Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens

ABSTRACT

1. This study elucidated the last-male sperm precedence (LMSP) mechanism in chickens by examining replacement in storage tubules (SSTs) after multiple artificial inseminations (AI) and the effects of seminal plasma (SP) and male breed on sperm replacement in SSTs.

2. Hens were artificially inseminated with fluorescent dye-labelled spermatozoa from White Leghorn (WL) chickens. Secondary AI was conducted 3 d later with sperm labelled with different nuclear fluorescent dye. Percentage of first and second inseminated sperm in SSTs and their logarithmic odds were calculated. The effect of SP on LMSP was examined using (1) Lake’s solution-washed sperm before second insemination, and (2) SP injected continuously after first insemination. Effect of breed difference on sperm replacement was investigated using Barred Plymouth Rock (BP) sperm.

3. Successive WL-sperm inseminations at three-day intervals caused > 70% stored sperm replacement in SSTs. Although SP removal from sperm from second insemination significantly decreased replacement, its intra-vaginal injection did not affect release. Secondary insemination using BP sperm significantly increased replacement.

4. Sperm replacement is a major factor favouring LMSP in domestic chickens. Two fluorescent staining of sperm, and intra-vaginal multiple AI technique have enabled visualisation, differentiation, and quantification of multiple inseminated sperm stored in the SSTs.

KEYWORDS:

• Sperm
• artificial insemination
• sperm storage tubules
• last-male sperm precedence
• chicken

Acknowledgments

The authors are grateful to Dr. A. Huang (Sichuan Agricultural University), Ms. H. Y. T. Bui, Ms. and Y. Nakamura for their technical assistance. They would like to thank Prof. Y. Yoshimura (Hiroshima University) and Prof H. Horiuchi (Hiroshima University) for allowing us to use fluorescent microscope and digital camera. They would like to thank Dr. K. Ichikawa (Hiroshima University) for helpful advice on the draft of this paper.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.

Additional information

Funding

This work was supported by a Grant-in-Aid for Scientific Research (B) (General) (17H03902 to TS) and a Grant-in-Aid for Early-Career Scientists (20K15648 to MM).