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

TESTING FOR REPRODUCTIVE TOXICITY 453disrupt the tissue. Bring the homogenate to the appropriate final volume (generally between 50 and100 ml), using deionized water. Record the final volume in the study records.2) Testicular Sample Preparation — For fresh tissue or after thawing the frozen tissue, weighand record the testis weight. Remove and weigh the tunica albuginea. Place the parenchyma in aglass vial containing 30 ml of diluent. Homogenize the parenchyma for 30 sec to 3 min to adequatelydisrupt the tissue. Bring the homogenate up to appropriate final volume (generally between 50 and100 ml), using deionized water. Record the final volume in the study records.3) Manual Sperm and Spermatid CountsLoading the Hemocytometer — Manual concentration analysis is conducted by producing ahomogenate of standard volume from a cauda epididymis or testis sample of known weight andcounting the number of sperm heads in a known volume of homogenate by use of a hemocytometer.Place the coverslip on the hemocytometer. Transfer 10 µl of the suspension into both chambers ofthe hemocytometer. Touch the center edge of the coverslip with the pipette tip and allow eachchamber to fill by capillary action. To facilitate counting, place the loaded hemocytometer in ahumid area (e.g., a Petri dish with moist gauze) for approximately 5 min so the sperm heads cansettle to a common focal plane.Counting using the Hemocytometer 96 — Count the four corner squares and the middle squarein each chamber. Include cells touching the middle line at the top and the left side. Do not countcells touching the middle line at the bottom and on the right side. Obtain two sets of hemocytometercounts (total of four chambers counted). If there are 15 sperm or less per counting chamber, recordall counts. If there are greater than 15 sperm, determine if a recount is necessary within each setof hemocytometer counts. Generally, if there is less than a 20% difference between hemocytometercounts, the sperm head counts are recorded and accepted. If there is a 20% to 50% differencebetween counts, the counts are recorded, the hemocytometer is cleaned and reloaded, and the newsample is counted and recorded. If greater than 50% difference exists between counts, the countis recorded but not used for tabulation. Then the hemocytometer is cleaned and reloaded, and anew sample is counted and recorded. (Note: Remember to always clean the hemocytometer andcover slip immediately after completion of counting by rinsing with deionized water and air dryingor drying with lens tissue.)4) Epididymal Calculations for Manual Sperm and Spermatid CountsSperm per Cauda EpididymisSperm per cauda epididymis =mean count multiplied by dilution volumevolume of counting chamberFor example, with a mean chamber count of 20, a dilution volume of 100 ml, and a chamber volumeof 0.0001 ml, the calculation would be (20 × 100)/0.0001 = 2 × 10 7 sperm per cauda epididymis.Sperm per Gram Cauda EpididymisSperm per gram cauda epididymis =sperm count per caudaweight of cauda (grams)For example, with a cauda sperm count of 2 × 10 7 and a cauda weight of 0.25 g, the calculationwould be 2 × 10 7 /0.25 = 8 × 10 7 sperm per gram epididymis.© 2006 by Taylor & Francis Group, LLC