209 FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

Abstract

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4 h. The cauda epididymis was dissected out and flushed with 2 mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1 mL aliquot was removed for analysis and the balance (9 mL; 2 mL fraction A + 7 mL sperm sample) mixed with an additional 27.2 mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2 mL of cooled fraction B were added (final sperm concentration = 150 × 10⁶ mL⁻¹). Sperm samples were loaded into 0.25-mL straws and frozen over LN₂ vapor (4 cm for 20 min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30 s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20 mM HEPES, 355 mM sucrose, 10 mM glucose, 2.5 mM KOH) and HEPES saline (197 mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5 min at 600 × g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50 ng mL⁻¹ in HEPES saline;; 10 min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5 μM in HEPES saline;; 30 min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100 mOsm/kg HEPES saline (1 : 20; 15 min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490 nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.