Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains

Short Communication Acta Vet Eurasia 2020; 46: 132-136

Nathan Isaac DIBAL , Sani Hyedima GARBA , Tamunotonye Watson JACKS Department of Human Anatomy, University of Maiduguri, Maiduguri, Nigeria Cite this article as: Dibal, N.I., Garba, S.H., Jacks, T.W., 2020. Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains. Acta Vet Eurasia 2020; 46: 132-136.

ORCID IDs of the authors: N.I.D. 0000-0002-1805-7553; S.H.G. 0000-0001-6165-0064; T.W.J. 0000-0002-1096-2997 Abstract

Spermatozoa are unique and highly specialized cells operat- blue, Alcian blue, Harris hematoxylin, and eosin stains. The re- ing at the microscopic level in a complex environment and sult showed that the head and the proximal and distal parts have various sizes, shapes, and forms. The sizes, shapes, and of the tail were stained excellently with crystal violet and H&E forms of the spermatozoa are dependent on the species but stains. Giemsa and Harris hematoxylin were also good but may be different even within the same species. Five Wistar rats could not stain the distal part of the tail. In conclusion, crystal were euthanized using ketamine injection. The epididymis of violet is the best stain for sperm morphology in Wistar rats fol- each rat was dissected, teased and diluted in normal saline. lowed by H&E stain. Several smears were prepared and stained with Giemsa, Leish- Keywords: Crystal violet, eosin, hematoxylin, morphology, man, hematoxylin and eosin (H&E), crystal violet, methylene sperm

Introduction copy (Sousa et al., 2013; Villaverde et al., 2008). Different stains may exert different effects on the spermatozoa. Therefore, it Spermatozoa are unique and highly specialized cells operating can significantly affect the results of morphometric measure- at the microscopic level in a complex environment and have ment and consequently, the sperm quality (Banaszewska et al., various sizes, shapes, and forms (Gage and Freckleton, 2003). 2015). The structure of the mammalian sperm is designed in The sizes, shapes, and forms of the spermatozoa are depen- such a way that it enables them to penetrate the ovum, giving dent on the species but may be different even within the same them the ability to transfer genetic information in the process species (Andraszek et al., 2018; Banaszewska et al., 2015). The of fertilization (Kondracki et al., 2017). Examination of sperm differences in the sperm sizes and shapes within different spe- morphology allows one to determine the fertility status in a cies, coupled with the fact that spermatozoa are always hetero- male (Phillips et al., 2004). geneous in a single ejaculate (normal and abnormal/damaged sperm), are the major obstacles in proper semen analysis (Ban- Staining of the sperm is important to study the affinity of the aszewska et al., 2015). Understanding the adaptive significance different parts of the spermatozoa to various histological stains. of sperm form and function in different species has been a chal- Several stains are used for sperm in different species, and they lenge to biologists; therefore, various staining techniques have include Giemsa-Wright (Perez-Marin et al., 2016), eosin-nigrosin been developed for different species (Gage, 1998). Sperm stain- (Oridupa et al., 2018), eosin and Leishman (Isaiah et al., 2018), ing is aimed at easy visualization of the cells and providing a eosin-gentian (Kondracki et al., 2017), Weigert’s ferrous hema- better identification of the abnormalities through light micros- toxylin, hematoxylin & eosin (H&E), Papanicolau, orange G, light

Address for Correspondence: Nathan Isaac DIBAL • E-mail: [email protected] This work is licensed under a Creative Received Date: 15.04.2020 • Accepted Date: 04.06.2020 • Available Online Date: 23.07.2020 • DOI: 10.5152/actavet.2020.20018 Commons Attribution-NonCommercial Available online at actavet.org 4.0 International License.

132 DIBAL et al. Morphological Assessment of Sperm of Wistar Rats Acta Vet Eurasia 2020; 46: 132-136

Figure 1. Spermatozoa stained with Giemsa at 400x Figure 2. Spermatozoa stained with hematoxylin and eosin magnification at 400x magnification green, Shorr, and Janus green (Aksoy et al., 2012). Microscopic ter ad libitum. The research was approved by the Department evaluation of sperm morphology is relatively simple but can of Human Anatomy ethical committee (Code No. UM/HA/ produce results similar to those obtained with more sophisti- PGR19.20-09900). The rats were euthanized using ketamine in- cated and expensive methods (Buendia et al., 2002). Papanico- jection. The whole epididymis of each rat was dissected, teased, laou staining is time consuming, multistage, and involves more and diluted in 5 mL of 10% normal saline at room temperature. than 10 different solutions (Van der Horst and Maree, 2009). Several smears were prepared on a glass slide, allowed to dry Therefore, the method has a limited applicability in case of at room temperature, and fixed with ethanol. The smears were urgent need. Eosin-nigrosin staining is a popular method for stained with crystal violet stain (C25H30ClN3), methylene blue semen evaluation in mammals and birds (Bjorndahl et al., 2004; stain (C16H18ClN3S), Alcian blue (C56H68Cl4CuN16S4), hematoxylin

Lącka et al., 2016). The method enables the analysis of sperm (C16H14O6), eosin Y (C20H6Br4Na2O5), Giemsa stain (buffered solu- viability and evaluation of its structure. Eosin-nigrosin staining tion of methylene blue, eosin Y, and Azure B in methanol and is easy to perform and allows detection of the morphological glycerol), Leishman stain (buffered solution of methylene blue abnormalities as well as determination of membrane integrity and eosin Y in alcohol), and H&E stain. The stains were washed of the sperm. after 5–10 minutes depending on the type of stain used and allowed to dry at room temperature. All the slides were observed Animals have been used for scientific studies mainly to develop under light microscope at 400x magnification. a better understanding of animal and human anatomy, physiol- ogy, and pathology (Andersen and Winter, 2019). Domesticated Results rats (Rattus norvegicus) were the first rodent species to be used Photomicrograph of the spermatozoa of Wistar rats showed a in research as early as 1828. In the 20th century, rats became the characteristic sickle-shaped head and a long slender tail. The preferred animal model in research. The first standard rat strain outline of the head and proximal part of the tail were seen (Wistar rat) was developed in 1909, and it is from this strain that clearly with Giemsa stain; both the outline of the head and most of the rats used in laboratories today are believed to have proximal part of the tail were stained blue (Figure 1). The heads descended. Wistar rats are the most frequently used rat model of the spermatozoa were stained red and blue with H&E; the in research (Franco, 2013; Lindsey and Baker, 2006). Therefore, proximal part of the tails was stained dark blue while the dis- understanding the sperm morphology of Wistar rats will pro- tal part was stained pale blue. The central part and outline of vide a clue for studying the sperm cells in different vertebrates, the head appeared red and blue, respectively (Figure 2). Harris including humans. The aim of this study was to assess the hematoxylin and Leishman stains gave an outline of both the morphology of epididymal sperm in Wistar rats using different head and tail of the spermatozoa; the middle part of the head stains. did not pick any stain. The tail appeared light blue with Harris Materials and Methods hematoxylin and dark blue with Leishman stain. The proximal part of the tail stained dark blue while the distal part stained A total of 5 male Wistar rats between 8 and 9 weeks old, weigh- light blue with Leishman stain (Figures 3 and 4). The heads and ing 118–126 g were used for the study. They were fed with tails of the spermatozoa were stained purple with crystal violet. grower mash (Vital Feed, Grand Cereal Jos, Nigeria) and wa- The heads were completely stained, and both the proximal and

133 DIBAL et al. Morphological Assessment of Sperm of Wistar Rats Acta Vet Eurasia 2020; 46: 132-136

Figure 3. Spermatozoa stained with Harris hematoxylin at Figure 5. Spermatozoa stained with crystal violet at 400x 400x magnification magnification

Figure 4. Spermatozoa stain with Leishman stain at 400x Figure 6. Spermatozoa stained with methylene blue at 400x magnification magnification distal parts of the tail were also stained (Figure 5). The sperma- of stallion (Serafini et al., 2013). Giemsa stain was reported to tozoa stained with methylene blue and Alcian blue were ob- give a clear head morphology of human spermatozoa, but the served as an outline of the head and proximal part of the tail; middle piece and the tail morphology were not clear (Aksoy et however, the distal part of the tail was not stained even with al., 2012). In this study, H&E showed a good staining of the head prolonged time of exposure to the stains (Figures 6 and 7). The as well as the tail. Earlier studies reported H&E as an excellent heads of the spermatozoa stained with eosin appeared bright stain for human spermatozoa morphology as it could stain the red with a dark outline, the proximal part of the tails stained head, acrosome, and tail with a clear distinction between all the blue, and the distal part of the tail did not pick any stain even parts (Kondracki et al., 2017). after prolonged exposure to the stain (Figure 8). Lingappa et al. (2015) identified the H&E stain as the best one Discussion for the assessment of human sperm head morphology. The stains gave a clear morphology of the head as purple conden- With the Giemsa stain, a clear outline of the sperm can be ob- sation; detailed morphology of the middle part and the tail served, which indicates that this stain is good for the cell mem- were also clearly identified. brane because it clearly displays the outline of both the head and tail with a clear cytoplasm. Trypan Blue-Giemsa was report- In this study, Harris hematoxylin and Leishman stains display an ed to be good for the evaluation of sperm membrane integrity outline of the head and tail of the spermatozoa. Harris hematox-

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Figure 7. Spermatozoa stained with Alcian blue at 400x Figure 8. Spermatozoa stained with eosin at 400x magnification magnification ylin was reported to be a good stain for spermatozoa as it could nation with other stains for a clear demonstration of cell com- stain the head, middle part, and the tail clearly for sperm mor- ponents or sperm morphology. phology evaluation (Ageep et al., 2009; Hidalgo et al., 2006). An earlier study reported Leishman stain as a poor stain for sperma- Conclusion tozoa because the head and tail could not be easily identified Crystal violet is the best stain for sperm morphology in Wistar in the Leishman stained sections (Ageep et al., 2009). However, rats followed by H&E stain. They provide a clear visualization of Leishman stain was reported as a good alternative to the Giemsa the sperm head and every part of the tail. Because of the differ- stain for the identification ofPlasmodium falciparum and Plasmo- ential staining of the head and tail with H&E stain, it is preferred dium vivax in blood smear and a good stain for red and white over crystal violet when the contrast between the head and tail blood cell morphology (Sathpathi et al., 2014). is needed. Other stains such as Giemsa and Harris hematoxylin Sperm heads and tails were properly stained with crystal violet were also good but could not stain the distal part of the tail. in this study, which implies that it can be used effectively to Therefore, an abnormality in the distal part of the tail could not study sperm morphology in Wistar rats. Crystal violet (gentian) be clearly seen with these stains. has a wide range of uses as it can be used for demonstration of lipofuscin and Nissl granules and for visualization of bacteria, such as Streptococcus pneumonia and Salmonella typhi (Pilati Ethics Committee Approval: Ethics committee approval was received et al., 2008; Smith and Hussey, 2016; Terr, 1986). It can also be for this study from the Department of Human Anatomy ethical commit- used in combination with other stains and was reported to be tee, University of Maiduguri (Code No. UM/HA/PGR19.20-09900). effective in the detection of gonococcal urethritis when used in combination with methylene blue stain (Taylor et al., 2011). Peer-review: Externally peer-reviewed. Eosin-gentian complex was reported as the routine stain for Author Contributions: Concept - N.I.D.; Design - N.I.D., S.H.G., T.W.J.; sperm morphology in boar; however, it significantly affects Supervision - S.H.G., T.W.J.; Resources - N.I.D.; Materials - N.I.D.; Data Col- sperm head parameters compared with the unstained sperm lection and/or Processing - N.I.D.; Analysis and/or Interpretation - N.I.D., (Czubaszek et al., 2019). S.H.G., T.W.J.; Literature Search - N.I.D.; Writing Manuscript - N.I.D.; Criti- cal Review - S.H.G., T.W.J. Methylene blue and Alcian blue give an outline of the head and proximal part of the tail. These stains are more effective Conflict of Interest: The authors have no conflicts of interest to declare. for staining the mucin, bacteria, and vaginal smears (Micke et al., 2019; Smith and Hussey, 2016; Zirlik et al., 2012) but not as Financial Disclosure: The authors declared that this study has received good for demonstration of sperm morphology. Eosin gives an no financial support. excellently stained head with an outline of the proximal part References of the tail and is mostly used in combination with hematoxylin for general histology, Nigrosin stain for sperm vitality assess- Ageep, A.K., Ali, S.A., Almobarak, A.O., 2009. The best stain for mor- ment, and gentian stain for morphology (Bjorndahl et al., 2003; phological study of human seminal fluid’s smears. Sudan Journal of Czubaszek et al., 2019). Therefore, eosin is best used in combi- Medical Sciences 4, 1-5. [Crossref]

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