Impact of cholesterol supplementation on Pantaneiro bovine semen cryopreservation: Insights into in vitro embryo production

This study aimed to evaluate the impact of cholesterol supplementation at various concentrations in cryopreserved Pantaneiro bovine semen on in vitro embryo production (IVEP). Grade I and II cumulus-oocyte complexes (COCs) were collected from ovaries retrieved from a commercial slaughterhouse and matured in vitro for 24 h. The matured COCs were divided into four groups based on the concentration of cholesterol -loaded cyclodextrin (CLC) during semen cryopreservation from a Pantaneiro breed bull: Control (C) - 0 mg/mL CLC, T1 - 0.5 CLC, T2 - 1 mg/mL CLC, and T3 - 1.5 mg/mL CLC. After 18 h of incubation, structures were denuded and transferred to in vitro culture (CIV) for 8 days. Cleavage rate was assessed on the second day (D2), and on the seventh day (D7), embryo classification and blastocyst production rate were evaluated. Additionally, total and apoptotic embryonic cell counts were conducted using differential staining for bovine embryos. Oxidative status was assessed in the FIV and CIV media of each treatment by measuring thiobarbituric acid reactive substances – TBARS. Concentrations of 1.0 mg/mL and 1.5 mg/mL of CLC significantly reduced the proportion of cleaved structures (P = 0.0029), as well as the percentages of cleaved structures and blastocysts (P < 0.001). Moreover, increasing CLC concentrations decreased the total number of embryonic cells (P < 0.001). No significant differences were noticed in other parameters. In conclusion, supplementing cryopreserved Pantaneiro cattle semen with cholesterol-loaded cyclodextrin (CLC) at concentrations of 0.5, 1, and 1.5 mg/mL adversely affects in vitro embryo production.