20Assisted reproductive technologies (ARTs) are of great importance for increasing the genetic diversity in captive animals. The use of bovine or murine oocytes in heterologous IVF provide advantages compared to homologous IVF in non-domestic animals, such as the accessibility to oocytes and the availability of well-developed in vitro maturation systems. The aim of this study was to determine the heterologous IVF 25 parameters using cryopreserved dolphin spermatozoa and zona-intact bovine or murine oocytes, as well as to examine the nuclear chromatin status of the dolphin spermatozoa.All the processes involved in the fertilization including embryo cleavage were observed by confocal microscopy and hybrid embryo formation was confirmed by PCR.Heterologous bovine IVF showed no polyspermy, lower percentages of pronuclear 30 formation and a lower cleavage rate compared to homologous IVF group (34.8 vs.89.3%). Heterologous murine IVF showed a lower cleavage rate than homologous IVF (9.6 vs. 77.1%). With respect to dolphin sperm chromatin, it was more stable, i.e. more resistant to EDTA-SDS decondensation than the bovine sperm chromatin. This study revealed the stability of the dolphin sperm chromatin and the ability of the dolphin 35 spermatozoa to penetrate zona-intact bovine and murine oocytes, leading to hybrid embryo formation.