Characterization of DNA Fragmentation Dynamics in the Refrigeration of Bovine Spermatoza

Refrigeration of bovine sperm provides a window of opportunity for storage/transportation and utilization of semen and provides a management option in combination with fixed-time artificial insemination. Evaluation of semen quality, which typically includes subjective variables such as morphology and motility, give an estimate of the overall population of sperm and only indicate gross functionality. Although practitioners will typically accept or reject a sample for artificial insemination based on microscopic parameters, the ability of these parameters to predict fertility varies. DNA fragmentation in ejaculated spermatozoa has been correlated with oxidative stress and with impaired sperm function and has recently gained attention as an indicator of fertilizing ability and pregnancy outcomes in humans.

The objective of this study was to describe the dynamic DNA fragmentation patterns for ambient temperature and refrigerated bovine sperm during a 72 h storage period and to compare sDFI findings with the microscopic parameters normally evaluated by practitioners in the field. Five Black Angus bulls (ages 2 to 8) of proven fertility in good body condition were used for the study. Ejaculates from all bulls were collected by electroejaculation, diluted in tris-buffered extender containing egg yolk (20% v/v) and glycerol (7% v/v), adjusted to pH of 6.8 and maintained at 32°C. Ejaculates from individual bulls were adjusted to equal number of spermatozoa (500x106 sperm), pooled into a heterospermic sample which was then divided into two groups: (i) ambient temperature (20-22°C), (ii) refrigerated (0.67°C/min) to a final maintenance and storage temperature of 5–7°C. Aliquots for analysis were taken at 0, 6, 12, 24, 48 and 72 h post-collection. Mass motility, progressive motility and membrane integrity (eosin, 1-step protocol, WHO) were blindly assessed by experienced technician. Uridine nick-end labeling (TUNEL) assay was used to study the extent of DNA damage.

Results were analyzed using analysis of variance in a measured repeated design with six replicates. Statistical analysis was performed using GraphPad Prism v.6, post hoc pairwise comparisons between treatments for each time period were conducted using Tukey’s test, p was considered significant at the 0.05 level. There were no significant differences (p?0.05) in individual progressive motility, membrane integrity and sDFI between ambient temperature and refrigerated groups at 0 and 6 h post-collection. Individual progressive motility differed significantly (p?0.05) at 12 (47 v. 30%), 24 (45 vs. 23%) 48 (40 vs. 18%) and 72 (35 vs. 10%) h post-collection. Membrane integrity differed significantly at 48 h (63 vs. 57%) and 72 h (61 vs. 45%) post collection. sDFI differed significantly at 48 h (25 vs. 31%) and 72 h (28 vs. 26%) post collection. Results of this study indicate a disparity between the microscopic characteristics commonly assessed by practitioners in the field and the patterns of dynamic DNA fragmentation of refrigerated bovine semen. Further studies should analyze DNA fragmentation thresholds and to study their impact on conceptions and pregnancy rates in the bovine species.

This paper was presented at the 1st International Congress of Embryonic Technologies, April 11-12, 2013 in Corrientes, Argentina.