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 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.

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

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2–6 μm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small ( 6 μm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos. Mol. Reprod. Dev. 61: 57–66, 2002. © 2002 Wiley-Liss, Inc.

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

The present study examined the penetrability of pig oocytes by frozen-thawed ejaculated boar spermatozoa, prepared by the pellet method, coincubated in a modified Tris-buffered medium. Subsequent embryonic development of fertilized oocytes was also determined. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml), and hormonal supplements (eCG and hCG: 10 IU/ml each) for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and oocytes were coincubated for 12 h with three different (1 x 10(5), 5 x 10(5) and 1 x 10(6)/ml) sperm concentrations (experiment 1). In experiment 2, oocytes were coincubated with sperm (5 x 10(5)/ml) for 3, 6, 9, and 12 h. In experiment 3, at 6 h after coincubation with sperm at 5 x 10(5)/ml concentration, oocytes were transferred into NCSU 23 + 0.4% BSA medium. At 48 and 144 h, cleavage and blastocyst formation rates, respectively, were evaluated. Insemination with 1 x 10(5)/ml resulted in a 40% sperm penetration rate of oocytes with 16% polyspermy. Mean number of sperm (MNS) per oocyte was 1.2 +/- 0.1. At 5 x 10(5) and 1 x 10(6)/ml, penetration rate (84-87%) and polyspermy (57-64%) increased (p < 0.001), with no difference between the two concentrations. However, MNS per oocyte increased (p < 0.05) with increasing sperm concentration. Penetration rate was 31% at 3 h and increased (p < 0.001) at 6-12 h (80-88%), with no difference between these time points. Polyspermy increased (p < 0.05) in a time-dependent manner up to 9 h, with no difference between 9 and 12 h. Compared to 3 h, MNS per oocyte increased (p < 0.05) at 9 and 12 h, with no mean difference at 6 h. At 48 after culture, the cleavage rate was 40%, and at 144 h, the blastocyst rate was 19%. This study describes the cryopreservation of ejaculated boar semen by the pellet method and the successful in vitro fertilization of pig oocytes by frozen-thawed spermatozoa with subsequent development to the blastocyst stage.

Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified tris-buffered medium with frozen-thawed ejaculated spermatozoa.

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

We have determined that the tolerance of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos to cryopreservation at the pre-morula stage can be improved by removal of cytoplasmic lipid droplets by centrifugation. Nucleus transfer was also performed using cryopreserved, delipated (lipid droplets removed) 8- to 16-cell-stage blastomeres of IVM/IVF embryos as donor nuclei. In vitro developmental ability of the delipated embryos to the blastocyst stage (20 of 126) was found to be equal to that of undelipated embryos (35 of 176); and of 53 delipated embryos cryopreserved at the 8- to 16-cell stage, 12 developed into blastocysts in vitro after thawing. On the other hand, only 2 of 43 undelipated embryos and 5 of 59 sham-operated embryos survived (p < 0.05). When blastomeres isolated from cryopreserved, delipated 8- to 16-cell-stage embryos were used for nucleus transfer, 57 of 80 successfully fused with enucleated oocytes, which was significantly lower than the fusion rate obtained with blastomeres of unfrozen, undelipated embryos (93 of 104, p < 0.01). However, the developmental rate to the blastocyst stage for nucleus transfer embryos reconstituted with frozen, delipated blastomeres (9 of 57) was not different from that of the nucleus transfer embryos with unfrozen, undelipated embryos (23 of 93). These results confirm that removal of cytoplasmic lipid droplets from bovine IVM/IVF zygotes allows for successful cryopreservation at the 8- to 16-cell stage and that blastomeres from these embryos can be used as donors of karyoplasts for nucleus transfer.

Cryopreservation of Bovine Pre-Morula-Stage In Vitro Matured/In Vitro Fertilized Embryos after Delipidation and before Use in Nucleus Transfer

Recent advances in cryopreservation of porcine embryos

To identify an in vitro culture procedure which can be used to assess porcine in vitro fertilized embryos, three culture systems were examined. A total of 103 in vivo fertilized zygotes were co-cultured in modified BMOC-3 medium with granulosa cells, or in modified BMOC-2 with oviduct cells, and 35 were cultured in Whitten's medium supplemented with 1.5% BSA. After 8 days in culture, 66.0 and 73.6% of the co-cultured zygotes, and 51.4% of the zygotes cultured in the modified Whitten's medium developed to blastocyst stage. In contrast, only 20.5% (18/88) and 1.4% (9/627) of in vitro fertilized zygotes derived from in vivo and in vitro matured oocytes developed to blastocysts in the co-culture with modified BMOC-3 and granulosa cells. It is concluded that the culture systems examined are competent to support development of porcine zygotes to blastocysts and evaluate in vitro fertilized porcine embryos.

Hiroshi Nagashima

Papers

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

Cryopreservation of Bovine Pre-Morula-Stage In Vitro Matured/In Vitro Fertilized Embryos after Delipidation and before Use in Nucleus Transfer

Recent advances in cryopreservation of porcine embryos

In Vitro Development of in Vivo and in Vitro Fertilized Porcine Zygotes

In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes