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This review presents evolution and current possibilities, state of knowledge and prospects for cryopreservation of pig embryos. In the early stages the development of this technology for use in the international pig industry was slow. Initially freezing technologies utilized the stepwise method to cryopreserve swine embryos. Conventional freezing methods will not work for pigs embryos, which are extremely sensitive to slow cooling below temperatures of approximately 15°C, since, as they cool, they undergo physiological and structural changes that leave them incapable of normal development. Using a rapid cooling processes – vitrification - is thought to outpace the damaging effects of slow cooling. It allows for an increase of the cryopreserved pig embryo survival rate to more than 80% in the laboratory. At present, vitrification is regarded as an alternative to traditional slow freezing procedures which do not offer satisfactory results for the cryopreservation of porcine embryos. Recently a novel approach consisting of a minimum sample size, increased cooling rate from 2500 C/min to 20.000 C/min, and the use propyleno and ethyleno glicol in the vitrification solution have been effective for crypreservation of pig embryos. Many factors such as the stage of embryonic development, cryoprotectant toxicity, the composition of vitryfication solution, cooling and warming rates can influence the survival of pig emryos after vitrification. Peri-hatching and hatched blastocysts tolerate slow freezing without special pretreatment, while morula and early blastocysts do not survive this cryopreservation procedure. Vitrification results in higher survival rates after warming when untreated early-to-hatched-blastocysts-stage embryos are used. An increase in cooling rate decreases sensitivity to slow freezing and may permit a reduction of cryoprotectant concentration.
Cryopreservation is not a routine procedure for the storage of equine embryos. Regardless of the method of cryopreservation, its efficiency in the transplantation of frozen embryos is relatively low. The success of freezing depends on the phase of embryo development, the type and concentration of the cryoprotectant and the freezing method. Equine embryos can be successfully cryopreserved by using conventional, slow-cooling methods or by vitrification, when embryos do not exceed 250 µm in diameter. In bigger equine embryos, changes in tolerance to cryopreservation are observed. These failures may result from the glycoprotein capsule, which appears in later stages of embryo development. New methods of vitrifying embryos in a reduced volume of fluid (2-10 µl) enable embryos to pass quickly through the critical stage of rapid temperature changes and make it possible to lower the concentration of cryoprotectants in the vitrification mixture. Vitrification is a rapid procedure that requires limited equipment and time. Further research is needed to increase the survival of equine embryos after slow-freezing or vitrification.
Surgically obtained early sheep embryos collected at morula, blastocyst, expanded blastocyst or hatching/ hatched blastocyst developmental stages were subjected to vitrification. The experiment was carried out by the traditional vitrification method, employing 0.25 ml insemination straws as carriers of embryos. The vitrification medium (VM) based on a modified phosphate-buffered solution (PB1) containing 3.0 g/L bovine serum albumin consisted of 1,2-propanediol, glycerol and sucrose (2.72 M, 1.36 M and 1.0 M - respectively). Embryos (each stage separately) were loaded into straws after 7-10 min of saturation in an equilibration solution (without sucrose). After 1 min period of saturation in VM embryos were plunged into liquid nitrogen. After warming, embryos were cultured in vitro and/or were surgically transferred to a surrogate mother for survival examination. Of 71 embryos tested, only 13 (18.3%) revealed development in vitro. The highest survival rate was observed in the most advanced group of embryos. Of 10 hatching/hatched embryos 6 (60%) survived (reexpanded). Three lambs were born after the transfer of 3 embryos from this group.
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