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Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana)

Summary. Electroejaculates from free-ranging, African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity. In Study I, each ejaculate was tested with each of 7 cryoprotective diluents. After cooling to 5°C and equilibration on ice (4°C) for 120 min, each aliquant was pellet frozen on solid CO₂, stored in liquid nitrogen and thawed (37°C) in saline or tissue culture solution. Amongst all diluents, post-thaw sperm motility, motility duration in vitro (37°C) and acrosomal integrity were greatest (P < 0·05) when diluent BF5F was used. Thawing medium had no effect on results. In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling–equilibration interval in an electronic cooler (5°C); however, post-thaw sperm motility rating and duration of motility in vitro were greater (P <0·01) with the pellet than the straw container freezing method. When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12h by maintaining thawed semen at 21 rather than 37°C (P <0·05) All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (1·5°C/min) compared to that of Study I (6·5°C/min). This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa must effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.

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