104 EFFECT OF FROZEN MEDIA ON IGF₂ EXPRESSION OF BOVINE EMBRYOS CULTURED ENTIRELY IN VITRO UNTIL DAY 14

Abstract

Freezing and stocking ready to use IVP medium, including hormones and fetal serum, is a practical alternative to rationalize work and reduce costs in in vitro embryo production. In our laboratory routine, embryo culture in frozen or fresh medium (produced weekly) has shown equal Day 7 blastocyst rates. Although morphological aspects were also similar, it is known that culture environment may alter gene expression patterns and embryo development on later stages. Thus, a preliminary study of mRNA expression of IGF₂ in Day 14 embryos produced in both culture conditions (frozen and fresh medium) was performed. For that, IVM (TCM 199 with hormones, antibiotics, l-glutamine and 10% fetal bovine serum) and IVC medium (SOFaaci) were split into 2-mL aliquots into Eppendorf tubes and frozen at −80°C temperature four weeks prior to use. The thawing was performed in a 4–5°C refrigerator overnight and the medium was stabilized in the incubator at least 4 h prior to use. Abattoir-derived oocytes were collected and randomly distributed into two culture groups: T1 (fresh IVM, fresh IVC) and T2 (frozen IVM, frozen IVC). On Day 7, blastocysts classified as Grades 1 and 2 (n = 12) were selected and continued in vitro culture in the Post Hatching Development system; PHD system (Brandao et al. 2004 Reprod. Fertil. Dev. 16, 123–124) under 38.5°C, 5% CO₂ in air. On Day 14, elongated embryos from T1 and T2 were removed from culture and analyzed by RT-PCR. Total RNA of each group was prepared from two D14 embryos of two distinct replicates using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol with modifications. The reverse transcription (RT) was done using the EZ-First Strand cDNA Synthesis Kit (Biological Industries, Israel). The β-actin gene was used as a constitutive control and PCR reactions for the two genes were carried out in triplicate using a PTC-100 MJ Research thermocycler. PCR products were electrophoresed on a 1.5% agarose gel. The two genes were detected in both samples; the relative expression of IGF₂ was higher in embryos cultivated in fresh medium (control). The consequences of IGF₂ levels in embryo development are still unknown, but highlight the late effects of culture conditions on embryonic gene expression. The perspective use of the PHD system might be an alternative embryo development for monitoring until further stages. In conclusion, IGF₂ appears to be a candidate marker gene for embryo development and, although frozen ready-to-use medium is a practical strategy, further studies on molecular trends are necessary to confirm its use.