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Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts
E C Curnow1, D Pawitri, E S Hayes
1Monash Institute of Reproduction and Development, Clayton, Australia.
Abstract:
In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.
Insights
A sequential culture system successfully produced Macaca fascicularis (Mf) blastocysts in vitro without cell support. This method improved Mf embryo development compared to nonsequential culture, overcoming previous limitations.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Primate Embryology
Background:
- In vitro blastocyst production in Macaca fascicularis (Mf) has historically required cell support.
- Nonsequential culture media (NSM) often lead to Mf embryo arrest at the morula stage.
- Serum supplementation and cell support are traditional factors influencing Mf embryo development.
Purpose of the Study:
- To demonstrate the in vitro production of Mf blastocysts without cell support using a sequential culture system.
- To compare the efficacy of a sequential medium (SM) versus a nonsequential medium (NSM) for Mf embryo development.
Main Methods:
- In vitro fertilization (IVF) of Macaca fascicularis oocytes (n=69).
- Culture of embryos in either a commercial sequential medium (SM) or a nonsequential medium (NSM) without cell support.
- Assessment of embryo cleavage, development to morula, blastocyst stage, and degeneration at various time points post-insemination (PI).
Main Results:
- Early cleavage (24 hr PI) was higher in NSM (65%) compared to SM (30%).
- By day 7-9 PI, blastocyst development was significantly higher in SM (20%) versus NSM (0%).
- Embryo degeneration was significantly higher in NSM (100%) compared to SM (56%) by day 7-9 PI.
Conclusions:
- The sequential culture system (SM) effectively supports in vitro development of Macaca fascicularis embryos to the blastocyst stage without cell support.
- Nonsequential culture medium (NSM) is inadequate for supporting Mf blastocyst development in vitro without cell support.
- Sequential culture represents a significant advancement for primate embryo production in vitro.