Epididymal bull spermatozoa morphometrics post cryopreservation in Moringa oleifera leaf extracts supplemented tris-egg yolk semen extender

Abstract

Bull semen preservation techniques, especially cryopreservation of epididymal sperm, are vital for genetic preservation and can enhance in vitro fertilization (IVF) efforts. Spermatozoa are prone to oxidative damage during assisted reproductive technologies, impacting fertility. However, adding Moringa oleifera leaf extract (MOLE) to a tris-egg yolk (TEY) extender improves sperm motility, viability, and post-thaw fertility. The objective is to establish a protocol for preserving semen from slaughtered bulls, allowing for sperm retrieval from genetically superior animals post-death, thus offering economic benefits and supporting a sustainable socioeconomic framework. The broad objective was to develop a suitable protocol for semen preservation from the epididymis of slaughtered bulls to enable the recovery of spermatozoa from genetically superior bulls after death. Testicles and ovaries were collected from a Makhado slaughterhouse and transported to the laboratory at a temperature of 5 °C. The flushing technique was employed to retrieve semen from the epididymis. The semen samples were then extended using a TEY solution supplemented with varying levels of MOLE. In the first experiment, the motility, viability, and morphology of spermatozoa were assessed in bull semen samples stored at 5 °C over different time intervals. The second experiment involved keeping bull testicles at 5 °C for varying durations (0, 2, and 4 h) before the recovery of semen from the epididymis. During the third experiment, the semen was diluted with a TEY extender containing different amounts of MOLE and was subsequently frozen using the LN2 vapour method. In the fourth experiment, the fertilization ability of cryopreserved semen (obtained at different heights of the LN2 vapour method) was evaluated through IVF utilizing various concentrations of MOLE. Data analysis was conducted using the General Linear Model (GLM) procedure in Minitab version 2019 to assess treatment effects with a 95% confidence interval. A Computer-Aided Sperm Analyzer (CASA) was employed to evaluate spermatozoa characteristics, while live and dead spermatozoa were manually assessed following staining procedures. The IVF workstation was utilized for the fertilization process. Different semen extenders significantly affected sperm motility (P<0.01). The TEY+0% MOLE extender had the highest total motility, similar to TEY+4% and TEY+8% MOLE but higher than TEY+12%. Live spermatozoa values were consistent at 0 and 24 h, but lower after longer storage. Total motility decreased with longer storage before recovery from the epididymis (0-4h). During cryopreservation, TEY+8% MOLE had the highest viability, followed by TEY+4% MOLE. xvii Sperm viability was higher at 5 cm compared to 7 cm above LN2. The highest fertilization rate was in TEY+12% MOLE, similar to TEY+0% and TEY+8% MOLE. LN2 levels did not affect total fertilization or zygote stages at 0 PN and >2 PN, but interactions did affect presumptive zygotes at stage 1 PN. In conclusion, adding MOLE to the TEY extender improves sperm motility and viability of epididymal post-slaughter bull sperm for up to 120 h at 5 °C. The TEY+MOLE extenders significantly enhanced motility parameters, with TEY+8% MOLE showing the best sperm characteristics. While prolonged storage decreases sperm quality, acceptable results were noted within 48 h before freezing. Slow cooling of semen straws at 5 cm above LN2 yielded better sperm survival than cooling at 7 cm. Different MOLE concentrations improved fertilization ability during IVF, with TEY+12% MOLE achieving the highest fertilization rate. However, significant effects on total fertilization and zygote development were not observed, though interactions between MOLE concentration and LN2 levels did impact zygotes at stage 1 PN.