HH5 double-carrier embryos fail to progress through early conceptus elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause preterm mortality. Holstein haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the transcription factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated with the inadvertent cross between carriers. To determine whether HH5 DC survive to maternal recognition of pregnancy, embryonic day (E) 14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. Double-carrier E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic reduction of their extra-embryonic membranes, which were >26-fold shorter than those of carrier or noncarrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos-functionally equivalent to DC embryos-were generated by clustered regularly interspaced short palindromic repeats (CRISPR) technology and cultured to the blastocyst stage, in vitro culture day (D) 8, and to the early embryonic disc stage, D12. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mitochondrial DNA (mtDNA) content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, which is required for pregnancy recognition.

Efficient and repeatable in vitro fertilization in rabbits.

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10-24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17-22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.