Reproductive Technology

Effect of Cryopreservation Methods and Pre-Cryopreservation Storage on Bottlenose Dolphin (Tursiops truncatus) Spermatozoa

Abstract
Research was conducted to develop an effective method for cryopreserving bottlenose dolphin semen processed immediately after collection or after 24 h liquid storage. In each of 2 experiments, 4 ejaculates were collected from 3 males. In Experiment 1, 3 cryopreservation methods (CM1, CM2, CM3), 2 straw sizes (0.25 ml, 0.5 ml), and 3 thawing rates (SLOW, MEDIUM, FAST) were evaluated. Evaluations were conducted at collection, pre-freeze and 0, 3 and 6 h post-thaw. A sperm motility index (SMI: total motility [TM] x % progressive motility [PPM] x kinetic rating [KR, 0 to 5]) was calculated and expressed as %SMI of initial ejaculate. For all ejaculates, initial TM and PPM were greater than 85% and KR was 5. At 0 h post-thaw, differences in %SMI among cryopreservation methods and thaw rates were observed (P < 0.05) but there was no effect of straw size. In Experiment 2, ejaculates were divided into 4 aliquots for dilution (1:1) and storage at 4°C with a skim milk-glucose or a TES-TRIS egg yolk solution, and at 21°C with a HEPES-TALP medium or no dilution. After 24 h, samples were frozen and thawed (CM3, 0.5 ml straws, FAST thawing rate) at 20 x 10⁶ spermatozoa ml^-1 (LOW) or 100 x 10⁶ spermatozoa ml^-1 (STD). Percent SMI at 0 h post-thaw was higher for samples stored at 4°C than 21°C (P < 0.001), and at 6 h post-thaw, %SMI was higher for samples frozen at STD than LOW concentration (P < 0.05). For both experiments, acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol applied to fresh or liquid-stored semen maintained high levels of initial ejaculate sperm characteristics.

Key words: Gamete Biology • Assisted Reproductive Technology • Testis • Male sexual function • Sperm

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