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.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

BACKGROUND: Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. OBJECTIVE: To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. STUDY DESIGN: Controlled and field study. METHODS: In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. RESULTS: Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). MAIN LIMITATIONS: Low number of mares in the fertility trial. CONCLUSION: Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

Conductivity of mammary gland secretions is a sensitive and specific predictor of parturition in mares.

BACKGROUND: Accurate prediction of parturition is paramount to ensuring monitoring of delivery and preventing complications. Assessing the pH and electrolytes of the mammary gland secretions (MGS) helps detect impending parturition. As conductivity is related to electrolyte concentrations and pH, it could be a useful alternative for predicting impending parturition; however, this hypothesis warrants a critical assessment. OBJECTIVES: To assess the ability of conductivity, pH, and Brix in the MGS to predict parturition and to investigate their associations. STUDY DESIGN: Field study. METHODS: The MGS of periparturient mares (n = 241) was assessed daily for conductivity, pH, and Brix index from 320d until parturition. Receiving operating curve cut-off values for conductivity (≤4.8 mS/cm), pH (≤6.4), and Brix index (>23.6%) were used to calculate sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for predicting parturition in ≤24 h. RESULTS: Impending parturition was associated with a reduction in conductivity and pH (p < 0.05), and conductivity was strongly correlated with pH (r = 0.88) and Brix (r = -0.80) (p < 0.05). Sensitivity, specificity, PPV, and NPV for parturition in ≤24 h for conductivity (82%, 91%, 77%, and 92%, respectively), pH (79%, 84%, 81%, and 71%, respectively), and Brix (72%, 79%, 66%, and 83%, respectively) were determined separated and pairwise. Of interest, the sensitivity, specificity, PPV, and NPV, of combining conductivity and pH, were 80%, 95%, 90%, and 88%, respectively. Conductivity (≤4.8 mS/cm) presented the greatest odds ratio for predicting parturition in ≤24 h, and coupling it with pH (≤6.4 pH units) doubled its odds ratio (i.e., 25.4-62.3). MAIN LIMITATIONS: Field study. CONCLUSION: The conductivity of MGS is a sensitive and specific method to predict parturition. This is the first large-scale study showing that a combination of conductivity and pH is useful for predicting parturition in mares. The methods employed can likely apply to other settings with similar results.

Mule embryos share identical morphological features to horse embryos.

This study aimed to compare the morphometry of horse and mule embryos. The study's hypothesis was that the micronuclei and nuclear fragmentation indexes are higher in mule embryos than in horse embryos. Twenty-two mares were randomly assigned in a crossover design to receive semen from a horse and a donkey; thirteen horse and thirteen mule embryos were obtained. Embryos were recovered eight days post-ovulation and classified according to the stage of development and quality with a score from 1 (excellent) to 4 (degenerate). Embryos were stained with Hoechst33342, and images were acquired with a fluorescence microscope. Nuclei were categorized as compact, mitotic, or fragmented; the fragmented and mitotic indexes were calculated based on their proportion over the total amount of nuclei counted. Embryo size and nuclear morphometry were assessed through ImageJ. Data analyses were carried out with GraphPad using ANOVA and T-test; significance was set at P < 0.05. The number of positive flushes in cycles bred with donkey or stallion semen did not differ when compared per cycle or per ovulation (13 vs. 12) (P > 0.05). One set of twins was recovered from a mare bred to the stallion that had a double ovulation; a mule and horse embryos were both recovered from eight mares. There was no difference in size between mule and horse embryos (915.5 ± 288 µm vs. 575.8 ± 69.6 µm) (P > 0.05) size of the study. The mule embryos scored between grade 1 (n = 9) and grade 2 (n = 4); similarly, the horse embryos scored between grade 1 (n = 6) and grade 2 (n = 7). The evaluation of the nuclear morphometry revealed that horse and mule embryos have a similar number of compact nuclei per sector (148.7 ± 6.8 nuclei/sector in mule embryos vs. 156.5 ± 8.5 nuclei/sector in horse embryos) (P > 0.05); however, the number of mitotic nuclei tended to be higher in mule embryos (5.2 ± 0.82) than in horse embryos (3.3 ± 0.3) (P = 0.08). The fragmented nuclei index was similar between mule (0.25 ± 0.1%) and horse (0.22 ± 0.1%) embryos (P = 0.4); the mitotic nuclei index was higher in mule embryos (3.2 ± 0.4%) than in horse embryos (2.2 ± 0.2%) (P = 0.02). In conclusion, embryo morphology of mares bred to a donkey and a horse shares similar nuclear ultrastructure features, except that mule embryos have a higher mitotic index.

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.

Characterization of rumen microbiome and metabolome from oro-esophageal tubing and rumen cannula in Holstein dairy cows.

Less invasive rumen sampling methods, such as oro-esophageal tubing, became widely popular for exploring the rumen microbiome and metabolome. However, it remains unclear if such methods represent well the rumen contents from the rumen cannula technique. Herein, we characterized the microbiome and metabolome in the rumen content collected by an oro-esophageal tube and by rumen cannula in ten multiparous lactating Holstein cows. The 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Untargeted metabolome was characterized using gas chromatography of a time-of-flight mass spectrometer. Bacteroidetes, Firmicutes, and Proteobacteria were the top three most abundant phyla representing ~ 90% of all samples. Although the pH of oro-esophageal samples was greater than rumen cannula, we found no difference in alpha and beta-diversity among their microbiomes. The overall metabolome of oro-esophageal samples was slightly different from rumen cannula samples yet more closely related to the rumen cannula content as a whole, including its fluid and particulate fractions. Enrichment pathway analysis revealed a few differences between sampling methods, such as when evaluating unsaturated fatty acid pathways in the rumen. The results of the current study suggest that oro-esophageal sampling can be a proxy to screen the 16S rRNA rumen microbiome compared to the rumen cannula technique. The variation introduced by the 16S rRNA methodology may be mitigated by oro-esophageal sampling and the possibility of increasing experimental units for a more consistent representation of the overall microbial population. Studies should consider an under or over-representation of metabolites and specific metabolic pathways depending on the sampling method.

Semen Quality of the First and Second Ejaculates Collected from Breeding Inactive Stallions after Cooling and Freezing.

This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After collection (n = 40 ejaculates), the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of each ejaculate was extended and cooled for 48 h; a second aliquot was cushion-centrifuged and cooled for 48 h; and a third aliquot was processed and then frozen. The total motility (TM) and progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP) were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The second ejaculate had a lower gel-free semen volume (p = 0.026). The sperm concentration was greater in the first than in the second ejaculate (p < 0.001). The sperm morphology was similar between the ejaculates (p > 0.05). Cushion-centrifugation prevented a reduction in the TM, PM, and PMI over time (p < 0.05). The TM, PM, and PMI decreased after freezing but not between the ejaculates (p > 0.05). The first and second ejaculates of the season, which were collected 1 h apart, varied in quantity but not in quality after cooling and freezing.

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.

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy.

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare.

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.

High-Risk Pregnancy Is Associated With Increased Alpha-Fetoprotein Concentrations in the Amniotic Fluid and Foal Plasma.

This study aimed to determine alpha-fetoprotein (AFP) concentrations in amniotic fluid, plasma of mares and respective foals: carrying normal pregnancies and delivering healthy foals (n = 20; Group 1); carrying apparently normal pregnancies and delivering sick foals (n = 15; Group 2); carrying high-risk pregnancies and delivering sick foals (n = 14; Group 3). High-risk pregnancy was defined by a history of premature udder development/lactation or increased of the combined thickness of the uterus and placenta, or vulvar discharge and/or mares' systemic illness. Sick foals were affected by neonatal encephalopathy, sepsis, prematurity/dysmaturity, or hypoxic-ischemic encephalopathy. Based on histological examination of the chorioallantois, AFP trend was analyzed in pregnancies with pathologic (PFM) and normal fetal membranes (NFM). Concentrations of AFP were measured using a commercially available immunoassay previously validated for horses. Mares' plasma AFP did not change during the last 15-20 days of pregnancy in the three groups, and there was no difference among them. Amniotic fluid AFP was higher in Group 3 (P = .014). Foals' plasma AFP concentration was higher from birth to 72hours in foals of Group 2 and 3 than in healthy ones, and foals of Group 3 had the highest value. The strong association (r = 0.84; P < .0001) between AFP in amniotic fluid and foals' plasma at birth is likely due to the presence of AFP in fetal urine. AFP was higher in pregnancy with PFM than with NFM in mare's plasma at admission (P = .031), amniotic fluid (P = .004), foal's plasma at birth (P = .002), at 24 (P = .005) and at 72 hours of life (P = .004). AFP is higher in pregnancy with histopathological lesions of the chorioallantois providing the evidence of the differences between pregnancy with a normal placental barrier and the more compromised ones. The increased AFP concentration in the amniotic fluid and plasma of high-risk foals suggests upregulation.

Evaluation of Recombinant Bovine Interleukin-8 (rbIL-8) as a Treatment for Chronic Intramammary Infection in Dairy Cows.

Mastitis is one of the main contributors to antimicrobial resistance in livestock, so alternative therapies are being investigated to address it. The present study assessed the capability of recombinant bovine interleukin-8 (rbIL-8) to improve neutrophil function in the mammary gland and resolve chronic high somatic cell count (SCC) in Holstein cows. Multiparous cows (n = 8) with more than 300,000 SCC per mL were allocated to one of two intramammary infusions: saline (10 mL of saline solution) or rbIL-8 (1.57 mg/mL of recombinant bovine IL-8 diluted in 9 mL of saline). In addition, there was an untreated control group (n = 2, SCC < 300,000 SCC/mL). Milk samples were collected post-treatment at 0, 4, 8, 12, 24, 48, and 144 h to quantify milk SCC, haptoglobin, and IgG concentrations. Neutrophil's phagocytosis in milk and blood was evaluated via flow cytometry at 0, 24, and 48 h. The log of SCC did not differ between the infused groups (p = 0.369). Neutrophils presented a similar log of cells with high fluorescence for propidium-iodide (PI) and dihydrorhodamine (DHR) in milk (p = 0.412) and blood samples (p = 0.766) in both infused groups. Intramammary infusion of 1.57 mg/mL of rbIL-8 did not improve neutrophils response and failed to resolve chronic high SCC.

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.

Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids.

This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentration was greater in donkeys than horses (684 ± 62.9 vs. 494 ± 50.9 million sperm/mL) (p = 0.02). The total sperm harvested was lower in SF (3.6 ± 0.7 billion) than RF (10.4 ± 1.5 billion) and in horses (4.6 ± 0.8 billion) than in donkeys (10.7 ± 1.8 billion) (p < 0.05). RF followed by SF resulted in 57% and 31% more sperm per harvest in donkeys and horses. Results of TM and PM before freezing were not affected by technique or species (p > 0.05). Post-thawing SMI and MMP did not vary with technique or species (p > 0.05); TM and PM were not influenced by the technique or the species (p > 0.05) but by their interaction (p = 0.005). In conclusion, using RF followed by SF enhances sperm recovery without affecting cryopreservation in equids.

Age-related changes in testicular morphometry and function in Egyptian donkeys.

This study aimed to assess the effects of age on testicular morphometry and function in donkeys. Testes and epididymides of 57 donkeys were harvested immediately after slaughtering. The donkeys were grouped: young (1-4 years old, n = 13); adult (5-15 years old, n = 25) and aged (>15 years old, n = 19). Each testis and epididymis were weighed separately. Testicular volume was calculated. Epididymal sperm was harvested by retrograde flushing method, and sperm parameters were evaluated. The testicular parenchyma was immunolabelled for BAX and COX2. Adult and aged donkeys had greater testicular weight and volume than young (p < .05). Epididymal sperm concentration, motility and viability were greater (p < .05) in adults and aged (931.8 ± 39.3 and 858.2 ± 33.2 × 106 /ml) than in young animals (316.3 ± 72.8 × 106 /ml). Aged donkeys had a higher percentage of morphological sperm defects than the other categories (p < .05). Histological examination revealed the presence of age-related degenerative changes in testicular tissue of donkeys. Aged donkeys had higher COX2 protein expression than adult and young donkeys. BAX protein was overly expressed in adults than aged or young animals. In conclusion, advancement of age affects the testicular morphometry and function in donkeys.

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.

Assessment of Fetal Aortic, Thoracic, and Heartbeat in Healthy Donkey Pregnancies.

Practitioners are frequently requested to diagnose and stage pregnancy in donkeys with unknown breeding dates; however, scant work has been done to stage pregnancy in the species. Therefore, this study aimed to determine the association between measurements of fetal aortic, thoracic, and heartbeat with gestational age in donkeys carrying and delivering healthy foals. Multiparous Dezhou donkeys (n = 50) ranging from 4 to 16 years were enrolled in the study by 150 days of gestation. Transabdominal ultrasonography coupled with a 3.5 MHz sectorial convex transducer was performed at 30 day-intervals until delivery to obtain fetal aortic, thoracic, and fetal heartbeat measurements. Data were tested for normality with Shapiro- Wilk's test and then ANOVA and Tukey's. Statistical significance was set at P < .05. The mean duration of pregnancy was 356.6 ± 10.6 days (339-368). There were significant associations between gestational age and fetal aortic (r = 0.89) and thoracic (r = 0.88) measurements. Fetal heartbeat (r = -0.76) was negatively correlated with gestation length. The fetal aortic and thoracic measurements increased from the seventh month of gestation to the term (P < .001). The fetal heartbeat remained steady from 150 days to 270 days of gestation, then continuously decreased from 270 days until parturition (P < .001). In conclusion, fetal aortic and thoracic measurements are strongly associated with gestational age in donkeys. Fetal heartbeat decreases with advanced pregnancy.

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.

Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive tract. Two alleles (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16  S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.

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.