Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.

Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure.

The feasibility of repeated collection and enzymatic isolation of large numbers of viable primordial and primary follicles from living donor cows were tested. Ovarian cortical biopsies were collected transvaginally by the Biopsy Pick-Up (BPU) device, a modification of an Ovum Pick-Up instrument. Follicles were enzymatically isolated from the retrieved cortical tissue samples, and follicle viability was determined by a live/dead fluorescent assay. Six cows were subjected to BPU once per week during 4 consecutive weeks, and in each BPU session 4 cortical tissue samples were collected per ovary. Over the 4-week trial period, a total of 1443 primordial and primary follicles were collected, 1358 (94%) of which were primordial and 85 (6%) were primary follicles. In each BPU session, an average 60.1 +/- 10.7 (mean +/- SEM) primordial and primary follicles were isolated per cow. The number of follicles varied considerably throughout the trial period and between cows. Statistical analysis of the data, however, did not support the presence of any distinct trends in the follicle yields over time or between cows. A total of 111 enzymatically isolated follicles were analyzed for viability with fluorescent probes. The vast majority of isolated follicles (92.8%) were totally viable. We conclude that the standardized BPU procedure generates sufficiently large numbers of vital primordial and primary follicles, thus validating BPU as a new tool for research into early bovine follicular development.