Why the WHO Recommendations for Eosin‐Nigrosin Staining Techniques for Human Sperm Vitality Assessment Must Change

Björndahl, Lars; Söderlund, Inger; Johansson, Sofia; Mohammadieh, Majid; Pourian, Mohammad R.; Kvist, Ulrik

doi:10.1002/j.1939-4640.2004.tb02839.x

Cited by 47 publications

(29 citation statements)

References 12 publications

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“…Studies by Kondracki et al (2006) and Banaszewska et al (2011) confirm that eosin staining is the standard technique for assessment of sperm morphology in farm animals, commonly applied at animal breeding and insemination centres. Eosin-nigrosin staining is also recommended by WHO as a basic method for evaluating human sperm morphology (Björndahl et al 2003(Björndahl et al , 2004. Apart from sperm morphology assessments, this method also enables the identification of living and dead sperm (Sprecher & Coe 1996, Zambelli & Cunto 2006, Freneau et al 2010.…”

Section: Discussionmentioning

confidence: 99%

Comparison of different chromatin staining techniques for bull sperm

Andraszek¹,

Banaszewska

Czubaszek³

et al. 2014

Arch. Anim. Breed.

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Morphological analysis of semen is a very important step in fertility assessment, but many semen defects are not detectable at the morphological level. These include pathological changes in sperm chromatin structure. During mammalian spermiogenesis, histone proteins associated with DNA structure are replaced by specific protamines, with which chromatin does not form nucleosomal complexes. In the fully developed, mature sperm, the histones are replaced with protamines. Disruptions of nucleoprotein structure can be treated as possible indicators of the biological value of spermatozoa. The experimental material consisted of sperm from one-and-a-half-year-old bulls, isolated post mortem from the tail of the epididymis. The smears were stained with silver nitrate (AgNO 3 ), acridine orange (AO), aniline blue (AB) and chromomycin A3 (CMA3). Sperm dimensions largely depend on individual variability among the bulls. In most cases, differences in sperm dimensions were identified between individuals, which was confirmed in the statistics. Sperm with elevated, abnormal histone levels were proportionally quite scarce (1.4 %). Studies of nuclear proteins in the context of infertility demonstrate the important influence of normal chromatin structure on sperm functions.

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Section: Discussionmentioning

confidence: 99%

Comparison of different chromatin staining techniques for bull sperm

Andraszek¹,

Banaszewska

Czubaszek³

et al. 2014

Arch. Anim. Breed.

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“…It has been reported that the eosin-nigrosin stain with low osmolality is associated with a significant increase in bent tails in canine spermatozoa (Johnson et al 1991). But, as described by Bjorndahl et al (2004), staining solutions used for morphological assessment in human sperm show wide osmotic variations. Cells exposed to hypertonic solutions may suffer a net leak/influx of non-permeating solutes into the cells.…”

Section: Discussionmentioning

confidence: 99%

Choice of staining technique affects the morphological assessment of epididymal feline sperm

Marı́n¹,

Jiménez²,

Agüera³

2016

Vet. Med. - Czech

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Morphological examination of spermatozoa remains a valuable tool for determining the quality of feline sperm for cryopreservation and/or for use in artificial insemination. Numerous staining techniques have been employed for feline sperm assessment, and several variations have also been reported. Forty-eight domestic cats were castrated and epididymal sperm samples were obtained. Sperm morphology was assessed using two staining methods, an air-dried stain (Giemsa-Wright) and a wet stain (eosin-nigrosin), to determine their relative abilities for estimation of the proportion of normal and abnormal spermatozoa. The effects of centrifugation and the cooling process on sperm morphology were also analysed. The mean percentage of morphologically normal spermatozoa was significantly lower in eosin-nigrosin-stained samples than in Giemsa-Wright-stained preparations (46.5% vs 53.4%; P < 0.01), both examined by conventional light microscopy. Results concerning head, midpiece and tail sperm abnormalities suggest that these methods differ in their capacity to identify sperm anomalies in cat. Although no significant differences were observed for secondary abnormalities, cytoplasmic droplets were significantly lower in Giemsa-Wright-stained samples (P < 0.01). Centrifugation or refrigeration of sperm samples did not affect the epididymal sperm morphology in evaluated samples. This study highlights the necessity of minimising morphological assessment variations in order to eliminate subsequent errors and to improve the reproducibility of sperm morphology assessment in different laboratories.

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“…The eosin-nigrosin staining method is widely used for the evaluation of sperm because it is simple and rapid, but this conventional staining method for the evaluation of sperm is very subjective because only a very limited number of dead sperm can be counted [18] . Dyeing time, sperm concentration, characteristics of the semen extenders or some factors affecting the stability of the sperm membrane intactness are involved in eosin dyeing processes could alter the ratio of intact sperm [2,19] . Chalah and Brillard [20] reported that in terms of sperm membrane integrity flow cytometric analysis was a more effective method compared to eosin-nigrosin staining.…”

Section: Discussionmentioning

confidence: 99%

Epididimal boğa Spermatozoonlarının Bazı Morfolojik Özelliklerinin Belirlenmesi Amacıyla Akım Sitometri ve Eozin-Nigrozin Boyama Yöntemlerinin Karşılaştırılması

Akyol¹,

Azkur²,

Ekici³

2015

Kafkas Univ Vet Fak Derg

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The aim of this study is to investigate necrosis and apoptosis in epididymal bull spermatozoa before freezing and after thawing using the flow cytometric method and to compare this with eosin-nigrosin dyeing, which is the conventional method used in assessing of spermatozoa. The testicles from fourteen bulls at local slaughterhouse were used for this study. The proportions of live spermatozoa, total apoptotic, necrotic and early necrotic spermatozoa levels were observed via flow cytometry. Annexin V/PI fluoruscence dyeing was used to investigate the proprotions of apoptotic, necrotic, early necrotic and live spermatozoa for flow cytometry. The proportion of dead spermatozoa and protoplasmic droplets were determined using the eosin-nigrosin conventional dyeing method in fresh and frozen-thawed spermatozoa. The average dead spermatozoa count with flow cytometry was less than with the eosin-nigrosin method (P<0.05). Some morphological characteristics such as protoplasmic droplets could be determined with the eosin-nigrosin method; however, sperm subpopulations entering the death process (apoptotic, necrotic and early necrotic) could be defined clearly only with the flow cytometric method. As a result, combination of eosin-nigrosin dyeing method and flow cytometric analysis of sperm morphological evaluation could give better results of bull epididymal semen in comparison to eosin-nigrosin dyeing method alone. Keywords: Flow cytometry, Eosin-nigrosin, Morphological characteristics, Epidydimal spermatozoa, Bull Epididimal boğa Spermatozoonlarının Bazı Morfolojik Özelliklerinin Belirlenmesi Amacıyla Akım Sitometri ve Eozin-Nigrozin Boyama Yöntemlerinin Karşılaştırılması ÖzetBu çalışmanın amacı, epididimal kaynaklı boğa spermatozoonlarının taze ve dondurulup çözdürüldükten sonra akım sitometri (Flow Cytometri) yöntemiyle analiz edilmesi, nekroz/apoptoz düzeylerinin incelenmesi ve klasik boyama yöntemi olan eozin-nigrozin boyama metoduyla kıyaslanması amaçlanmıştır. Bu amaçla yerel mezbahada kesilen 14 boğanın epididimislerinden elde edilen spermatozoonlar kullanılmıştır. Canlı spermatozoa, toplam apoptotik, nekrotik ve erken nekrotik spermatozoa düzeyleri akım sitometri yöntemiyle tespit edilmiştir ve bu amaçla AnnexinV+PI (Propidium İyodür) floresan boyama yöntemi kullanılmıştır. Taze ve çözdürülmüş sperma içerisindeki, ölü spermatozoa ve protoplazmik damlacık taşıyan spermatozoonların oranları eozin-nigrozin boyama yöntemi ile belirlenmiştir. Taze spermada, akım sitometri ile ölü spermatozoon sayısı eozin-nigrozin boyamada bulunana göre daha az olduğu görülmüştür (P<0.05). Protoplazmik damlacık gibi bazı morfolojik parametrelerin mikroskopik muayenelerle belirlenebildiği ancak ölüm sürecine giren spermatozoonların alt kategorilerinin (apoptotik, nekrotik, erken nekrotik) akım sitometri ile net olarak tespit edilebildiği görülmüştür. Sonuç olarak, klasik yöntemlere göre spermatozoa canlılığı tespitinde akım sitometri yönteminin klasik boyama yöntemlerine destek olarak ve daha etkin bir şekilde kullanılabileceği...

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Why the WHO Recommendations for Eosin‐Nigrosin Staining Techniques for Human Sperm Vitality Assessment Must Change

Cited by 47 publications

References 12 publications

Comparison of different chromatin staining techniques for bull sperm

Comparison of different chromatin staining techniques for bull sperm

Choice of staining technique affects the morphological assessment of epididymal feline sperm

Epididimal boğa Spermatozoonlarının Bazı Morfolojik Özelliklerinin Belirlenmesi Amacıyla Akım Sitometri ve Eozin-Nigrozin Boyama Yöntemlerinin Karşılaştırılması

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