Research Article

Assessment of three generations of mice derived by ICSI using freeze-dried sperm

Ming-Wen Li^a2a3, Brandon J. Willis^a2, Stephen M. Griffey^a4, Jimmy L. Spearow^a5 p1 and K. C. Kent Lloyd^a1a2a3 c1

^a1 Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis, California 95616, USA.

^a2 Mouse Biology Program, School of Veterinary Medicine, University of California, Davis, California, USA.

^a3 Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis, California, USA.

^a4 Comparative Pathology Laboratory, School of Veterinary Medicine, University of California, Davis, California, USA.

^a5 Section of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, California, USA.

Summary

Although the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 °C for 1–2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocysts in vitro (C57BL/6J and B6D2F1/J) and liveborn pups in vivo (B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.

(Received August 10 2008)

(Accepted January 10 2009)

Keywords:

• Freeze drying;
• ICSI;
• Microsatellite;
• Mouse;
• Sperm

Correspondence:

^c1 All correspondence to: K.C. Kent Lloyd, Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis, California 95616, USA. Tel: +1 530 752–6865. Fax: +1 530 752 7914. e-mail: kclloyd@ucdavis.edu

^p1 Present address: Department of Toxic Substances Control, California EPA, Sacramento, California, USA.