Different fixatives influence morphology, antigen preservation, and TUNEL staining in chicken (Gallus gallus) testis.

The optimized fixative for testis is still controversial. This study investigated the effects of Modified Davidson's Fluid (mDF), 4% Paraformaldehyde (4% PFA), and Bouin's Fluid (BF) fixatives on chicken testes in normal/cadmium (Cd) feeding groups using hematoxylin and eosin (HE), immunohistochemistry (IHC), and Terminal Transferase dUTP Nick End Labeling (TUNEL) staining. Compared to the mDF, we established that the testes fixed with 4% PFA and BF in the normal group had severe shrinkage in tubular and interstitial compartments. Moreover, compared with 4% PFA, the number of GATA4-positive Sertoli cells/mm2 reduced by 67.61% in mDF and 80.57% in BF for one seminiferous tubule. The TUNEL assay illustrated that more positive cells/mm2 in mDF group (28.47 ± 11.38) than in 4% PFA (10.49 ± 7.89). In Cd-treated testes, mDF showed more morphological details than 4% PFA and BF. In contrast, the number of GATA4-positive Sertoli cells/mm2 of 4% PFA was higher than that of mDF by 65.78% and BF by 64.80% in a seminiferous tubule. The number of TUNEL positive cells/mm2 in mDF (272.60 ± 34.41) were higher than in 4% PFA (175.91 ± 19.87). These results suggest that mDF fixative is suitable for normal and Cd-treated testis fixation for HE and TUNEL staining in chicken, whereas 4% PFA fixative is better for IHC examination.

Postnatal differentiation and regional histological variations in the ductus epididymidis of the Congjiang Xiang pig.

The Congjiang Xiang pig is a rare Chinese miniature breed whose epididymal histologic features through the postnatal development are poorly understood. To clarify the histomorphological differences between each region of epididymis during postnatal development, 24 male Congjiang Xiang pigs aged from neonatal (15 d), peri-puberty (30 d), puberty (60 d) to adult (180 d) stages, were examined. Postnatal differentiation of the different regions (I-V) of the epididymis started from birth and continued until maturity that showed regional variations. Developmental progression was disto-proximal. At the neonatal stage, Wolffian duct differentiation starts in the distal region, then ascends to the middle region which forms regions V, IV and III, respectively. A simple lined cuboidal in the epidydimal epithelial, which gradually differentiated into a pseudostratified columnar with stereocilia from neonatal to post-pubertal. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility.

Effects of 4% paraformaldehyde and modified Davidson's fluid on the morphology and immunohistochemistry of Xiang pig testes.

Modified Davidson's fluid (mDF) is a good fixative for morphological and antigen preservation. However, recent studies have shown that 4% paraformaldehyde (PFA) can better preserve the actin structure in rodent testes. It remains controversial which of these fixatives is best for testicular tissue. This study investigated the effects of both mDF and 4% PFA on the morphology and antigen preservation of Xiang pig testes using hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). The stronger testis penetration of mDF compared with that of 4% PFA was primarily manifested as testicular color change and decrease in tissue weight loss. Testes fixed with 4% PFA displayed a severe shrinkage of both the tubular and interstitial compartments and the seminiferous tubule area decreased by 12.02% compared with that in mDF-fixed tissues. In contrast, IHC results showed that 4% PFA fixation achieved better IHC-positive performance than mDF fixation for antigens specifically expressed in germ cells, Leydig cells and Sertoli cells. Due to this improved antigen preservation by 4% PFA fixation, the relative immunoreactions intensity significantly increased by 39.8%, 27.8%, and 76.4%, respectively, compared with that in mDF fixation. In summary, fixation of Xiang pig testes with mDF was suitable for HE staining, while fixation with 4% PFA was more suitable for IHC.