Timing of Mating, Sperm Dynamics: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)Calculation of Sperm Number in Male and Female Reproductive Tracts

As marsupial spermatozoa are more fragile than their eutherian counterparts and are prone to disintegration if given harsh chemical or mechanical treatments, a more gentle, manual extraction procedure was employed to separate spermatozoa from reproductive tract segments. antibiotics levaquin

For females, the luminal surface of the lateral vagina and uterine segments was exposed by longitudinal incision, and spermatozoa were removed by gently stripping the exposed inner surfaces with blunt-nosed forceps. Spermatozoa from the oviductal segments were forced into the surrounding medium by repeated squeezing and stripping along the length of the segments with blunt-nosed forceps as described previously in A. stuartii. The total number of spermatozoa was then calculated using a hemocytometer.

For males, spermatozoa were removed from the testis and from each of the 12 segments of the epididymis using the cut-and-squeeze method described for A. stuartii previously. Sperm number was then calculated using a hemocytometer.

Estimation of Ovulation Date

If, after examination of the ovaries, ovulation was found to have occurred, the uteri were dissected free, placed in a small Petri dish containing MEM, and carefully dissected open. Each uterus was then everted and any uterine embryos were flushed out into the surrounding medium. The diameter of all embryos was then calculated with the aid of a hemocytometer, and the date of ovulation was determined by comparing the mean diameter of all embryos from both uteri per animal with data on embryo development published previously for A. stuartii.


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