Fluids and Sperm Collection
Experiments on animals were conducted according to the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction. Testis and epididymis were obtained from adult Ile de France and Romanov rams (at least 10 different animals). Testicular fluid was obtained after puncture of the rete testis. Epididymal fluids were collected by microperfusion with PBS from the 10 different zones defined in the three main regions (caput, corpus, cauda) and the caudal fluid (CEP) by retroperfusion of zones 8 and 9 from the deferent duct (see Fig. 1).
Ejaculates were obtained from five intact adult animals or two vasectomized animals using an artificial vagina.
The spermatozoa were separated from the fluid or seminal plasma by two successive centrifugations (15 min, 15 000 X g, 4°C). Where indicated, the sperm from different zones were diluted in PBS and layered on a discontinuous percoll gradient (40% and 90%) and centrifuged at 3000 X g for 10 min at 4°C. The sperm pellets were carefully collected, diluted with PBS, and centrifuged again to remove the percoll.
FIG. 1. Photograph of an epididymis from adult ram with the different zones of microperfusion indicated. Caput, corpus, and cauda indicate the gross morphological regions. (Real size, 15 cm.)