263 EVALUATION OFALTERNATIVE CRYOPROTECTANTS AND CHOLESTEROL-LOADED CYCLODEXTRINS FOR INCREASING CRYOSURVIVAL OF SEXED REFROZEN STALLION SPERMATOZOA

Abstract

Commercial use of sex-sorted stallion sperm will depend upon the ability to cryopreserve the sperm. In many cases the desired sperm for sorting is already cryopreserved; therefore, this will necessitate the need to thaw, sex-sort, and subsequently refreeze the sperm for future use in intracytoplasmic sperm injection (ICSI). Experiments were conducted to determine if previously frozen stallion spermcould be thawed, sex-sorted, and cryopreserved. Ejaculates from 6 stallions were frozen in FR5 cryopreservation medium at 400 million sperm mL^–1 (0.5-mL straws). Samples were thawed for 30 s in 37°C water and diluted (100 million sperm mL^–1) in Kenny's modified Tyrode's medium (KMT). Samples (2 mL) were stained with 36.53 µm Hoechst 33342 in 34°C water for 45 min and then diluted to 75 million sperm mL^–1 with KMT containing 0.002% food coloring dye (FD&C #40). Sperm were sorted using an SX MoFlo^™ (Dako, Fort Collins, CO, USA), and the sorted samples centrifuged at 850g for 10 min. The sperm were suspended to 4.6 million sperm mL^–1 in FR5 + 0.54 m glycerol (control) or the following treatments: FR5 + 0.52 m dimethylformamide (DMF); FR5 + 0.54 m glycerol + 1.5 mg cholesterol-loaded cyclodextrins (CLC)/120 million cells; FR5 + 0.52 m DMF + 1.5 mg CLC/120 million cells. Non-sorted control sperm (NSC) were removed from original frozen/thawed samples and suspended to 4.6 million sperm mL^–1 in FR5 + 0.52 m DMF. Sorted and non-sorted samples were cooled from 22°C to 4°C over 2 h, loaded into 0.25-mL straws, and frozen in liquid nitrogen vapor. Straws were thawed in 37°C water for 30 s and the percentages of motile and viable sperm were determined using computer-assisted sperm analysis and flow cytometry (5 million cells were stained with 10 µL propidium iodide and 20 µL SYBR-14), respectively. Data were analyzed by analysis of variance and treatment means were separated using the Student-Newman-Keuls mean separation test. The percentages of total motile spermatozoa were higher for frozen/thawed sexed refrozen (FTR) sperm refrozen in DMF + CLC (10%) than for the glycerol control (2%; SEM ± 1; P < 0.05), but this motility was similar to that of the DMF control (5%), the glycerol + CLC treatment (4%), and the NSC (7%; SEM ± 1; P > 0.05). The percentages of viable spermatozoawere higher for FTR (glycerol and DMF) sperm treated with CLC prior to freezing (68% and 71%) than for control sperm (58% and 56%; SEM ± 3; P < 0.05), respectively. Frozen/thawed sexed refrozen samples resulted in significantly higher percentages of viable spermatozoa compared to NSC (42%; SEM ± 3; P < 0.05). These findings indicate that cryopreserved stallion sperm can be thawed, sex-sorted, and refrozen and still maintain a high percentage of viable spermatozoa; treating sperm with CLC prior to refreezing improved cryosurvival for both cryoprotectants. This may provide adequate numbers of viable sperm for use in intracytoplasmic sperm injection procedures.

This work was funded by XY, Inc.