Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at-196 degrees C

This study reports the development of a reliable method for cryopreservatio n of rat epididymal spermatozoa and the production of live young by artific ial insemination using these cryopreserved spermatozoa. The motility and me mbrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations o f glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generat e normal offspring by intrauterine insemination was also evaluated. Rat spe rmatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition , after centrifugation three times the percentage of membrane-intact sperma tozoa decreased to approximately 0%. The percentage of membrane-intact sper matozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycer ol or with 3% glycerol did not influence rates of sperm motility after free zing and thawing, the percentage of membrane-intact spermatozoa was improve d by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containi ng 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adju sted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rat s were inseminated into the oviductal end of both uterine horns with frozen -thawed spermatozoa. Forty-one normal live offspring were obtained from nin e of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedur e is the first successful production of offspring using spermatozoa cryopre served in liquid nitrogen.