Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture
Introduction
If bovine IVM, IVF, and IVC systems are to be commercially successful, it is essential to produce a high rate of viable embryos that result in normal pregnancies. Traditionally, the commercial development of bovine embryos derived from IVM, IVF and IVC has employed co-culture of somatic cell lines such as buffalo rat liver cells (BRL) or vero cells in complex tissue culture media supplemented with serum for embryo culture [1], [2], [3]. While these culture systems have been shown to support high rates of blastocyst development, the commercial applications of these techniques have been hampered by problems associated with resultant pregnancies. The use of co-culture with somatic cells and serum for embryo development is associated with high rates of fetal loss, pregnancy complications and high rates of neonatal loss associated with abnormally large calves [4], [5]. There are an increasing number of studies that indicate that these abnormalities can be attributed to the use of serum [6], [7]. It has been reported that co-culture systems can still give rise to good rates of blastocyst development if the serum is restricted to the period after the first 72 h of culture. However, this did not reduce pregnancy complications [8]. Inclusion of serum in the culture medium has direct detrimental effects on the embryo itself resulting in perturbations in metabolism [9], disruptions in the ultrastructure of the mitochondria, and development of large lipid vesicles that displace the normal ultrastructure [5], [6], [8], [10], [11]. Therefore, if the application of this technology is to continue, it is essential to use serum-free culture systems that support high rates of development in culture, enable subsequent cryopreservation of the blastocysts and result in the establishment of normal pregnancies in a commercial application.
One such serum-free culture system is the sequential media G1.2 and G2.2 system [12]. These media were formulated specifically to prevent intracellular stress to the embryo thereby maintaining embryo viability [12]. Additionally, these media take into account the changing carbohydrate and amino acid requirements of the embryo. As a result these media are able to support high rates of blastocyst development in culture of embryos from many species [12].
The aim of this study was to assess the ability of G1.2/G2.2 sequential media to support bovine embryo development and pregnancy rates in a commercial embryo transfer program.
Section snippets
Oocyte recovery and in vitro maturation
Ovaries were obtained from a slaughterhouse and transported to the lab in a plastic bag at 26–30 °C. Upon arrival in the laboratory, ovaries were rinsed with tap water at 28 °C and then washed with 1% Nolvasan solution (Aveco Co. Inc., Fort Dodge, IA, USA) and 1% 7X (ICN Biochemicals Inc., Costa Mesa, CA, USA) in 28 °C tap water. Ovaries were then rinsed very well with tap water and maintained at 23–27 °C until aspiration. Cumulus–oocytes complexes (COC) were aspirated from 2 to 10 mm follicles
Comparison of bovine embryo development in co-culture and sequential G1.2/G2.2 culture systems from slaughterhouse oocytes
Oocytes were matured and fertilized under standard conditions and randomly allocated to culture in either BRL co-culture system or media G1.2/G2.2 at 5% CO2:5% O2:90% N2. Development to the blastocyst stage, blastocyst cell number, allocation to the ICM and survival following cryopreservation were assessed after 144 h of culture.
Effect of oxygen tension on embryo development in sequential media G1.2/G2.2
Oocytes were matured and fertilized in standard conditions and then randomly allocated to culture in G1.2/G2.2 at either 5% CO2 in air or 5% CO2:5% O2:90% N2.
Experiment 1
Development to the blastocyst and expanded blastocyst stages was equivalent for zygotes cultured in either the BRL co-culture system or G1.2/G2.2 sequential media system in 5% CO2:5% O2:90% N2 (Table 2). There was also no difference in the cell numbers of the early blastocysts or mid blastocysts between the two culture systems (Table 3). However, expanded blastocysts grown in sequential media G1.2/G2.2 had significantly higher total cell numbers and ICM cell numbers compared to expanded
Discussion
The data presented in this manuscript demonstrate that it is possible to obtain equivalent rates of blastocyst development in a commercial IVM, IVF, and IVC production system using a physiologically based sequential serum-free culture system (G1.2/G2.2) to those obtained using a BRL co-culture system. Most importantly, however, the blastocysts developed in a serum-free system had equivalent rates of cryosurvival and gave rise to equivalent rates of pregnancies after transfer. These data
Acknowledgments
We thank the staff of Em Tran Inc. for helping to make this study possible and Vitrolife AB for funding.
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