The gene pool of various animals can be maintained either by collecting, breeding and housing the animals or by preserving the gametes (sperm and oocytes) or embryos in vitro. The latter method is more flexible and usually more cost-effective as well. In vitro gamete and embryo preservation has also been used in connection with in vitro cultures of gametes and embryos to reproduce human and non-human animals. For example, in in vitro fertilization (IVF) for the mammalian species, the collected sperm and oocytes are preserved and cultured in vitro before the oocytes are fertilized. After fertilization, the resultant embryos are cultured in vitro until a certain stage of maturity (usually blastocysts) is reached, after which the embryos are implanted to induce pregnancy.
The in vitro preservation and culture of gametes and embryos has been used more successfully in some species than in others. A freeze-thaw cycle reduces the percentage of viable sperm more dramatically in avian semen samples than in mammalian semen samples. On a related note, the freeze-thaw process can also reduce the percentage of viable oocytes and embryos. The frequency of productive pregnancy induced in bovines is 80% lower when the in vitro fertilized embryos implanted were frozen and thawed before being implanted. In addition, mammalian embryos are very sensitive to temperature change. For example, less than one-half of a degree temperature change can kill a mammalian embryo. In contrast, natural bird embryos, which are natively in contact with white yolk, are fairly resistant to temperature change (e.g., fertilized chicken eggs can be held at 32° F. for 10 hours without significant effect on hatching rate).
Moreover, IVF pregnancies also yield offspring having a higher rate of abnormalities than natural pregnancies. IVF procedures have yielded larger bovine calves and smaller human babies at a much higher frequency than natural pregnancies. These abnormalities may arise from poorly characterized differences between the environments encountered in vivo and in vitro by gametes and embryos. Also, many supplements of conventional culture systems do not naturally contact embryos in vivo during early embryo development. More particularly, the proteins encountered by gametes and embryos in vitro (e.g., bovine serum albumin, amniotic fluid and fetal calf serum) come from fully formed individuals that have homeostatic organs and adaptations to handle waste, control pH, transport food, and accommodate temperature insult.
In birds, reptiles, marsupials and egg-laying mammals such as monotremes (Hughes and Hall, 1998), natural embryos are in contact with white yolk, a minor egg yolk constituent (less than 2%) that differs in composition, structure, and physical properties from yellow yolk (Burley and Vadehra, 1989). White yolk, but not yellow yolk, contacts the early embryo and there is no clear demarcation between embryo and white yolk (Lillie, 1919) because of the meroblastic cleavage wherein some of the developing cells appear to have no membrane between the cytoplasm and the white yolk.
Growth of the avian ovum is known as vitellogenesis. As in most mammals, an avian chick has its full complement of oocytes at hatching. Marza and Marza (1935) divided oogenesis in the hen into three phases. The first, which can last for several years, is a quiescent period in which the primordial yolk is laid down and maintained. White yolk slowly accumulates in the second period, which lasts for about 2 months. In the final phase, which lasts for 5–9 days just prior to ovulation, yellow yolk is rapidly deposited.
White yolk surrounds and appears to compartmentalize yellow yolk. From a bulb-shaped white yolk latebra at the center of the egg yolk, an elongated stem extends toward the blastoderm if an embryo is present (or toward the blastodisc if the egg is unfertilized or if embryogenesis did not commence). The stem flares out at its distal end into the Nucleus of Pander. Contiguous with the Nucleus of Pander is a small amount of white yolk lying just below the vitelline membrane. Fabian (1982) determined that the Nucleus of Pander was directly over the center of the yolk in 90% of 181 White Leghorn eggs examined. Romanoff and Romanoff (1949) identified light colored rings within the mass of yellow yolk as white yolk, but these rings are not white yolk. Rather, these rings should be called light yolk as they are identical to yellow yolk except for color and probably reflect some diurnal aspect of yellow yolk deposition (Gilbert, 1971; Burley and Vadehra, 1989).
Yellow yolk protein and lipid are synthesized in the liver and then are transported via circulation to the ovary (Burley and Vadehra, 1989). White yolk generally has a non-liver origin. White yolk remains liquid after a freeze-thaw cycle whereas yellow yolk becomes firm and gelatinous, a change known as yolk gelling (Burley and Vadehra, 1989). Tanabe-Yuji et al. (2000) determined that 14 hours after oviposition one small white follicle of the ovary has a layer of yellow yolk spheres deposited just under the perivitelline membrane. Such follicles are called transition follicles. A hen in lay has a series of developing follicles from small white follicles to one the size of a laid egg.
White yolk can be collected in at least two ways. It can be separated from yellow yolk after one or more freeze-thaw cycles. It can also be collected from small immature follicles (diameter between 1 and 8 mm) where yellow yolk has not been deposited.