148 • Technologies To Maintain Biological Diversity 



MAINTENANCE TECHNOLOGIES 



Storage Technologies 



Cryogenic storage of gametes and embryos 

 introduces a new level of complexity to the pro- 

 cedures already discussed, but it also holds the 

 promise of greatly facilitating conservation of 

 genetic diversity. For both semen and embryos, 

 a critical element for cryopreservation involves 

 development of media to protect cells when 

 they are frozen in liquid nitrogen at —196° C. 

 Likewise, procedures must be developed to reg- 

 ulate the rate of freezing and thawing of this 

 material in a way that will maintain the integrity 

 of the cells. 



Photo credit: Zoological Society of San Diego 



In a vial, frozen cells may be stored in suspended 



animation and later resuscitated. Technologies for 



storing sperm, ova, and embryos are being developed 



for domestic and nondomestic species. 



The ability to freeze semen successfully re- 

 sulted from the accidental discovery in 1949 

 of the cryoprotective action of glycerol. To date, 

 semen has been frozen from at least 200 differ- 

 ent species, but little has actually been thawed 

 and tested. Commercial use of artificial insemi- 

 nation with frozen semen is a reality today only 

 for domestic species. Current media for freez- 

 ing of semen usually include buffering agents, 

 a cryoprotectant such as glycerol, antibiotics, 

 and either egg yolk or milk. Many variations 

 of these media exist, and a somewhat different 

 mix usually must be developed for different 

 species. 



The first successful freezing of mammalian 

 embryos with a subsequent live birth was re- 

 ported in 1972 with mice (50,51). Since then, 

 embryos of 10 mammalian species have been 

 successfully frozen, and the procedure has be- 

 come routine with the mouse, cow, and rabbit. 

 As with semen, a variety of freezing media and 

 of freezing and thawing procedures are avail- 

 able and are being evaluated. Rapid increases 

 in efficiency have occurred in the bovine em- 

 bryo transfer industry, and frozen embryos can 

 now be transferred in a manner analogous to 

 artificial insemination. As in the freezing of se- 

 men, specific procedures and media appear to 

 be required for each species. Yet the procedure 

 in general rests on a firm mechanistic under- 

 standing of the processes responsible for cell 

 injury during freezing, thawing, and dilution. 

 Previous detailed work with mice and primates 

 can act as a model for extension of these tech- 

 niques to other mammals. Thus, given appro- 

 priate research, cryogenic storage of embryos 

 could be developed for a range of species. 



Cryopreservation is probably the most prom- 

 ising area of reproduction research today. The 

 potential exists to hold a well-constructed sam- 

 ple of the genetic diversity of a population in 

 suspended animation indefinitely. In practice, 

 frozen semen or embryo storage would prob- 

 ably be used with living populations for a num- 

 ber of reasons: to augment the genetic varia- 

 tion within breeding populations, to allow 

 periodic comparisons between original and cur- 

 rent populations, and to validate the viability 



