89 SURVIVAL OF CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS

Abstract

A general objective of our studies on cat sperm is to enhance methods for both short- (+4°C) and long-term (–196°C) cryostorage, with particular focuses on improving compatibility with sex sorting and conforming to regulations for international shipment. Here, our specific aims were to a) determine the ability of cat sperm to survive during temporary cool storage in defined extenders (Exp. 1), and b) compare sperm survival after cryopreservation in the optimal defined extender v. TEST buffered extender + 2% egg yolk (TYB, Exp. 2). Testes from local veterinary clinics were transported in HEPES saline. Epididymides were dissected in HEPES 199 medium (He199), repeatedly sliced, and held at 37°C for ∼20 min. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL He199 and centrifuged for 5 min at 250 × g. In Exp. 1 (5 replicates), aliquots of the sperm pellet were extended in either of 2 defined extenders, Bioxcell® (BXC; IMV, Minneapolis, MN, USA) or HypoThermosol®-FSR (HTS; BioLife Solutions Inc., Bothell, WA, USA) or in TYB. Motility (Mot, Hamilton Thorne Sperm Analysis System CEROS 12, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at days 0, 1, 2, and 3 (Exp. 1), or after cooling (4°C) and post-thawing (p.t.), after 0 and 3 h incubation at 37°C (Exp. 2). In Exp. 2 (10 replicates), the sperm pellet was extended in BXC or TYB and gradually cooled to 4°C. Then, BXC or TYB + 12% glycerol was added (1:1) using a modified fixed osmolarity method (1995 Hum. Reprod. 10, 1109). Samples were loaded into 0.25-mL straws and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed in air (∼22°C) for 5 s and immersed in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps, centrifuged at 800 × g for 5 min, and pellets resuspended in He199. In Exp. 1, sperm in TYB, BXC, and HTS maintained 93, 69, and 56%, respectively, of initial motility (71%) after 3 days at 4°C (TYB > BXC and HTS; P < 0.05, 1-way ANOVA). Initially, 75 and 86% of sperm had membrane integrity and intact acrosomes, respectively. At 72 h, ∼80% of membrane intact sperm retained integrity in the two defined extenders v. nearly 90% in TYB (P > 0.05). At 24 h, all groups had high percentages of sperm with intact acrosomes (87 to 93%), but at 72 h, there was a difference between HTS (96%) and BXC (79%; P < 0.05). In Exp. 2 (Table 1), motility in TYB and BXC at 0 h p.t. was 77 and 70% of pre-freeze values – 77% (TYB) and 73% (BXC), respectively. Motility at 3 h p.t. was similar (BXC = 35% v. TYB = 37%). Membrane integrity and acrosomal status at 3 h p.t. ranged from 60% (BXC) to 72% (TYB) and from 65% (BXC) to 68% (TYB) of pre-freeze values, respectively. At 3 h p.t. M.I. of sperm in TYB was higher (P < 0.05) than in BXC. In summary, we have shown that cat epididymal sperm can be stored temporarily and cryopreserved successfully in a defined extender without animal proteins. Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage