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The effect of seasonal variation and lunar cycles on reproductive parameters in dogs is unknown. Lunar cycles have important effects on several biological events. Controversy exists about the influence of lunar cycles on offspring sex ratio. This study examined the sex offspring distribution of 973 puppies (48% females and 52% males) from 150 bitches in Italy between 2015 and 2020. A two-way ANOVA followed by post-hoc t-test (Bonferroni correction) was used to analyze the influence of season and lunar phase on offspring sex ratio at conception. Sex offspring distribution was not affected by season, whereas lunar phase had a significant effect (p<.05). During the new moon, the proportion of male puppies born was significantly lower than during the full moon phase (p <.05). We conclude that season had no effect on sex offspring distribution. The new moon phase at conception appeared to be related to a lower male sex ratio at birth. Further studies assessing additional factors will help provide a better understanding of the lunar cycle differences observed in the sex ratio distribution of dogs. [ABSTRACT FROM AUTHOR]