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Lipid characterization of in vitro-produced bovine embryos with distinct kinetics of development

Published online by Cambridge University Press:  30 September 2019

Kelly Annes
Center of Natural and Human Sciences, Universidade Federal do ABC, Av. dos Estados, 5001, 09210-580, Santo André, São Paulo, Brazil
Mateus José Sudano
Center of Natural and Human Sciences, Universidade Federal do ABC, Av. dos Estados, 5001, 09210-580, Santo André, São Paulo, Brazil
Katia Roberta A. Belaz
ThoMSon Mass Spectrometry Laboratory, Chemistry Institute, University of Campinas, Cidade Universitária Zeferino Vaz s/n, CP 6154, 13083-970, Distrito de Barão Geraldo–Campinas, São Paulo, Brazil.
Alessandra Tata
ThoMSon Mass Spectrometry Laboratory, Chemistry Institute, University of Campinas, Cidade Universitária Zeferino Vaz s/n, CP 6154, 13083-970, Distrito de Barão Geraldo–Campinas, São Paulo, Brazil.
Vanessa Gonçalves Santos
ThoMSon Mass Spectrometry Laboratory, Chemistry Institute, University of Campinas, Cidade Universitária Zeferino Vaz s/n, CP 6154, 13083-970, Distrito de Barão Geraldo–Campinas, São Paulo, Brazil.
Aldcejam Martins da Fonseca Junior
Center of Natural and Human Sciences, Universidade Federal do ABC, Av. dos Estados, 5001, 09210-580, Santo André, São Paulo, Brazil
Érika Cristina dos Santos
Center of Natural and Human Sciences, Universidade Federal do ABC, Av. dos Estados, 5001, 09210-580, Santo André, São Paulo, Brazil
Marcos Nogueira Eberlin
ThoMSon Mass Spectrometry Laboratory, Chemistry Institute, University of Campinas, Cidade Universitária Zeferino Vaz s/n, CP 6154, 13083-970, Distrito de Barão Geraldo–Campinas, São Paulo, Brazil.
Marcella Pecora Milazzotto*
Center of Natural and Human Sciences, Universidade Federal do ABC, Av. dos Estados, 5001, 09210-580, Santo André, São Paulo, Brazil
Address for correspondence: Marcella Pecora Milazzotto. Center of Natural and Human Sciences, Universidade Federal do ABC: Avenida dos Estados, 5001, 09210-580, Bloco A, Torre 3, Lab502-3, Bairro Bangu, Santo André, SP, Brazil. Fax: +55 11 4996 8390. E-mail:


Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8–16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

Research Article
© Cambridge University Press 2019 

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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, 5766.CrossRefGoogle ScholarPubMed
Alikani, M, Sadowy, S and Cohen, J (2002) Human embryo morphology and developmental capacity. In: Van Soom, A and Boerjan, M (eds). Assessment of Mammalian Embryo Quality: Invasive and Non-invasive Techniques. Dordrecht, The Netherlands: Kluwer’s Academic Publishers, pp. 131.Google Scholar
Annes, K, Soares, CA, Lima, CB and Milazzotto, MP (2017) Effective individual culture system for in vitro production of bovine embryos. Braz J Vet Res Anim Sci 54, 209.CrossRefGoogle Scholar
Aparicio, B, Cruz, M and Meseguer, M (2013) Is morphokinetic analysis the answer? Reprod BioMed Online 27, 654–63.CrossRefGoogle Scholar
Baumann, CG, Morris, DG, Sreenan, JM and Leese, HJ (2007) The quiet embryo hypothesis: molecular characteristics favoring viability. Mol Reprod Dev 74, 1345–53.CrossRefGoogle ScholarPubMed
Camargo, LS, Boite, MC, Wohlres-Viana, S, Mota, GB, Serapiao, RV, As, WF, Viana, JH and Nogueira, LA (2011) Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro . Cryobiology 63, 256–62.CrossRefGoogle ScholarPubMed
Ciray, HN, Ulug, U, Tosun, S, Erden, HF and Bahceci, M (2006) Outcome of 1114 ICSI and embryo transfer cycles of women 40 years of age and over. Reprod Biomed Online 13, 516–22.10.1016/S1472-6483(10)60639-6CrossRefGoogle ScholarPubMed
Das, S and Rand, RP (1984) Diacylglycerol causes major structural transitions in phospholipid bilayer membranes. Biochem Biophys Res Commun 124, 491–6.10.1016/0006-291X(84)91580-8CrossRefGoogle ScholarPubMed
Dode, MAN, Dufort, I, Massicotte, L and Sirard, MA (2006) Quantitative expression. of candidate genes for developmental competence in bovine two-cell embryos. Mol Reprod Dev 73, 288–97.CrossRefGoogle ScholarPubMed
dos Santos, EC, Varchetta, R, de Lima, CB, Ispada, J, Martinho, HS, Fontes, PK, Nogueira, MFG, Gasparrini, B and Milazzotto, MP (2019) The effects of crocetin supplementation on the blastocyst outcome, transcriptomic and metabolic profile of in vitro produced bovine embryos. Theriogenology 123, 30–6.CrossRefGoogle ScholarPubMed
Edidin, M (2003) Lipids on the frontier: a century of cell-membrane bilayers. Nat Rev Mol Cell Biol 4, 414–8.CrossRefGoogle ScholarPubMed
Fair, T, Murphy, M, Rizos, D, Moss, C, Martin, F, Boland, MP and Lonergan, P (2004) Analysis of differential maternal mRNA expression in developmentally competent and incompetent bovine two-cell embryos. Mol Reprod Dev 67 136–44.CrossRefGoogle ScholarPubMed
Ferguson, EM and Leese, HJ (2006) A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development. Mol Reprod Dev 73, 1195–201.CrossRefGoogle ScholarPubMed
Ferguson, EM and Leese, HJ (1999) Triglyceride content of bovine oocytes and early embryos. J Reprod Fertil 116, 373–8.CrossRefGoogle ScholarPubMed
Ferreira, CR, Saraiva, SA, Catharino, RR, Garcia, JS, Gozzo, FC, Sanvido, GB, Santos, LF, Lo Turco, EG, Pontes, JH, Basso, AC, Bertolla, RP, Sartori, R and Eberlin, MN (2010) Single embryo and oocyte lipid fingerprinting by mass spectrometry. J Lipid Res 51, 1218–27.10.1194/jlr.D001768CrossRefGoogle ScholarPubMed
Garcia, SM, Rafagnin Marinho, LS, Lunardelli, PA, Seneda, MM and Meirelles, FV (2015) Developmental block and programmed cell death in Bos indicus embryos: effects of protein supplementation source and developmental kinetics. PLoS One 10, e0119463.CrossRefGoogle ScholarPubMed
Ghanem, N, Ha, AN, Fakruzzamn, MD, Bang, J, Lee, SC and Kong, IK (2014) Differential expression of selected candidate genes in bovine embryos produced in vitro and cultured with chemicals modulating lipid metabolism. Theriogenology 82, 238–50.CrossRefGoogle ScholarPubMed
Goñi, FM and Alonso, A (1999) Structure and functional properties of diacylglycerols in membranes. Prog Lipid Res 38, 148.CrossRefGoogle ScholarPubMed
González-Serrano, AF, Pirro, V, Ferreira, RG, Oliveri, P, Eberlin, LS, Heinzmann, J, Lucas-Hahn, A, Neimann, H and Cooks, RG (2013) Desorption electrospray ionization mass spectrometry reveals lipid metabolism of individual oocytes and embryos. PLoS One 8, e74981.CrossRefGoogle ScholarPubMed
Hasler, JF (2003) The current status and the future of the commercial embryo transfer in cattle. Anim Reprod Sci 15, 245–64.CrossRefGoogle Scholar
Herrero, J and Meseguer, M (2013) Selection of high potential embryos using time-lapse imaging: the era of morphokinetics. Fertil Steril 15, 1030–4.10.1016/j.fertnstert.2013.01.089CrossRefGoogle Scholar
Ispada, J, Lima, CB, Sirard, MA, Fontes, PK, Nogueira, MFG, Annes, K and Milazzotto, MP (2018) Genome-wide screening of DNA methylation in bovine blastocysts with different kinetics of development. Epigenetics Chromatin 11, 113.CrossRefGoogle ScholarPubMed
Jung, J, Shin, H, Bang, S, Mok, HJ, Suh, CS, Kim, KP and Lim, HJ (2014) Analysis of the phospholipid profile of metaphase II mouse oocytes undergoing vitrification. PLoS One 9, 37.CrossRefGoogle ScholarPubMed
Kuleshova, LL and Lopata, A (2002) Vitrification can be more favorable than slow cooling. Fertil Steril 78, 449–54.CrossRefGoogle ScholarPubMed
Leão, BCS, Rocha-Frigoni, NAS, Cabral, EC, Franco, M, Ferreira, CR, Eberlin, MN, Filgueiras, PR and Mingoti, GZ (2014) Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos. Zygote 23, 732–41.CrossRefGoogle ScholarPubMed
Leese, HJ (2012) Metabolism of the preimplantation embryo: 40 years on. Reproduction 143, 417–27.CrossRefGoogle Scholar
Leibo, SP, Martino, A, Kobayashi, S and Pollard, JW (1996) Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 42, 4553.CrossRefGoogle Scholar
Lemmen, JG, Agerholm, I and Ziebe, S (2008) Kinetic markers of human embryo quality using time-lapse recordings of IVF/ICSI-fertilized oocytes. Reprod Biomed Online 17, 385–91.CrossRefGoogle ScholarPubMed
Lonergan, P, Khatir, H, Piumi, F, Rieger, D, Humblot, P and Boland, MP (1999) Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J Reprod Fertil 117, 159–67.10.1530/jrf.0.1170159CrossRefGoogle ScholarPubMed
Lundin, K, Bergh, C and Hardarson, T (2001) Early embryo cleavage is a strong indicator of embryo quality in human IVF. Hum Reprod 16, 2652–7.CrossRefGoogle ScholarPubMed
Market-Velker, BA, Denomme, MM and Mann, MR (2012) Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture. Biol Reprod 86, 143.CrossRefGoogle ScholarPubMed
Meseguer, M, Herrero, J, Tejera, A, Hillidsoe, KM, Ramsing, NB and Remohí, J (2011) The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 26, 2658–71.CrossRefGoogle ScholarPubMed
Milazzotto, MP, Goissis, MD, Chitwood, JL, Annes, K, Soares, CA, Ispada, J and Assumpção, MEOA (2016) Early cleavages influence the molecular and metabolic pattern of individually cultures boine blastocysts. Mol Reprod Dev 83, 324–26.CrossRefGoogle Scholar
Milhas, D, Clarke, CJ and Hannun, YA (2010) Sphingomyelin metabolism at the plasma membrane: implications for bioactive sphingolipids. FEBS Lett 584, 1887–94.CrossRefGoogle ScholarPubMed
Milne, S, Ivanova, P, Forrester, J and Brown, HA (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39, 92103.CrossRefGoogle ScholarPubMed
Mio, Y and Maeda, K (2008) Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos. Am J Obstet Gynecol 199, 660.e1–5.CrossRefGoogle ScholarPubMed
Parrish, JJ (1988) Capacitation of bovine sperm by heparin. Biol Reprod 38, 1171–80.CrossRefGoogle ScholarPubMed
Ripamonte, P, Mesquita, LG, Cortezzi, SS, Balieiro, JCC, Merigue, GKF, Watanabe, WF, Caetano, AR and Meirelles, FV (2012) Differential gene expression and developmental competence in in vitro produced bovine embryos. Zygote 20, 281–90.CrossRefGoogle ScholarPubMed
Rizos, D, Ward, F, Duffy, P, Boland, MP and Lonergan, P (2002) Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev 61, 234–8.CrossRefGoogle ScholarPubMed
Robertson, I and Nelson, RE (2010) Certification and identification of embryo. In: Stringfellow, DA and Givens, MD (eds). Manual of the International Embryo Transfer Society. Champaign, (IL) USA. International Embryo Transfer Society, pp. 86105.Google Scholar
Salumets, A, Hyden-Granskog, C, Suikkari, A., Tiitinen, A and Tuuri, T (2001) The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality. Hum Reprod 16, 2177–81.CrossRefGoogle ScholarPubMed
Shrestha, B, Sripadi, P, Reschke, BR, Henderson, HD, Powell, MJ, Moody, SA and Vertes, A (2014) Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry. PLoS One 9, e115173 CrossRefGoogle ScholarPubMed
Silva, T, Santos, EC, Annes, K, Soares, CA, Leite, RF, Lima, CB and Milazzotto, MP (2016) Morphokinetic-related response to stress in individually cultured bovine embryos. Theriogenology 86, 1308–17.CrossRefGoogle ScholarPubMed
Sirard, MA (2012) Factors affecting oocyte and embryo transcriptomes. Reprod Domest Anim 47(Suppl 4), 148–55.CrossRefGoogle ScholarPubMed
Sudano, MJ, Rascado, TDS, Tata, A, Belaz, KRA, Santos, VG, Valente, RS, Mesquita, FS, Ferreira, CR, Araújo, JP, Eberlin, MN and Landim-Alvarenga, FDC (2016). Lipidome signatures in early bovine embryo development. Theriogenology 86, 472–84.CrossRefGoogle ScholarPubMed
Sudano, MJ, Paschoal, DM, Maziero, RRD, Rascado, TD, Guastali, MD, Crocomo, LF, Magalhães, BA, Martins, A Jr, Machado, R and Landim-Alvarenga, FDC (2013) Improving postcryopreservation survival capacity: an embryo-focused approach. Anim Reprod 10, 160–7.Google Scholar
Sudano, M J, Santos, VG, Tata, A, Ferreira, CR, Paschoal, DM, Machado, R, Buratini, J, Eberlin, MN and Landim-Alvarenga, FD (2012) Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Biol Reprod 87, 130.CrossRefGoogle ScholarPubMed
Sudano, MJ, Paschoal, D, Rascado, TCM, Crocomo, L, Lima-Neto, J and Landim-Alvarenga, F (2011) Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification. Theriogenology 75, 1211–20.10.1016/j.theriogenology.2010.11.033CrossRefGoogle ScholarPubMed
Sugimura, S, Akami, T, Hashiyada, Y, Somfail, T, Yasushi, I, Hirayama, M, Yamanouchi, T, Matsuda, H, Kobayashil, S, Aikawa, Y, Ohtakel, M, Kobayashil, M, Konishil, K and Imail, K (2012) Promising system for selecting healthy in vitro-fertilized embryos in cattle. PLoS One 7, e36627.CrossRefGoogle ScholarPubMed
Tata, A, Sudano, MJ, Santos, VG, Landim-Alvarenga, FD, Ferreira, CR and Eberlin, MN (2013) Optimal single-embryo mass spectrometry fingerprinting. J Mass Spectrom 48, 844–9.CrossRefGoogle ScholarPubMed
Vajta, G, Peura, TT, Holm, P, Paldi, A, Greve, T, Trounson, AO and Callesen, H (2000) New method for culture of zona-included or zona-free embryos: the well of the well (WOW) system. Mol Reprod Dev 55, 256–64.3.0.CO;2-7>CrossRefGoogle ScholarPubMed
Van Meer, G, Voelker, DR and Feigenson, GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9, 112–24.CrossRefGoogle ScholarPubMed
Vandaele, L, Mateusen, B, Maes, DG, de Kruif, A and Van Soom, A (2007) Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity. Reproduction 133, 709–18.CrossRefGoogle ScholarPubMed
Zullo, G, Albero, G, De Canditiis, C, Bifulco, G, Campanile, G and Gasparrini, B (2016) L-Ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos. Theriogenology 85, 688–97.CrossRefGoogle ScholarPubMed
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