Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

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Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

The use of pigs in neuroscience: modeling brain disorders.

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

https://www.rbmojournal.com/article/S1472-6483(10)61091-7/pdf

Are programmable freezers still needed in the embryo laboratory? Review on vitrification

While much is known about the metabolism of exogenous nutrients such as glucose, lactate, pyruvate, amino acids by oocytes and pre-implantation mammalian embryos, the role of endogenous stores, particularly lipid, has been largely overlooked. The presence of lipid within oocytes and early embryos has been long known, and comparisons between species indicate that the amounts and types of lipid present vary considerably. Large amounts of intracellular lipid can compromise the success of cryopreservation and the removal of such lipid has been the subject of considerable effort. In this review, we present evidence that strongly suggests a metabolic role for lipid, specifically with regard to energy provision, in the late-stage oocyte and the pre-implantation embryo. We focus initially on oxygen consumption as a global indicator of metabolic activity, before reviewing different approaches that either have been designed to investigate directly, or have revealed indirectly the role of endogenous lipid in energy generation. These fall under five headings: (i) fatty acid oxidation; (ii) inhibition of triglyceride oxidation; (iii) culture in the absence of exogenous substrates; (iv) cytoplasmic organization; and (v) delipidation. On the basis of the data derived from these studies, we conclude that there is strong evidence for the utilization of endogenous lipid as an energy substrate by oocytes and early embryos.

/pdf/role-of-fatty-acids-in-energy-provision-during-oocyte-70kyyvsd9c.pdf

Role of Fatty Acids in Energy Provision During Oocyte Maturation and Early Embryo Development

Improving cryopreservation systems.

Porcine embryos are cryopreserved using a method for reducing the level of lipid within cells of the embryo followed by freezing the embryo. Reduction of the level of lipid can be achieved either by polarizing the lipid within the embryo by centrifugation of the embryo, followed by rapid freezing or by microsurgical removal of the polarized lipid. Alternatively the level of lipid may be reduced by culturing the embryo in vitro. The reduction of the level of lipid before freezing results in good survival rates and can be used on zona intact porcine embryos.

Cryopreservation of porcine embryos

The lipid content of porcine 1-cell stage embryos was reduced (delipated) through the use of micromanipulation to remove the lipid layer formed after centrifugation Of 94 delipated embryos chilled to 4 degrees C for 1 h at the 1-cell or 2- to 4-cell stage, 60 (64%) cleaved in culture with development to the morula-blastocyst stage, whereas all of the control embryos lysed within 24 h Significantly more embryos developed beyond the 8-cell stage when they were chilled at the 2- to 4-cell stage compared with chilling at the 1-cell stage (44%, 20 of 45 vs 18%, 9 of 49) Fewer embryos developed after chilling if they were only partially rather than fully delipated Developmental rates of partially delipated embryos to the 8-cell and blastocyst stages were 33% (13 of 40) and 8% (3 of 40), rates significantly (p or = blastocyst: 40%, 19 of 48 vs 57%, 17 of 30) These data demonstrate that the sensitivity of porcine embryos to chilling is related to their high lipid content and that they can become tolerant to chilling if their lipid content is reduced

Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling

Recent advances in cryopreservation of porcine embryos

Successful cryopreservation of porcine early cleavage stage embryos following removal of cytoplasmic lipid

Viability of pig blastocysts following in vitro culture for 0-3 day(s) was assessed after transfer to recipients. Embryos collected on Day 4, 5 and 6 were cultured in a modified Brinster's medium +10% fetal calf serum for 3, 2 and 1 day(s) respectively. Embryos were transferred to Day 6 recipients. Day 7 noncultured blastocysts were transferred as a control. Nine (90%) of 10 recipients with noncultured embryos, 6 (75%) of 8 recipients with embryos cultured for 1 day, 5 (83%) of 6 recipients with embryos cultured for 2 days, and 2 (29%) of 7 recipients with embryos cultured for 3 days farrowed. Embryo survival was 50%, 34%, 35% and 12%, respectively. Sur-vival of noncultured embryos was higher (P<0.05) than that of embryos cultured for 1-3 day(s). Survival of embryos cultured for 3 days was lower (P<0.01) than that of those cultured for 1 or 2 day(s). These results indicate that the viability of cultured pig embryos following transfer de-creases with increasing length of culture in vitro.

Naomi Kashiwazaki

Papers

Cryopreservation of porcine embryos

Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling

Recent advances in cryopreservation of porcine embryos

Successful cryopreservation of porcine early cleavage stage embryos following removal of cytoplasmic lipid

Development to Term of Pig Blastocysts Cultured In Vitro for 0-3 Days