A Practical Approach, Second Edition=Ronald D. Ho.pdf

TESTING FOR REPRODUCTIVE TOXICITY 439should be verified as contaminant-free (e.g., free of dirt, dust, spermatozoa, epithelial cells). Thefemale rat is removed from the cage and its identification is verified. One or two drops (approximately0.25 ml) of physiological (0.9%) saline are withdrawn from the aliquot into a new, cleandropping pipette. The tip of the dropping pipette is gently inserted into the vaginal canal. Thepipette bulb is firmly but gently depressed to expel the saline from the pipette into the vagina. Thesaline is gently drawn back into the dropping pipette, and the pipette is removed from the vaginalcanal. If the flush appears very clear, it is recommended that the lavage be repeated. Care must betaken not to insert the tip too far into the vaginal canal, because excessive stimulation of the cervixcan result in pseudopregnancy (prolonged diestrus). The female is returned to her cage. The contentsof the pipette are placed onto a clean glass slide. Each slide is numbered, a clear top and bottomof each slide is identified, and a data sheet is marked to indicate the location of each vaginal smear.All slides should be handled carefully to avoid mixing vaginal lavage samples from differentanimals. If samples become mixed, they should be discarded and the sampling procedure repeatedfor those animals. After vaginal lavage sampling has been completed for the day, the saline shouldbe reevaluated for contamination that may have occurred during sample collection.The vaginal lavage slides are allowed to air dry and are examined microscopically. When spermare present in the vaginal smear, a positive (+) is recorded. When no sperm are present in thevaginal smear, a negative (–) is recorded. A second vaginal lavage sample may be required to moreconclusively determine mating status (e.g., when the cage pan plugs are present but the vaginallavage sample is not sperm positive or only a few sperm were present). When an additional vaginallavage sample is collected and examined, an “a” for additional sample is noted, and the results ofthe second lavage are recorded on the cohabitation sheet.D. Reproductive Indices1. Male Mating and Fertility Indexa. Male Mating IndexThe male mating index is reported in studies where the male is the test system. It is calculatedusing the following formula:Male mating index =number of males with confirmed matingnumber of males cohabitated× 100Historical control values for the male mating index are approximately 94% with a range of 80%to 100%. In fertility or reproduction studies where both sexes are treated, this index can be usedas an indicator of reproductive toxicity, but not of specific male or female reproductive toxicity.Confirmation of male mating is usually based on the presence of a copulatory plug or sperm in thevaginal smear. Mating and pregnancy can also be confirmed by the unexpected delivery of a litteror by the discovery of resorbed uterine implantation sites in the postmortem uterine evaluation ofpresumed nonpregnant females (no confirmed mating and considered not pregnant). The malemating index can be influenced by many factors, including physical impairment, acute intoxication,alterations to the neuroendocrine-gonadal axis affecting libido, hormonal imbalance, etc. Otherfactors that might affect the male mating index include estrous cycle disruption (prolonged cyclesor persistent estrus or diestrus) and lack of female receptivity.b. Male Fertility IndexThe male fertility index is calculated using the following formula:© 2006 by Taylor & Francis Group, LLC