Abstract: 【Aim】 To address the issue of low transfering rate of sperm after artificial insemination of Apis cerana cerana and to extend the time during which inseminated queens lay fertilized eggs within the colony.【Methods】A. c. cerana gynes at 7 d after eclosion were inseminated with 1, 3 and 5 μL of semen. For each dosage, there were treatment groups and control groups, with 5 gynes in each group. After insemination, a plastic device was immediately fitted onto the end of abdomen of queen in the treatment group, while no plastic device was fitted onto the end of abdomen of queen in the control group. After 24 h, the number of sperms in the spermatheca of each queen was counted. 【Results】Physical isolation via the fitted plastic device significantly improved the transferring rate of sperm of A. c. cerana to spermatheca after artificial insemination. It demonstrated a particularly notable advantage under low semen dosage, with the transferring rate of sperm increasing from approximately 11% under conventional artificial insemination to 43%. This effectively extended the time during which the queen laid fertilized eggs within the colony. The application of this method successfully elucidated the inheritance pattern of body color in A. c. cerana, demonstrating that body color is controlled by a pair of alleles, with yellow being dominant and black recessive. The three castes in the offspring resulting from the backcrossing of the heterozygous queen (F₁) with the black drone exhibited a stable 1∶1 segregation ratio of body color, consistent with Mendelian inheritance. 【Conclusion】 The optimized sperm transfer method developed in this study significantly enhances the transferring rate of sperm of A. c. cerana from lateral oviduct to spermatheca in artificially inseminated queens, particularly under low semen dosage, such as single-drone insemination. This method can be successfully applied in the study of the inheritance pattern of body color, providing both a theoretical foundation and technical support for the selective breeding and germplasm conservation of A. c. cerana.

Key words: Apis cerana cerana, transferring rate of sperm, artificial insemination, fertilized eggs, spermatheca