New insights into sperm rheotaxis, agglutination and bundle formation in Sharkasi chickens based on an in vitro study

Fertility in birds is dependent on their ability to store adequate populations of viable sperm for extended durations in sperm storage tubules (SSTs). The exact mechanisms by which sperm enter, reside, and egress from the SSTs are still controversial. Sharkasi chicken sperm showed a high tendency to agglutinate, forming motile thread-like bundles comprising many cells. Since it is difficult to observe sperm motility and behavior inside the opaque oviduct, we employed a microfluidic device with a microchannel cross-section resembling close to that of sperm glands allowing for the study of sperm agglutination and motility behavior. This study discusses how sperm bundles are formed, how they move, and what role they may have in extending sperm residency inside the SSTs. We investigated sperm velocity and rheotaxis behavior when a fluid flow was generated inside a microfluidic channel by hydrostatic pressure (flow velocity = 33 µm/s). Spermatozoa tended to swim against the flow (positive rheotaxis) and sperm bundles had significantly lower velocity compared to lonesome sperm. Sperm bundles were observed to swim in a spiral-like motion and to grow in length and thickness as more lonesome sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Scanning and transmission electron microscopy revealed that sperm bundles were supported by a copious dense substance. The findings show the distinct motility of Sharkasi chicken sperm, as well as sperm's capacity to agglutinate and form motile bundles, which provides a better understanding of long-term sperm storage in the SSTs.