141 Enhanced developmental potential of bovine embryos generated by piezo-intracytoplasmic sperm injection with sorted capacitated spermatozoa by fluorescence-activated cell sorting

Intracytoplasmic sperm injection (ICSI) in cattle has a lower embryonic development compared with other species. One of the factors responsible for this low efficiency is sperm capacitation. Sperm capacitation involves all post-ejaculation biochemical and physiological changes that render mammalian spermatozoa capable of fertilizing the oocyte. This well-orchestrated process includes the removal of cholesterol from the plasma membrane, an increase of membrane permeability and fluidity, the influx of Ca²⁺ and bicarbonate, an increase of intracellular pH, activation of kinases, and increase in tyrosine phosphorylation. However, during the ICSI procedure, these important physiological events are bypassed because non-capacitated spermatozoa are frequently used for sperm injection in most species. Furthermore, even if capacitated sperm are used for ICSI, there is a possibility to inject non-capacitated or not fully capacitated spermatozoa. To tackle this problem, we evaluated different bovine sperm capacitation conditions to identify a treatment that allows the sorting of capacitated spermatozoa by fluorescence-activated cell sorting (FACS), for subsequent use in ICSI experiments. The effect of different capacitating inducers (heparin, methyl-β-cyclodextrin, and dibutyryl cyclic AMP) were evaluated singly or in combination in basal sperm TALP medium. Assessed parameters included plasma and acrosomal membrane integrity, membrane fluidity, and intracellular calcium levels. We chose the treatment with the highest capacitation levels assessed by flow cytometry to sort capacitated sperm by FACS, generating two populations of sperm of the same sample: (1) high and (2) low capacitation levels. Cumulus–oocyte complexes were aspirated from abattoir ovaries and matured in TCM-199 medium for 20–24 h at 38.5°C and 5% CO₂. Piezo-ICSI was performed with sorted sperm. Embryos were cultured in 50-μL drops of KSOM medium at 38.5°C and 5% CO₂, 5% O₂, and 90% N₂. Cleavage was recorded at 72 h, and blastocyst rate at 192 h. Data were transformed to arcsine, analysed by analysis of variance, and means were compared using Tukey’s test with Stat graphics Plus 2 software. An increase in acrosomal membrane damage, membrane fluidity, and Ca²⁺ levels was observed in all capacitating treatments compared with the non-capacitating control (P < 0.05). However, a higher level in capacitation parameters was observed in the heparin treatment, and therefore we chose this treatment for sorting and for the generation of embryos. Results demonstrated a higher cleavage and blastocyst rate (46% and 24.5%, respectively) when capacitated sorted sperm were used for ICSI, compared with uncapacitated sperm (31.2% and 12.5%, respectively). In conclusion, heparin treatment improved the capacitation status of frozen-thawed bovine spermatozoa incubated in Sp-TALP medium. Cell sorting allowed the selection of highly capacitated sperm that significantly improved the embryo developmental potential in bovine ICSI.

Funding support was from FONDECYT 1201166 ANID and slaughterhouse Frigorifico Temuco, Chile.