Effect of Two Different Sperm Selection Methods on Boar Sperm Parameters and In Vitro Fertilisation Outcomes.

Porcine in vitro embryo production (IVP) protocols have conventionally used density gradient selection (DGS) by centrifugation to prepare sperm samples and achieve successful fertilisation. However, the possible toxicity of the solutions used and the potential damage caused by the centrifugation step may have a negative effect on the quality of the sample. Microfluidic chip-based sperm (MCS) sorting has been proposed as an alternative technique for the selection of high-quality sperm with the purpose of improving reproductive outcomes in IVF. This device does not require centrifugation or any toxic solution to prepare the sample for fertilisation. The sample is not subjected to unnecessary stress, and the process is less operator-dependent. In this study, we compared the sperm parameters of unselected extender-diluted boar semen samples with selected samples using DGS and MCS methods. The results show an expected reduction in sperm concentration after both methods. All the groups were significantly different from one another, with MCS being the group with the lowest concentration. Though the three groups had a similar overall motility, significant differences were found in progressive motility when comparing the unselected group (control, 19.5 ± 1.4%) with DGS and MCS. Progressive motility in DGS was also significantly higher than in MCS (65.2 ± 4.9% and 45.7% ± 5.3, respectively). However, MCS selection resulted in enriched sperm samples with a significantly lower proportion of morphologically abnormal sperm compared to DGS. After fertilisation, no statistical differences were found between the two methods for embryological parameters such as cleavage rates, blastulation rates, and embryo quality. The number of cells in blastocysts derived from MCS was significantly greater than those derived from DGS sperm. Thus, we demonstrate that MCS is at least as good as the standard DGS for most measures. As a more gentle and reproducible approach for sperm selection, however, it could improve consistency and improve IVP outcomes as mediated by a greater proportion of morphologically normal sperm and manifested by a higher cell count in blastocysts.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant.

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in "pearls" and in "mini straws" using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Preimplantation Genetic Testing for Aneuploidy Improves Live Birth Rates with In Vitro Produced Bovine Embryos: A Blind Retrospective Study.

Approximately one million in vitro produced (IVP) cattle embryos are transferred worldwide each year as a way to improve the rates of genetic gain. The most advanced programmes also apply genomic selection at the embryonic stage by SNP genotyping and the calculation of genomic estimated breeding values (GEBVs). However, a high proportion of cattle embryos fail to establish a pregnancy. Here, we demonstrate that further interrogation of the SNP data collected for GEBVs can effectively remove aneuploid embryos from the pool, improving live births per embryo transfer (ET). Using three preimplantation genetic testing for aneuploidy (PGT-A) approaches, we assessed 1713 cattle blastocysts in a blind, retrospective analysis. Our findings indicate aneuploid embryos have a 5.8% chance of establishing a pregnancy and a 5.0% chance of given rise to a live birth. This compares to 59.6% and 46.7% for euploid embryos (p < 0.0001). PGT-A improved overall pregnancy and live birth rates by 7.5% and 5.8%, respectively (p < 0.0001). More detailed analyses revealed donor, chromosome, stage, grade, and sex-specific rates of error. Notably, we discovered a significantly higher incidence of aneuploidy in XY embryos and, as in humans, detected a preponderance of maternal meiosis I errors. Our data strongly support the use of PGT-A in cattle IVP programmes.