Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum)

• Effective rhino sperm sex-sorting and preservation methodologies were developed.
• 69% of fractions contained urine contamination as determined by creatinine analysis.
• Urine contamination (≥21.0 μg creatinine/ml) was detrimental to sperm quality.
• Directional freezing resulted in enhanced quality of non-sorted and sorted sperm.

White rhinoceros ejaculates (n = 9) collected by electroejaculation from four males were shipped (10 °C, 12 h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0 μg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5 °C up to 54 h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54 h post-collection, some motility parameters were higher (P < 0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P < 0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89 ± 6%) or Y-bearing (86 ± 3%) spermatozoa was achieved using moderate sorting rates (2540 ± 498 X-spermatozoa/s; 1800 ± 557 Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI.