Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

doi:10.1016/S0093-691X(00)00430-1

Theriogenology

Volume 54, Issue 8, 1 November 2000, Pages 1239-1247

https://doi.org/10.1016/S0093-691X(00)00430-1 Get rights and content

Abstract

The objective of this study was to test a new co-culture system of bovine embryos, which we call “mixed co-culture.” This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 μL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P≤0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P≤0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.

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Several types of conditions have been studied in order to improve embryo development rate and quality in vitro. Among these, coculture with somatic cells has been amply investigated with the purpose of finding a condition resembling as much as possible that of the embryo's maternal environment [2–4]. Our study focuses on the use of a monolayer made with bovine oviduct epithelial cells (BOEC) as feeders.
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Plus qu’un simple conduit, l’oviducte joue un rôle actif dans différents mécanismes de la reproduction et, en particulier, dans la régulation du développement du jeune embryon et son transport vers le site d’implantation. Plusieurs études ont mis en évidence l’existence de communications entre l’oviducte et les embryons préimplantatoires au niveau fonctionnel et moléculaire. L’étude de ces interactions étant délicate in vivo, de nombreux travaux s’appuient sur des systèmes in vitro mimant le milieu maternel. Ces différentes approches ont permis de mettre en évidence une action des cellules sur la qualité du développement embryonnaire (taux et viabilité des blastocystes). En retour, les embryons en développement émettent des signaux qui modulent l’activité des cellules maternelles.
Beyond being a pipe between ovary and uterus, the oviduct is an active player in different aspects of early reproductive processes, in particular in the transport of embryos to the site of implantation and the regulation of its early development. Different studies evidenced a communication between oviduct and early embryo at the molecular and functional levels. Since the study of these interactions is difficult in vivo, different in vitro systems have been developed to mimic the maternal milieu during early development. These systems allowed to confirm the action of the cells on the quality of early development (blastocyst rate and viability). In turn, the embryos are producing signals that are able to modify and adapt the activity of maternal cells.
Transvaginal ultrasound-guided cumulus oocyte complexes aspiration and in vitro embryo production in suckled beef and lactating dairy cattle on pasture-based management conditions
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The overall COC recovery rate and blastocyst production obtained in our study were similar to those reported by Gibbons et al. (1994) and Garcia and Salaheddine (1998) but higher than those documented by Aller et al. (2010). In addition, IVP of bovine embryos was similar to previously reported studies using media supplemented with aminoacids (Aller et al., 2000), bovine serum (Galli and Lazzari, 1996) or supported by feeder cells (Duszewska et al., 2000; Gutierrez-Adam et al., 2001). However, pregnancy rates in the present study were lower than that reported from in vivo produced embryos (Spell et al., 2001).
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A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams.
The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams.
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Embryo-somatic cell co-culture was devised over 40 years ago in an attempt to improve the development and viability of mammalian preimplantation embryos generated and cultured in vitro. While initial endeavours were successful in this respect, other studies soon highlighted a number of significant long-term detrimental impacts of this approach. Surprisingly little is known about the mechanisms underlying the beneficial effects of co-culture, although the production of embryotrophic compounds, modulation of nutrient profile, protection against culture-induced stress and/or toxin clearance are all contenders. The extent to which the inadvertent exposure of embryos to serum accounts for many of these effects remains open to question. Although the popularity of somatic cell co-culture has recently declined in favour of the use of sequential media due to concerns associated with its risk of disease transmission and long-term sequelae, we argue that complete dismissal of this technique is ill advised, given that our limited understanding of basic somatic cell interactions has prevented us from fully exploiting its potential. In this respect, there is some merit in focussing future research strategies based on reconstructed maternal tract tissue. Although the use of co-culture in clinical practice is unacceptable and its implementation in domestic species for commercial purposes should be viewed with diffidence, this technique can still provide a wealth of information on the development of novel, more physiological embryo in vitro culture systems. The proviso for acquiring such information is to gain a fuller understanding of the culture requirements/biochemistry of somatic cells and their interaction with the early conceptus.
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Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

Abstract

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Proceedings of the Fourteenth World Congress on Fertility and Sterility (Caracas)

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J Biol Chem

J Biol Chem

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Dev Biol

Theriogenology