120 IMPACT OF SEASON ON SEMINAL CHARACTERISTICS AND SPERM CRYOPRESERVATION IN THE TUFTED DEER (ELAPHODUS CEPHALOPHUS)

Abstract

The Tufted deer (Elaphodus cephalophus), a small deer species native to China, is listed as near threatened on the IUCN Red List and >70 animals are managed in North American zoos as a hedge against extinction. In this study, we 1) characterized the seminal traits, 2) assessed the impact of season on ejaculate traits and testosterone level, and 3) examined sperm sensitivity to cryopreservation. Semen (24 ejaculates) were obtained from five males (1–2 ejaculates/male per season) by electro-ejaculation and evaluated for volume, osmolality, pH as well as sperm concentration, motility (%M), forward progression (FP, scale = 0–5) and acrosomal integrity (%AI). Ejaculates were divided into two aliquots and cryopreserved (4% vol/vol glycerol final concentration; 50–200 × 106 sperm mL–1) over liquid nitrogen vapor using Beltsville extender (BF5F; Howard et al. 1986) or Triladyl® (TRIL; Minitüb GmbH, Germany) extender. Sperm motility and %AI were assessed immediately (subjective) upon thawing and following swim-up processing (SU; 30 min) using computer-assisted semen analysis after 1, 2, 3, and 4 h of incubation (37°C). Fecal samples were collected 3–5 times weekly for 2 years and analyzed for testosterone (T) metabolites using enzyme immunoassay as a function of season (autumn, September–November; winter, December–February; spring, March–May; and summer, June–August). Data were analyzed using Proc GLM or ANOVA with Tukey multiple mean comparison. Significance was set at P < 0.05. Male reproductive and semen traits peaked in autumn (volume, 2.0 mL; concentration, 207.6 × 106 mL–1; pH, 7.6; osmolality, 310.8 mOsm; %M, 76%; FP, 3.5; and %AI, 82.3). Mean testicular length and neck girth in autumn were 4.9 and 43.2 cm, respectively. Mean T concentration (~1.23 µg g–1 of dry feces) was higher (P < 0.05) in summer compared with winter (1.07 µg g–1 of dry feces) or spring (1.06 µg g–1 of dry feces). Sperm motility and %AI were lower (P < 0.05) immediately after thawing (BF5F, 34.6 and 34.7%, respectively; and TRIL, 23.1 and 29.1%, respectively) compared with fresh semen (76.9 and 74.6%, respectively). Motility characteristics immediately after SU (computer-assisted semen analysis) were higher (P < 0.05) in BF5F compared with TRIL: %M (56.4 v. 44.9%), progressive motility (42.5 v. 21.9%), %AI (41.2 v. 31.3%), straight-line velocity (68.0 v. 53.2 µm s–1), straightness (85.1 v. 75.1%), and linearity (54.3 v. 45.0%). At the end of 4 h of incubation, sperm %M and FP declined (P > 0.05) in both BF5F and TRIL (47 and 30%, respectively) but the %AI was higher (P < 0.05) in BF5F (32%) than TRIL (21%). Results indicate that tufted deer ejaculates exhibit seasonal variations in reproductive traits and cryopreservation in BF5F better preserves sperm motility and acrosomal membrane integrity compared with TRIL.