Fixation of Testes and Eyes Using a Modified Davidson's Fluid: Comparison with Bouin's Fluid and Conventional Davidson's Fluid

Abstract

Most recent revisions of regulatory guidelines for testing effects of chemicals on reproduction recommend Bouin's fluid (BF) or a "comparable fixative" instead of formalin to preserve the morphologic detail of testes for histopathological evaluation. However, picric acid in BF is a health and safety hazard, as well as a laboratory waste disposal problem. Furthermore, use of BF is labor intensive, requiring multiple alcohol rinses to remove picric acid for optimum preservation and immunohistochemical (IHC) detection of testicular antigens that may potentially be used to identify and quantify cells and functional proteins with critical roles in spermatogenesis. Recently a modified Davidson's fluid (mDF) has been reported as an alternative to BF to fix testes for routine histopathological examination. This study compared the overall histomorphologic clarity and the immunoand histochemical staining of testicular specimens fixed in BF and mDF. Additionally, because conventional Davidson's fixative (DF) is used routinely for optimum fixation of eyes, preservation of ocular histomorphology by DF and mDF was compared. mDF resulted in noticeably less shrinkage of the seminiferous tubules and superior overall morphologic detail compared to BF. Unlike DF, the mDF also supported excellent staining of acrosomes with periodic acid-Schiff (PAS) reagent when staging of spermatogenesis was required. IHC detection of androgen receptor and PCNA (to directly and indirectly identify Sertoli cells) as well as protein gene product 9.5 (to label spermatogonia) was superior in mDF compared to BF-fixed specimens. For histopathological examination of the eye, apposition and preservation of rods and cones, and nuclear layers of the retina were slightly inferior with mDF compared to DF. This paper has demonstrated that mDF provides comparable, and in many respects superior preservation of the testes to that of BF, both for IHC staining and for detailed histopathological examination. It also provides an acceptable fixative for eyes, although the quality of cellular preservation is inferior to that of DF.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Male Reproductive System

“…Accumulation of interstitial fluid (which is modified lymph) commonly occurs as a result of fixing the testes in hyperosmotic fixatives such as Bouin's and Modified Davidson's which cause tubular contraction and postmortem diffusion of proteinaceous fluid into the interstitial space surrounding the contracted tubules. When due to fixation, the fluid accumulation is typically more prominent in the center of the testis (Latendresse et al 2002). In some cases of severe tubular atrophy, there appears to be a true edema, which likely existed ante mortem, but most accumulation of proteinaceous fluid in the interstitium is probably a postmortem fixation artifact.…”

“…Proper microscopic assessment of testicular tissue in toxicity studies requires use of sexually mature animals, appropriate fixation, sampling and processing (Foley 2001;Lanning et al 2002;Latendresse et al 2002;Kittel et al 2004), and an understanding of spermatogenesis and its histologic presentation in the species under consideration ).…”

Development of strips of ovine testes after xenografting under the skin of mice and co-transplantation of exogenous spermatogonia with grafts

“…Pieces of each of the donor testes, grafts, and vesicular glands were fixed overnight at 4 8C in modified Davidson's fluid (Latendresse et al 2002). Fixed tissue was embedded in paraffin, sectioned at 5 mm and stained with hematoxylin and eosin using standard procedures.…”

The thermal effect of 2.45 GHz microwave radiation on rat testes

“…The whole right testis was obtained from each animal and processed for light microscopy. The samples were fixed in mDF solution (Latendresse et al 2002) for 24 h, dehydrated and finally embedded in paraffin. Five µm thick tissue sections were stained with haematoxylin and eosin (HE), examined under the light microscope Zeiss Axio Lab A1 (Zeiss, Germany) and documented with a camera Axio Cam ERc 5 (Zeiss, Germany).…”