PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - B

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(404) IMPROVED ACROSOMAL INTEGRITY OF CRYOPRESERVED CHEETAH Acinonyx jubatus SPERM AFTER CENTRIFUGATION THROUGH ACCUDENZ.

Crosier, Adrienne^{1, 2}, Pukazhenthi, Budhan¹, Henghali, Josephine², Howard, JoGayle¹, Marker, Laurie², Wildt, David¹, ¹ Smithsonian Institution's National Zoological Park, Washington, DC² Cheetah Conservation Fund, Otjiwarongo, Namibia

ABSTRACT- Acrosomal membranes of cheetah sperm are exquisitely sensitive to cryopreservation, and integrity is lost in up to 50% of sperm following the best felid-specific cryopreservation protocols. Removal of cryoprotectant slowly after thawing may minimize acrosomal damage. Utilizing glycerol as a cryoprotectant, we examined the impact of: multi-step dilution vs. centrifugation through an Accudenz gradient on sperm motility and acrosomal integrity in thawed cheetah sperm. Electroejaculates (n = 6) were washed in Ham's F10 + HEPES (HF10) and pellets resuspended in TEST Yolk-Buffer (TYB) with 0% glycerol. An equal volume of TYB + 8% glycerol was added after cooling (3.5 h) to 4°C. Samples were cryopreserved over liquid nitrogen, thawed and assessed for percent motility (% M), forward progression (FP; scale 0-5) and percent intact acrosomes (% IA). Sperm motility index (SMI) was calculated as [(%M) + (FP x 20)]/2. Thawed samples were divided into 3 treatments: 1) control: aliquot diluted drop-wise in 300 &uscript;l HF10 over 1 min; 2) multi-step: aliquot diluted in 1,400 &uscript;l HF10 added in 7 aliquots over 3 min; and 3) Accudenz: aliquot centrifuged (100 g; 8 min) in a 2-step Accudenz gradient (4% and 10%) and interphase sperm resuspended in 1 ml HF10. Samples were centrifuged and sperm resuspended in HF10 and assessed for % M, FP and % IA post dilution, post centrifugation and every hour for 4 h. Both % IA and SMI decreased (P<0.05) upon thawing (% IA, 47.2±1.8; SMI, 55.8±2.7) compared to raw ejaculates (% IA, 87.9±1.9; SMI, 70.0±3.2). Centrifugation through Accudenz increased (P<0.05) % IA immediately after washing (37.0±1.3%) compared to the control (31.6±1.3%) and multi-step treatments (32.0±1.3%) and at each subsequent time of evaluation (ranges: Accudenz, 32-35%; control, 23-28%; and multi-step, 22-29%). Both post dilution and post centrifugation, SMI was increased (P<0.05) in control (61.6±1.9 and 56.5±1.3, respectively) compared to multi-step treatment (53.3±1.9 and 51.1±1.3), but was not different from Accudenz treatment (56.6±1.9 and 54.4±1.3). There was no effect (P>0.05) of treatment on SMI at any other time point. Results reaffirm the sensitivity of cheetah sperm acrosomes to cryopreservation and suggest that centrifugation of thawed sperm through Accudenz improves acrosomal integrity 5% without affecting motility.

KEY WORDS: spermatozoa, cryopreservation, cheetah, acrosome