The interaction between seminal plasma, sperm, and endometrium in inter- and intra-species breeding in equids.

In brief: In some instances, extra-species breeding in equids is more successful than intraspecies breeding; however, little is known about the immunomodulatory effect of donkey semen and seminal plasma on the mare's endometrium. This study compared the mare uterine inflammatory response during extra- and intraspecies breeding. Abstract: Anecdotal experience suggests horse mares have less post-breeding inflammation and better fertility when bred with donkeys. This study aimed to compare the post-breeding inflammatory response of mares exposed to donkey and horse semen and seminal plasma and evaluate the proteome and metabolome of donkey and horse sperm and seminal plasma. Uterine edema, intrauterine fluid accumulation, polymorphonuclear neutrophils on cytology, and concentrations of progesterone, and pro- and anti-inflammatory cytokines (IL1A, IL1B, IL4, IL6, CXCL8, IL10) were assessed pre- and post infusion of semen and seminal plasma (donkey and horse). The metabolome and proteome were analyzed by LC-MS/MS. Mare cycles bred with horse semen had a greater progesterone concentration than those bred with donkey semen at 8 days post ovulation (P = 0.046). At 6 h post infusion, the inflammatory response due to the donkey semen tended to be lower (P = 0.074). Donkey seminal plasma had anti-inflammatory properties compared to horse semen and seminal plasma, as determined by fewer neutrophils on uterine cytology (P < 0.05). Horse semen resulted in greater concentrations of IL6 and lesser concentrations of IL1B (P < 0.05). PGE1, PGE3, and lactoferrin concentrations were significantly more abundant in donkey sperm and seminal plasma. Prostaglandins play an important role in immunomodulation and might contribute to the response triggered in interspecies breeding. In conclusion, breeding horse mares with donkey semen induces similar post-breeding endometritis as observed with horse semen. Donkey seminal plasma results in a lower post-infusion inflammatory response compared to other combinations in the immediate post-breeding.

Luteal tissue blood flow and side effects of horse-recommended luteolytic doses of dinoprost and cloprostenol in donkeys.

This study assessed luteolysis and side effects in jennies receiving standard horse-recommended doses of cloprostenol and dinoprost. Sixteen cycles of eight jennies were randomly assigned in a sequential crossover design to receive dinoprost (5 mg, i.m.) and cloprostenol (0.25 mg, i.m.) at 5-d post-ovulation. B-mode and Doppler ultrasonography were employed to assess luteal tissue size and blood flow before (-15 min and 0h) and after (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 48h) administering PGF2α. Immunoreactive progesterone concentrations were assayed at similar timepoints via RIA. Side effects such as sweating, abdominal discomfort, and diarrhea were scored at 15-min-intervals for 1h after PGF2α. Data normality was assessed with the Shapiro-Wilk's test. Luteal tissue size and blood flow were analyzed using PROC-MIXED and post-hoc by Tukey. Non-parametric tests analyzed side effect variables. The luteal blood flow increased overtime by 27% at 45 min and peaked by 49% at 3 h for dinoprost, and conversely, it increased by 14% at 30 min and peaked at 39% at 5h for cloprostenol (P<0.05). Luteal blood flow was reduced by 50%, 25%, and 10% on both groups at 8, 12, and 24h (P<0.05). Immunoreactive progesterone concentrations decreased in 0.5h for dinoprost and 1h for cloprostenol and gradually decreased by 48h (P<0.05). Dinoprost induced greater sudoresis scores, while cloprostenol resulted in greater abdominal discomfort and diarrhea scores (P<0.05). In conclusion, dinoprost and cloprostenol effectively induced luteolysis with distinct side effects; this could guide practitioners' case selection to use one or another PGF2α.

The Temporal Associations of B-Mode and Power-Doppler Ultrasonography, and Ovarian Steroid Changes of the Periovulatory Follicle and Corpus Luteum During Luteogenesis and Luteolysis in Jennies.

This study aimed to determine the associations between B-mode and Power-doppler ultrasonography and ovarian steroids of the periovulatory follicle and respective corpus luteum (CL) during luteogenesis and luteolysis in jennies. Twenty-four periovulatory follicles/estrus of correspondent one inter-ovulatory interval (n = 12 jennies) were assessed in the study. B-mode ultrasonography and teasing were carried out once day until the detection of a periovulatory follicle (≥28 mm, uterine edema, and signs of estrus). Thereafter, jennies were monitored at 4-hour-intervals by B-mode and Power-doppler ultrasonography. Closer to ovulation, jennies were hourly checked. Each CL was checked daily from luteogenesis to luteolysis. Plasma concentrations of estradiol and progesterone were assessed daily with chemiluminescence immunoassay. Granulosa echogenicity and thickness increased from -36 hour to -1 hour before ovulation in 70% of follicles (P < .05) and were strongly associated with impending ovulation (r = 0.80 and r = 0.70, respectively). The follicular-wall blood flow increased from -72 to -24 hour pre-ovulation, while the estradiol concentration declined from 42 pg/mL by -72 hour to 31.6 pg/mL by 24 hour before ovulation (P < .05). The vascularization of the periovulatory follicle decreased from 62% (-36 hour) to 37% (-1 hour) before ovulation (P < .05). The CL vascularization and progesterone concentration gradually increased, reaching the peak at 11- and 10-day after the ovulation, respectively (P < .05). The CL vascularization started to decline 3 day before luteolysis, while progesterone concentrations started to drop 4 day before luteolysis (P < .05). In conclusion, the structural changes of the periovulatory follicle detected on B-mode and Power-doppler can be used to detect impending ovulation in donkeys; however, Power-doppler, but not B-mode ultrasonography, can be used to assess CL function in jennies.

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.

Uterine Inflammatory Response After Prostaglandin E1 (Misoprostol) Infusion Prebreeding or Immediately After Embryo Flushing in Commercial Donor Mares.

Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +200 µg) or sham (3 mL of lactate Ringer's solution) infusions, bilaterally deposited via deep-horn, at least 72 hours prebreeding (experiment 1) or immediately after embryo flushing (experiment 2). Each mare had one cycle for misoprostol and sham in both experiments and a breeding cycle (no sham or misoprostol) between experiments. Uterine edema, fluid accumulation, and the number of uterine PMN were assessed before each infusion and then daily for 72 hours. Uterine lavage was performed the day after each infusion across groups and experiments. Ovulation was hastened with a GnRH agonist and confirmed at 24 hour-intervals. Mares were bred with semen from one of six stallions per owner's choice. Embryo flushing was performed 8 to 9 days postovulation. In either experiment, misoprostol did not affect uterine edema or fluid accumulation (P > .05). However, both the sham and misoprostol infusions increased the number of PMN up to 48 hours postinfusion in both experiments. Embryo recoveries were similar between sham (45%, 5/11) and misoprostol cycles in experiments 1 (45%, 5/11; P > .05) and 2 (sham, 68%, 7/11; misoprostol, 45%, 5/11; P > .05). In conclusion, misoprostol did not induce exacerbated uterine inflammation in mares or systemic adverse reactions when infused prebreeding or immediately after embryo flushing.

Luteal Tissue Area and Immunoreactive Concentration of Progesterone in Plasma of Bred and Non-bred Mares.

Progesterone is pivotal to maintain pregnancy in the first trimester and low concentration (<4 ng/mL) has been associated with early pregnancy loss. Measurement of progesterone is widely used in practice to determine whether a mare needs progestin supplementation. Therefore, the objective of the present study was to determine progesterone concentration and the luteal tissue area in mares non-bred, and those bred becoming pregnant, and those failing to become pregnant. We hypothesized that pregnant mares have greater progesterone concentration than non-pregnant mares. Fifty-two cycles of mares (n = 14) were monitored by ultrasonography every other day until detection of a pre-ovulatory follicle. Then deslorelin acetate was administered to induce ovulation. Twenty-four hours later, mares were bred (∼2 billion progressively motile sperm extended in 50 mL; n = 37 cycles) or a sham-bred (50 mL of extender; n = 15 cycles). Ovulation was confirmed and number of corpora lutea and the luteal tissue area were recorded daily until 10-days post-ovulation. Progesterone concentration was assessed daily from the day of the ovulation up to 10-days post-ovulation. Pregnancy diagnosis was carried out at 10- and 13-days post-ovulation. Of the bred mares, 20 of them became pregnant and 17 did not. Data were analyzed with a mixed model, Tukey's test as post-hoc, and Pearson's coefficient of correlation. Progesterone concentration and luteal tissue area varied with time (P = .001) but not with group (P > .05). Multiple ovulations were associated with greater progesterone concentration and luteal tissue area (P = .0001). There was a moderate positive association between the number of ovulations and luteal tissue area (r = 0.54; P = .0001). The lack of change in the progesterone concentration and luteal tissue area between bred and non-bred mares suggests that horse seminal plasma does not affect luteal function in mares. As all mares had progesterone above 4 ng/mL after 5-days post-ovulation; it is possible that if mares with abnormal progesterone concentration were used, the results could have been different. In conclusion, pregnancy was not associated with greater progesterone concentration or luteal tissue area.

Fetoplacental Unit Ultrasonography and Immunoreactive Steroid Concentrations in Jennies Experiencing Late-Term Pregnancy Loss.

This study aimed to compare ultrasonographic features and steroid concentrations of jennies undergoing late-term pregnancy loss (n = 5) with gestationally age-matched health controls (n = 5). Transrectal ultrasonography of the combined thickness of uterus and placenta (CTUP) and fetal eyeball diameter was carried out at 15-day-intervals. Fetal heartbeat, aortic, and thorax diameters were determined by transabdominal ultrasonography at 30-day-intervals. Blood samples were collected simultaneous with each transrectal ultrasonography to determine progestogen and estradiol concentrations. Data were assessed for normality with Shapiro-Wilks. Data were log-transformed and analyzed with a mix model. Non-normally distributed data were analyzed with Kruskal-Wallis. Post-hoc analyzes were performed with Sidák's or Dunn's tests based on distribution. Gestational length between groups was compared with a t-test. Statistical significance was set at P < .05. The gestational length was shorter in jennies experiencing pregnancy loss (345 ± 32.3 vs. 365.4 ± 10.4 d P = .0009). Increasing gestational age (P < .0001) and pregnancy loss group (P = .004) had greater CTUP measurements with an interaction between them (P = .01). Fetal eyeball diameter increased with gestational age (P < .0001) but did not vary with group (P = .26), and there was no interaction between gestational age and group (P = .71). Fetal aortic and thorax diameters increased with gestational age (P < .0001), but an interaction between gestational age and group was only present with thorax diameter (P = .01). No effect of group was found for aortic (P = .78) or thorax (P = .86) diameters. Group (P = .06) and gestational age (P = .07) tended to be associated with an increased fetal heartbeat, but there was no interaction between them (P = .98). There was no effect of gestational age (P = .31), group (P = .19), or interaction between them for progestogens concentrations (P = .21). Estradiol concentration was not affected by gestational age (P = .76) or group (P = .51). In conclusion, late-term pregnancy loss was associated with increased CTUP measurements in donkeys.

Ovulatory response to GnRH agonist during early and late fall in mares.

This study aimed to assess the ovulatory response of deslorelin acetate during the fall and the response to PGF2α 8 d post-ovulation. One hundred estrous cycles from 22 mares kept in 40° latitude were evaluated. Mares were checked by transrectal ultrasonography until a preovulatory follicle was detected and ovulation induced with deslorelin acetate. Ovulation was confirmed by ultrasonography performed at 24, 36 h post-induction and then repeated at 2-h intervals post-induction. Serum progesterone concentrations and luteal tissue area were determined daily to assess CL function. A dose of PGF2α was administered 8 d post-ovulation and interval to the subsequent ovulation was observed; each mare completed up to five cycles. The effects of local climate on endpoints were analyzed. Cycles were grouped as early (Sept 13, 2020-Oct 31, 2020; n = 55; 22 mares) and late fall (Nov 1, 2020-Dec 31, 2020; n = 45; 20 mares) based on the date of induction. The overall number of cycles with ovulations between 24 and 48 h was 90%. The number of multiple ovulations were similar between early (n = 5) and late (n = 4) fall (P = 0.87). There were no differences in deemed spontaneous ovulations occurring before 24 h between early (n = 6) and late (n = 2) fall (P = 0.29). Two failures to respond to deslorelin by 48 h were recorded in early fall and none in the late fall. The interval from induction to ovulation was similar in early (40.6 ± 0.4 h) and late (41.2 ± 0.5 h) fall (P = 0.55). The percentage of mares ovulating between 36 and 48 h post-deslorelin did not vary between early and late fall (91 vs. 95%, P = 0.21), as did not for ovulation occurring between 38 h and 44 h (62 vs. 60%, P = 0.69). Edema scores varied with time relative to ovulation (P < 0.001) and were lower in late fall (P = 0.01). Progesterone concentrations varied with time (P < 0.001) but did not differ between early and late fall (P = 0.73) and correlated weakly with the luteal area (r = 0.13; P = 0.031). Follicles <35 mm at the PGF2α had a shorter interval to the next ovulation than follicles ≥ 35 mm (9.2 ± 0.5 d vs. 10.6 ± 1.2 d) (P = 0.03). Lower temperature was associated with a smaller follicle size at induction (P = 0.0021) and ovulation (P = 0.009) and lower relative humidity was associated with a larger follicle size at ovulation (P = 0.032). In conclusion, cycling mares displayed a highly efficacious response to deslorelin acetate and apparently normal luteal function during the fall, despite lower edema scores in late fall.

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare.

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo's signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10-13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.

Steroidogenic Enzyme and Steroid Receptor Expression in the Equine Accessory Sex Glands.

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3ß-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3ßHSD), P450,17α hydroxylase, 17-20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-ß, and 3ßHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p > 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p < 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3ß-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed.

Epididymal Sperm Granuloma and Antisperm Antibodies in Donkeys.

This study aimed to describe and compare semen parameters (pre-freeze and post-freezing) and antisperm antibodies (ASA) of donkeys with epididymal sperm granuloma and healthy controls. Feral donkeys (n = 10) castrated in a concurrent study were enrolled in the present experiment. Three donkeys had unilateral granulomas, two donkeys had bilateral granulomas, whereas the remaining five had grossly normal epididymides. The granulomas were either single or multiple, firm, well-circumscribed, tan to red, and 1-5 mm in size. Upon incision, abundant, thick, tan to white-yellow fluid was recovered. Histopathology revealed epithelioid macrophages, multinucleated giant cells, and abundant sperm cell fragments with mineralized cellular debris. Each epididymis was dissected, and semen harvested for cryopreservation. Semen was assessed for sperm motility parameters, plasma membrane integrity, and mitochondrial membrane potential. All donkeys had semen cryopreserved in a standard manner. In addition, post-thaw semen from all donkeys was assessed for ASA (IgG and IgA), acrosome integrity and morphology. Post-thaw, the progressive sperm motility and the percentage of sperm with an intact plasma membrane were reduced in donkeys with sperm granuloma (P = 0.04). There was no difference in total sperm motility, morphology, or damaged acrosome across groups (P > 0.05). Three donkeys with sperm granuloma (60%) displayed increased IgG and IgA ASA. In conclusion, sperm granulomas only marginally affected sperm quality and resulted in IgG ASA binding to sperm with damaged plasma membrane. It remains to be determined if sperm granuloma and ASA affect fertility in donkeys.

Equine endometrial development during late fetal and postnatal periods.

Equine uterine development, including endometrial histogenesis, begins prenatally and is completed postnatally. Little is known about this process in the horse. Uterine tissue was acquired from 38 foals, ranging in developmental age from gestational day (GD) 300 to postnatal day (PND) 180, for assessment of endometrial histogenesis. Patterns of endometrial cell proliferation were evaluated by multispectral imaging of uterine tissue sections stained immunofluorescently for Ki-67. Labeling index (LI, % labeled cells) for Ki-67 was calculated for each endometrial cell compartment (luminal epithelium, glandular epithelium, stroma). Histologically, nascent endometrial glands were present in all pre- and postnatal uterine tissues. Overall, Ki-67 LI increased (P < 0.0001) from the pre-to postnatal periods, and was higher (P < 0.0001) in epithelium as compared to stroma. Postnatally, endometrial Ki-67 LI increased (P < 0.0001) from week 1 to week 24. Our findings confirm that, in contrast to neonatal patterns of uterine development described for domestic ungulates, equine endometrial histogenesis begins prenatally, marked by the appearance of uterine glands as early as GD 300. Epithelial proliferation associated with maturation of the equine endometrium is pronounced by postnatal week 24.

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing.

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey's test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.

Administration of nerve growth factor-ß to heifers with a pre-ovulatory follicle enhanced luteal formation and function and promoted LH release.

The objective of this study was to determine the effects of bovine nerve growth factor-ß (NGF) on pre-ovulatory follicle vascular area, LH release, ovulation, and luteal function when administered systemically to heifers. Post-pubertal Holstein heifers (n = 12) received an intravaginal progesterone-releasing device (CIDR) and GnRH agonist (100 µg IM). The CIDR was removed 5 d later, and heifers were given dinoprost (25 mg IM) at CIDR removal and 24 h later, followed by a second dose of GnRH agonist 48 h later. Heifers were randomly assigned to treatments using a cross-over design. For example, heifers assigned to NGF (250 µg reconstituted in 12 mL PBS IM) in replicate 1 were assigned to control (12 mL PBS IM) in replicate 2. Transrectal ultrasonography was performed before treatment and repeated every 4 h up to 32 h to determine the pre-ovulatory follicle diameter, vascular area, and ovulation. Serum samples were obtained to assess LH concentrations during the periovulatory period and every 2 d post-ovulation for measuring progesterone concentrations. A subset of heifers had luteal biopsies performed on days 9 (n = 6 per treatment) and 14 (n = 6 per treatment) post-ovulation to count luteal cell numbers and measure relative mRNA abundance for steroidogenic and angiogenic enzymes and LH receptor. Treatment with NGF increased pre-ovulatory follicle diameter (P = 0.02) and serum LH concentrations (P = 0.03) but did not affect time to ovulation (P = 0.42). Heifers treated with NGF had increased serum progesterone concentrations in the subsequent luteal phase (P = 0.03), but no change in vascular area of the follicle (P = 0.16) or CL (P = 0.20). Heifers treated with NGF had a greater number of small luteal cells (P < 0.01) and a tendency for increased LH receptor (LHR) mRNA abundance in the CL (P = 0.10). There was also increased steroidogenic acute regulatory protein (STAR; P = 0.05) and a tendency for increased cytochrome P450 family 11 (CYP11A1; P = 0.10) mRNA abundance in the CL of NGF-treated heifers. There was decreased prostaglandin E2 synthase (PGES; P = 0.03) and its receptor (PGER; P = 0.05) mRNA abundance and a tendency for decreased cytochrome P450 family 17 subfamily A member 1 (CYP17A1; P = 0.08) and hydroxysteroid 17-beta dehydrogenase (HSD17B; P = 0.06) mRNA abundance in the CL of NGF-treated heifers. Administration of NGF improved CL function in heifers potentially as a result of increased LH release.

Biomarkers for placental disease in mares.

Placentitis is an important cause of abortion, stillbirth, and neonatal death in horses. The diagnosis of placentitis is based on occurrence of clinical signs (premature mammary gland development and vulvar discharge) and ultrasonography of the caudal placental pole. However, early and subtle cases can be missed. In the last few years, several studies have provided objective means of diagnosing placentitis in mares with single or serial measurements of blood markers. Among the markers evaluated the steroids produced by the fetoplacental unit have been shown to change in association with placentitis. Mares with chronic placentitis have an increase in peripheral progestogens; however, mares acutely infected will display a reduction in peripheral concentrations of progestogens. Estradiol-17ß (free- and conjugated form) concentrations are drastically reduced in plasma of mares with placentitis. Acute-phase proteins, particularly serum amyloid A, are increased in plasma of mares suffering from placentitis, and this increase is due to endometrial and chorioallantoic secretions, and minimally from the fetus. Alpha-fetoprotein, a protein expressed in the fetoplacental unit, was shown to be increased in plasma of mares suffering from placentitis. A plephora of microRNA have been identified in plasma and tissues of mares undergoing experimentally induced placentitis, but have not been tested in spontaneous cases. Unique proteomic signatures were found in the fetal fluids of mares undergoing experimentally induced ascending placentitis, making the fetal fluids potentially useful to diagnose placentitis in mares. However, currently the lack of use of transabdominal fetal fluid sampling prevents wide use of the fetal fluids as diagnostic techniques. This manuscript aimed to discuss recent discoveries regarding biomarkers for placentitis in mares.

Assessment of peripheral markers and ultrasonographic parameters in pregnant mares receiving intramuscular or intrauterine cloprostenol.

The present study aimed to compare two methods of prostaglandin-induced abortion in mares by determining blood markers (progesterone, estradiol-17ß, alpha-fetoprotein, 13,14-dihydro-15-keto-prostaglandin-F2α (PGFM)), B-mode ultrasonographic parameters, and time until loss of fetal heartbeat. It was hypothesized that intrauterine infusion of cloprostenol results in earlier fetal compromise than intramuscular administration. Ovarian structures (number and sizes of follicles and corpora lutea area), fetal heartbeat, and fetal mobility of thirteen singleton pregnancies were assessed daily by transrectal ultrasonography until induction of pregnancy termination (60 ±â€¯2 days of gestation). Mares received 500 µg of cloprostenol intramuscularly every 12 h (IM, n = 7) or once transcervically (TC, n = 6). After initial cloprostenol administration, ultrasonographic examinations were repeated at 6-h intervals until loss of fetal heartbeat was detected. Plasma progesterone, estradiol-17ß, and alpha-fetoprotein were assessed for five days before and after pregnancy loss. In addition, plasma PGFM concentrations were assessed immediately before cloprostenol administration (0 min), and then 15, 30, and 45 min, and 1, 2, 3, 4, 6, 12 h after administration. Data were analyzed using the MIXED procedure with repeated measures in SAS. Significance was set at P < 0.05. All mares lost their pregnancies within 48 h after initial cloprostenol administration, with no difference in time to pregnancy loss. There were significant effects of time starting by 12 h post-induction of pregnancy termination but there was no time by group interaction for progesterone concentrations. Estradiol-17ß and alpha-fetoprotein concentrations were not altered upon impending abortion. Concentrations of PGFM increased significantly by 2 h after cloprostenol administration, but there were no differences between groups. No time effects or time by group interaction for fetal mobility and heartbeat was detected. Expectedly, the number and area of corpora lutea decreased significantly after cloprostenol administration with no significant differences between groups. In conclusion, intrauterine administration of cloprostenol was not different from repeated systemic administration to terminate the pregnancy. Both models for early fetal loss were equivalent for the endpoints assessed herein. The present study provides evidence that transcervical cloprostenol administration technique is repeatable in different settings and results in negligible side effects. While systemic administration results in colic-like signs and may result in severe reaction.

Uterine responses and equine chorionic gonadotropin concentrations after two intrauterine infusions with kerosene post early fetal loss in mares.

Pregnancy loss during the normal lifespan of endometrial cups (∼37-120-150 days of gestation) may affect a mare's ability to conceive again in the same breeding season, as equine chorionic gonadotropin (eCG) secretion by retained endometrial cups can lead to abnormal ovulations and follicular growth. While intrauterine kerosene infusion has anecdotally been proposed as a treatment for endometrial cup retention, there are no controlled studies evaluating kerosene's ability to enhance endometrial cup regression following abortion. The objectives of this study were to assess uterine response, systemic side effects, and efficacy of intrauterine kerosene infusions after abortion. We hypothesized that kerosene infusions would hasten regression of endometrial cups without detrimental effects on the endometrium and the mare's general health. Twelve light-breed mares were enrolled in the study after an experimentally induced abortion with cloprostenol (n = 12) by 60 ± 2 days of gestation. Mares were randomly allocated to receive an intrauterine infusion with 500 mL of kerosene (n = 6) or 500 mL saline (n = 6) on days 21 and 35 after pregnancy termination. Uterine biopsies were collected at days 7, 21, 35, and 49 post-abortion to evaluate the degree of endometrial fibrosis with Picrosirius Red Stain and to be graded according to the Kenney & Doig 1986 classification. Furthermore, histomorphometry analysis of the endometrium lining, glandular epithelium and glandular density was performed. Endometrial lymphocyte B CD20+, lymphocyte T CD3+, and macrophage IBA-1+ cell populations were characterized by immunohistochemistry. Physical examinations, blood cell counts, and serum biochemistry were performed before, and for 2 days after each uterine infusion. Serum samples were collected for assessment of eCG concentrations. Continuous data were analyzed with MIXED procedure with repeated measures in SAS, categorical data with LOGISTIC procedure of SAS. Significance was set at p < 0.05. Kerosene infusion did not affect complete blood cell counts, serum chemistry parameters, or physical examinations. Concentrations of eCG decreased over time (p < 0.001), but there were no differences between groups or time by group interactions (p = 0.72). Histological evaluation of the uterus showed no signs of increased fibrosis or degeneration in the treatment group. In conclusion, while kerosene infusions did not appear to have detrimental effects on mare health, our findings suggest that the use of kerosene in the uterus does not enhance the regression of endometrial cups by 49 days post-abortion.

Fetal Death Associated With Premature Mammary Gland Development and Lactation in a Mare Treated With Weekly Injections of Long-Acting Progesterone.

A 14-year-old, 530-kg, multiparous, pregnant Quarter Horse mare was referred for evaluation of premature mammary gland development and lactation. The mare was in the seventh month of gestation. The mare had a history of subfertility and was receiving weekly injections of long-acting progesterone, prescribed by the referring veterinarian. The last dose had been administered four days before presentation. Upon presentation, the mare had vital signs within normal limits, a moderately developed, nonpainful udder with galactorrhea, and no vulvar discharge. Transrectal palpation revealed a well-toned uterus and cervix and discreetly palpable fetal parts, however, ballottement of the fetus did not result in appreciable fetal movement. Transrectal ultrasound was unremarkable, but transabdominal ultrasound revealed one underdeveloped, immotile fetus in the left uterine horn with no heartbeat. Abortion was induced with repeated doses of cloprostenol. Fifty-four hours after the first cloprostenol injection, the mare displayed signs of labor, the cervix was manually dilated, and the fetus and fetal membranes were expelled with gentle manual manipulation. Standard postabortion care included uterine lavage and oxytocin for 48 hours before being discharged to the care of her owners. Fetal crown-rump length (53 cm) was consistent with a 6-month fetus rather than its gestational age of 7 months. The umbilical cord was edematous, and a distended, fluid-filled structure surrounded the cord at the intersection of the allantoic and amniotic segments of the umbilical cord. This structure was determined to be the severely dilated urachus. Microscopic findings included placental stromal mineralization, distended umbilicus adventitia, and dilated umbilical lymphatics with no other significant findings. Remaining abortion diagnostic tests were unremarkable. The mare recovered well and was discharged to the care of her owner two days after abortion. The following breeding season the mare carried a healthy foal to term.

Cortisol, progesterone, 17αOHprogesterone, and pregnenolone in foals born from mare's hormone-treated for experimentally induced ascending placentitis.

This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no treatment (NO TREAT n = 10). At delivery, foals were classified as high-risk or low-risk based on clinical and hematologic findings, and survival rates were recorded during the first week of life for comparisons across groups. Cortisol, progesterone, 17αOHprogesterone, and pregnenolone concentrations were determined via immunoassays in 31 of the 46 foals immediately after foaling (0 h), at 12, 24, 48 h, and seven days post-partum (168h). At birth, serum cortisol concentrations were higher in Control and TMS+FM+ECP foals than in remaining groups (p < 0.05). Foals in TMS+FM+ALT and TMS+FM groups had higher 17αOHprogesterone concentrations at 24 h and 48 h, respectively (p < 0.05). Pregnenolone concentrations were higher in TMS+FM than TMS+FM+ALT+ECP foals at 7 days (p < 0.05). High-risk and non-surviving foals had decreased concentrations of cortisol at parturition, but increased concentrations of progesterone from 0 h to 48 h. Pregnenolone and 17αOHprogesterone concentrations were increased and pregnenolone after 12 h in high-risk and non-surviving foals (p < 0.05). In conclusion, adding ECP to the treatment of experimentally-induced placentitis appears to improve foal viability and endocrine response. Cortisol and progestogen profiles were abnormal in high-risk and non-surviving foals, and those treated with ALT or TMS+FM only.

Endocrine and metabolic profile of peripubertal Standardbred colts.

The objectives of this study were to determine the concentrations of reproductive and metabolic hormones during the peripubertal period and to assess their relationship with testicular development and body fat deposition. Blood samples were collected from 23 healthy Standardbred colts every four weeks for twelve months. Colts were weighed monthly, and percent of body fat and testicular volume estimated by ultrasound. Onset of puberty was determined as the month when testosterone was two standard deviations above the previous mean. Plasma FSH, LH, leptin, estradiol-17ß, androstenedione, IGF-1, insulin, inhibin-A, and inhibin-B were analyzed for a seven month peripubertal period. Spring born Standardbred colts underwent puberty at 13 months of age; onset of puberty coincided with exponential testicular growth but did not coincide with an increase in cutaneous body fat deposition or leptin (p > 0.05). Plasma inhibin-B concentrations were significantly increased in the postpubertal period (p < 0.05), but no increase was seen in inhibin-A, androstenedione, FSH, LH, or estradiol-17ß. In conclusion, the rise in testosterone and subsequent onset of puberty coincides with rapid testicular growth but is not correlated with an increase in gonadotropins, IGF-1, cutaneous body fat or leptin in the horse.