Colony-stimulating factor 2 (CSF2) does not significantly affect cellular and molecular parameters, or blastocyst rates under in vitro culture conditions with reduced nutrient concentrations.

Colony-stimulating factor 2 (CSF2) is an embryokine used between days 5 and 7 of culture to improve embryonic development. We evaluated its use from Day 4 and throughout the culture period in a reduced nutrient concentrations culture medium. Presumptive zygotes were assigned to three treatments: Control - synthetic oviduct fluid (SOF), CSF2 D1 (SOF + CSF2 from Day 1-7), CSF2 D4 (SOF + CSF2 from Day 4-7). Cleavage rates on Day 4 and blastocyst rates on days 6 and 7 were evaluated. On Day 7, expanded blastocysts were used to assess mitochondrial activity, lipid quantification, total cell number, gene expression and DNA methylation patterns. Embryo development data were analyzed using the chi-square test (P ≤ 0.05). Data normally distributed were analyzed with ANOVA and Tukey's test, while non-normally distributed data were evaluated using the Kruskal-Wallis and Mann-Whitney tests. Cleavage and blastocyst rates on Day 7 were similar (P > 0.05) among treatments. Of 11 genes evaluated, only Placenta-specific 8 presented a lower number of transcripts (P ≤ 0.05) in embryos cultured with CSF2 from Day 4. Assessments of DNA methylation, lipid droplet staining, mitochondrial activity and total cell number showed no significant differences among treatments (P > 0.05). In conclusion, CSF2 had no effect on the assessed parameters when a culture medium with reduced nutrient concentrations was used, and can be used throughout the entire culture period without affecting embryo production and developmental kinetics compared to CSF2 added on Day 4.