10 Segmental cervical aplasia in a mare with mosaic X-chromosome aneuploidy

Segmental cervical aplasia is a congenital Müllerian abnormality characterised by the complete or partial failure of cervical development resulting from abnormal fusion of the Müllerian ducts to the urogenital sinus. In the present case report, we describe a congenital segmental cervical aplasia in a Colombian creole mare. The mare was presented to the Animal Reproduction Clinic of Universidad Nacional de Colombia for diagnosis because the external orifice of her cervix was not detected when a uterine lavage was attempted as a therapy for detectable uterine fluid accumulation. The mare had a history of 20-day oestrous cycles confirmed by receptivity to a mature stallion and had no history of natural service or artificial insemination. A complete breeding soundness evaluation of the mare including transrectal palpation, ultrasonography, vaginoscopy, endoscopy, transvaginal aspiration of the uterine fluid and cytogenetic analysis, and an oestrous cycle follow-up were performed. Clinical and ultrasonographic evaluation of the genital tract revealed normal-size ovaries with structures suggestive of regular ovarian activity. Ovulation was confirmed by the formation of a corpus haemorrhagicum followed by a mature corpus luteum in diestrus. In addition, granular free-floating fluid material distending the uterus was detected. Upon vaginal examination, the organ ended in a blind bag with a small papilla with no evident external os cervix. Cytology of the uterine fluid obtained by transvaginal aspiration showed predominant neutrophils with diplococcus bacteria and inflammatory cells compatible with inflammatory content. Cytogenetic analysis of 134 metaphase lymphocytes showed that the mare had an abnormal karyotype [64,XX]/[63,XO]/[65,XXX] with a ratio of 45%, 45%, and 10%, respectively. G- and C-banded analysis was conducted for the X chromosome. Mosaicism of the X chromosome was diagnosed, and the observed congenital segmental cervical aplasia was proposed as the clinical consequence of the mosaicism detected. To our knowledge, this is the first case of this reproductive pathology in a Colombian mare with regular ovarian activity and X chromosome aneuploidy in mosaic form. The cause of the persistently contaminated uterine content in this mare was not clear; it is possible that via the systemic or transcervical route, bacterial contamination could have colonized the uterus, resulting in chronic inflammation and fluid accumulation. This case report demonstrates the importance of performing an adequate routine gynaecological examination in mares to determine their reproductive health. In most cases, the diagnosis of congenital pathologies of the cervix is an incidental finding when performing breeding programs or therapeutic strategies for managing uterine inflammatory conditions. Furthermore, cytogenetic analysis is an important complementary tool for clinical reproductive examination, to accurately identify causes of congenital malformations, and to determine additional causes of reproductive failure in mares.

124 Effect of transvaginal oocyte aspiration on equine blood and peritoneal fluid parameters

Transvaginal aspiration of oocytes (TVA) in the equine industry has gained more relevance and become a valuable technique to produce offspring from subfertile mares. TVA is a semi-invasive procedure and requires handling the ovaries transrectally to position them closely to an ultrasound probe located in the mare’s vagina. Once the ovary lies in close apposition to the ultrasound probe, a 12G needle is inserted through the needle guide, puncturing, aspirating, and scraping each follicle to recover the oocyte. Potential complications described include rectal tears, puncturing of blood vessels, ovarian abscesses, and peritonitis. Occasionally, problems occur after uneventful procedures, such as colic, peritonitis, pain, and anorexia. However, the source of these complications is not fully known. We hypothesize that blood and peritoneal fluid parameters would differ pre- and post-TVA in mares. A few reports provide some parameters after TVA (e.g. peritoneal protein, neutrophils, nucleated cells) without reference to pre-TVA values. These studies have not identified an effect in peritoneal fluid variables due to multiple abdominocenteses. Therefore, our aim was to analyse blood and peritoneal fluid in mares pre- and post-TVA, and to identify changes in parameters of the procedure (duration, number of pokes, number of follicles) and the mares’ clinical responses. Ten healthy mares were selected to undergo the procedure. Thirty minutes before starting TVA, a blood sample was drawn for complete blood count (CBC) and blood chemistry, and abdominocentesis was performed to obtain abdominal fluid and assess the cytology. This same protocol was repeated 24 hours after TVA. Physical exams were performed pre- and post-TVA. Paired t-tests were used to identify differences between groups (pre- and post-TVA). Spearman correlations (ρ) were used to assess the relationship between variables. There was a significant increase in peritoneal lactate (5.65-fold), peritoneal total protein (2.4-fold), and total nucleated cells (46-fold) between pre- and post-samples. These parameters were not associated with operator, number of times the needle was introduced into the ovaries, or number of aspirated follicles. The remaining parameters evaluated in CBC and blood chemistry did not differ. A positive correlation between total peritoneal protein and blood albumin was found post-TVA (ρ=0.72, P=0.01) but not pre-TVA (ρ=−0.1, P=0.65), suggesting an increase in protein level due to bleeding. Clinically, 9 mares were healthy throughout the study except one that presented signs of pain (facial grimace, anorexia, hyperthermia) the day following TVA. In conclusion, we showed changes in the peritoneal fluid during uneventful TVA procedures. The information provided by this research gives further insight into changes potentially caused by a TVA in abdominal fluid parameters. Further studies are necessary to determine expected standards and the duration of the changes after TVA.

Spontaneous Ovarian Hyperstimulation Syndrome: Looking Beyond the Ovary

We report a case of a 22-year-old single female with spontaneous ovarian hyperstimulation syndrome (s-OHSS) referred for transvaginal aspiration of follicles. Investigations revealed primary hypothyroidism, mild hyperprolactinaemia and unelevated levels of both follicle stimulating hormone (FSH) and estradiol. Supplementation with L-thyroxine lead to euthyroid status and gradual resolution of signs and symptoms of ovarian hyperstimulation syndrome (OHSS) over 4 months.

Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes

Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxide (DMSO) or 3.5% glycerol. Immature equine oocytes were obtained from ovarian follicles of mares during diestrus by transvaginal aspiration (n=128). After invitro maturation, MII oocytes (n=90) were fertilised by ICSI with thawed stallion spermatozoa (n=45 in both the DMSO and glycerol groups). The embryo cleavage rate was greater in the DMSO than glycerol group (73.3% vs 46.7% respectively; P=0.0098), but the blastocyst development rate per fertilised oocyte was similar between the two groups (28.9% vs 15.6% respectively; P=0.128), as was the blastocyst production rate per cleaved embryo (39.4% vs 33.3% respectively; P=0.653). In this study, cryopreservation of equine spermatozoa in 1M DMSO was correlated with significantly higher cleavage rates in IVM oocytes fertilised by ICSI compared with spermatozoa cryopreserved using 3.5% glycerol. Although not statistically significant in this small number of stallions, increased blastocyst production and individual stallion variability was observed among CPA treatments. This warrants further critical examination of cryoprotectants used in equine sperm subpopulations used for ICSI in a larger number of stallions.

ТРАНСВАГИНАЛЬНАЯ АСПИРАЦИЯ ФОЛЛИКУЛОВ И СПОСОБНОСТЬ OPU-ООЦИТОВ КОРОВ К ЭМБРИОНАЛЬНОМУ РАЗВИТИЮ В УСЛОВИЯХ IN VITRO

Представлены результаты трансвагинальной аспирации ооцитов коров, а также оценен их потенциал к эмбриональному развитию после оплодотворения в условиях in vitro. Донорами яйцеклеток являлись половозрелые телки симментальской породы в возрасте 1619 мес. Животныедоноры (n7) перед проведением процедуры Ovum Pickup (OPU) были гормонально обработаны с целью стимуляции роста фолликулов. Количество выделенных ооцитов от индивидуальных доноров составило в среднем 7,7 ооциткумулюсных комплексов (ОКК), что соответствовало степени извлечения 54,57,7. Доля ОКК хорошего качества, рассчитанная от общего числа извлеченных ОКК, между отдельными животными существенно не различалась (значения варьировали от 60,0 до 75,0) и в среднем составила 67,21,9. ОКК с признаками нормальной морфологии подвергали in vitro процедурам созревания, оплодотворения и последующего культивирования до стадии бластоцисты. Доля раздробившихся ооцитов и выход бластоцист после in vitro осеменения яйцеклеток коров равнялась 75,7 и 24,3, соответственно. В целом от одного донора за сессию OPU было получено 1,3 эмбриона на стадии бластоцисты, содержащих в среднем 89,8 ядра. Оцененный способ экстракорпорального оплодотворения OPUооцитов коров позволяет получать эмбрионы, пригодные для замораживания и трансплантации реципиентам и может быть использован в программах по воспроизводству желаемых генотипов у крупного рогатого скота.In the present work, we report the data on transvaginal aspiration of bovine ovarian follicles and estimation of in vitro embryo development competence of collected oocytes. The oocytes were collected by ovum pickup OPU from seven 1619 monthold Simmental heifers, previously hormonallytreated in order to stimulate ovarian follicular growth. In average, 7.7 oocytecumulus complexes (OCCs) per heifer per OPU session were collected that corresponded to 54.57.7 of recovery rate. Morphologically, 60.075.0 of OCCs were the good quality and this rate did not significantly differ between the animals. Good quality OCCs (total n37) were then subjected to in vitro maturation, in vitro fertilization and in vitro embryo development up to blastocyst stage. Cleavage and blastocyst rates were 75.7 и 24.3 , respectively. In total, 1.3 blastocysts were obtained per cow per OPU session in average these blastocysts contained 89.9 cells. In conclusion, we developed the methodology of in vitro fertilization of bovine OPUcollected oocytes that allowed obtaining the blastocysts potentially suitable for freezing and transplantation to recipients. This approach can be used to multiply desired genotypes in cattle reproduction.