Effects of spawning group sex ratio and stocking density on the outcome of captive reproduction in golden cuttlefish Sepia esculenta

The supplemental release of hatchery-reared animals is important for maintaining or enhancing fishery resources. The golden cuttlefish, Sepia esculenta, is a commercially important cephalopod utilized in stock enhancement activities throughout East Asia. The long-term success of such activities requires proper breeding regimes to ensure the efficient use of resources and the preservation of genetic diversity in the offspring. For captive-bred cephalopods, information concerning the effects of different mating regimes on the paternity of offspring is scarce. However, it could be of particular importance for polyandric species such as golden cuttlefish. As previous studies have suggested that both the number and mating order of males are related to female fecundity, we investigated the effects of stocking density and sex ratio on female fecundity and determined the paternity of the offspring in golden cuttlefish. We used two experimental approaches. First, four different female (F): male (M) sex ratios (1F:1M, 1F:2.5 M, 1F:3M, 1F:6M) were set up to investigate the effects of operational sex ratio on mating behavior, female fecundity, and hatchling paternity. Next, we investigated female fecundity and hatchling paternity patterns under low and high stocking densities (5 or 10 individuals ∙ m⁻³), following a standard sex ratio setting that is widely employed in cuttlefish hatcheries (1F:2M). With an increasing proportion of males, the average fecundity of females and the participation rate of males in paternity decreased. With increasing stocking density, the females had lower average daily egg production, and the males had a high variance in reproductive success and decreased participation rate in spawning. The estimated effective number of breeders (Nₑ) was 19.29 (Nₑ/N ratio = 0.54) at high-density and 10.17 (Nₑ/N ratio = 0.57) at low density. To achieve high reproductive performance and assure genetic diversity of golden cuttlefish offspring, our study suggests that equal (1F:1M) or slightly male-biased (1F:2.5 M) sex ratios and a low stocking density may be useful when breeding golden cuttlefish in captivity.