corpora lutea and 28% of atretic follicles. Concentration of AMH in plasma averaged 6.8 ± 0.5 ng/mL (range 1.3-21.7 ng/mL). The AFC increased with increasing AMH concentration with a moderate positive correlation between AFC and AMH (r = 0.286, p < 0.01). Plasma AMH concentration was not affected by season or cats' age, weight, stage of the estrous cycle and presence of follicular cysts. In conclusion, AMH protein is expressed in all endocrine structures of the cat ovary. While AMH is a marker for the presence of ovarian tissue, its usefulness to assess ovarian function in individual female cats is of limited value.

Psychological stress induces an increase in cholinergic enteric neuromuscular pathways mediated by glucocorticoid receptors.

Introduction: Repeated acute stress (RASt) is known to be associated with gastrointestinal dysfunctions. However, the mechanisms underlying these effects have not yet been fully understood. While glucocorticoids are clearly identified as stress hormones, their involvement in RASt-induced gut dysfunctions remains unclear, as does the function of glucocorticoid receptors (GR). The aim of our study was to evaluate the involvement of GR on RASt-induced changes in gut motility, particularly through the enteric nervous system (ENS). Methods: Using a murine water avoidance stress (WAS) model, we characterized the impact of RASt upon the ENS phenotype and colonic motility. We then evaluated the expression of glucocorticoid receptors in the ENS and their functional impact upon RASt-induced changes in ENS phenotype and motor response. Results: We showed that GR were expressed in myenteric neurons in the distal colon under basal conditions, and that RASt enhanced their nuclear translocation. RASt increased the proportion of ChAT-immunoreactive neurons, the tissue concentration of acetylcholine and enhanced cholinergic neuromuscular transmission as compared to controls. Finally, we showed that a GR-specific antagonist (CORT108297) prevented the increase of acetylcholine colonic tissue level and in vivo colonic motility. Discussion: Our study suggests that RASt-induced functional changes in motility are, at least partly, due to a GR-dependent enhanced cholinergic component in the ENS.

Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses.

High progesterone concentrations in the early luteal phase support pregnancy, whereas subphysiological progesterone concentrations delay embryonic development at least until placentation. In this study, fetal growth and development of pregnancy was investigated in pregnancies with prostaglandin F2α-induced low progesterone concentrations (PGF) in the early luteal phase and control pregnancies (CON) in the same mares (n = 12). Mares were inseminated and in PGF pregnancies received the prostaglandin F2α analogue cloprostenol (62.5 µg) on days 0-3 after ovulation to induce subphysiological progesterone concentrations; CON pregnancies remained untreated. Mares were assigned to PGF or CON treatments in alternating order and received the opposite treatment in the following year. Blood was collected and conceptus size determined repeatedly by transrectal (≤day 101) and transabdominal (>day 101) ultrasonography. After birth, foals were weighed, measured and submitted to a clinical examination. Treatment PGF resulted in fewer pregnancies than CON treatment. All foals born from CON pregnancies were healthy and mature, whereas 4/7 PGF pregnancies were either lost (one embryonic death, one abortion) or resulted in the birth of compromised foals (P = 0.018). Size of the conceptus (e.g., diameter day 49: PGF 6.6 ± 0.7, CON 7.7 ± 0.7 cm, P = 0.006) and embryo proper (e.g., crown rump length day 54; PGF 4.4 ± 0.8, CON 5.8 ± 0.6 cm, P = 0.015) differed between treatments. These size differences decreased over time and at birth PGF foals did not differ significantly from CON foals. In conclusion, reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.

"Fine feathers make fine birds" - The mammalian sperm plasma membrane lipid composition and effects on assisted reproduction.

The sperm plasma membrane is important in modulating many sperm functions. The sperm membrane is composed of a complex mixture of lipids including phospholipids, glycolipids and sterols. There are differences of sperm membrane composition among mammalian species with two groups differing in the most abundant polyunsaturated fatty acid (PUFA), either docosahexaenoic (ω-3 PUFA) or docosapentaenoic acid (ω-6 PUFA). During testicular and epididymal maturation, composition of the sperm plasma membrane evolves with spermatozoa gaining the capacity for fertilization. The importance of fatty acid metabolism for complete spermatogenesis has been elucidated using gene knockout mice. During epididymal transit cholesterol content decreases and PUFA content increases, conferring more fluidity properties to the sperm membrane. The relatively lesser content of antioxidant enzymes and the relatively larger content of PUFA make the spermatozoa particularly susceptible to lipid peroxidation during sperm preservation. In numerous studies, there was adding of PUFA and antioxidants to the diet of animals or to semen extenders with the aim to improve sperm membrane integrity. This review highlights the current knowledge on the sperm membrane composition and effects on sperm function in mammalian domestic animals.

Low plasma progesterone concentration during the early luteal phase delays endometrial development and the beginning of placentation in mares.

While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to treatment (TREAT) or control (CON) during two pregnancies. In TREAT pregnancies, mares received a PGF2α analogue for four consecutive days starting on the day of ovulation with the aim to reduce progesterone secretion. Mares were left untreated in CON pregnancies and thus served as their own controls. Endometrial biopsies for analysis of histomorphology, epidermal growth factor (EGF) and EGF receptor (EGFR) mRNA and protein expression in the endometrium, AC, and CG as well as abundance of regulatory T lymphocytes (Tregs) were collected on day 34 of pregnancy. Histomorphometric analysis revealed a higher luminal endometrium and a higher CG epithelium in CON compared to TREAT pregnancies. Abundance of mRNA for EGF and EGFR was large in the endometrium, AC and CG but did not differ between TREAT and CON pregnancies. The number of endometrial regulatory T lymphocytes was reduced in TREAT compared to CON pregnancies, adding further aspects to the potentially detrimental effects of reduced progesterone concentrations on equine pregnancy.