Effect of vitrification on global gene expression dynamics of bovine elongating embryos
Emilio Gutierrez-Castillo A , Hao Ming
A , Brittany Foster A , Lauren Gatenby A , Chun Kuen Mak B , Carlos Pinto B , Kenneth Bondioli A C and Zongliang Jiang A C
+ Author Affiliations
- Author Affiliations
A School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA, USA.
B Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
C Corresponding authors. Email: zjiang@agcenter.lsu.edu; kbondioli@agcenter.lsu.edu
Reproduction, Fertility and Development 33(5) 338-348 https://doi.org/10.1071/RD20285
Submitted: 30 October 2020 Accepted: 13 January 2021 Published: 19 February 2021
Abstract
Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, in vitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n = 80) and vitrified (n =80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell–sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.
Keywords: transcriptome, vitrification, blastocyst, trophectoderm, elongation.
References
Abe, H., Yamashita, S., Satoh, T., and Hoshi, H. (2002). Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Mol. Reprod. Dev. 61, 57–66.| Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.Crossref | GoogleScholarGoogle Scholar | 11774376PubMed |
Aksu, D. A., Agca, C., Aksu, S., Bagis, H., Akkoc, T., Caputcu, A. T., Arat, S., Taskin, A. C., Kizil, S. H., Karasahin, T., Akyol, N., Satilmis, M., Sagirkaya, H., Ustuner, B., Nur, Z., and Agca, Y. (2012). Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts. Mol. Reprod. Dev. 79, 613–625.
| Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts.Crossref | GoogleScholarGoogle Scholar | 22778065PubMed |
Amoushahi, M., Salehnia, M., and Mowla, S. J. (2017). Vitrification of Mouse MII Oocyte Decreases the Mitochondrial DNA Copy Number, TFAM Gene Expression and Mitochondrial Enzyme Activity. J. Reprod. Infertil. 18, 343–351.
| 29201664PubMed |
Awasthi, P., Foiani, M., and Kumar, A. (2015). ATM and ATR signaling at a glance. J. Cell Sci. 128, 4255–4262.
| ATM and ATR signaling at a glance.Crossref | GoogleScholarGoogle Scholar | 26567218PubMed |
Bakkenist, C. J., and Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506.
| DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Crossref | GoogleScholarGoogle Scholar | 12556884PubMed |
Bartolac, L. K., Lowe, J. L., Koustas, G., Grupen, C. G., and Sjöblom, C. (2018). Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts. Cryobiology 80, 70–76.
| Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts.Crossref | GoogleScholarGoogle Scholar | 29221996PubMed |
Bedzhov, I., Leung, C. Y., Bialecka, M., and Zernicka-Goetz, M. (2014). In vitro culture of mouse blastocysts beyond the implantation stages. Nat. Protoc. 9, 2732–2739.
| In vitro culture of mouse blastocysts beyond the implantation stages.Crossref | GoogleScholarGoogle Scholar | 25356584PubMed |
Berthelot, F., Martinat-Botté, F., Vajta, G., and Terqui, M. (2003). Cryopreservation of porcine embryos: state of the art. Livest. Prod. Sci. 83, 73–83.
| Cryopreservation of porcine embryos: state of the art.Crossref | GoogleScholarGoogle Scholar |
Blockeel, C., Campbell, A., Coticchio, G., Esler, J., Garcia-Velasco, J. A., Santulli, P., and Pinborg, A. (2019). Should we still perform fresh embryo transfers in ART? Hum. Reprod. 34, 2319–2329.
| Should we still perform fresh embryo transfers in ART?Crossref | GoogleScholarGoogle Scholar | 31803911PubMed |
Boonkusol, D., Gal, A. B., Bodo, S., Gorhony, B., Kitiyanant, Y., and Dinnyes, A. (2006). Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos. Mol. Reprod. Dev. 73, 700–708.
| Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos.Crossref | GoogleScholarGoogle Scholar | 16541460PubMed |
Burkuš, J., Kačmarová, M., Kubandová, J., Kokošová, N., Fabianová, K., Fabian, D., Koppel, J., and Čikoš, Š. (2015). Stress exposure during the preimplantation period affects blastocyst lineages and offspring development. J. Reprod. Dev. 61, 325–331.
| Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.Crossref | GoogleScholarGoogle Scholar | 25985793PubMed |
Callis, J. (2014). The ubiquitination machinery of the ubiquitin system. Arabidopsis Book 12, e0174.
| The ubiquitination machinery of the ubiquitin system.Crossref | GoogleScholarGoogle Scholar | 25320573PubMed |
Chatterjee, A., Saha, D., Niemann, H., Gryshkov, O., Glasmacher, B., and Hofmann, N. (2017). Effects of cryopreservation on the epigenetic profile of cells. Cryobiology 74, 1–7.
| Effects of cryopreservation on the epigenetic profile of cells.Crossref | GoogleScholarGoogle Scholar | 27940283PubMed |
Choi, Y.-H., and Hinrichs, K. (2017). Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program. Theriogenology 87, 48–54.
| Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program.Crossref | GoogleScholarGoogle Scholar | 27634397PubMed |
Dai, J., Wu, C., Muneri, C. W., Niu, Y., Zhang, S., Rui, R., and Zhang, D. (2015). Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability. Cryobiology 71, 291–298.
| Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability.Crossref | GoogleScholarGoogle Scholar | 26247316PubMed |
de Oliveira Leme, L., Dufort, I., Spricigo, J. F. W., Braga, T. F., Sirard, M.-A., Franco, M. M., and Dode, M. A. N. (2016). Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos. Theriogenology 85, 733.e1.
| Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.Crossref | GoogleScholarGoogle Scholar | 26553569PubMed |
Deglincerti, A., Croft, G. F., Pietila, L. N., Zernicka-Goetz, M., Siggia, E. D., and Brivanlou, A. H. (2016). Self-organization of the in vitro attached human embryo. Nature 533, 251–254.
| Self-organization of the in vitro attached human embryo.Crossref | GoogleScholarGoogle Scholar | 27144363PubMed |
Desai, N., Szeptycki, J., Scott, M., AbdelHafez, F. F., and Goldfarb, J. (2008). Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts. Cell Preserv. Technol. 6, 181–190.
| Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts.Crossref | GoogleScholarGoogle Scholar |
Diaz, F., Bondiolli, K., Paccamonti, D., and Gentry, G. T. (2016). Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification. Theriogenology 85, 894–903.
| Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.Crossref | GoogleScholarGoogle Scholar | 26639642PubMed |
Diaz, F. A., Gutierrez, E. J., Cramer, E., Paccamonti, D. L., Gentry, G. T., and Bondioli, K. R. (2018). Pregnancy Rates Following Low-Temperature Storage of Large Equine Embryos Before Vitrification. J. Equine Vet. Sci. 64, 12–16.
| Pregnancy Rates Following Low-Temperature Storage of Large Equine Embryos Before Vitrification.Crossref | GoogleScholarGoogle Scholar | 30973146PubMed |
Dochi, O. (2019). Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management. J. Reprod. Dev. 65, 389–396.
| Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management.Crossref | GoogleScholarGoogle Scholar | 31189772PubMed |
Dos Santos-Neto, P. C., Cuadro, F., Barrera, N., Crispo, M., and Menchaca, A. (2017). Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos. Cryobiology 78, 8–14.
| Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos.Crossref | GoogleScholarGoogle Scholar | 28803846PubMed |
Frijters, A. C. J., Mullaart, E., Roelofs, R. M. G., van Hoorne, R. P., Moreno, J. F., Moreno, O., and Merton, J. S. (2009). What affects fertility of sexed bull semen more, low sperm dosage or the sorting process? Theriogenology 71, 64–67.
| What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?Crossref | GoogleScholarGoogle Scholar |
Garcia-Dominguez, X., Peñaranda, D. S., Estruch, G., Blanca, J., García-Carpintero, V., Cañizares, J., Marco-Jiménez, F., and Vicente, J. S. (2018). Long-term phenotypic effects following vitrified-thawed embryo transfer in a rabbit model. bioRxiv , 410514.
| Long-term phenotypic effects following vitrified-thawed embryo transfer in a rabbit model.Crossref | GoogleScholarGoogle Scholar |
Gómez, E., and Muñoz, M. (2015). Multiple-embryo transfer for studying very early maternal–embryo interactions in cattle. Reproduction 150, R35–R43.
| Multiple-embryo transfer for studying very early maternal–embryo interactions in cattle.Crossref | GoogleScholarGoogle Scholar | 25918434PubMed |
Grothmann, H. (2019) 46th Annual Conference of the IETS. In ‘Embryo Technology Newsletter. Vol. 37.’ (International Embryo Technology Society Champaign, IL)
Gupta, A., Singh, J., Dufort, I., Robert, C., Dias, F. C. F., and Anzar, M. (2017). Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage. PLoS One 12, e0187268.
| Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage.Crossref | GoogleScholarGoogle Scholar | 29236768PubMed |
Harada, H., Andersen, J. S., Mann, M., Terada, N., and Korsmeyer, S. J. (2001). p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD. Proc. Natl. Acad. Sci. USA 98, 9666–9670.
| p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD.Crossref | GoogleScholarGoogle Scholar | 11493700PubMed |
Hernández, G., and Vazquez-Pianzola, P. (2005). Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families. Mech. Dev. 122, 865–876.
| Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families.Crossref | GoogleScholarGoogle Scholar | 15922571PubMed |
Jeffrey, I. W., Bushell, M., Tilleray, V. J., Morley, S., and Clemens, M. J. (2002). Inhibition of protein synthesis in apoptosis: differential requirements by the tumor necrosis factor alpha family and a DNA-damaging agent for caspases and the double-stranded RNA-dependent protein kinase. Cancer Res. 62, 2272–2280.
| 11956083PubMed |
Jiang, Q., Li, F., Shi, K., Wu, P., An, J., Yang, Y., and Xu, C. (2014). Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2α/ATF4 axis in selenite-treated NB4 cells. Cell Death Dis. 5, e1270.
| Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2α/ATF4 axis in selenite-treated NB4 cells.Crossref | GoogleScholarGoogle Scholar | 25356875PubMed |
Krisher, R. L., and Prather, R. S. (2012). A role for the Warburg effect in preimplantation embryo development: metabolic modification to support rapid cell proliferation. Mol. Reprod. Dev. 79, 311–320.
| A role for the Warburg effect in preimplantation embryo development: metabolic modification to support rapid cell proliferation.Crossref | GoogleScholarGoogle Scholar | 22431437PubMed |
Lei, T., Guo, N., Tan, M.-h., and Li, Y.-f. (2014). Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution. Journal of Huazhong University of Science and Technology [Medical Sciences] 34, 99–102.
| Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution.Crossref | GoogleScholarGoogle Scholar |
Leibo, S. P. (2008). Cryopreservation of oocytes and embryos: Optimization by theoretical versus empirical analysis. Theriogenology 69, 37–47.
| Cryopreservation of oocytes and embryos: Optimization by theoretical versus empirical analysis.Crossref | GoogleScholarGoogle Scholar | 18023472PubMed |
Li, L., Zhang, X., Zhao, L., Xia, X., and Wang, W. (2012). Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing. Mol. Reprod. Dev. 79, 229–236.
| Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing.Crossref | GoogleScholarGoogle Scholar | 22213487PubMed |
Mahmoudzadeh, A. R., Van Soom, A., Ysebaert, M. T., and de Kruif, A. (1994). Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology 42, 1389–1397.
| Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos.Crossref | GoogleScholarGoogle Scholar |
Miermont, A., Antolović, V., Lenn, T., Nichols, J. M. E., Millward, L. J., and Chubb, J. R. (2019). The fate of cells undergoing spontaneous DNA damage during development. Development 146, dev174268.
| The fate of cells undergoing spontaneous DNA damage during development.Crossref | GoogleScholarGoogle Scholar | 30975700PubMed |
Moussa, M., Bersinger, I., Doligez, P., Guignot, F., Duchamp, G., Vidament, M., Mermillod, P., and Bruyas, J. F. (2005). In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification. Theriogenology 64, 1619–1632.
| In vitro comparisons of two cryopreservation techniques for equine embryos: Slow-cooling and open pulled straw (OPS) vitrification.Crossref | GoogleScholarGoogle Scholar | 15907992PubMed |
Moussa, M., Yang, C.-Y., Zheng, H.-Y., Li, M.-Q., Yu, N.-Q., Yan, S.-F., Huang, J.-X., and Shang, J.-H. (2019). Vitrification alters cell adhesion related genes in pre-implantation buffalo embryos: Protective role of β-mercaptoethanol. Theriogenology 125, 317–323.
| Vitrification alters cell adhesion related genes in pre-implantation buffalo embryos: Protective role of β-mercaptoethanol.Crossref | GoogleScholarGoogle Scholar | 30502624PubMed |
Nazmara, Z., Salehnia, M., and HosseinKhani, S. (2014). Mitochondrial distribution and ATP content of vitrified, in vitro matured mouse oocytes. Avicenna J. Med. Biotechnol. 6, 210–217.
| 25414783PubMed |
Nedambale, T. L., Dinnyés, A., Groen, W., Dobrinsky, J. R., Tian, X. C., and Yang, X. (2004). Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology 62, 437–449.
| Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification.Crossref | GoogleScholarGoogle Scholar | 15226000PubMed |
Orlowski, R. Z. (1999). The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ. 6, 303–313.
| The role of the ubiquitin-proteasome pathway in apoptosis.Crossref | GoogleScholarGoogle Scholar | 10381632PubMed |
Pollard, T. D., Earnshaw, W. C., Lippincott-Schwartz, J., and Johnson, G. T. (2017) Chapter 31 - Intercellular Junctions. In ‘Cell Biology (Third Edition).’ (Eds TD Pollard, WC Earnshaw, J Lippincott-Schwartz and GT Johnson) pp. 543–553. (Elsevier)
Rall, W. F., and Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at -196 [deg]C by vitrification. Nature 313, 573–575.
| Ice-free cryopreservation of mouse embryos at -196 [deg]C by vitrification.Crossref | GoogleScholarGoogle Scholar | 3969158PubMed |
Raynes, R., Brunquell, J., and Westerheide, S. D. (2013). Stress Inducibility of SIRT1 and Its Role in Cytoprotection and Cancer. Genes Cancer 4, 172–182.
| Stress Inducibility of SIRT1 and Its Role in Cytoprotection and Cancer.Crossref | GoogleScholarGoogle Scholar | 24020008PubMed |
Rho, G.-J., Kim, S., Yoo, J.-G., Balasubramanian, S., Lee, H.-J., and Choe, S.-Y. (2002). Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes. Mol. Reprod. Dev. 63, 464–470.
| Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes.Crossref | GoogleScholarGoogle Scholar | 12412049PubMed |
Rodriguez, R. M., Fernandez, A. F., and Fraga, M. F. (2013). Role of sirtuins in stem cell differentiation. Genes Cancer 4, 105–111.
| Role of sirtuins in stem cell differentiation.Crossref | GoogleScholarGoogle Scholar | 24020001PubMed |
Sandeep, R. K. D., Pratap, S. A., Mohan, J. L., and Chandra, B. C. (2018). Developmental Potential and Apoptosis Incidence of In Vitro Produced Buffalo Embryos Vitrified by Solid Surface Technique. Journal of Animal Research 8, 571–577.
| Developmental Potential and Apoptosis Incidence of In Vitro Produced Buffalo Embryos Vitrified by Solid Surface Technique.Crossref | GoogleScholarGoogle Scholar |
Saxton, R. A., and Sabatini, D. M. (2017). mTOR Signaling in Growth, Metabolism, and Disease. Cell 168, 960–976.
| mTOR Signaling in Growth, Metabolism, and Disease.Crossref | GoogleScholarGoogle Scholar | 28283069PubMed |
Schmitt, E., Naveau, M., and Mechulam, Y. (2010). Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier. FEBS Lett. 584, 405–412.
| Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier.Crossref | GoogleScholarGoogle Scholar | 19896944PubMed |
Senger, P.L. (2012) ‘Pathways to pregnancy & parturition (Third Edition).’ (Current Conceptions, Inc.)
Shahbazi, M. N., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., Bolton, V., Fogarty, N. N. M., Campbell, A., Devito, L., Ilic, D., Khalaf, Y., Niakan, K. K., Fishel, S., and Zernicka-Goetz, M. (2016). Self-organization of the human embryo in the absence of maternal tissues. Nat. Cell Biol. 18, 700–708.
| Self-organization of the human embryo in the absence of maternal tissues.Crossref | GoogleScholarGoogle Scholar | 27144686PubMed |
Sheikh, M. S., and Fornace, A. J. (1999). Regulation of translation initiation following stress. Oncogene 18, 6121–6128.
| Regulation of translation initiation following stress.Crossref | GoogleScholarGoogle Scholar | 10557103PubMed |
Sjunnesson, Y. (2019). In vitro fertilisation in domestic mammals: a brief overview. Ups. J. Med. Sci. 125, 68–76.
| In vitro fertilisation in domestic mammals: a brief overview.Crossref | GoogleScholarGoogle Scholar | 31833449PubMed |
Spencer, T. E., Forde, N., Dorniak, P., Hansen, T. R., Romero, J. J., and Lonergan, P. (2013). Conceptus-derived prostaglandins regulate gene expression in the endometrium prior to pregnancy recognition in ruminants. Reproduction 146, 377–387.
| Conceptus-derived prostaglandins regulate gene expression in the endometrium prior to pregnancy recognition in ruminants.Crossref | GoogleScholarGoogle Scholar | 23966582PubMed |
Stolboushkina, E. A., and Garber, M. B. (2011). Eukaryotic type translation initiation factor 2: Structure–functional aspects. Biochemistry (Mosc.) 76, 283–294.
| Eukaryotic type translation initiation factor 2: Structure–functional aspects.Crossref | GoogleScholarGoogle Scholar | 21568863PubMed |
Stracker, T. H., Roig, I., Knobel, P. A., and Marjanović, M. (2013). The ATM signaling network in development and disease. Front. Genet. 4, 37.
| The ATM signaling network in development and disease.Crossref | GoogleScholarGoogle Scholar | 23532176PubMed |
Vicente, J. S., Saenz-de-Juano, M. D., Jiménez-Trigos, E., Viudes-de-Castro, M. P., Peñaranda, D. S., and Marco-Jiménez, F. (2013). Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation. Cryobiology 67, 321–326.
| Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation.Crossref | GoogleScholarGoogle Scholar | 24080489PubMed |
Wek, R. C., Jiang, H. Y., and Anthony, T. G. (2006). Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7–11.
| Coping with stress: eIF2 kinases and translational control.Crossref | GoogleScholarGoogle Scholar | 16246168PubMed |
Wong, K. M., Mastenbroek, S., and Repping, S. (2014). Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil. Steril. 102, 19–26.
| Cryopreservation of human embryos and its contribution to in vitro fertilization success rates.Crossref | GoogleScholarGoogle Scholar | 24890275PubMed |
Xu, J., Chaubal, S. A., and Du, F. (2009). Optimizing IVF with sexed sperm in cattle. Theriogenology 71, 39–47.
| Optimizing IVF with sexed sperm in cattle.Crossref | GoogleScholarGoogle Scholar | 18947864PubMed |
Zhao, X. M., Fu, X. W., Hou, Y. P., Yan, C. L., Suo, L., Wang, Y. P., Zhu, H. B., Dinnyés, A., and Zhu, S. E. (2009). Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos. Mol. Reprod. Dev. 76, 1056–1063.
| Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.Crossref | GoogleScholarGoogle Scholar | 19551710PubMed |
Zhao, X.-M., Du, W.-H., Wang, D., Hao, H.-S., Liu, Y., Qin, T., and Zhu, H.-B. (2011). Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture. Mol. Reprod. Dev. 78, 942–950.
| Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture.Crossref | GoogleScholarGoogle Scholar | 21919110PubMed |
