Control Mares Research Articles

Effect of human chorionic gonadotropin treatment on the duration of oxytocin-induced prolonged corpus luteum function in mares

Oxytocin is used increasingly in mares to prolong corpus luteum (CL) function to suppress estrus. When mares develop prolonged CL function in response to oxytocin treatment, the CL generally remains functional for 60–90 days. However, a longer duration of CL function can make this method of estrus suppression even more clinically useful. We hypothesized that human chorionic gonadotropin (hCG) treatment during the period of oxytocin-induced prolonged CL function would extend the duration of CL function (i.e. beyond 90 days). Prolonged CL function was induced in mares (n = 14) by treating them with 60 units of intramuscular oxytocin once daily on days 7–14 after ovulation. Mares were then randomly assigned equally to a control group that received no additional treatment and an hCG-treated group that received 2,500 units of intramuscular hCG on days 30, 45, 60, 75, and 90 after ovulation. Jugular blood samples were collected for progesterone concentration determination on the day of ovulation and then 3 times (M, W, and F) weekly for 120 days. Duration of CL function was not different (p > 0.05) and it was 78.0 ± 7.5 and 91.4 ± 20.4 days (mean ± standard deviation [SD]) in control and hCG-treated mares, respectively. Therefore, hCG treatment (during the period of oxytocin-induced prolonged CL function) did not extend CL function, hence alternative methods should be explored.

Evaluation of dietary arginine supplementation to increase placental nutrient transporters in aged mares.

Nine pregnant mares (18.2 ± 0.7 yr; 493.82 ± 12.74 kg body weight [BW]) were used to test the hypothesis that dietary supplementation of l-arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and assigned randomly to dietary treatments of either supplemental l-arginine (50 mg/kg BW; n = 7) or l-alanine (100 mg/kg BW; n = 6; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal Bermudagrass hay. Treatments began on day 14 of gestation and were terminated at parturition. Mare BW, body condition score (BCS), and rump fat were determined, and body fat percentage was calculated every 28 d and concentrate adjusted accordingly. Doppler blood flow measurements including resistance index (RI) and pulsatility index for uterine artery ipsilateral to the pregnant uterine horn were obtained beginning on day 21 and continued every 7 d until day 154 of gestation, and prior to parturition. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn was analyzed histologically to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (SLC7A1) proteins. Semiquantitative analyses were performed using 10 nonoverlapping images per sample fixed in a 10× field (Fiji ImageJ v1.2). Mare performance data were analyzed using PROC MIXED in SAS and foaling and placental data were analyzed using PROC GLM. Gestation length at parturition was not influenced (P > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P < 0.01) and greater percent body fat (P = 0.03), and BCS (P < 0.01) at parturition. Arginine-supplemented mares had a lower RI than control mares prior to parturition (P < 0.01). Body length, height, and BW of foals at birth, as well as placental weight and volume, and immunohistochemical staining for VEGF and SLC7A1 at parturition, were not affected (P > 0.05) by maternal arginine supplementation. These results indicate that dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares. A larger number of mares is required to extend knowledge of effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Reimmunization intervals for application of GnRH immunocontraceptive vaccine (GonaCon-Equine) in free-roaming horses (Equus ferus caballus) using syringe darts

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.

A multicenter retrospective case-cohort study on the prevalence of incisional morbidities in late pregnant mares following exploratory celiotomy (2014-2019): 579 cases.

To report the prevalence and risk factors for incisional morbidities in late pregnant and nonpregnant/early pregnant control mares following colic surgery. Multicenter, retrospective, cohort study from January 2014 to December 2019. Two university teaching hospitals and 1 private referral center. Five hundred and seventy-nine fillies and mares ≥2 years old that underwent celiotomy. Pregnant mares (n=54) were >240days in gestation from the last known breeding date and were compared to control females (n=525) undergoing colic surgery. None. Morbidity rates were not different between mare groups with 56% of pregnant mares and 51% of control mares reporting at least 1 morbidity. Incisional swelling was the most common reported complication in both groups. Incisional swelling was associated with shorter hospital stays (odds ratio [OR], 0.18; P<0.01), and drainage was associated with a longer hospital stay (OR, 1.27; P≤0.01) and with use of an abdominal bandage (OR, 4.4; P<0.01). Herniation was associated with hypercapnia under anesthesia (OR, 1.1; P=0.048), previous abdominal surgery (OR, 8.3; P=0.003), and with use of an abdominal bandage (OR, 56; P=0.006). Body wall dehiscence was associated with longer hospital stay (OR, 1.2; P<0.01). Nonsurvival was higher in pregnant mares (13%) compared to control mares (5%; P=0.02). The prevalence of incisional morbidities did not differ between pregnant and control mares undergoing colic surgery. Several factors were associated with incisional morbidities, including the duration of surgery and anesthesia, anesthetic variables, abdominal bandage use, previous ventral abdominal incision, and longer duration of hospitalization.

The effect of dexamethasone and flunixin-meglumine on ovulation, endometrial oedema, and inter-ovulatory interval length in the mare

The use of flunixin-meglumine (a potent non-steroidal anti-inflammatory drug) during the critical period of intrafollicular prostaglandin production before ovulation (24 and 36 h after hCG treatment) results in a high rate of ovulatory failure and formation of haemorrhagic anovulatory follicles (HAF) in the mare. Dexamethasone is commonly used to prevent persistent mating-induced endometritis in susceptible mares, but the effect on ovulation blockage within the pre-ovulatory critical window of intrafollicular prostaglandins production following hCG administration has not been determined. Six mares were followed during four consecutive cycles in a crossover design; once in oestrus with a follicle of >32 mm in diameter, mares were treated with hCG (Hour 0) and assigned to one of 4 groups randomly: 1) FM, mares received 1.7 mg/kg flunixin-meglumine at Hour 24 and 36; 2) CON, mares received no further treatment. 3) DEX1, mares received 0.1 mg/kg dexamethasone at Hour 24, and 4) DEX2, mares received 0.1 mg/kg dexamethasone at Hour 24 and 36. For all groups, ovulation and HAF rates, endometrial oedema profiles and the inter-ovulatory intervals (IOI) were determined and compared statistically. All CON and DEX mares ovulated normally and did not form any HAF. On the contrary, FM mares developed a HAF in 83% of cycles (P < 0.01). The endometrial oedema score was lower following DEX administration than FM (P < 0.05). The mean IOI was longer (P < 0.05) in DEX1 and DEX2 groups (26.5 and 26 days, respectively) than in CON and FM groups (21.5 and 22 days, respectively). In conclusion, dexamethasone treatment given either once or twice during the critical window of hCG-induced ovulation did not block or delay ovulation, but had a similar ovulation rate than untreated control mares. However, the inter-ovulatory intervals of dexamethasone treated mares was longer than control and FM treated mares. Finally, dexamethasone treatment was more effective in reducing endometrial oedema than FM.

219 Evaluation of Dietary Arginine Supplementation to Increase Placental Nutrient Transporters of Aged Mares

Abstract Nine pregnant mares (18.2 ± 0.7 y; 493.82 ± 13 kg BW) were used to test the hypothesis that dietary supplementation of arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and randomly assigned to one of two dietary treatments consisting of supplemental L-arginine (50 mg/kg BW; n=5) or L-alanine (100 mg/kg BW; n=4; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal bermudagrass hay. Treatments began on d 14 of gestation and were terminated at parturition. Mare BW, BCS, and rump fat were determined every 28 d and concentrate adjusted accordingly. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn underwent histological analyses to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (CAT1) proteins. Semiquantitative analyses were performed using 10 non-overlapping images per sample fixed in a 10X field (Fiji ImageJ v1.2). Data were analyzed using SAS PROC MIXED procedure. BW increased (P &amp;lt; 0.01) in all mares with advancing gestation. BW, BCS, and gestation length of mares were not influenced (P &amp;gt; 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P &amp;lt; 0.01) at parturition. Body length, height, and BW of foals at birth, as well as placental weight, volume, and positive staining for VEGF and CAT1 at parturition were not affected (P &amp;gt; 0.05) by maternal arginine supplementation. These results indicate dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares and promotes mobilization of white adipose tissue. A larger number of mares is required to determine effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Uterine extracellular vesicles as multi-signal messengers during maternal recognition of pregnancy in the mare

In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare.

Development of Foals Until One Year of Age When the Dam was Exposed to Blue Monochromatic Light Directed at One Eye During Late Pregnancy

In horses, blue LED light directed at one eye of pregnant mares shortens gestation length and results in the birth of foals with lower wither heights, similar weight and reduced hair length compared to controls. In this study, we have therefore analysed postnatal development of foals born to either blue LED light-treated (n = 20) or control mares (n = 20). Size, weight and hair coat changes were determined for 1 year and heart rate, heart rate variability (HRV) and selected haematology parameters for 1 month. Haematocrit decreased (P < .001) and leukocyte and lymphocyte counts increased (P < .001) but none of the parameters differed between groups. Heart rate (P < .001) and HRV (P < .01) increased until day 6 but did not differ between groups, indicating that foals born to blue LED light-treated mares were mature and healthy. The guard hair was shorter in foals born to treated mares compared to control foals at birth (P < .001) but no differences in hair coat length were observed beyond the age of 2 months. At birth and 6 days thereafter, wither height (P < .01) and elbow to carpus distance (P < .05) of control foals were increased relative to foals born to blue LED light-treated mares. Height differences decreased over time and and for elbow-to-carpus distance there was a time x group interaction (P < .005) In conclusion, blue LED light treatment of pregnant mares is without detrimental effects on postnatal foal growth and development.

Oocyte metabolic function, lipid composition, and developmental potential are altered by diet in older mares.

Dietary supplementation is the most feasible method to improve oocyte function and developmental potential in vivo. During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant support (reproductive support supplement (RSS)) intended to increase antioxidant activity and lipid oxidation. RSS was modified for individual experiments with additional antioxidants or altered concentrations of n-3 to n-6 fatty acids. Oocytes from mares supplemented with RSS when compared to COB had higher basal oxygen consumption, indicative of higher aerobic metabolism, and proportionately more aerobic to anaerobic metabolism. In the second experiment, oocytes collected from the same mares prior to (CON) and after approximately 8 weeks of RSS supplementation had significantly reduced oocyte lipid abundance. In the final experiment, COB was compared to RSS supplementation, including RSS modified to proportionately reduce n-3 fatty acids and increase n-6 fatty acids. The ability of sperm-injected oocytes to develop into blastocysts was higher for RSS, regardless of fatty acid content, than for COB. We demonstrated that short-term diet supplementation can directly affect oocyte function in older mares, resulting in oocytes with increased metabolic activity, reduced lipid content, and increased developmental potential.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes

Simple SummaryIn pregnant individuals, maternal overnutrition is associated with disturbances in the expression of specific genes and nutrient transporters in the early embryo, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of several genes, nutrient transporters and DNA methyltransferases (DNMTs; play an important role in regulating gene expression) were determined in extra-embryonic membranes after recovery on day 28 of gestation. The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after embryo removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven embryos were assessed. Maternal overfeeding resulted in elevated expression of several genes, DNMTs and nutrient transporters following embryo transfer from an HE to a control mare. The expression of two amino acid transporters was also elevated in the endometrium after embryo transfer from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in day-seven embryos, or glucose uptake by membranes of day-28 embryos. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes (IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN), DNMTs (DNMT1 and DNMT3B), glucose (SLC2A1), fructose (SLC2A5) and amino acid (SLC7A2) transporters following ET from an HE to a control mare. Expression of amino acid transporters (SLC1A5 and SLC7A1) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Effect of a long-term high-energy diet on cardiovascular parameters in Shetland pony mares.

BackgroundChanges in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown.ObjectivesDetermine the order in which EMS‐associated changes in cardiovascular parameters arise.AnimalsTwenty Shetland pony mares.MethodsHigh‐energy (HE) diet mares were fed 200% of net energy requirements for 1 (n = 3) or 2 (n = 7) consecutive diet‐years, with 17 weeks of hay‐only between years. Noninvasive BP measurements and echocardiograms were performed during both years. Resting 24‐hour ECGs and measurements of autonomic tone (splenic volume and packed cell volume [PCV]) were performed at the end of diet‐year 1. Results were compared to control mares receiving a maintenance diet for 1 (n = 7) or 2 (n = 3) consecutive years.ResultsIn year 1, HE mares had significantly higher values than control mares for mean relative left ventricular wall thickness (P = .001). After 2 diet‐years, mean systolic (P = .003), diastolic (P < .001) and mean arterial BP (P = .001), heart rate (HR; P < .001), and mean left ventricular wall thickness (P = .001) also were significantly increased in HE compared to control mares. No pathological arrhythmias or differences in splenic volume or PCV were detected.Conclusions and Clinical ImportanceIngesting a HE diet first induced minor changes in BP, and progressed to left‐sided cardiac hypertrophy in Shetland pony mares. These findings are of interest given the increasing incidence of obesity in horses.

Hemodynamic changes in cytokines, chemokines, acute phase proteins and prostaglandins in mares with subclinical endometritis

Apparently healthy mares with conception failure or embryonic loss may have subclinical endometritis (SE). Our objective was to document evidence of systemic immune responses against SE in mares. In apparently healthy (control) mares as well as those with chronic endometritis (ChE) or subacute suppurative endometritis (SSE), both considered classes of SE, serum concentrations of cytokines (IL-1β, IL-6, IL-10 and TNF-α), chemokines (IL-8), acute phase protein (SAA), and plasma concentrations of prostaglandins (PGF2α and PGE2) were measured using validated enzyme linked immunoassays (EIA). Mixed-breed mares of known reproductive history, were used. Based on an endometrial cytological examination, mares were allocated into the following groups: healthy (control), ChE, and SSE (12, 26 and 11 mares, respectively). Serum concentrations of IL-6, IL-8 and IL-10 and plasma PGF2α concentrations were increased (P < 0.01) in mares with SSE compared to healthy mares. Furthermore, serum concentrations of IL-6 (P < 0.05) and IL-10 (P < 0.05) were elevated in the ChE group compared to the healthy group whereas serum SAA (P < 0.01) and plasma PGE2 (P < 0.05) were higher in healthy mares compared to ChE and SSE mares. However, serum concentrations of IL-1β and TNF-α were not significantly different among groups. In conclusion, there were two novel findings: (1) development of serum testing for a set of biochemical markers has promise to explore the pathogenesis of inflammation in mares with SE; and (2) none of the individual biomarkers studied, excluding IL-8, was a significant predictor of SE. However, serum IL-6:IL-10 and IL-6:TNF-α concentrations, as well as plasma PGE2:PGF2α ratios, may yield a novel diagnostic marker for chronic subclinical endometritis.

24 Antioxidant, reactive oxygen species, and Coenzyme Q10 concentrations and expression of electron transfer proteins in thoroughbreds with recurrent exertional rhabdomyolysis

In the mitochondria, complexes I and III of the electron transport system (ETS) generate reactive oxygen species (ROS) during exercise that are reduced by antioxidants to mitigate oxidative stress. Among these antioxidants are thiol-based antioxidants such as glutathione as well as Coenzyme Q10 (CoQ10), which also transports electrons from complex I and II to complex III. Recurrent exertional rhabdomyolysis (RER) is associated with aberrant calcium regulation with the potential for excessive mitochondrial calcium uptake impacting mitochondria and ROS. We hypothesized that there would be a decrease in CoQ10 concentrations, as well as an increase in ROS and peptide subunits of ETS complexes in thoroughbreds with RER. Concentrations of CoQ10 (LC/MS/MS), thiol-based antioxidants (LC/MS/MS), ROS (OxiSelect, Cell Biolabs), and citrate synthase activity (CS fluorometry) were determined in gluteal muscle biopsie from thoroughbred mares with a history of, but not currently experiencing, rhabdomyolysis (n = 20, RER; mean ± SD age 3.3 ± 1.3 yr), compared with healthy race trained mares at the same facility (n = 8, control; age 2.9 ± 0.8 yr). RER and control were compared by unpaired t -test. Subunit peptide components of the ETS were analyzed in 5 RER and 5 control mares using tandem mass tag proteomic analysis (FDR < 0.05). CoQ10 ( P = 0.36), ROS ( P = 0.36), and thiol ( P = 0.99) concentrations were not significantly different between RER and controls. CS activity, a marker for mitochondrial volume density, did not differ between RER and controls ( P = 0.39). ETS subunits were, however, differentially expressed in RER versus controls, with decreased expression of 11 complex I, 2 complex II, 2 complex III, 4 complex IV and 9 ATP synthase peptide subunits ( P < 0.01). In conclusion, antioxidant concentrations and mitochondrial volume density did not differ in control versus RER mares between episodes of rhabdomyolysis. Expression, however, of specific peptide subunits within ETS complexes was consistently decreased in RER mares, which could be related to impacts from mitochondrial calcium buffering. Further investigation into ETS function in RER racehorses is warranted.

106 Maternal recognition of pregnancy (MRP) and mares: Is the epithelial sodium channel (ENaC) involved?

Maternal recognition of pregnancy (MRP) describes the prevention of luteolysis via the early conceptus. By prolonging the presence of the corpus luteum (CL), the conceptus ensures an environment capable of supporting pregnancy. In mares, MRP is enigmatic; the horse is one of few domesticated species wherein the MRP signal has yet to be discovered. Equine conceptus mobility throughout the uterine horns, termed migration, is known to be essential for pregnancy establishment. However, while the MRP signal in several other species is conceptus-derived, such a signal has yet to be observed from the equine embryo. We hypothesize that the migration of the equine conceptus itself activates MRP, potentially through epithelial sodium channel (ENaC). ENaC has been previously detected in multiple equine organs, observed in the uteri of various mammalian species, and also found to be upregulated during preimplantation in mice. However, neither the presence of ENaC within the mare uterus nor its potential involvement with the equine pregnancy has been researched. The aim of this study was 1) to determine if ENaC was present within the endometrium of the mare uterus and 2) to describe the location of ENaC if observed. Estrus synchronization was completed in mares (n = 14) during peak breeding season. Mares were subsequently blocked by age and breed and then placed into one of 2 groups: control mares (C; n = 7) and treatment mares (T; n = 7). Transrectal ultrasonography (US) was completed in all mares daily when one or more ovarian follicles reached 25 mm. When a mare had a 35 mm or larger follicle present, she received 2,000 international units of human chorionic gonadotropin intramuscularly to induce ovulation (OV) and US continued daily. Additionally, T mares were artificially inseminated (AI) with fresh, extended semen every day until the day after OV and were subsequently checked for pregnancy 12 d post-OV. C mares had the cervix manually manipulated the day after OV to replicate physical stimulation that occurs to the cervix when AI; however, no semen was deposited in C mare reproductive tracts. Endometrial biopsies were collected from all mares before OV, after OV, and during early pregnancy (when applicable) and samples were then processed for ENaC immunohistochemistry analysis. ENaC protein was immunodetected in all C and T mare endometrial samples and was localized to both uterine luminal epithelium and uterine glands. While further analysis will be conducted to quantify endometrial ENaC expression in C versus T (open versus pregnant) mares, the present histological data confirms that ENaC is present within the equine uterus.

59 Late gestation supplementation of long chain fatty acids increases foal docosahexaenoic acid concentrations at birth

Maternal supplementation of omega-3 fatty acids during gestation has a variety of benefits on the dam and resulting offspring in several species, including reduced latency to stand and suckle. The objective of the study is to test the hypothesis that foals born to mares supplemented with marine-derived algae during late gestation will have greater plasma DHA concentrations than foals born to either control mares or mares supplemented with flaxseed-derived ALA. The current study utilized 13 American Quarter Horse mares and their foals, assigned to one of 3 dietary treatments: a control basal diet (CON, n = 5), the basal diet plus 242.4 g of a supplement providing marine-derived omega-3 fatty acids (DigestaWell Ⓡ Omega; MAR, n = 5), or the basal diet with the addition of 190 g of a flaxseed supplement (Smartpak Simple Flax; FLAX, n = 3). Supplements were fed twice daily and were designed to deliver 40 g of omega-3 fatty acids. Mares began treatments 30 d before expected foaling dates and continued through d 5 post-parturition (PP). Plasma was collected from mares before supplementation, and on d 0, 5, and 30 PP. Milk samples were collected from mares at d 0 and 5 PP. Plasma samples were collected from foals at birth, and on d 5 and 30 PP. Fatty acid compositions of samples were determined using lipid extraction and gas chromatography. Data were analyzed using MIXED procedure of SAS, using repeated measures for milk and plasma. Simple effect differences were detected via Tukey test, and α = 0.05. The CON diet provided 29 g of α-linolenic acid (ALA), 0 g of eicosapentaenoic acid (EPA), and 4.5 g of docosahexaenoic acid (DHA), the MAR and FLAX diets provided 27 and 34 g of ALA, 0 and 0.03 g of EPA, and 17.4 and 4.5 g of DHA to mares, daily. Fatty acids are represented as g of fatty acid per 100 g of lipid. DHA (1.9 ± 0.2 g/100g) and EPA (0.3 ± 0.1 g/100g) contributed to a greater percent of plasma lipids in foals born to MAR supplemented mares compared with CON foals (DHA = 0.6 ± 0.2; EPA = 0 g/100g; P < 0.05). These fatty acids were not different between FLAX and CON foals ( P > 0.1) Samples obtained from foals at birth before nursing had the highest DHA levels (1.90 ± 0.18 g/100g), compared with all other samples, and DHA declined at d 5 (0.6 ± 0.2 g/100g) and 30 (0.1 ± 0.2 g/100g; P < 0.001) PP. No differences were observed in mare plasma or milk. Maternal supplementation of marine-derived omega-3 fatty acids in late gestation increased foal plasma DHA concentrations more so than that of foals from mares fed flax.

Transcriptome Profiling of the Retained Fetal Membranes-An Insight in the Possible Pathogenesis of the Disease.

Simple SummaryRetained fetal membranes (RFM) in mares is a disease of a multifactorial etiology with not fully understood pathogenesis. Profound analysis of genes expressed in the placenta may reveal pathways and processes which might be comprised in mares with this disease and hence help to explain the pathogenesis of RFM. This work employed RNA sequencing to identify and compare genes differentially expressed (DEGs) in the placenta of mares that retained fetal membranes and those that released them physiologically. Results showed that within DEGs genes important for apoptosis, inflammatory-related processes, and metabolism of extracellular matrix were identified.Retained fetal membranes (RFM) is one of the most common post-partum diseases of a complex etiology. Moreover, its pathogenesis is still not elucidated. Detailed transcriptomic analysis of physiological and retained placenta may bring profound insight in the pathogenesis of the disease. The aim of the study was to compare the transcriptome of the retained and physiologically released placenta as well as biological pathways and processes in order to determine the possible pathogenesis of the disease. Samples of the endometrium and the allantochorion were taken within 2 h after parturition from control mares (n = 3) and mares with RFM (n = 3). RNA sequencing was performed with the use of all samples and mRNA expression of chosen genes was validated with Real Time PCR. Analysis of RNA-seq identified 487 differentially expressed genes in the allantochorion and 261 in the endometrium of control and RFM mares (p < 0.0001). Within genes that may be important in the release of fetal membranes and were differentially expressed, our report pinpointed BGN, TIMP1, DRB, CD3E, C3, FCN3, CASP3, BCL2L1. Gene ontology analysis showed possible processes which were altered in RFM that are apoptosis, inflammatory-related processes, and extracellular matrix metabolism and might be involved in the pathogenesis of RFM. This is the first report on the transcriptome of RFM and physiologically released placenta in mares.

Interleukin-6 pathobiology in equine placental infection.

Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. Sub-acute ascending placentitis was induced via trans-cervical inoculation of Szooepidemicus, and various fluids/serum/tissues collected 8days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n=6) and control (n=6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Simple SummaryObesity has been associated with altered reproductive activity in mares and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

The contraceptive efficacy of a self-assembling intra-uterine device in domestic mares.

The primary aim of this study was to investigate the contraceptive efficacy of a self-assembling uterine device (iUPOD™) in the mare. In addition, the effects of iUPODs on oestrous cyclicity, uterine health and circulating concentrations of cortisol were evaluated. Domestic mares underwent oestrous monitoring and artificial insemination. After subsequent ovulation, mares underwent either placement (n = 7) or sham placement (n = 7; controls) of an iUPOD device. Devices were left in place for at least 3 months. Pregnancy diagnoses were carried out 14 days post-ovulation, with any pregnancies terminated at 28 days post-ovulation. All mares underwent weekly blood sampling with or without reproductive examinations throughout the study. Towards the end of the study, multiple serum samples collected over three consecutive days were analysed for concentrations of cortisol. Endometrial biopsies were collected before artificial insemination and during the subsequent breeding season. Endometrial cytology and bacterial cultures were performed before device removal (iUPOD mares) or at the end of the study (control mares). Pregnancies were diagnosed in 0 of 7 iUPOD mares versus 7 of 7 control mares. Placement of iUPODs was associated with extended luteal phases and variable accumulations of intra-uterine fluid. Bacterial culture results suggested that the mild endometritis associated with iUPODs was sterile in six of seven mares. Short-term placement of iUPODs had no detrimental effects on endometrial architecture. Mean serum cortisol concentrations were significantly lower in iUPOD mares than control mares. iUPODs represent a promising means of fertility control in the mare.