Penfold, Linda Margaret; (1993) Cryopreservation of mouse spermatozoa. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A new protocol for the cryopreservation of mouse spermatozoa was developed for the CBA strain. With the aid of a cryomicroscope, several cryoprotectants were screened. The glycerol concentration and osmolarity of an egg yolk TES/Tris diluent resulting in optimal cryoprotection were 1.25% and 675 mOsm respectively. The optimal rate of cooling was 50°C/min from 4°C to -70°C. The percentage of motile sperm following cryopreservation in the modified diluent was assessed subjectively as 55 ± 7.4% Acrosome integrity was investigated on permeabilized cells using a specific monoclonal antibody to the acrosome. The proportion of acrosome-intact spermatozoa after freezing and thawing was 55 ± 13%. The developed protocol was transferred to a cell freezer and the diluent was further modified by the inclusion of 0.1% sodium lauryl sulphate which facilitated recovery of frozen-thawed spermatozoa. This diluent was designated Mouse Sperm Cryoprotectant (MSC). Incubation of oocytes with frozen-thawed spermatozoa resulted in 51% developing to the 2-cell stage, of which 67% developed to the morula/blastocyst stage. Transfer of 2-cell stage embryos to the oviducts of pseudopregnant recipients resulted in a total implantation rate of 39%, with 16% developing to fetuses. Replacement of 2-cell stage embryos to the oviducts of pregnant recipients resulted in 17% developing to live offspring. Further experiments with cryopreserved oocytes and cryopreserved sperm from two strains of mice, CBA and (C57blxCBA) F1, resulted in fertilization rates of 5% and 13% respectively. Transfer of 2-cell stage embryos from oocytes fertilized with CBA and F1 cryopreserved spermatozoa to pseudopregnant recipients resulted in implantation rates of 67% and 92%, with 22% and 25% developing to fetuses respectively. This protocol, employing a novel cryoprotectant, will provide a consistent and reproducible method for preserving valuable strains of mice.

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