212 EFFECT OF CRYOPRESERVATION METHODS AND PRE-CRYOPRESERVATION STORAGE ON BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATES) SPERMATOZOA

Abstract

In conjunction with artificial insemination (AI) and sperm preservation, sperm sexing technology has great potential as a population management strategy for captive bottlenose dolphins. Successful AI using fresh spermatozoa (Robeck TR et al. 2001 CRC Marine Mammal Medicine 193–226) and flow cytometric analysis of bottlenose dolphin spermatozoa (Garner DL and Seidel GE Jr 2002 CSAS Symposium 2–13) support this approach. For sperm sexing, methods for short-term storage of semen in a liquid state are required to enable transport of spermatozoa to the sorting laboratory. In addition, cryopreservation techniques must be optimized for long-term storage of sexed spermatozoa. Our objectives were to assess: (i) 3 cryopreservation methods × 2 straw sizes × 3 thawing rates (Exp. 1) and (ii) effects of liquid storage for 24 h (pre-cryopreservation) and sperm concentration at freezing (Exp. 2) on post-thaw characteristics (PT) of bottlenose dolphin spermatozoa. For Exp. 1 and 2, 4 ejaculates (collected by manual stimulation) × 3 males (aged 14–34 yr) × 4 replicates were used. For Exp. 1, semen was frozen in 0.25-mL (SM) and 0.5-mL straws (LG) by 3 methods (Mt) (Mt1: lactose, egg yolk, −32°C min⁻¹; Mt2: lactose, egg yolk, 1.5% Equex STM (Nova Chemical, Calgary, Canada), −19.7°C min⁻¹; Mt3: Test yolk buffer (TYB), −116°C min⁻¹). All Mt had 3% glycerol. Samples were thawed using a slow (S: 2.8°C s⁻¹), medium (M: 8.8°C s⁻¹) or fast (F: 21°C s⁻¹) rate. In Exp. 2, ejaculates were divided into 4 aliquots for dilution (1:1) and stored at 4°C with EquiPro^® (EP4°C, Minitube, Verona, WI, USA) and TYB (TYB4°C) or at 21°C with Androhep Enduraguard^TM (AH21°C, Minitube) or no dilution (NEAT21°C). After 24 h, samples were frozen and thawed using Mt3 × SM × F at 10 × 10⁶ sperm mL⁻¹ (LOW) or 100 × 10⁶ sperm mL⁻¹ (STD). PT evaluations of motility (total motility [TM], % progressive motility [PPM], kinetic rating [KR, 0 to 5]) and acrosomal status (Spermac^® , Minitube) were performed at 30 min and 6 h after dilution (1:1) with AH at 21°C. For statistical analysis (ANOVA), a sperm motility index (SMI = TM × PPM × KR) was calculated and expressed as % of initial SMI. For all ejaculates, initial TM and PPM were greater than 85% and KR was 5. In Exp. 1, at 6 h PT, %SMI was highest for Mt3 × LG × M (45.5 ± 8.7) and Mt3 × SM × F (44.8 ± 11.9). For Exp. 2, %SMI at 0 h PT was higher for samples stored at 4°C than at 21°C (TYB4°C 41.0 ± 8.4, EP4°C: 36.7 ± 7.7, NEAT21°C: 23.8 ± 8.6, AH21°C: 14.8 ± 8.6, P < 0.001) and, with the exception of AH21°C, was similar between the LOW and STD concentration. At 6 h PT, %SMI for all treatments was higher for STD than LOW concentration (P < 0.05). Acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol maintained high levels of the initial characteristics of ejaculated spermatozoa. Transport of semen for sex pre-selection and cryopreservation within 24 h may be feasible, but impact of storage time on functional capacity of dolphin spermatozoa is unknown.