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Liquid storage of porcine in vitro-produced blastocysts; a practical approach for short storage

Published online by Cambridge University Press:  08 June 2023

Linda Marijke Haug ,
Reina Jochems Open the ORCID record for Reina Jochems [Opens in a new window] ,
Ann Helen Gaustad ,
Elisabeth Kommisrud ,
Frøydis Deinboll Myromslien ,
Eli Grindflek  and
Anne Hege Alm-Kristiansen Open the ORCID record for Anne Hege Alm-Kristiansen [Opens in a new window]
Linda Marijke Haug
Affiliation:
Department of Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway
Reina Jochems
Affiliation:
Norsvin SA, Hamar, Norway
Ann Helen Gaustad
Affiliation:
Norsvin SA, Hamar, Norway
Elisabeth Kommisrud
Affiliation:
Department of Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway
Frøydis Deinboll Myromslien
Affiliation:
Department of Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway
Eli Grindflek
Affiliation:
Norsvin SA, Hamar, Norway
Anne Hege Alm-Kristiansen*
Affiliation:
Department of Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway
*
Corresponding author: Anne Hege Alm-Kristiansen; Email: anne.almkristiansen@inn.no
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Summary

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

Keywords

ApoptosisCO2 independent storageEmbryo qualityIVFRate of development
Type
Research Article
Information
Zygote , Volume 31 , Issue 5 , October 2023 , pp. 441 - 450
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Copyright
© The Author(s), 2023. Published by Cambridge University Press

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References

Berthelot, F., Martinat-Botté, F., Locatelli, A., Perreau, C. and Terqui, M. (2000). Piglets born after vitrification of embryos using the open pulled straw method. Cryobiology, 41(2), 116–124. doi: 10.1006/CRYO.2000.2273 CrossRefGoogle ScholarPubMed
Bó, G. A. and Mapletoft, R. J. (2013). Evaluation and classification of bovine embryos. Animal Reproduction, 10(3), 344–348.Google Scholar
Bodri, D., Sugimoto, T., Yao Serna, J., Kawachiya, S., Kato, R. and Matsumoto, T. (2016). Blastocyst collapse is not an independent predictor of reduced live birth: A time-lapse study. Fertility and Sterility, 105(6), 1476–1483.e3. doi: 10.1016/J.FERTNSTERT.2016.02.014 CrossRefGoogle Scholar
Bonesmo, H. and Enger, E. G. (2021). The effects of progress in genetics and management on intensities of greenhouse gas emissions from Norwegian pork production. Livestock Science, 254, 104746. doi: 10.1016/J.LIVSCI.2021.104746 CrossRefGoogle Scholar
Cambra, J. M., Martinez, E. A., Rodriguez-Martinez, H., Gil, M. A. and Cuello, C. (2021). Transcriptional profiling of porcine blastocysts produced in vitro in a chemically defined culture medium. Animals: An Open Access Journal from MDPI, 11(5), 1–19. doi: 10.3390/ani11051414 CrossRefGoogle Scholar
Campbell, A., Fishel, S., Bowman, N., Duffy, S., Sedler, M. and Hickman, C. F. L. (2013). Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reproductive Biomedicine Online, 26(5), 477–485. doi: 10.1016/J.RBMO.2013.02.006 CrossRefGoogle ScholarPubMed
Chen, Y. N., Dai, J. J., Wu, C. F., Zhang, S. S., Sun, L. W. and Zhang, D. F. (2018). Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts. Animal Reproduction Science, 198, 137–144. doi: 10.1016/J.ANIREPROSCI.2018.09.012 CrossRefGoogle ScholarPubMed
Chen, P. R., Redel, B. K., Kerns, K. C., Spate, L. D. and Prather, R. S. (2021). Challenges and considerations during in vitro production of porcine embryos. Cells, 10(10), 1–23. doi: 10.3390/cells10102770 CrossRefGoogle ScholarPubMed
Cuello, C., Martinez, C. A., Nohalez, A., Parrilla, I., Roca, J., Gil, M. A. and Martinez, E. A. (2016). Effective vitrification and warming of porcine embryos using a pH-stable, chemically defined medium. Scientific Reports, 6, 33915. doi: 10.1038/srep33915 CrossRefGoogle ScholarPubMed
Fowler, K. E., Mandawala, A. A., Griffin, D. K., Walling, G. A. and Harvey, S. C. (2018). The production of pig preimplantation embryos in vitro: current progress and future prospects. Reproductive Biology, 18(3), 203–211. doi: 10.1016/j.repbio.2018.07.001 CrossRefGoogle ScholarPubMed
Gomis, J., Cuello, C., Sanchez-Osorio, J., Gil, M. A., Parrilla, I., Angel, M. A., Maside, C., del Olmo, D., Vazquez, J. M., Roca, J. and Martinez, E. A. (2012). Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: Evaluation of critical steps of the procedure. Theriogenology, 78(6), 1339–1349. doi: 10.1016/J.THERIOGENOLOGY.2012.05.035 CrossRefGoogle ScholarPubMed
Gonzales, D. S., Jones, J. M., Pinyopummintr, T., Carnevale, E.M., Ginther, O. J., Shapiro, S. S. and Bavister, B. D. (1996). Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts. Human Reproduction, 11(12), 2739–2745. https://academic.oup.com/humrep/article/11/12/2739/714605 10.1093/oxfordjournals.humrep.a019201CrossRefGoogle ScholarPubMed
Hao, Y., Lai, L., Mao, J., Im, G. S., Bonk, A. and Prather, R. S. (2004). Apoptosis in parthenogenetic preimplantation porcine embryos. Biology of Reproduction, 70(6), 1644–1649. doi: 10.1095/biolreprod.103.026005 CrossRefGoogle ScholarPubMed
Haouzi, D. and Hamamah, S. (2009). Pertinence of apoptosis markers for the improvement of in vitro fertilization (IVF). Current Medicinal Chemistry, 16(15), 1905–1916. doi: 10.2174/092986709788186075 CrossRefGoogle ScholarPubMed
Jochems, R., Gaustad, A. H., Zak, L. J., Grindflek, E., Zeremichael, T. T., Oskam, I. C., Myromslien, F. D., Kommisrud, E. and Krogenæs, A. K. (2022). Effect of two ‘progressively motile sperm–oocyte’ ratios on porcine in vitro fertilization and embryo development. Zygote, 30(4), 543–549. doi: 10.1017/S0967199422000053 CrossRefGoogle ScholarPubMed
Lin, Q., Le, Q. A., Takebayashi, K., Hirata, M., Tanihara, F., Thongkittidilok, C., Sawamoto, O., Kikuchi, T. and Otoi, T. (2022). Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature. Reproduction in Domestic Animals, 57(5), 556–563. doi: 10.1111/rda.14095 CrossRefGoogle Scholar
Mandawala, A. A., Harvey, S. C., Roy, T. K. and Fowler, K. E. (2016). Cryopreservation of animal oocytes and embryos: current progress and future prospects. Theriogenology, 86(7), 1637–1644. doi: 10.1016/J.THERIOGENOLOGY.2016.07.018 CrossRefGoogle ScholarPubMed
Marcos, J., Pérez-Albalá, S., Mifsud, A., Molla, M., Landeras, J. and Meseguer, M. (2015). Collapse of blastocysts is strongly related to lower implantation success: A time-lapse study. Human Reproduction, 30(11), 2501–2508. doi: 10.1093/humrep/dev216 CrossRefGoogle ScholarPubMed
Marsico, T. V., de Camargo, J., Valente, R. S. and Sudano, M. J. (2019). Embryo competence and cryosurvival: molecular and cellular features. Animal Reproduction, 16(3), 423–439. doi: 10.21451/1984-3143-AR2019-0072 CrossRefGoogle ScholarPubMed
Martinez, E. A., Cuello, C., Parrilla, I., Rodriguez-Martinez, H., Roca, J., Vazquez, J. L., Vazquez, J. M. and Gil, M. A. (2013). Design, development, and application of a non-surgical deep uterine embryo transfer technique in pigs. Animal Frontiers, 3(4), 40–47. doi: 10.2527/af.2013-0032 CrossRefGoogle Scholar
Martinez, E. A., Angel, M. A., Cuello, C., Sanchez-Osorio, J., Gomis, J., Parrilla, I., Vila, J., Colina, I., Diaz, M., Reixach, J., Vazquez, J. L., Vazquez, J. M., Roca, J. and Gil, M. A. (2014). Successful non-surgical deep uterine transfer of porcine morulae after 24 hour culture in a chemically defined medium. PLOS ONE, 9(8), e104696. doi: 10.1371/journal.pone.0104696 CrossRefGoogle Scholar
Martinez, E. A., Cuello, C., Parrilla, I., Martinez, C. A., Nohalez, A., Vazquez, J. L., Vazquez, J. M., Roca, J. and Gil, M. A. (2016). Recent advances toward the practical application of embryo transfer in pigs. Theriogenology, 85(1), 152–161. doi: 10.1016/j.theriogenology.2015.06.002 CrossRefGoogle ScholarPubMed
Martinez, C. A., Nohalez, A., Parrilla, I., Lucas, X., Sanchez-Osorio, J., Roca, J., Cuello, C., Rodriguez-Martinez, H., Martinez, E. A. and Gil, M. A. (2018). Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence. Theriogenology, 108, 229–238. doi: 10.1016/j.theriogenology.2017.12.001 CrossRefGoogle Scholar
Martinez, C. A., Cambra, J. M., Nohalez, A., Parrilla, I., Roca, J., Vazquez, J. L., Rodriguez-Martinez, H., Gil, M. A., Martinez, E. A. and Cuello, C. (2019a). Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 °C. Scientific Reports, 9(1), 6219. doi: 10.1038/s41598-019-42712-x CrossRefGoogle ScholarPubMed
Martinez, E. A., Martinez, C. A., Cambra, J. M., Maside, C., Lucas, X., Vazquez, J. L., Vazquez, J. M., Roca, J., Rodriguez-Martinez, H., Gil, M. A., Parrilla, I. and Cuello, C. (2019b). Achievements and future perspectives of embryo transfer technology in pigs. Reproduction in Domestic Animals, 54(Suppl. 4), 4–13. doi: 10.1111/rda.13465 CrossRefGoogle ScholarPubMed
Mito, T. and Hoshi, H. (2019). In vitro culture of late stage pig embryos in a chemically defined medium, porcine blastocyst medium (PBM). In Methods in Molecular Biology Herrick, J. (ed.), Comparative Embryo Culture. Humana Press, pp. 105–113.Google Scholar
Mito, T., Yoshioka, K., Noguchi, M., Yamashita, S., Misumi, K., Hoshi, T. and Hoshi, H. (2015). Birth of piglets from in vitro-produced porcine blastocysts vitrified and warmed in a chemically defined medium. Theriogenology, 84(8), 1314–1320. doi: 10.1016/j.theriogenology.2015.06.024 CrossRefGoogle Scholar
Morató, R., Castillo-Martín, M., Yeste, M. and Bonet, S. (2016). Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification. Reproduction, Fertility, and Development, 28(7), 886–892. doi: 10.1071/RD14203 CrossRefGoogle ScholarPubMed
Niimura, S. (2003). Time-lapse videomicrographic analyses of contractions in mouse blastocysts. Journal of Reproduction and Development, 49(6), 413–423. doi: 10.1262/jrd.49.413 CrossRefGoogle ScholarPubMed
Nohalez, A., Martinez, C. A., Parrilla, I., Roca, J., Gil, M. A., Rodriguez-Martinez, H., Martinez, E. A. and Cuello, C. (2018). Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes. Theriogenology, 113, 113–119. doi: 10.1016/j.theriogenology.2018.02.014 CrossRefGoogle ScholarPubMed
Ohlweiler, L. U., Mezzalira, J. C. and Mezzalira, A. (2019). Vitrification of in vitro produced porcine blastocysts: Influence of cryoprotectants toxicity and embryo age. Acta Scientiae Veterinariae, 47(1), 1–8. doi: 10.22456/1679-9216.93774 CrossRefGoogle Scholar
Petters, R. M. and Wells, K. D. (1993). Culture of pig embryos. Journal of Reproduction and Fertility. Supplement, 48, 61–73. doi: 10.1530/biosciprocs.14.005 Google ScholarPubMed
Ramos-Ibeas, P., Gimeno, I., Cañón-Beltrán, K., Gutiérrez-Adán, A., Rizos, D. and Gómez, E. (2020). Senescence and apoptosis during in vitro embryo development in a bovine model. Frontiers in Cell and Developmental Biology, 8, 619902. doi: 10.3389/fcell.2020.619902 CrossRefGoogle Scholar
Rubio Pomar, F. J., Ducro-Steverink, D. W. B., Hazeleger, W., Teerds, K. J., Colenbrander, B. and Bevers, M. M. (2004). Development, DNA fragmentation and cell death in porcine embryos after 24-h storage under different conditions. Theriogenology, 61(1), 147–158. doi: 10.1016/s0093-691x(03)00204-8 CrossRefGoogle Scholar
Sanchez-Osorio, J., Cuello, C., Gil, M. A., Almiñana, C., Parrilla, I., Caballero, I., Garcia, E. M., Vazquez, J. M., Roca, J. and Martinez, E. A. (2008). Factors affecting the success rate of porcine embryo vitrification by the open pulled straw method. Animal Reproduction Science, 108(3–4), 334–344. doi: 10.1016/J.ANIREPROSCI.2007.09.001 CrossRefGoogle ScholarPubMed
Sciorio, R., Thong, K. J. and Pickering, S. J. (2020). Spontaneous blastocyst collapse as an embryo marker of low pregnancy outcome: a time-lapse study. JBRA Assisted Reproduction, 24(1), 34–40. doi: 10.5935/1518-0557.20190044 Google ScholarPubMed
Stringfellow, D. A. and Givens, M. D. (2009). Manual of the International Embryo Transfer Society (4th edn). International Embryo Transfer Society.Google Scholar
Sudano, M., Paschoal, D., Maziero, R., Rascado, T., Crocomo, L., Magalhães, L., Monteiro, B., Martins, A. Jr, Machado, R. and Landim-Alvarenga, F. (2013). Improving postcryopreservation survival capacity: an embryo-focused approach. Animal Reproduction, 10(3), 160–167. http://www.iets.org/comm_data.asp Google Scholar
Van Soom, A., Ysebaert, M. T. and De Kruif, A. (1997). Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos. Molecular Reproduction and Development, 47(1), 47–56. doi: 10.1002/(SICI)1098–2795(199705) 3.0.CO;2-Q>CrossRefGoogle ScholarPubMed
Viñals Gonzalez, X., Odia, R., Cawood, S., Gaunt, M., Saab, W., Seshadri, S. and Serhal, P. (2018). Contraction behaviour reduces embryo competence in high-quality euploid blastocysts. Journal of Assisted Reproduction and Genetics, 35(8), 1509–1517. doi: 10.1007/s10815-018-1246-x CrossRefGoogle ScholarPubMed
Waterhouse, K. E., Hofmo, P. O., Tverdal, A. and Miller, R. R. (2006). Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction, 131(5), 887–894. doi: 10.1530/rep.1.01049 CrossRefGoogle ScholarPubMed
Yoshioka, K., Suzuki, C. and Onishi, A. (2008). Defined system for in vitro production of porcine embryos using a single basic medium. Journal of Reproduction and Development, 54(3), 208–213. doi: 10.1262/jrd.20001 CrossRefGoogle ScholarPubMed