Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets.

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies.

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.