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Glucose transport and utilization play a crucial role during preimplantation development of the embryo, especially during the transition into the blastocyst stage. In the earlier developmental stages embryos do not use glucose as the main fuel but prefer more oxidized sources of energy such as pyruvate and lactate. The increased glucose transport indicated in mouse, bovine, porcine, rat, rabbit and human blastocysts is probably caused by the elevated energetic demands resulting from the morphological changes occurring in this stage of development. Moreover, the decreased glucose transport causes apoptotis in blastocyst cells resulting in further malformations of the fetus and even miscarriages. Glucose is transported into the embryo mainly by the membrane glucose transporters glycoproteins (GLUTs) GLUT 1, 2, 3, 4, 8, 9 that are specifically expressed during preimplantation development. GLUT 1, 2, 3, 9 are expressed in the cell membranes while GLUT 4 and 8 - insulin/IGF-I dependent transporters - are localized intracellularly and translocated into the membrane in response to insulin action. Contrary to the above mentioned transporters amongst which GLUT 1 is expressed as early as in oocytes, the expression of GLUT 4 and 8 begins from the blastocyst stage. The increased expression of glucose transporters on the blastocyst stage and especially the specific localization in blastocyst cells indicate their important role in preimplantation development.
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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