Abstract:Culter alburnus gynogenetic diploids were created by activating eggs with ultraviolet-irradiated commoncarp sperm and preventing extrusion of the second polar body using cold shock to induce chromosome duplicationin the eggs. A three-factor five-level quadratic orthogonal rotation combined design was used to determinethe optimal combination of three experimental factors, including time after fertilization (x1), treatment duration(x2), and shock temperature (x3) to induce artificial gynogenesis in C. alburnus, using gynogenesis rate (y1)and hatching rate (y2) as indicators in the quadratic regression model. Furthermore, the response surface methodwas used to determine the effect of single factors and their interactive effect on the response value. We found thatthe optimal combination of factors to induce gynogenesis (18.00%) was cold shock for 18 min at 7℃, applied 6min after fertilization, whereas that for maximal egg hatch (33.00%) was cold shock for 23 min at 5℃, applied 6min after fertilization. The relationship between the various factors on the response values was parabolic with adownward opening. According to the factor contribution rates, cold shock start time had a highly significant effecton cold shock, followed by shock temperature and treatment duration. This is the first study to use a quadraticorthogonal rotation combined design to determine the optimal combination of factors to induce gynogenesis in C.alburnus. These results provide a basis to explore the sex-determining mechanism in C. alburnus.