Blastulation time measured with time-lapse system can predict in vitro viability of bovine blastocysts.

The objective of this study was to evaluate the time of blastulation monitored by time-lapse technology to predict in vitro viability of bovine blastocysts. This technology can be a powerful tool for bovine embryos selection with higher implantation capacity and competence. Also, in humans an early blastulation is associated with higher quality and pregnancy rate. Cumulus oocyte complexes (COCs) were matured for 20 to 22 h and then fertilized by co-incubation of COCs and spermatozoa (10,000 sperm per oocyte) for 18 h. Presumptive zygotes were placed individually in microwells, in droplets of commercial culture medium. The Primo Vision TL system (EVO+; Vitrolife) captured digital images of developing embryos every 15 minutes. The time frame from IVF to the start of blastulation (tSB) and to blastocyst development (tB) was recorded. After day 7.5, the blastocysts were in vitro culture for 48 h until day 9.5 after IVF to evaluate post hatching development. In vitro viability was evaluated at day 9.5: those with a diameter greater than 200 µm and a total cell count greater than 180 were classified as viable (value 1), while the rest were classified as non in vitro viable (value 0). The area under the ROC curve (AUC) was estimated to determine the predictive power of in vitro viability through blastulation time. In addition, binary logistic regression analysis was used to generate a mathematical model with morphokinetic variables that allow the best prediction of in vitro viability. In 13 sessions, the blastocyst production rate was 46.2% (96/208). The cut-off time to discriminate early or late blastulation was 149.8 h. The post-hatching development of the embryos with early blastulation was 63.3% (31/49), being statistically superior (p = 0.001) than the late blastulation group 14.9% (7/47). Likewise, the time of blastulation showed an accuracy of 90.8% (p < 0.001) in predicting in vitro viability of bovine blastocysts. In conclusion, the selection of blastocysts based on blastulation time (< 155 h) and blastocyst diameter measured on day 7.5 after IVF (> 180 µm) maximizes the in vitro viability.

Heritability of Locomotor Stereotypies in Chilean Horses.

The Chilean horse is a breed of closed registry. Stall-walking and weaving are locomotor stereotypies that affect this breed, and genetic predisposition has been suggested for both conditions. The objective of the present study was to estimate heritability of stall-walking and weaving in Chilean horses. Owners of 2,098 horses registered in the Chilean horse Stud Book, which were or had been stabled for at least 1 year, were asked to provide for identification data of the animal and presence or absence of stall-walking and/or weaving. The Chilean Horse Stud Book was accessed online, to collect information on name and registration number of the sire and dam of each horse. The prevalence of stall-walking and weaving was calculated from the sample (n = 2,098). The database to estimate the heritability (h2) included all the sample horses (n = 2,098) and their sires and dams. Additionally, all the ancestors available in the Chilean Horse Stud Book were included in the database for 297 sampled horses including those with locomotor stereotypies. The genealogical database consisted of 7,187 individuals. The prevalence of stall-walking was 2.05% and prevalence of weaving was 1.43%, being more frequent in males (P < .05) for stall-walking. Heritability of stall-walking was low (h2 = 0.213 ± 0.08) and moderate for weaving (h2 = 0.435 ± 0.06). Heritability and prevalence of locomotor stereotypies found in the Chilean horse suggest that they are hereditary disorders, highly influenced by environmental factors.

Extracellular vesicles secreted during blastulation show viability of bovine embryos.

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 µm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86-91%) and snoRNA (9-14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.