HH5 Double-Carrier Embryos Fail to Progress through Early Conceptus Elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause pre-term 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 to the inadvertent cross between carriers. To determine if HH5 DC survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. DC E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic (>26-fold) reduction in the proliferation of extraembryonic membranes compared with carrier or non-carrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos -functionally equivalent to DC embryos- were generated by CRISPR technology and cultured to the blastocyst stage -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 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, 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.

Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10-30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.