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

468 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONHistopathological examination should detect qualitative depletion of the primordial folliclepopulation; however, reproductive study guidelines require a quantitative evaluation of primordialfollicles of the F 1 females. The number of animals, ovarian section selection method, and sectionsample size should be statistically appropriate for the evaluation procedure used. The guidelinescite ovarian follicular counting methodologies 159–161 that use a differential count of the primordialfollicles, growing follicles, and antral follicles. The current EPA Multigeneration ProtocolModification 10 requires ovaries from 10 randomly selected females from highest treatment andcontrol groups. Five 5-µm ovarian sections are made from the inner third of each ovary, at least100 µm apart. The number of primordial follicles (and small growing follicles) is counted, and thepresence or absence of growing follicles and corpora lutea is confirmed for comparison of highesttreatment group and control group ovaries. If statistically significant differences occur, the nextlower treatment group will be evaluated.Differential follicular counts are associated with age of the female, number of animals evaluated,number and location of the ovarian sections, the number of sections per ovary, section thickness,differences between evaluators, and the criteria for defining primordial follicles. Ovarian folliclecount appears to be more sensitive endpoint than ovarian weight. In 18 RACB studies using Sprague-Dawley rats, 161 the number of small follicles counted had a wide variation, ranging from 147 ± 57to 556 ± 144, and another investigator reported 301 ± 13 small follicles. 131 A decrease in ovarianfollicle count is usually considered a biomarker of an adverse reproductive effect because norecovery is possible. A recent review of follicle count historical control data in rats 162 suggests that:(1) with a large number of animals, there is relatively good replication within a generation (whenevaluated by the same person); (2) there is great variability within individual ovaries and sections;(3) random selection of ovarian sections has a high probability of biasing effects; (4) ovarian folliclecounts do not appear to be correlated with either ovarian weight or body weight at termination;and (5) ovarian follicle counts should not be the sole endpoint used in risk assessment. However,even with the great variability between reported values, a detectable decrease in follicle count ina treated group when compared with the control group is considered an adverse effect.A. ParturitionV. PARTURITION AND LITTER EVALUATIONSFemale rats should be housed to litter in boxes with bedding material no later than day 20 ofpresumed gestation (2 d prior to the day of expected delivery; earlier if required by protocol). Thedams are observed periodically throughout the day for stretching, visible uterine contractions,vaginal bleeding, and/or placentas in the nesting box. These signs are strong indicators of parturitiononset, and indicate that dams should be monitored closely for delivery of the first pup.As soon as a pup is found, parturition is considered initiated. If required by protocol, the timeshould be recorded. A marker (e.g., a tape “flag”) may be placed on the outside of the nesting box,indicating a delivery in progress. Once delivery has begun, the dams are checked periodically duringthe day for evidence of difficulty in labor or delivery (e.g., a pup only partially delivered, a damcold to the touch and pale). Any apparent difficulty is recorded, and a supervisor or veterinarianshould be notified if the difficulty seems extreme or unusual.The calendar date on which delivery of a litter appears to be completed is defined as lactationday 0 (LD 0), postpartum day 0 (PND 0), and day 0 of age. The following maternal behaviorsoffer evidence that parturition is complete: (1) removal of amniotic sacs, placentas, and umbilicalcords, and grooming of the pups by the dam; (2) self-grooming by the dam; (3) nesting behaviorby the dam; and (4) nursing. However, parturition may be complete without all of these behaviorspresent. Gentle palpation of the dam may also be used. If the time of completion of parturition is© 2006 by Taylor & Francis Group, LLC