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

Ushijima, Hitoshi; Yamakawa, Hirohito; Nagashima, Hiroshi

doi:10.1095/biolreprod60.2.534

Cited by 68 publications

(47 citation statements)

References 30 publications

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“…Such problems are not encountered after blastocyst expansion when, coincident with significant increases in energy needs, blastocyst neutral lipid content is reduced (Nagashima et al, 1992). Physical removal of excess lipid from ova of cattle (Tominaga et al, 1998;Ushijima et al, 1999) and pigs (Nagashima et al, 1994) at earlier developmental stages also reduces sensitivity to chilling. Moreover, Ferguson and Leese (1999) reported that triglyceride concentrations in cattle embryos remain stable from the two-cell to the blastocyst stage in vivo (32.2 versus 33 ng per embryo), whereas in vitro, in embryos exposed to serum (10%, v/v) from the four-cell stage, the triglyceride reserves can double by the blastocyst stage.…”

Section: Discussionmentioning

confidence: 99%

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

McEvoy

Coull²,

Broadbent³

et al. 2000

Reproduction

262

212

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Cattle, pig and sheep oocytes isolated from healthy cumulus-oocyte complexes were pooled, within species, to provide samples of immature denuded oocytes with intact zona pellucida (n = 1000 per sample) for determination of fatty acid mass and composition in total lipid, constituent phospholipid and triglyceride. Acyl-containing lipid extracts, transmethylated in the presence of a reference penta-decaenoic acid (15:0), yielded fatty acid methyl esters which were analysed by gas chromatography. Mean (± SEM) fatty acid content in samples of pig oocytes (161 ± 18 µg per 1000 oocytes) was greater than that in cattle (63 ± 6 µg; P < 0.01) and sheep oocytes (89 ± 7 µg; P < 0.05). Of 24 fatty acids detected, palmitic (16:0; 25-35%, w/w), stearic (18:0; 14-16%) and oleic (18:1n-9; 22-26%) acids were most prominent in all three species. Saturated fatty acids (mean = 45-55%, w/w) were more abundant than mono-(27-34%) or polyunsaturates (11-21%). Fatty acids of the n-6 series, notably linoleic (18:2n-6; 5-8%, w/w) and arachidonic acid (20:4n-6; 1-3%), were the most abundant polyunsaturates. Phospholipid consistently accounted for a quarter of all fatty acids in the three species, but ruminant oocytes had a lower complement of polyunsaturates (14-19%, w/w) in this fraction than pig oocytes (34%, w/w) which, for example, had a three-to fourfold greater linoleic acid content. An estimated 74 ng of fatty acid was sequestered in the triglyceride fraction of individual pig oocytes compared with 23-25 ng in ruminant oocytes (P < 0.01). It is concluded that the greater fatty acid content of pig oocytes is primarily due to more abundant triglyceride reserves. Furthermore, this speciesspecific difference, and that in respect of polyunsaturated fatty acid reserves, may underlie the contrasting chilling, culture and cryopreservation sensitivities of embryos derived from pig and ruminant (cattle, sheep) oocytes.

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Section: Discussionmentioning

confidence: 99%

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

McEvoy

Coull²,

Broadbent³

et al. 2000

Reproduction

262

212

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“…Moreover, post-thaw survival of bovine blastocysts was improved after removal of lipid droplets at an earlier stage [18][19][20]. However, Ushijima et al [19] r e p o r t e d t h a t c y t o c ha l a s in t r ea t m e n t a nd centrifugation without micromanipulation did not improve the cryotolerance of bovine zygotes or embryos. We have investigated the effect of polarization of lipid droplets on cryotolerance of 16-cell stage embryos without micromanipulation.…”

Section: Cytoplasmic Lipids Responsible For Cryoinjurymentioning

confidence: 92%

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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“…Likewise, Nagashima et al [215] demonstrated that the high lipid content of pig embryos was responsible for their higher chilling sensitivity and also that embryos delipidated by centrifugation and microaspiration of polarized lipids became more tolerant to chilling. The lipid content was also pointed out as responsible for the chilling and freezing sensitivity of IVP cattle embryos, and when lipid droplets were displaced by ultracentrifugation (mechanical delipidation), their cryosurvival was improved [216,217]. On the contrary, Romão et al [8] found a reduced viability in IVP sheep embryos submitted to an ultracentrifugation process mostly due to zona pellucida and membrane fracture.…”

Section: Methods Of Improving the Cryopreservation Of Sheep Embryosmentioning

confidence: 99%

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

Romão¹,

Marques²,

Bettencourt³

et al. 2016

Insights From Animal Reproduction

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Due to economical and scientific limitations, sheep embryo reproductive technologies are less commercially applied than in other animal species. However, it is very clear that, in the near future, those techniques are expected to have a central role in animal production as a consequence of genetic and reproductive demands. One drawback is that results obtained after sheep embryo cryopreservation are unattractive for commercial purposes. It is expected that a successful cryopreservation of sheep embryos can push forward all other reproductive biotechnologies in this species, such as multiple ovulation and embryo transfer (MOET), artificial insemination, or in vitro production of embryos. This paper tries to discuss the current and future perspectives of cryopreservation of in vivo-and in vitro-produced sheep embryos concerning advantages and limitations for its practical use and possible solutions for improving methods to allow a higher survival rate of cryopreserved embryos.

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

Cited by 68 publications

References 30 publications

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Cryopreservation of Sheep Produced Embryos – Current and Future Perspectives

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