101 CRYOPRESERVATION OF RAT EPIDIDYMAL SPERM: COMPARISON OF TWO COOLING PROTOCOLS

Abstract

The present study examined post-thaw motility, plasma membrane integrity and fertility of rat epididymal sperm cooled by two procedures to +5°C, and then cryopreserved by controlled-rate freezing. Wistar rats were used in the present study. In protocol-I (2001 Reproduction 122, 463), epididymides were collected from a mature male and placed in a plastic dish containing 2 mL of freezing medium I [23% (v/v) egg yolk, 8% (w/v) lactose monohydrate and antibiotics]. The epididymides were dissected with scissors to release epididymal sperm. The semen was kept at 15°C for 30 min and then held at 5°C for 30 min. The cooling rate from 15°C to 5°C was 0.3°C min⁻¹. The cooled semen was diluted with 2 mL of freezing medium II [freezing medium I with 1.4% (v/v) Equex Stm (ES, Nova Chemical Sales, Inc., Scituate, MA, USA)]. Mixed semen was aspirated into 0.25-mL straws and exposed to liquid nitrogen (LN) vapor for 10 min. The straws were then plunged into LN. In protocol-II, epididymides were collected from a mature male and placed in 4 mL of freezing medium III [freezing medium I and 0.7% (v/v) ES]. The epididymides were dissected with scissors and held for 10 min at room temperature to release epididymal sperm. The semen was loaded into 0.25-mL straws and kept at 15°C for 15 min and then held at 5°C for 15 min. The cooling rate from 15°C to 5°C was 0.7°C min⁻¹. The cooled straws were then exposed to LN vapor for 10 min and plunged into LN. Straws were thawed in a 37°C water bath for 10 s. Thawed semen in a straw was diluted with 1 mL of KRB medium with 0.4% (w/v) bovine serum albumin (BSA, fraction V, Sigma, Tokyo, Japan) at 37°C and then incubated at 37°C in 5% CO₂ in humidified air. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37°C under a light microscopy at 100×. The sperm membrane integrity was determined using a commercial Live/Dead sperm viability kit (Molecular Probes, Inc., Eugene, OR, USA) which differentiates between cells with intact plasma membranes and those with damaged membranes by fluorescent staining patterns observed with a fluorescence microscope (Olympus, IX-71, Tokyo, Japan). Similar levels of sperm motility were observed immediately after thawing of sperm from both protocols. However, after 2 h of incubation, the post-thaw motility of sperm frozen by protocol-II was significantly (P < 0.01) higher than that of protocol-I. Sperm membrane integrity immediately after thawing was also higher for sperm frozen by protocol-II (22.1% v. 9.3%, P < 0.01). Sperm frozen/thawed by protocol-II was inseminated into the top of the uterine horns of recipient females to confirm fertility. Two of three inseminated females became pregnant and gave birth to 5 pups. These results suggest that loading sperm into straws before cooling and subsequent slow cooling at 5°C to 0.7°C min⁻¹ increases post-thaw survival of rat epididymal sperm.