60.Apply cell technology and cryopreservation technology for safe gene bank

Cryobiologic principles

Cryopreservation allows virtually indefinite storage of biological material without deterioration over a time scale of at least several thousands of years, but probably much longer. Important progress in cryobiology was achieved in the second half of the previous century. Much progress resulted from empirical studies. In later years, progress was also strongly stimulated by the development of fundamental theoretical cryobiology.

In so-called “slow cooling” methods, the biological material is cooled at a range of cooling rates that are fast enough to prevent “slow cooling damage” but slow enough to allow sufficient dehydration of the cells to prevent intracellular ice formation. The dehydrated cells in the “unfrozen fraction” that remains between the masses of ice will ultimately reach a stable glassy state, or “vitrify”. In so-called “vitrification methods”, the water content is lowered before cooling by adding high concentrations of cryoprotective agents (CPA). Thus, no ice is formed at all, and the entire sample will vitrify. This allows fast cooling rates without risk of IIF. The CPA concentration of vitrification solutions can be minimized by using very high cooling and thawing rates. By using extremely high cooling rates, vitrification is possible even in complete absence of CPAs.

5.3.2 Semen

Semen of most livestock species can be frozen adequately. In addition, dedicated freezing media and equipment for collecting, packing, freezing and inseminating semen have been developed and are available commercially for a large number of bird and mammal livestock species. In the cattle artificial insemination (AI) industry, in which bulls are selected for “freezability” of their semen, the post-thaw semen quality is quite good, featuring 50 to 70 percent motile spermatozoa. Pregnancy or calving rate is the same as that of fresh semen, provided that higher sperm dosages are used for frozen sperm. For other mammalian species, the percentage of post-thaw motile sperm or membrane-intact sperm is generally somewhat lower, but a fair post-thaw viability can be expected for most species. For many species, the fertility of frozen semen is found to be lower than that of fresh semen. This may depend on the site of semen deposition, the morphology of the female genital tract, and the ability to detect heat or ovulation. For instance in sheep, very poor results are obtained with cervical semen deposition when using frozen rather than fresh ram semen. There may be considerable differences between breeds and between males in the “freezability” of the semen. As a consequence, frozen semen of some genetically interesting breeds or males may not be suitable as a gene bank resource, or can be used only with a poor efficiency.

As regards avian livestock species, semen-freezing techniques for fowl, turkey, goose, and duck render a fair post-thaw sperm survival of up to 60 percent live spermatozoa. Reasonable insemination results with frozen-thawed semen have been reported for the major avian livestock species. More than 200 fish species with external fertilization have been tested for sperm cryopreservation. The present state of the art for many species of fish seems to be adequate for the purpose of gene banking. The insemination ratios used may vary according to species and procedure between 104 and 107 spermatozoa per egg. Even in fish species like the African catfish, in which semen can only be obtained by testis destruction or death of the male, enough semen can be obtained from one single male to produce close to 106 larvae. Thus, for gene bank purposes, storage of only one single vial or straw would be sufficient to generate plenty of progeny of that male.

The freezing media vary widely between the classes (mammals, birds, fish), but also between species within a class. Most media feature a saline or saccharide bulk osmotic support, a suitable CPA at concentrations varying from 0.2–1.5 M, and various protective macromolecular additives, mostly milk and egg yolk components, or lipid components from vegetal origi. Milk or egg yolk is often used in media for mammalian semen. In mammalian semen, the egg yolk and milk components protect the spermatozoa during cooling, freezing and thawing (Watson, 1976). These additives are generally not used in freezing media for avian and fish species, although in a few studies with fish semen, egg yolk was found to confer protection against cryodamage.

5.3.3 Oocytes

In the last ten years, considerable progress has been made with cryopreservation of oocytes. Viable oocytes have been recovered after freezing and thawing in a great number of species. Successes have been reported in post-thaw oocyte maturation, fertilization, and embryo development in a number of species. Live-born young from embryos produced from cryopreserved oocytes have been reported in cattle,mice, rats, horses and humans (Stachecki and Cohen, 2004). The present efficiency and reliability of using frozen thawed oocytes for generating offspring is still much lower than with cryopreserved embryos. Freezing oocytes of avian and fish species is not successful however, largely because of the large size, the high lipid content, and the polar organization of bird and fish ova.

5.3.4 Embryos or embryonic cells

In cattle, cryopreservation of embryos is highly successful. Both slow freezing and vitrification protocols are effective. The success of cryopreservation depends on the stage of the embryo, that is, especially good results are obtained with blastocysts. Cryopreservation of embryos resulting in live offspring has been reported for most of the important (mammalian) livestock species. Cryopreservation of pig embryos has long been problematic due to extreme chilling sensitivity and high lipid content of the pig embryos. However, recent studies have focused on overcoming these problems and produced successful vitrification methods for cryopreservation of pig embryos.

5.3.5 Somatic cells

Cryopreservation of somatic cells proved to be possible for a number of cell types. In early studies, the methods came down to adding 5 to 10 percent of a suitable cryoprotectant, such as glycerol or DMSO to the suspension of cells in culture medium, and placing tubes with a few millilitres of that suspension at -80 °C in a mechanical freezer. In fact, this simple procedure is still effectively used today. Obviously, with this simple procedure the rate of cooling cannot be controlled; in fact in many publications the cooling rate is unknown. There are only a few studies in which controlled rate freezers were used, e.g. with skin fibroblast.

61 Rules on keeping and breeding facilities in the laboratory. Proper housing and management of animal facilities are essential to animal well-being, to the quality of research data and teaching or testing programs in which animals are used.The primary enclosure (usually a cage, pen, or stall) provides the limits of an animal's immediate environment.* Allow for the normal physiologic and behavioral needs of the animals, including urination and defecation, maintenance of body temperature, normal movement and postural adjustments, and, where indicated, reproduction. * Allow conspecific social interaction and development of hierarchies within or between enclosures. * Make it possible for the animals to remain clean and dry (as consistent with the requirements of the species). * Allow adequate ventilation. * Allow the animals access to food and water and permit easy filling, refilling, changing, servicing, and cleaning of food and water utensils. * Provide a secure environment that does not allow escape of or accidental entrapment of animals or their appendages between opposing surfaces or by structural openings. * Are free of sharp edges or projections that could cause injury to the animals. * Allow observation of the animals with minimal disturbance of them. * Room air is mixed with at least 50% fresh air (that is, the supply air does not exceed 50% recycled air). * Husbandry practices, such as bedding-change and cage-washing frequency, and the preparation of recycled air used are sufficient to minimize toxic gases and odors. * Recycled air is returned only to the room or area from which it was generated, except if it comes from other than animal-housing areas. * Recycled air is appropriately conditioned and mixed with sufficient fresh air to address the thermal and humidity requirements of animals in that space. In general, lighting should be diffused throughout an animal holding area and provide sufficient illumi nation for the well-being of the animals and to allow good housekeeping practices, adequate inspection of animals including the bottom-most cages in racks and safe working conditions for personnel. Some conditions might require increased environmental temperatures, such as postoperative recovery, maintenance of chicks for the first few days after hatching, housing of some hairless rodents, and housing of neonates that have been separated from their mothers. Noise produced by animals and animal-care activities is inherent in the operation of an animal facility. Therefore, noise control should be considered in facility design and operation. Noisy animals such as dogs, swine, goats, and nonhuman primates should be housed away from quieter animals, such as rodents, rabbits, and cats. Radios, alarms, and other sound generators should not be used in animal rooms unless they are parts of an approved protocol or an enrichment program.