Timing of Mating, Sperm Dynamics: MATERIALS AND METHODS(2)

Tissue Preparation

Females were killed on the day of capture (i.p. injection of pentobarbitone sodium, 60 mg/kg; Nembutal: Abbott Laboratories, Asquith, NSW, Australia) either if spermatozoa were detected in their urine samples or, in the later half of August, if few cornified epithelial cells and no sperm were seen. The reproductive tract was then removed, gently dissected free of connective tissue, and placed in Modified Eagle’s Medium (MEM, Sigma-Aldrich, Pty. Ltd.; Castle Hill, NSW, Australia). The ovaries were examined to determine whether females had ovulated.

If ovulation had not occurred, one side of the reproductive tract was divided into the lateral vagina, uterus, and uterine neck, lower isthmus, upper isthmus, and ampulla (Fig. 1) and rapidly isolated into 200-^l drops of MEM in order to determine sperm number in each segment. The contralateral side was then immersion fixed for either structural (light microscopy; Bouin’s fixative) or ultrastructural studies (electron microscopy; glutaraldehyde-based fixative in 0.1 M cacodylate buffer). ampicillin antibiotic

In order to obtain data on the number of spermatozoa remaining in the male reproductive tract at the conclusion of the mating season, five adult male agile Antechinus were captured and killed (see above) between 28 August and 5 September. The testes and epididymides of these animals were removed, dissected free of connective tissue, and weighed. One testis and epididymis was fixed and processed for routine light and electron microscopy. The remaining epididymis was cut into 12 segments (segments 1-5 caput, 6-9 corpus, 10-12 cauda epididymidis) as outlined previously for this species. Each segment was then placed in a drop of MEM, and the number of spermatozoa in each segment was determined (see below).

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