208EFFECT OF REFRIGERATION AND CRYOPRESERVATION ON THE QUALITY OF LION
EPIDIDYMAL SPERMATOZOA
Authors: Malo A.F. , Martinez-Pastor F. , Olivier F. , Spies T. , Roldan E.R.S. , Bartels P. 
Published: February 2004

Epididymal spermatozoa from harvested wild animals is potentially useful for conservation purposes, as it can be used for subsequent artificial insemination or stored in Biological Resource Banks for future use. The potential of sperm banking is of particular interest for use in lion (Panthera leo) populations maintained in small National Parks, as translocation of males to effect gene-flow is often problematic, resulting in the translocated lion being killed by resident pride males. We measured the change in sperm quality over time during cool storage (at 4°C) and after thawing of samples cryopreserved at −196°C. Also, we present a correlation between sperm plasma membrane integrity and mitochondrial activity as measured by fluorescent analysis. The testes from a pride lion were removed and transported to the laboratory (at 4°C) within 6h. The epididymides were removed and both cauda epididymides were flushed with 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany). The sample containing 2930×106 cells mL−1 was washed (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma, South Africa) and after centrifugation (5min. at 600g), the pellet was resuspended in 0.5mL of washing solution (with 197mM NaCl instead of sucrose). One aliquot of spermatozoa was kept at 4°C and evaluated at 24h intervals for 7 days. A second aliquot of the sperm sample was extended in Tris-citrate egg yolk extender with glycerol (Fraction B, Biladyl), frozen in liquid nitrogen (LN) vapor and stored in LN. The frozen sample was later thawed and evaluated as for the cooled samples. Percentages of motile (MS) and progressive (PS) spermatozoa were assessed using a phase contrast microscope (×200; stage at 37°C). Sperm plasma membrane damage was assessed by determining the percentage of cells exhibiting red fluoresence after staining with propidium iodide (PI, 50ng/mL; 10min RT). Spermatozoa that did not stain red in PI were classified as plasma membrane intact (PMI). Resilience to hypo-osmotic shock and plasma membrane integrity were evaluated by incubating a portion of the sample in a 100mOsm/kg solution (10nM glucose, 20nM HEPES, 30nM NaCl) containing PI for 15min at room temperature. The percentage of sperm cells with active mitochondria (MIT) was determined by counting spermatozoa showing orange fluoresence over the mid-piece after staining with JC-1(7.5 uM Sigma) for 30min at 37°C. At collection, MS was 15% and did not show a significant decrease during the 7-day storage period. Initially, PS was 10% and dropped to 5% after 7 days, with values fluctuating during the storage period. Both PMI and HOSPMI were 80% on Day 1, gradually decreasing to 75% on Day 7 of storage. PMI and MIT showed a highly significant correlation (r=0.88; P=0.003; n=8). In frozen-thawed sperm samples, MS fell from a pre-freeze value of 15% to 5% after thawing. Similarly, PS fell from 10% in pre-freeze to 3% in frozen-thawed samples. Likewise, PMI, HOSPMI and MIT values were 80% and 45%, 87% and 45% and 89% and 49%, respectively. Our study showed that lion sperm PMI and MIT remained high after 7 days at 4°C. MS and PS, although low, did not vary during this same period. PI and JC-1 showed a significant correlation, suggesting that both might be affected by the same deleterious factors. Although PMI, HOSPMI and MIT values decreased approximately 40% after freezing, we feel that such sperm samples could be used for in vitro embryo production, if not by IVF, by ICSI. Of course, additional studies are needed to validate our suggestion.