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

240 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONleft longitudinally and the right transversely) so that the renal papilla and renal pelvis may beexamined. Any abnormal dilation of the ureters or the renal pelvis (more common in rats) is noted.The urinary bladder should be checked at this time.The sex of each fetus is then determined by carefully inspecting the gonads. Rodent testes aredefined by their round to bean shape and the tortuous spermatic artery that runs along the side ofeach testis, with associated epididymides (caput [head], corpus [body], and cauda [tail]) on thelateral side of the testes. The testes should be located low in the pelvis on either side of the urinarybladder. Additional reproductive structures can be seen in the term rat fetus with a magnifying lensor dissecting microscope. For males, these include the white, threadlike efferent ducts (vasaefferentia) from the testes to the caput epididymis, the single, thin ductus deferens (vas deferens,Wolffian duct) from the cauda epididymis to the urethra and the thick gubernaculums, which extendscaudally from the cauda epididymis to the inguinal ring. In rabbits, the testes are in the lowerquadrant; they are slightly oval, and their superficial blood vessels can be seen.Ovaries are small and elongated. Their location in rodents is high in the pelvis, just inferior tothe kidneys, and they are cupped by the funnel-shaped ends of the oviducts (uterine tubes, fallopiantubes). The bicornuate (“two horned”) uterus is continuous with the proximal ends of the oviducts.For females, in addition to the ovaries, oviducts, and uterus, the cranial suspensory ligament canbe seen (with magnification) extending from each ovary anteriorly to the diaphragm along thedorsal body wall. Rabbit ovaries are pinkish and elongated but, like the testes, are lower in theabdomen than is the case for rodents. Once the visceral examination is concluded, the viscera areremoved in toto, and the eviscerated carcass is prepared for skeletal staining.e. Visceral Examination—Wilson’s TechniqueAn alternative method for visceral examination of rodent fetuses involves fixation and decalcificationin Bouin’s solution of fetuses selected for visceral examination. 17, 69–74 Each fetus is then seriallycross-sectioned through the head (see head examination, below) and trunk regions by freehandcutting with a razor or scalpel blade. The advantages to this technique include examination of thefetuses at the convenience of the technical staff (since the fetuses are fixed) and retention of sectionsfor documentation or subsequent histologic confirmation of a lesion. The disadvantages include:(1) inability to examine the same fetus for visceral and skeletal alterations (since Bouin’s fixativedecalcifies the skeleton and the serial sections preclude such examination), 75 (2) inability to usecolor changes in fresh specimens and/or blood flow through the heart and great vessels as aids indetection and diagnosis of circulatory alterations, (3) difficulty in sectioning each fetus in a litterand every litter in precisely the same way (e.g., through the heart) to assure comparability ofsections, and (4) difficulty in visualizing morphological alteration from serial cross-sections. Acomparison of Staples’s versus Wilson’s technique in rat fetuses indicated that Staples’s techniqueidentified more heart and great vessel malformations than Wilson’s technique did. 76 An alternativemicrodissection technique after fixation has been presented by Barrow and Taylor. 77Surface staining of Wilson’s sections can enhance detection of visceral alterations. 78 SinceBouin’s fixative contains picric acid, which is reactive, unstable, explosive, and also a strong irritantand allergen (and therefore a safety and health hazard), and Bouin’s fixative can result in fragilefriable soft tissues, alternative fixatives that do not contain picric acid have been suggested. Theuse of a modified Davidson’s fixative for fetuses has been reported 79 (and for fixation of testes andeyes 80 ), with subsequent serial sectioning of fetuses by Wilson’s technique. The authors 79 reportthat side-by-side comparisons of results with the two fixatives indicate Davidson’s fixative producessuperior contrast and definition of organs and vessels in the cranial, thoracic, and abdominal regions,with the tissues remaining moist for a longer time. The procedure reported is as follows. Fetusesare immersed in modified Davidson’s fixative (14 ml ethanol, 6.25 ml glacial acetic acid, 37.5 mlsaturated commercial grade 37% formaldehyde, and 42.25 ml distilled water for 100 ml of fixative)© 2006 by Taylor & Francis Group, LLC