Management of overabundant free-roaming horse (Equus ferus caballus) populations has become a divisive and challenging effort for natural resource managers worldwide. Controlling these populations requires land managers to balance socio-political issues, multiple use management concepts, and endearing affection of horses to some stakeholders against negative ecological alterations created by excessive densities of these animals. One potential solution to this dilemma is controlling the fertility of female horses. During 2013–2020, research was conducted to develop and test a prototype dart configuration system to deliver an effective dose of GonaCon-Equine to free-roaming horses. This remote dart delivery system was demonstrated to be safe and reliable in free-ranging horses with a 90% (71/79) success rate for first attempts and provided the opportunity to design an experiment to determine the long-term effectiveness, reversibility, and physiological side effects of different reimmunization schedules. Five experimental groups of mares were established consisting of a control (n = 25), and a 4-yr reimmunization interval (n = 25) of mares from a previous experiment that were treated with this same vaccine by hand injection in 2013. Additional booster intervals of horses were (2-yr (n = 11), 1-yr (n = 15), and 0.5-yr (n = 14) and vaccinated by remote dart delivery during 2013–2016. Weekly ground observations were utilized to estimate foaling proportions, foal survival, body condition, and injection site reactions related to treatment groups. All GonaCon-Equine booster intervals reduced (P ≤ 0.001) foaling proportions between treated and control mares for all years of the experiment. Effective contraception was also achieved when GonaCon-Equine was applied by remote dart delivery at post-primary intervals shorter than 4 years. Reversibility of GonaCon-Equine immunization occurred in all treatment groups and averaged 0.40 (26/65). The only detectable adverse side effect of vaccination was intramuscular swelling observed in 89% (115/129) of mares and occasional draining abscesses at the site of vaccine injection. However, regardless of method of vaccine delivery (hand vs dart), none of these mares displayed any evidence of lameness, altered gait, or abnormal range of movement during the 6 years of this study. These results support previous findings that effective contraception of free-roaming horses will require a primary vaccination followed by reimmunization but that timing from 6 months to 4 years result in similar success and together can achieve greater reduction in population growth rates.

Feral horse populations continue to grow, and management is focused on removal both at a substantial cost in the process and their long-term care. Fertility control has been identified as a means to provide a solution. Two EPA registered immunocontraceptives (Gonacon and ZonaStat-H) are available for equids, but both require an initial dose and at least one booster ranging from weeks to years later making this relatively impractical. Our aim was to develop a single dose long-lasting contraceptive targeting 2 oocyte-specific growth factors (OGF) GDF-9 and BMP-15. In our proof-of-concept study, GDF-9 antigen representing 14 amino acids of the mature protein, and a BMP-15 antigen representing 24 amino acids in the mature sequence were produced. Both were conjugated to keyhole limpet hemocyanin (KLH), and the experimental vaccine was manufactured using Pet-Gel A as an adjuvant. We reported that domestic mares were treated with an initial dose and 4 boosters at 6 week intervals. All treated mares developed antibodies and demonstrated infertility for an entire year, and 88% of the mares remained infertile for the following year. The current investigation was conducted to examine the effects of a single dose with a potentially long-lasting immunostimulant. We enrolled 32 mares and 4 stallions with proven fertility that had been removed from the Nevada range that year. All mares had produced foalsthe year before. In May, 16 of these mares were treated with a new OGF vaccine formulation using the same antigens conjugated to KLH and AdjuVac adjuvant containing liposomes. Another 16 mares received a sham injection of adjuvant alone. Serum samples were collected monthly for titer analysis via enzyme-linked immunosorbent assay and progesterone assays via radioimmunoassay. Pregnancy determination was also conducted monthly via transrectal ultrasonography. All mares were then housed with fertile stallions in August. Four treated and 4 control mares were housed in each of 4 pens with a single stallion. All (16/16) of control mares became pregnant. One treated mare was euthanized for reasons unrelated to treatment. Of the remaining treated mares 67% (10/15) became pregnant within 60 d while 20% (3/15) became pregnant much later (180–300 d). Two mares (13%) remained anovulatory for 2 years. The mares with delayed and inhibited fertility demonstrated the greatest (P < 0.001) and longest lasting (P < 0.0001) anti-OGF titers. While this specific vaccine attenuated ovulation it failed to sufficiently alter fertility as pregnancy rates were not different. Our goal is to now find an adjuvant that will result in a single long-lasting vaccine.

Laminitis associated with equine metabolic syndrome causes significant economic losses in the equine industry. Diets high in non-structural carbohydrates (NSC) have been linked to insulin resistance and laminitis in horses. Nutrigenomic studies analyzing the interaction of diets high in NSCs and gene expression regulating endogenous microRNAs (miRNA) are rare. This study's objectives were to determine whether miRNAs from dietary corn can be detected in equine serum and muscle and its impacts on endogenous miRNA. Twelve mares were blocked by age, body condition score, and weight and assigned to a control (mixed legume grass hay diet) and a mixed legume hay diet supplemented with corn. Muscle biopsies and serum were collected on Days 0 and 28. Transcript abundances were analyzed using qRT-PCR for three plant-specific and 277 endogenous equine miRNAs. Plant miRNAs were found in serum and skeletal muscle samples with a treatment effect (p < .05) with corn-specific miRNA being higher than control in serum after feeding. Endogenous miRNAs showed 12 different (p < .05) miRNAs in equine serum after corn supplementation, six (eca-mir16, -4863p, -4865p, -126-3p, -296, and -192) previously linked to obesity or metabolic disease. The results of our study indicate that dietary plant miRNAs can appear in circulation and tissues and may regulate endogenous genes.

Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. We examined the effects of a high-fat diet (HFD) during pregnancy on fetal skeletal muscle metabolism and metabolic risk parameters using an ovine model. White-faced ewes were fed a standardized diet containing 5% fat wt/wt (CON), or the same diet supplemented with 6% rumen-protected fats (11% total fat wt/wt; HFD) beginning 2 wk before mating until midgestation (GD75). Maternal HFD increased maternal weight gain, fetal body weight, and low-density lipoprotein levels in the uterine and umbilical circulation but had no significant effects on circulating glucose, triglycerides, or placental fatty acid transporters. Fatty acid (palmitoylcarnitine) oxidation capacity of permeabilized hindlimb muscle fibers was >50% higher in fetuses from HFD pregnancies, whereas pyruvate and maximal (mixed substrate) oxidation capacities were similar to CON. This corresponded to greater triacylglycerol content and protein expression of fatty acid transport and oxidation enzymes in fetal muscle but no significant effect on respiratory chain complexes or pyruvate dehydrogenase expression. However, serine-308 phosphorylation of insulin receptor substrate-1 was greater in fetal muscle from HFD pregnancies along with c-jun-NH2 terminal kinase activation, consistent with prenatal inhibition of skeletal muscle insulin signaling. These results indicate that maternal high-fat feeding shifts fetal skeletal muscle metabolism toward a greater capacity for fatty acid over glucose utilization and favors prenatal development of insulin resistance, which may predispose offspring to metabolic syndrome later in life.NEW & NOTEWORTHY Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. This study examined the effects of a high-fat diet during pregnancy on metabolic risk parameters using a new sheep model. Results align with findings previously reported in nonhuman primates, demonstrating changes in fetal skeletal muscle metabolism that may predispose offspring to metabolic syndrome later in life.

A core group of 27 equine nutritionists and physiologists joined together in the late 1960s to formally address and enhance the direction of equine research, creating the Equine Nutrition and Physiology Society. In 2003, that growing society transformed into the Equine Science Society, which now serves as the preeminent, internationally recognized scientific equine organization. In recent years, it has been appreciated that equine science encompasses a wide range of focus areas, including exercise science, nutrition, genetics, reproductive physiology, teaching and extension, production and management, and mix of other specialties, qualified as biosciences. Additionally, trainees are highly valued in the society, with the clear understanding that young people are the future of equine science. Amongst tightening budgets, equine researchers must focus on timely dissemination of high-quality research studies and development of strong, interdisciplinary, cross-species, and multi-institutional collaborations to ensure sustainability of academic research programs. With a little creativity, equine science will continue to thrive for the betterment of the horse and all involved in the equine industry.

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.

The management of free-roaming horses (Equus ferus) and burros (E. asinus) in the United States has been referred to as a "wicked problem " because, although there are population control options, societal values will ultimately determine what is acceptable and what is not. In the United States, free-roaming equids are managed by different types of organizations and agencies, and the landscapes that these animals inhabit vary widely in terms of access, size, topography, climate, natural resources, flora, and fauna. This landscape diversity, coupled with contemporary socioeconomic and political environments, means that adaptive management practices are needed to regulate these free-roaming populations. The Bureau of Land Management (BLM) currently manages free-roaming equids on 177 herd management areas in the United States by applying fertility control measures in situ and/or removing horses, which are either adopted by private individuals or sent to long-term holding facilities. The BLM off-range population currently includes > 50,000 animals and costs approximately $50 million USD per year to maintain; on-range equid numbers were estimated in March 2022 to be approximately 82,384. On-range populations can grow at 15-20% annually, and current estimates far exceed the designated appropriate management level of 26,715. To reduce population recruitment, managers need better information about effective, long-lasting or permanent fertility control measures. Because mares breed only once a year, fertility control studies take years to complete. Some contraceptive approaches have been studied for decades, and results from various trials can collectively inform future research directions and actions. Employing 1 or more fertility control tools in concert with removals offers the best potential for success. Active, iterative, cooperative, and thoughtful management practices can protect free-roaming horses while simultaneously protecting the habitat. Herein, we review contraceptive vaccines, intrauterine devices, and surgical sterilization options for controlling fertility of free-roaming horses. This review provides managers with a "fertility control toolbox " and guides future research.

Antioxidant supplementation decreases postexercise oxidative stress but could also decrease muscle protein synthesis. This study compared the effects of three diets: low antioxidant (control, CON), high antioxidant (AO), and branched-chain amino acid high antioxidant (BCAO) supplementation on postexercise protein synthesis and oxidative stress. We hypothesized that supplementing antioxidants with branched-chain amino acids(BCAA) would reduce oxidative stress without hindering muscle protein synthesis. Eighteen mixed-breed polo horses (11 mares and 7 geldings, with age range between 5 and 18 years, were on CON diet for 30 days (from day -45 until day 0) and then were assigned to one of the treatments after the first lactate threshold test (day 0, LT). LT were also conducted on days 15 and 30 of supplemenation. Oxidative stress was assessed by measuring blood glutathione peroxidase, superoxide dismutase, and malondialdehyde concentrations before 2 and 4 hours after each LT. Muscle biopsies were taken before and 4 hours after each LT and analyzed for gene expression of protein synthesis by RTqPCR. Data were analyzed by ANOVA and compared by least-square means. A reduction in oxidative stress occurred over time (P < .05), from day 0 to day 30. An up-regulation in the abundance of muscle protein mRNA transcripts was found for CD36, CPT1, PDK4, MYF5, and MYOG (P < .05) after all lactate threshold tests, without a treatment effect. A treatment-by-exercise effect was observed for MYOD1 (P = .0041). Transcript abundance was upregulated in AO samples post exercise compared to other treatments. MYF6 exhibited a time-by-treatment effect (P = .045), where abundance increased more in AO samples from day 0 to day 15 and 30 compared to other treatments. Transcript abundance for metabolic and myogenic genes was upregulated in post exercise muscle samples with no advantage from supplementation of antioxidants with branched-chain amino acids compared to antioxidants alone.

Normal physiological levels of androgens are important for female reproductive health. However, high levels of androgens, as seen in women with Polycystic Ovarian Syndrome (PCOS) (8%-13% of reproductive age women) [1] or gestational diabetes (incidence in US of 10%) [2] are associated with reproductive complications. Pathologies accompanying significantly elevated testosterone levels involve impaired ovulation and early pregnancy maintenance. Formation of the Corpus Luteum (CL) requires a normal ovulation process to occur. The CL plays a central role in the establishment and early maintenance of a pregnancy. Here we present a brief report on the function of the CL and our current understanding on the potential role and function of testosterone and the activated androgen receptor.

Abstract Histone lysine demethylase 1A is a master regulator of genes necessary for trophoblast cell proliferation. A proper functioning placenta is critical for pregnancy, fetal growth and development and postnatal health. Trophoblast cell proliferation and differentiation is critical for placental development and function. Recently we demonstrated that the histone lysine demethylase KDM1A binds to androgen receptor (AR) in human and sheep trophoblast cells, and targets the same promoter region of vascular endothelial growth factor A (VEGFA), suggesting a role for KDM1A and AR in early placental angiogenesis. The goal of this study was to determine the function of KDM1A during early placental development. We hypothesized that KDM1A regulates genes that are necessary for trophoblast cell proliferation, and early placental development. To this end, both in vitro and in vivo approaches were used in this study. ACH-3P cells (human first trimester trophoblast cells (CT and EVT) fused with the choriocarcinoma cell line AC1-1) were used, and a KDM1A knock out (KO) cell line was generated using CRISPR-Cas 9 based genome editing. KDM1A KO in ACH-3P cells led to significant (P<0.05) reduction in AR and VEGFA. Furthermore, factors important for cell proliferation and trophoblast cell development high mobility group AT-hook 1 (HMGA1), LIN28, and MYC protooncogene (cMYC) were significantly (P<0.05) lower in KDM1A KO ACH-3P cells. Cell proliferation assays revealed a significant (P<0.05) reduction in KDM1A KO ACH-3P cells compared to scramble controls. An in vivo experiment was conducted to demonstrate a role for KDM1A in placental development, using the sheep as a model. Day 9 hatched blastocysts were flushed and infected with a Lenti-CRISPRv2 KDM1A target construct (n=4) to knockout KDM1A specifically in the trophectoderm, or with SC (n=5). Infected embryos were transferred to recipient ewes and embryos were collected at gestational day 16. Data suggests that KDM1A KO in trophoblast cells is necessary for conceptus elongation. Current experiments are ongoing to determine the effects of KDM1A and AR knockdown using shRNA lentiviral target vectors on conceptus elongation and pregnancy. Collectively these results indicate that KDM1A plays a central role in regulating genes necessary for trophoblast cell proliferation. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29000 from the USDA National Institute of Food and Agriculture.

To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 mu g iv), or a combination thereof every 4 h for 3 days. The following observations were made: 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are G alpha(q/11) coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells: one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH. (C) 2020 Published by Elsevier Inc.

Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production invitro from Day 30 CG and AC cells. Three mares (n=3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured invitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7±2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42ngmL−1) invitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042ngmL−1) invitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced invitro as early as Day 30 from CG cells.

Reproductive efficiency is critically dependent on embryo survival, establishment of a successful pregnancy and placental development. Recent advances in gene editing technology have enabled investigators to use gene knockdown and knockout approaches to better understand the role of hormone signaling in placental function and fetal growth and development. In this review, an overview of ruminant placentation will be provided, including recent data highlighting the role of histone lysine demethylase 1A and androgen signaling in ruminant placenta and pregnancy. Studies in ruminant placenta establish a role for histone lysine demethylase 1A in controlling genetic networks necessary for important cellular events such as cell proliferation and angiogenesis, as well as androgen receptor signaling during early placentation.

Abstract The objective of this study was to determine the effect of corn supplementation on muscle microRNA (miRNA) profiles. Twelve mares were blocked by weight and BCS and assigned to one of two treatments (6 hd/treatment): 1) control, (basal diet: 9 Kg/hd/d of chopped mixed grass hay and ad libitum mixed grass hay), 2) basal diet supplemented with 454 g/d steam flaked corn individually using feed bags. All mares were placed on the basal diet and in the same pen 14 days prior to the beginning of corn supplementation. Mares were weighed and BCS on d -7 and 28. Muscle biopsies of the gluteus medius were taken from all horses on d0 and d26. Muscle samples were analyzed using real time RT-qPCR for 277 endogenous miRNAs. Raw CT values were normalized with the geometric mean of three consistently appearing endogenous miRNAs. Sixteen miRNAs that consistently appeared in at least 8 horses including 3 relating to metabolic disorders appearing at d0, d26, or both were analyzed. Differential abundances of miRNAs using log transformed fold change and differences between BW and BCS of treatment groups were determined by ANOVA and LS means analysis. There was no difference between BW (P < .05) and BCS (P < .05) between the two treatment groups. Feeding corn lead to higher quantities (P < .05) of mir 133a, 1515p, 6155p, 770, and 99b within muscle compared to control group. Lower quantities of mir 382 and 433 were found within muscle of horses fed corn compared to the control group (P < .05). Based upon the results of this study corn supplementation appears to influence endogenous miRNA expression profiles within muscle of horses.

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.

Objective To compare pain-related responses in mares receiving topical or injected anesthesia of the ovarian pedicle prior to standing unilateral laparoscopic ovariectomy. Study design Prospective randomized, blinded, placebo-controlled study. Animals Fifteen healthy research mares. Methods Mares were restrained in stocks and administered sedation. A right or left paralumbar ovariectomy was performed by using a laparoscopic portal and two instrument portals. Mares were divided into two treatment groups, and equal volumes of mepivacaine anesthesia were administered either topically (n = 8) or by injection into the ovarian pedicle (n = 7). Saline controls were simultaneously administered topically (n = 7) or by injection (n = 8), and surgeons were blinded to the treatment group. Ovarian removal was performed with traumatic forceps and a blunt tip vessel sealer and divider. Pain responses were measured by operative visual analog scale (VAS) scoring and perioperative serum cortisol response. Visual analog scale and serum cortisol were compared between groups by using Mann-Whitney testing. Serum cortisol concentrations were evaluated using repeated-measures one-way analysis of variance. Results Ovaries were removed in all mares by using the described technique without operative complications. Quantity of sedation required to complete the procedure, operative VAS scores, and perioperative cortisol concentrations did not differ between treatment groups. Conclusion Application of topical mepivacaine to the ovary provided intraoperative analgesia similar to injection of the ovarian pedicle when performing unilateral standing laparoscopic ovariectomy in mares. Clinical significance Topical anesthesia application to the ovary could provide an alternative to laparoscopic needle use, reducing the risk of inadvertent trauma to the pedicle or other visceral organs during laparoscopic ovariectomy.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP.

New biomarkers are necessary to improve detection of the risk of infection in heart transplantation. We performed a multicenter study to evaluate humoral immunity profiles that could better enable us to identify heart recipients at risk of severe infections.We prospectively analyzed 170 adult heart recipients at 8 centers in Spain. Study points were before transplantation and 7 and 30 days after transplantation. Immune parameters included IgG, IgM, IgA and complement factors C3 and C4, and titers of specific antibody to pneumococcal polysaccharide antigens (anti-PPS) and to cytomegalovirus (CMV). To evaluate potential immunologic mechanisms leading to IgG hypogammaglobulinemia, before heart transplantation we assessed serum B-cell activating factor (BAFF) levels using enzyme-linked immunoassay. The clinical follow-up period lasted 6 months. Clinical outcome was need for intravenous anti-microbials for therapy of infection.During follow-up, 53 patients (31.2%) developed at least 1 severe infection. We confirmed that IgG hypogammaglobulinemia at Day 7 (defined as IgG <600 mg/dl) is a risk factor for infection in general, bacterial infections in particular, and CMV disease. At Day 7 after transplantation, the combination of IgG <600 mg/dl + C3 <80 mg/dl was more strongly associated with the outcome (adjusted odds ratio 7.40; 95% confidence interval 1.48 to 37.03; p = 0.014). We found that quantification of anti-CMV antibody titers and lower anti-PPS antibody concentrations were independent predictors of CMV disease and bacterial infections, respectively. Higher pre-transplant BAFF levels were a risk factor of acute cellular rejection.Early immunologic monitoring of humoral immunity profiles proved useful for the identification of heart recipients who are at risk of severe infection.