Authors’ address (for correspondence): María Alejandra Stornelli, Laboratorio de Reproducción Animal, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118, La Plata B1900VW, Buenos Aires, Argentina. E-mail: firstname.lastname@example.org
The aim of this study was to assess the histological and ultrastructural changes in cat epididymides (n = 22) stored at 4°C in two different media [saline solution (SAL) or tris-egg yolk (TEY)]. Our hypothesis was that epididymides stored in TEY would have delayed epithelial cell autolysis. Four epididymides were fixed and processed immediately, and the remaining 18 epididymides were stored at 4°C in SAL or TEY for 24, 48 or 72 h. In histological sections, the nuclear features and stereocilia morphology were scored from 0 to 3. Ultrastructurally, nuclear chromatin and stereocilia morphology were scored from 0 to 3. In addition, using transmission electron microscopy nuclear number, nuclear area, mitochondrial number and mitochondrial area were recorded. In the histological study, parameters changed with time and media (p < 0.01). A significant effect of time was observed (p < 0.01), and the morphological changes were greatest when the storage time increased. Morphological changes were higher in SAL compared with TEY (p < 0.01). In the ultrastructural study, nuclear chromatin and stereocilia morphology decreased with time and media as in the histological study (p < 0.01). In addition, nuclear number and nuclear area changed with time (p < 0.004; p < 0.001) but not with media. Conversely, mitochondrial number and mitochondrial area did not change with media or time (p > 0.05). In conclusion, these results show that TEY preserved epididymal epithelial cells better than SAL; this finding could help improve sperm quality of stored epididymides.
Reproductive research in the domestic cat may be used as a model for wild felids and to improve the tools for assisted reproductive techniques involved in conservation programmes of endangered felids. The recovery and freezing of sperm from the epididymides obtained either from dead animals (post-mortem recovery) or after orchiectomy is a viable option for preserving male gametes and thus for maintaining germ banks. On many occasions, the only option for salvaging gametes is to transport the testes and epididymides to a laboratory that is equipped for sperm processing. The conditions (time, temperature and environment) under which epididymides are handled could cause important changes in the viability of sperm samples. Cat epididymal sperm parameters are significantly affected by the epididymal storage time and media (Tittarelli et al. 2006; Gañán et al. 2009). Sperm cells can survive for some time in the epididymides of dead animals with changes in semen quality being related to body decomposition (Songsasen et al. 1998). It is very likely that autolytic changes within the epididymides could be responsible for a reduction in sperm quality (Tittarelli et al. 2006). Autolysis takes place in all cells post-mortem and induces histological and ultrastructural changes (Myers et al., 2007). The aim of this study was to assess histological and ultrastructural changes in principal cells of cat epididymides stored at 4°C for 24, 48 or 72 h in two different media. The hypothesis was that epididymides stored in TEY would have delayed epithelial cell autolysis, decreasing cell morphological changes.
Materials and Methods
Testes of cats (n = 13) aged between 0.6 and 3 years were obtained. The animals participated in a voluntary programme for control of urban feline reproduction in a pet public shelter. Four right epididymides of four cats were fixed immediately after orchiectomy (0 h). Eighteen epididymides of nine cats were stored at 4°C in two different media. After bilateral orchiectomy, the left testis with adjacent epididymis from each animal was placed in a TEY extender (Tittarelli et al. 2006) and the right in SAL supplemented with penicillin at 100 IU/mL and both stored at 4°C. Testes and epididymides from each animal placed in each medium were allocated to one of three storage times (24, 48 or 72 h) and were evenly distributed among treatments (three animals/treatment time). The tissue samples from the cauda epididymis were fixed by immersion in 2% glutaraldehyde in phosphate buffer pH 7.3 for 2 h at 4°C. Following primary fixation, specimens were post-fixed for 1 h in 1% osmium tetroxide (OsO4), dehydrated through a graded series of ethanol solution, cleared in acetone and embedded in epoxy resin. Semi-thin sections (1 μm) were stained with toluidine blue for light microscopy (1000×). The ultrathin sections (90 nm) from selected areas were cut using Super Nova Reichert-Jung Ultra-microtome (Wien, Austria) and were mounted on a 200-mesh copper grid, and uranyl acetate and lead citrate were used as contrast. The sections were then examined with a JEM 1200 EX II transmission electron microscope (JEOL Ltd., Tokio, Japan) at 80 kV. In all samples, mayor cell types of epididymal epithelium were observed. The histological study was undertaken on the semi-thin sections, and the ultrastructural study was performed on photomicrographs taken from the ultrathin sections. In the histological study, the nuclear features and stereocilia morphology were scored from 0 to 3 (Table 1). In the ultrastructural study, the nuclear chromatin and stereocilia morphology samples were scored from 0 to 3 (Table 2, Figure 1). In addition, on photomicrographs, ultrastructural morphometry was used to calculate the nuclear number, nuclear area, mitochondrial number and mitochondrial area using image j 1.43e (National Institute of Health, Bethesda, MD, USA). Data were analysed by Genmod procedure of sas® (SAS 1989).
Table 1. Score of nuclear features and stereocilia morphology used in histological study
Adapted from Arrighi et al. (1986), Myers and McGavin (2007).
Clumping of chromatin
Clumping of chromatin and irregular nuclear morphology
Putting in tuft
Chromatin loss ‘ghost nucleus’
Stereocilia breaking, destruction or loss
Table 2. Score of nuclear chromatin and stereocilia morphology used in ultrastructural study
Adapted from: Arrighi et al. (1986), Myers and McGavin (2007).
Normal nucleus euchromatin
Large clumping of chromatin in part dispersed and in part marginal
Putting in tuft
Small clumping of chromatin in part dispersed and in part marginal
Destroyed and lost
In the histological study, parameters changed with time and media (p < 0.01). A significant effect of time was observed in nuclear features and stereocilia morphology (0 vs 72 h; 0 ± 0.12 vs 2.66 ± 0.13, p < 0.01; 0 ± 0.26 vs 2.66 ± 0.31, p < 0.01; respectively), and the score of morphological changes was higher when the stored time increased. When we studied the effect of stored media, morphological changes in nuclear features and stereocilia morphology were higher in SAL compared with TEY (1.33 ± 0.12 vs 1.16 ± 0.13, p < 0.05; 1.71 ± 0.30 vs 1.38 ± 0.3, p < 0.05; respectively). In the ultrastructural study, nuclear chromatin and stereocilia morphology changed with time (0 vs 72 h, 0.00 ± 0.13 vs 1.50 ± 0.15, p < 0.01; 0.00 ± 0.17 vs 1.66 ± 0.20, p < 0.01; respectively) and media as in the histological study (SAL vs TEY, 0.93 ± 0.09 vs 0.73 ± 0.09, p < 0.01; 1.40 ± 0.12 vs 0.86 ± 0.12 p < 0.01; respectively). In addition, nuclear number and nuclear area changed with time (p < 0.004; p < 0.001) but not with media. Conversely, mitochondrial number and mitochondrial area did not change with media or time (p > 0.05).
The results of previous studies that have examined sperm quality upon recovery after various days of epididymal storage at 4 or 5°C have shown that sperm parameters decrease as storage interval increases (Tittarelli et al. 2006; Gañán et al. 2009). This observation could be related with the development of autolytic process on the epididymides (Tittarelli et al. 2006; Gañán et al. 2009). In agreement with these studies, our results show that the storage time increased changes in epididymal cell morphology. The histological and ultrastructural features of epididymal principal cells in non-stored organs (0 h) agree with those previously described by others authors (Arrighi et al. 1986; Axner et al. 1999). Similarly, morphological and ultrastructural changes observed in stored epididymides are in agreement with cell changes related with the cell death process (Myers and McGavin 2007). In the histological study, our result show that cell morphological changes were higher in epididymides stored compared with non-stored organs, and these changes were higher when the storage time increased. Our results appear to agree with data obtained in previous studies that found that the acrosome integrity, motility, velocity and vital stain were lower in sperm recovery of epididymides stored 72 h at 4°C compared with cells recovery of organs stored 24 h (Tittarelli et al. 2006). In that work, Tittarelli et al. (2006) found that sperm recovered from epididymides stored in TEY had higher sperm parameters that those recovered from epididymides stored in SAL (Tittarelli et al. 2006). Those results appear to concur with the present work in which histological and ultrastructural changes were higher in SAL compared with TEY.
In conclusion, these results show that epididymides stored for 24–72 h in TEY have delayed epithelial cell autolysis and decreased cell morphological changes; it seems likely that this would improve the quality of sperm recovered from these epididymides.
This study was supported in part by UNLP grant V11/162 and V11/200 to RLS and MAS. In addition, R Nuñez-Favre was supported with a scholarship from CONICET, and MC Bonaura with a scholarship from CIC. The authors would like to thank Mrs. Roxana Peralta for technical assistance in processing the light and electron microscopy samples of this study.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
Tittarelli CM helped to design, conduct the experiment and process the samples, analysed data and drafted paper. Jurado SB processed the ultrastructural study samples. Bonaura MC and Núñez-Favre R helped to conduct the experiment and process the samples. De la Sota RL and Stornelli MA helped to design and conduct the experiment and critically revised the paper.