The objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.