Ibeh Nnanna Isaiah¹*, Otabor Florence¹, Oyedeji Jelilat Taiwo², Emili Augustina¹ and Omorodion Nosa Terry

¹Health Services Department, University of Benin, Nigeria
²Department of Anatomy, Faculty of Basic Medical Sciences, University of Benin, Nigeria

*Corresponding Author: Ibeh Nnanna Isaiah, Health Services Department, University of Benin, Nigeria.

Received: December 04, 2017; Published: December 11, 2017

DOI: 10.31080/ASMI.2018.01.0004

Citation: Ibeh Nnanna Isaiah., et al. “Improvised Eosin and Leishman as Morphological Stain for Sperm Cell Analysis; Adult Male Wister Rats and Rabbit as a Model of Study”. Acta Scientific Microbiology 1.1 (2018).

Abstract

Introduction: Over the years improvement has been made on the staining techniques for the morphological analysis of sperm cells ranging from the normal eosin and nigrosine combination to other more simple or complex single staining technique.

Aim: Using improvised combination of Eosin and Leishman stain as morphological stain for detection abnormalities and viability studies towards future applications in assisted reproductive science.

Method: 22 Wister rats and rabbits sperm cells where stained to analyze their morphology, using both the conventional technique and the hibiscus flower extract to determine the reproducibility of this improved staining technique.

Results: From the staining technique the morphology of the sperm cells where properly noticeable from the head, body and tail which is represented in the pictures as shown.

Conclusion: In a third world country this hibiscus plants are accessible stains for quick and accurate morphological observation of Wister rats and Rabbit spermatozoa as an intervention tool in absence of complex staining techniques and since it is natural may have little or no effect on sperm cells.

Keywords: Wister rats; Rabbits; Spermatozoal; Eosin; Nigrosin; Leishman

Introduction

   The morphology of spermatozoa is a device critical for an effec- tive preparation and early embryonic advancement in helped con- ceptive systems [1] . Last Tygerberg Classification Criteria depicted by Kruger in 1986 and the WHO grouping are the most vital sper - matozoa morphological characterizations. As per Kruger’s sperm, the head, neck and tail segments ought to be ordinary, the limits of the head ought to be smooth and oval and 70% of head ought to be covered by acrosome. The leader of the sperm head length ought to be 4 - 5m and must have a width of 2.5 - 3.5m. The length of the tail ought to be a normal of 45m. The center part should be thin and long and width ought to be under 1 mm, length ought to be of 1.5 times the length of the head. The tail ought to be more slender than center piece, ought to be level and not wrinkled, and ought not contain broken parts and cytoplasmic flotsam and jetsam. Also, the sperm ought not have the neck, center piece, tail variations from the norm and an expansive cytoplasmic bead the greater part of the spermatozoon head in the neck range [2,3] .

  Morphological highlights of spermatozoa are communicated in numerical esteems. Techniques utilized as a part of the recoloring may cause a slight change in the estimation estimations of sperma- tozoa in light of the fact that fixatives can make cells contract a bit. For instance, 3 - 5m length and 2 - 3m wide estimations are viewed as typical for the head of spermatozoa in the strategy for Papani - colaou. These qualities change 5 - 6 and 2.5m - 3.5m in the Diff- Quick recoloring technique. In both the center piece ought to be 1m thick, the length of this esteem ought to be up to 1.5 times of this esteem. The tail segment ought to be 45m length decreasing bit by bit [4] . As a result of these distinctions, we analyzed morphology of spermatozoa with various recoloring techniques and planned to locate the better recoloring strategies for research facilities in our examination.

Materials and Methods

  In this examination randomized 10 Wister rats and 10 rabbit’s semen tests were acquired from the Wister rats the sperm cells where reaped from the vas deferens from the rabbits, the sperm cells where gathered utilizing a fake vagina. The examination was completed in the University of Benin Health Services Department.

   Spread were set up from the Wister rats and rabbits under - study; the smears were dried at room temperature and settled in methyl liquor.

   In the initial segment of the investigation, smears were recol- ored with the blend of Eosin and Leishman recolor in a propor - tion 1:5 and it was a homogenized, sifted and left to remain for 24 hours. The stain was poured on the slide and left for (10) ten min- utes and it was last flushed in supported typical saline, scratched out and left to air dry.

Result

   Head, center piece and tails of spermatozoa were seen obvi- ously with GE. Acrosome center rate was to a great degree great as light and dim pinkish (buildup). The stain was homogeneous - ly appropriated on the head and dim pinkish shading was seen. The center piece and tail were seen unmistakably and can be as- sessed extremely well (Figure 1a and 1b). The perfect picture was acquired by recoloring spermatozoa with Giemsa and Eosin. The buildup of the head can be picked by the whitish blue shading. Buildup and morphology of the head can be chosen obviously (Fig - ure 2a and 2b). (Figure 3).

   The spermatozoa were recolored dull blue purple with Eosin and Leishman recolor with expanded grouping of Eosin recolor without washing in cradled typical saline (Figure 4a and 4b). Head morphology and buildup could be seen extremely well however the center piece and the tail did not appear to be clear.

Discussion

   Appraisal of spermatozoa morphology is an imperative pa- rameter in the analysis of barren men. Because of the utilization of expanding measures of IVF (in vitro preparation) procedures, contemplates are likewise centered around the morphology of spermatozoa and its critical part in treatment. Assessment should be possible, recoloring spermatozoa with an assortment of tech - niques from the new semen by electron, quick differentiation and light magnifying lens. There are many recoloring strategies to as- sess the morphology of spermatozoa [5] . García-Herreros., et al. [6] utilized Hemaklor, Harris haematoxylin and PanOptic quick recoloring strategies to influence institutionalization of the ex - amining techniques, to test planning and recoloring for the leader of the spermatozoa with PC helped morphometric investigation (CASMA). They presumed that Haematoxylin and Hemaklorun are the best recoloring techniques for assessment of sperm heads.

  Soler., et al. [7] looked at 3 changed color techniques for ap- praisal of spermatozoa morphometry with mechanized Integrated Semen Analysis System (ISAS). They assessed 200 spermatozoa cells recolored with Hemacolor, Diff-Quik and harris haematoxy - lin. While they discovered all strategies were likewise valuable in that utilized as a part of recoloring of spermatozoa yet Diff-Quick stain demonstrated a critical distinction.

   TB is utilized as a part of the assessment of the atomic chro- matin of spermatozoa by recognizing the nonattendance or crack of disulfide bonds. Beletti and Mello [8] inspected connection between spermatozoa morphology and spermatozoa chromatin buildup with TB and Feulgen response. TB is a successful stain for the assessment of changes in spermatozoa chromatin. Our exami- nation is good with the learning of the writing. TB color recoloring technique is a straightforward strategy and permit synchronous assessment for morphology and buildup of spermatozoa. TB is a perfect color for routine use around there.

   Morel., et al. [9] arbitrarily chose 47 patients connected for se - men examination, they analyzed the decent variety of basic mor - phological issue in the human spermatozoa among people. Keep- ing in mind the end goal to demonstrate their association with the atomic development, sperms recolored with Aniline Blue, Sper- matozoa morphology deserts contrasts seen amongst people and they found a critical connection between regular of deformities and level of atomic maturate. Our examination is perfect with the information of the writing. Aniline Blue color gives the perfect pic- ture to gauge the rate of acrosome head. Buildup and morphology can be evaluated together.

   Tartaglione and Ritta [10] inspected the prognostic sperma - tological estimations of solidified broke down spermatozoa to evaluate in vitro richness. Assessing plasma film and acrosome trustworthiness are critical esteems for typical elements of sper - matozoa, for this point, they inspected smears recolored with Trypan Blue and Giemsa. They reasoned that these stains can be utilized for the guess of the ripeness in the semen utilized for IVF.

   Our assessment was predictable with the writing of Giemsa and Wright stains. Buildup and head morphology of spermatozoa were well-selectable. The center piece and tail could be seen. An unforeseen finding that tail and head totally recolored pink in the a portion of the spermatozoa which had harmed head morphol - ogy. Additionally think about was recommended to clarify the rea- son. Spermatozoa morphology and buildup were clear and smooth with Orange G recolor. Be that as it may, recoloring process was too long that is the reason Orange G recolor had no prevalence.

   Shorr procedure is favored in numerous research centers be- cause of the inadequate relationship in the consequences of IVF. Henkel., et al. [11] assessed diminished motility of spermatozoa in elderly men with Shorr strategy. The level of recolored sper - matozoa with ordinary and irregular flagella’s was assessed and discovered a negative connection between the recolored strange flagella’s and speed proportion and motility of the spermatozoa in elderly man.

   Convincingly In our investigation, spermatozoa heads showed up as Pinkish globules with the adjust of fixation with time light pale pinkish shaded, center segments and tails were recolored dim pinkish. Our examination was had all the earmarks of being perfect with the writing and as the aftereffect of our investigation. Sperma- tozoa center area which is a rich segment for mitochondria’s were uncovered plainly and was shown by and by the Eosin and Leish - man recolor which is proposed by this examination as a straight- forward and available stain for spermatozoal with Wister rats and Rabbits as a model [12-23].

Acknowledgement

   I wish to register my profound gratitude to the National Acad- emy for the Advancement of Science, for the immense support in terms of grants, I also wish to thank all member and staff of the Health Services Unit of the University of Benin, Benin city.

Conflict of Interest

The were no conflicting interest during this study.

Bibliography

1. Kuster CE., et al . “Determining sample size for the morphologi - cal assessment of sperm”. Theriogenology 61.4 (2004): 691- 703.
2. WHO. “Laboratory Manual for the Examination on Human Semen and Spermatozoa-Cervical Mucus Interaction”. Cam- bridge, Cambridge University Press (1999).
3. Mehta RH., et al . “Prevalences of oligozoospermia and azo- ospermia in male partners of infertile couples from different parts of India”. Asian Journal of Andrology 8.1 (2006): 89-93.
4. Irez T., et al . “Semen analysis. Andrology Laboratory Hand- book”. Istanbul, Nobel Medical Publishing (2007)
5. Uven M., et al . “Ultrastructural Changes in the Spermatozoa of Infertile Men”. Turkiye Klinikleri Journal of Medical Sciences 16 (1998): 103-105.
6. García-Herreros M., et al . “Standardization of sample prepa- ration, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa”. Interna- tional Journal of Andrology 29.5 (2006): 553-563.
7. Soler C., et al . “Comparison of three different staining methods for the assessment of epididymal red deer spermatozoa mor - phometry by computerized analysis with ISAS”. Theriogenology 64.5 (2005): 1236-1243.
8. Beletti ME and Mello ML. “Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology”. Theriogenology 62.3-4 (2004): 398-402.
9. Morel F., et al . “Interindividual variations in the disomy fre- quencies of human spermatozoa and their correlation with nuclear maturity as evaluated by aniline blue staining”. Fertility and Sterility 69.6 (1998): 1122-1127.
10. Tartaglione CM and Ritta MN. “Prognostic value of spermato- logical parameters as predictors of in vitro fertility of frozen- thawed bull semen”. Theriogenology 62.7 (2004): 1245-1252.
11. Henkel R., et al . “Molecular aspects of declining sperm motility in older men”. Fertility and Sterility 84.5 (2005): 1430-1437.
12. Anafarta K., et al . “Principles of Urology”. Ankara, Sun Pub- lishing (2007): 222
13. Avcı B. “Different fixation protocols with chromatin conden - sation of spermatozoa with sperm chromatin anomaly evalu - ation protocols”. Uludag University, Medical Faculty Journal 32 (2006): 55-59
14. Gago C., et al . “Standardization of sampling and staining methods for the morphometric evaluation of sperm heads in the Cynomolgus monkey (Macaca fascicularis) using comput- er-assisted image analysis”. International Journal of Androl - ogy 21.3 (1998): 169-176.
15. Hammadeh ME., et al . “Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility”. Archives of Andrology 46.2 (2001): 99-104.
16. et al . “Relationship between human sperm mor - phology and acrosomal function”. Journal of Assisted Repro- duction and Genetics 20.10 (2003): 432-438.
17. Menkveld R., et al . “Effects of different staining and washing procedures on the results of human sperm morphology eval - uation by manual and computerised methods”. Andrologia 29.1 (1997): 1-7.
18. Michael AY., et al . “Complete staining of human spermatozoa and immature germ cells combined with phase contrast mi- croscopy”. Archives of Andrology 19.3 (1987): 217-221.
19. Milingos S., et al . “The value of semen characteristics and tests of sperm function in selecting couples for intra-uterine insemination”. European Journal of Obstetrics and Gynecology and Reproductive Biology 64.1 (1996): 115-118.
20. Palermo G., et al . “Pregnancies after intracytoplasmic injec - tion of single spermatozoon into an oocyte”. Lancet 340.8810 (1992): 17-18.
21. Root Kustritz MV., et al . “The effects of stains and investiga- tors on assessment of morphology of canine spermatozoa”. Journal of the American Animal Hospital Association 34.4 (1998): 348-352.
22. Sánchez R., et al . “Evaluation of nuclear maturity in human spermatozoa obtained by sperm- preparation methods”. An - drologia 26.3 (1994): 173-176.
23. Spira A. “Epidemiology of human reproduction”. Human Reproduction 1.2 (1986): 111-115.

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