Lung Function Variation during the Estrus Cycle of Mares Affected by Severe Asthma.

While the prevalence of asthma is higher in boys than in girls during childhood, this tendency reverses at puberty, suggesting an effect of sex hormones on the disease pathophysiology. Fluctuations of asthma severity concurring with the estrus cycle are reported in women, but this phenomenon has never been investigated in mares to date. The objective of this exploratory study was to determine whether the estrus cycle modulates airway obstruction in severe equine asthma (SEA). Five mares with SEA during exacerbation of the disease were studied. The whole breath, expiratory and inspiratory resistance, and reactance were compared during the follicular and luteal phases of the estrus cycle. The reproductive tract was evaluated by rectal palpation, ultrasound, and serum progesterone levels. The inspiratory resistance and reactance improved during the luteal phase of the estrus cycle, and variation in progesterone levels and the dominant follicle size correlated with several lung function parameters. The fluctuation of airway dysfunction during the estrus cycle is noteworthy as deterioration of the disease could perhaps be expected and prevented by horse owners and veterinarians. Further studies are required to determine if the equine species could be a suitable model to evaluate the effects of sex hormones on asthma.

Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change.

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30-60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.

A combination of taurine and caffeine maintains sperm quality in equine semen during chilled storage.

OBJECTIVE: The objective of this study was to evaluate the effects of caffeine and taurine on the motility and viability of chilled equine semen. MATERIALS AND METHODS: A total of 12 ejaculates were collected from three mature stallions with proven fertility during the breeding season. The gel-free spermatic fraction of each ejaculate was divided into two aliquots and diluted with a semen extender (either INRA 96® or BotuSemen Gold®). The aliquots were then split and assigned to one of the six treatment groups: control (no supplement), caffeine (2 and 4 mM), taurine (25 and 50 mM), and a combination of caffeine (2 mM) plus taurine (25 mM). Samples were stored at 4°C and analyzed at different time points (0, 24, 48, 72, and 96 h) to evaluate total (TMOT) and progressive (PMOT) motility and viability by computer-assisted sperm analysis. RESULTS: Regardless of the extender, PMOT and TMOT decreased over time. However, compared with the control, the treatment with 4 mM caffeine significantly mitigated the decrease in PMOT at 72 h. Additionally, semen treated with a combination of caffeine plus taurine maintained a significantly higher PMOT at 96 h, with improved viability at all time points. CONCLUSIONS: The combination of caffeine plus taurine helps maintain chilled equine semen viability and progressive motility up to 96 h independently of the extender used.

Transvaginal uterine evisceration during labor in a Bengal queen.

CASE SUMMARY: A 2.5-year-old Bengal queen was admitted with a 12-h history of a mass protruding from the vulva during labor. At that time, three healthy kittens had already been delivered. Physical examination identified the mass as a portion of the uterus that was eviscerated without eversion of the mucosa. Exploratory laparotomy revealed a vaginal vault rupture with a large portion of the uterus herniated through the tear and eviscerated through the vulva. Ovariohysterectomy was performed, and a dead fetus was removed with the uterus. Reconstruction of the vaginal rupture required careful dissection and urethral catheterization. The queen recovered without complications. RELEVANCE AND NOVEL INFORMATION: Uterine evisceration through a vaginal tear is a very rare condition that sometimes is erroneously referred to as 'prolapse'. Uterine prolapse and uterine evisceration may have similar presenting signs; however, proper identification and surgical correction is key when the uterus is eviscerated. This case highlights the importance of differentiating these two conditions and of rapid identification and surgical intervention for successful patient survival.

Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes.

Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at cold (∼4 °C) temperatures. If so, this might allow longer holding periods that would ease shipping requirements. In this study, we compared oocyte maturation rates, as well as cleavage and blastocyst rates after ICSI, for COCs held at either room or cold temperatures overnight before the onset of in vitro maturation. In Exp. 1, COCs were shipped overnight in a commercial embryo holding medium, ViGRO (Vg), in insulated containers designed to hold at either room temperature (RT, ∼22 °C) or cold temperatures (Cold, ∼7 °C). Subsequent rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (39%, 90% and 41%, respectively) than in the Cold treatment (23%, 60% and 17%, respectively, P < .05). In Exp. 2, we compared Vg medium with a second commercial embryo holding medium, SYNGRO (Sy). There was no significant difference between Vg and Sy groups in any evaluated parameter within either RT or Cold treatments. Within each medium group and for both media combined, the rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (42%, 81% and 42%, respectively for the combined media) than in the Cold treatment (29%, 54% and 10%, respectively for the combined media, P < .05). We conclude that shipment of immature equine COCs at cold temperatures (∼7 °C) is detrimental to subsequent in vitro maturation and embryo production.

Effects of staining method and clinician experience on the evaluation of stallion sperm morphology.

Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification differs depending on the evaluator's experience. Semen was obtained from various horse breeds; 40 samples from stallions between 2 and 15 years of age were collected during the 2016 breeding season and stored in a 2% buffered formaldehyde solution until processing. For each sample, 3 semen smears were made and stained with Diff-Quik, eosin-nigrosin, and SpermBlue. All morphological parameters were then evaluated blindly using a light microscope by a novice evaluator and a more experienced evaluator. For each slide, 200 spermatozoa were examined randomly and classified according to their characteristics. For the identification of morphologically normal spermatozoa, no significant difference between evaluators was found with any of the staining methods used. In contrast, significant differences between evaluators were observed in the classification of some anomalies affecting mainly the midpiece and the tail. Poor dye fixation was observed with SpermBlue.

Blastocyst development after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage.

We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured were fertilized by ICSI. The maturation rate was similar between timing groups (29-36%), but was significantly lower than that for controls (73%). The 1m treatment yielded one blastocyst (11%), vs. 19% in controls. In Experiment 2, propylene glycol (PG) and trehalose (T) were also used. We compared two base solutions: M199 with 10% FBS (M199+), and 100% FBS; three cryoprotectant combinations: D-EG-S; PG-EG-S; and PG-EG-T; and two timings in vitrification solution: ∼30 s (30s) and 1 min (1m). The most effective treatment (FBS/PG-EG-T/30s) yielded 42% maturation, 80% cleavage and 1 blastocyst (10%), vs. 49%, 93% and 29%, respectively for controls (P > 0.1). In Experiment 3, we evaluated the toxicity of the M199/D-EG-S/1m and FBS/PG-EG-T/30s treatments, without actual vitrification. These treatments did not affect maturation but both significantly reduced blastocyst development (0% and 0%, vs. 21% for controls). This represents the second report of blastocyst development after vitrification of GV-stage equine oocytes, and presents the highest developmental competence yet achieved; however, more work is needed to increase the efficiency of this system.

Placental abnormalities in equine pregnancies generated by SCNT from one donor horse.

Placental changes associated with SCNT have been described in several species, but little information is available in this area in the horse. We evaluated the ultrasonographic, gross, and histopathological characteristics of placentas from three successful and five unsuccessful equine SCNT pregnancies, established using cells from a single donor horse. Starting at approximately 6-month gestation, the pregnancies were monitored periodically using transrectal (TR) and transabdominal (TA) ultrasonography (US) to examine the placentas, fetal fluids, and fetuses. Of the five mares that aborted, one mare did so suddenly without any abnormal signs detected by US and four had enlarged umbilical vessels visible on TA-US before abortion. Placental edema (TR-US) and intravascular thrombi in the umbilical cords were seen (TA-US) in two of these four mares; one mare aborted shortly after acute placental separation was identified on TA-US. In three mares that delivered live foals, TA-US showed engorged allantoic vessels and enlarged umbilical vessels. Two of these mares had placental thickening visible on TR-US, interpreted as a sign of placentitis, that subsided after aggressive medical treatment. Seven of the eight placentas were submitted for gross and histopathological examinations after delivery. All placentas had some degree of edema, abnormally engorged allantoic vessels, and enlarged umbilical vessels. Placentitis, large allantoic vesicles, cystic pouches in the fetal part of the cord, and hemorrhages and thrombi in the umbilical vessels were detected only in placentas from mares that aborted. Equine pregnancies resulting from SCNT may be associated with placental pathologies that can be detected using ultrasonography. However, interpreting their severity is difficult. Although placental abnormalities have been observed in SCNT pregnancies in other species, to the best of our knowledge, placentitis has not been previously reported and may be an important complication of equine SCNT pregnancies, leading to pregnancy loss.