Table Number of COCs and presumptive zygotes were used in each replicates are mentioned below

Trials / TCM-199 MCR2aa Defined
COCs
/ Presumptive zygotes / Presumptive zygotes / Presumptive zygotes
Trail 1 / 258 / 68 / 71 / 74
Trail 2 / 348 / 96 / 88 / 85
Trail 3 / 404 / 125 / 138 / 55
Trail 4 / 243 / 78 / 60 / 53
Trail 5 / 256 / 82 / 55 / 58
Trial 6 / 215 / 66 / 32 / 65
Trial 7 / 186 / 30 / 43 / 73
Trail 8 / 156 / 32 / 25 / 64

Developmentally important gene amplification in preimplantation embryos cultured in three different embryo culture media through RT-PCR analysis

PCR reaction was carried out in a 50 μL final volume containing 45 μL platinum PCR supermix (Invitrogen, Carlsbad, CA, USA), and 5 μ L of primer (200 nM each) and template DNA solution. A set of reaction without template cDNA was used as negative control for PCR. GAPDH was used as the reference gene. The primers sequences were used for GLUT-1, MnSOD, BAX, BCL-2, IGF-2, IGF-2R, DNMT-3A and IFNT are mentioned in Table 1. The PCR conditions were same except the annealing temperature (Table 1), as 94 °C for 2 min (Initial denaturation), denaturation at 94 °C for 30 s, elongation at 72 °C for 1 min (35 cycles). The amplified DNA fragments were resolved on 2 % agarose gel containing 0.5 μg⁄ml ethidium bromide against a 50-bp ladder and visualized under gel documentation system (Alpha Imager, Alpha Innotech,San Leandro, CA, USA).

RT-PCR analysis of developmentally important genes in buffalo embryos derived from undefined and defined media are shown in supplementary Fig. Amplicons of GLUT-1 with an expected length of 194 bp were detected in embryos at 2-cell, 4-cell, 8- to16- cell, morula and hatched blastocyst stages from undefined and defined media (Fig. A). The RT-PCR analysis detected, amplicons of MnSOD with an expected length of 193 bp, in embryos at 2-cell, 4-cell, 8- to16- cell, morula and hatched blastocyst stages from undefined and defined media (Fig. B). An expected 82 bp fragment encoding pro-apoptosis related genes (BAX) mRNA was detected in embryos at 2-cell, 4-cell, 8- to16- cell, morula and hatched blastocyst stages from undefined and defined media (Fig. C). ). An expected 97 bp fragment encoding anti-apoptosis related gene (BCL-2) was detected at all stages of pre-implantation embryos from undefined and defined media (Fig. D). An expected 107 bp fragment encoding IGF-2 mRNA was detected at all stages of pre-implantation embryos from undefined and defined media (Fig. E). An expected 186 bp fragment encoding IGF-2R mRNA was detected at all stages of pre-implantation embryos from undefined and defined media (Fig. F). Amplicons of DNMT-3A with an expected length of 134 bp, were detected in all stages of pre-implantation embryos from undefined and defined media (Fig. G). Transcripts for IFNT were detected only at blastocysts stage in embryos (Fig. H)

Fig. RT-PCR analysis of developmentally important genes in buffalo pre-implantation embryos at 2- cell, 4- cell, 8- to 16- cell, morula and hatched blastocyst stages obtained from TCM-199 (a), mCR2aa (b) and defined culture media (c). Agarose gel electrophoresis of analysis RT-PCR product revealed 194, 193, 82, 97, 107, 186, 134, and 106 bp amplicons, respectively for (A) GLUT-1, (B) MNSOD, (C) BAX, (D) BCL-2, (E) IGF-2, (F) IGF-2R, (G) DNMT3A and (H) IFNT, (I) GAPDH has been employed as reference gene, where (a) TCM-199, (b) mCR2aa, and (c) defined medium. DNA marker is 50 bp. The sizes of the expected PCR products are shown in bp