Appropriate fertilization practices play a crucial role in the maintenance of genetic diversity within captive fish broodstocks. We assessed the influence of in vitro storage of gametes (0-, 7- and 14-days post-stripping) at 1 °C on ovarian fluid pH, sperm motility, and embryo viability in the critically endangered Finnish landlocked salmon (Salmo salar m. sebago) population. A repeated full-factorial design used in both sperm-ovarian fluid activation analyses and fertilizations allowed us to separate the effects of female, male and their interaction on gamete quality and embryo survival across storage times. The ovarian fluid pH decreased most significantly during the first week of storage and showed considerable variation among females. The decrease of sperm motility varied across males, females and male–female (sperm-ovarian fluid) combinations. The mean proportion of viable embryos was initially 96.4%, but dropped to 45.5% after 7 days, and to 13.1% after 14 days of gamete storage. Embryo viability responses to gamete storage time predominantly increased among- and within-female variation but also showed major differences between female × male combinations (non-additive genetic effects). Interestingly, embryo viability showed virtually complete re-ranking (change of order) among full-sib families between the first two fertilization times (rs =  − 0.03) but consistency between the first and last times (rs = 0.59). Overall, our results indicate that even when the primary aim is to maximize the genetic diversity of endangered fish populations, it may be advisable to prioritize minimal egg storage time rather than maximizing the number of parental combinations in fertilizations.