Recent improvements in cryopreservation of mammalian eggs enable the long-term preservation of female germ cells in several mammalian species. Nevertheless, cryopreservation of porcine oocytes is still considered as a challenge. Although the use of vitrification techniques result in reasonable survival rates, developmental competence of vitrified oocytes has been compromised. Alterations of zona characteristics, cytoskeleton, mitochondrial functions and antioxidant-defense ability caused by vitrification are among the most frequently observed malformations which may be responsible for the low developmental competence of cryopreserved porcine oocytes. Furthermore, in vitro maturation, fertilization and embryo culture technologies, which are indispensable for generating embryos from cryopreserved oocytes, generate high rates of abnormal fertilization (polyspermy) and additional stress in resultant embryos further compromising their developmental competence. As a result, embryo development of porcine cryopreserved oocytes is still at low level and to date no piglet has been produced from such oocytes. The aim of the present review is to summarize knowledge on viability and developmental competence of vitrified porcine oocytes and to give ideas for future perspectives for the improvement of porcine oocyte cryopreservation technology.

Factors affecting cryopreservation of porcine oocytes.

Advances on in vitro production and cryopreservation of porcine embryos.

The following selection markers for in vitro-produced porcine embryos were investigated: the timing, pattern and evenness of the first cleavage and the timing of the second cleavage. The embryos that cleaved by 30 h post-insemination (hpi) developed to blastocysts at a significantly higher rate (60.9%) and with a significantly higher cell number (33.6 cells) than those of embryos cleaved by 36 hpi (26.4% and 23.6 cells, respectively, P 4-cell embryos (46.3 and 42.6%, respectively, P 4-cell embryos (23.6-27.8 cells, P<0.05). Among embryos cleaved by 30 hpi, the blastocysts derived from evenly cleaved embryos (40.6 cells) were of significantly better quality than those derived from unevenly cleaved embryos (33.2 cells, P<0.05), although their blastocyst rates did not differ. The evenly cleaved embryos that underwent subsequent cleavage within 18 h had significantly higher blastocyst rates (72.7-81.0%) and quality (36.2-40.9 cells) than those without subsequent cleavage (48.3% and 22.5 cells, respectively, P<0.05) during the same period. In conclusion, the timing, pattern and evenness of the first cleavage and the timing of the second cleavage affected the developmental competence and quality of in vitro-produced porcine embryos.

Evaluation of Developmental Competence of In Vitro-produced Porcine Embryos Based on the Timing, Pattern and Evenness of the First Cleavage and Onset of the Second Cleavage

Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions. Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition. They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate. Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.

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Culture of pig embryos

Factors and methods of pig oocyte and embryo quality improvement and their application in reproductive biotechnology.

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.

Carlos Manuel Ulloa Ullo

Papers

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos.