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

TESTING FOR REPRODUCTIVE TOXICITY 473separation occurs dorsolaterally and then ventrally on the penis and down the shaft of the penis. 54The prepuce remains attached to the glans penis on its ventral surface by the frenulum. (Note: Theprocess of development of the prepuce in humans is different from that of the rat.) Male rodentsare examined for balanopreputial separation beginning on PND 22 (mouse), PND 27 (hamster), orPND 35 to 40 (rat). Published preputial separation age ranges from PND 32 to 42 (Japanesestudies) 167–169 or PND 41 to 46 (American studies), 54,170 depending on the observation criterion. TheJapanese preputial separation criterion is based on the age that the prepuce separates from the glanspenis, while the American criterion is met when the prepuce completely retracts from the glanspenis and the foreskin is cuffed around the base of the penis. Therefore, it is important to definethe criterion in the protocol.Each male rodent is removed from its cage and held in a supine position. Because manipulationof the prepuce can accelerate the process of preputial separation, the males must be examinedgently. Gentle digital pressure is applied to the sides of the prepuce, and the criterion is met whenthe prepuce completely retracts from the head of the penis (see above for alternate criterion). Theforeskin can be attached along the shaft of the penis, but it cannot be attached to the opening ofthe urethra. Each male rodent is examined daily until acquisition or until PND 55, whichever isearlier. Body weight should be recorded on the day the criterion is met.2. Vaginal PatencyAfter the canalization of the vagina occurs in rats, the vaginal opening remains covered by a septumor membrane. The age when the septum is broken or no longer evident is described as the age ofvaginal patency, and vaginal patency is the most readily determined marker for puberty in rats. 54Published Sprague-Dawley rat vaginal patency values range from PND 30.8 to 38.4. 169,171 Femalerats are examined beginning a few days prior to expected age of maturation (e.g., PND 25 to 28),and the examinations are continued until the criterion for patency has been achieved or until PND43, whichever comes first. The female is removed from the cage and held in a supine position,exposing the genital area. Pressure is gently applied to the side of the vaginal opening to see if theseptum or membrane remains. When the membrane is present, the area has a slight “puckered”appearance; however, when the membrane has broken, the vaginal opening is about the size of apinhead. The criterion has been met when the vagina is completely open. Body weight should berecorded on the day the criterion is met.3. Day of First EstrusAfter vaginal opening has been determined, vaginal smears can be taken daily to determine theday of first estrus (defined as the first day on which only cornified epithelial cells are observed ona vaginal smear). First estrus normally occurs within a few days after vaginal opening (approximatelyPNDs 40 to 45) in the Sprague-Dawley rat. 1724. Pinna UnfoldingBeginning on PND 1, each pup in a litter is examined individually until all pups in the litter meet thecriterion for pinna unfolding (i.e., the first day that the point of a pinna [earflap] is detached from arodent pup’s head). Each pup is examined closely for detachment of either the right or left pinna fromthe side of the head, and criterion is met when the point of either pinna is detached. This usually occurswithin PND 1 164 to 7 165 in Sprague-Dawley rats, with the average acquisition around PND 2 or 3.5. Hair GrowthBeginning on PND 1, rodent pups are examined for the appearance of hair. All pups in each litterare examined daily until bristles appear on the dorsal surface of all pups in the litter. Criterion is© 2006 by Taylor & Francis Group, LLC