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ANIREP 5229 1–8

Animal Reproduction Science xxx (2015) xxx–xxx 1

Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Review article

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The effect of selected staining techniques on bull sperm morphometry

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Q2

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Dorota Banaszewska a , Katarzyna Andraszek b,∗ , Magdalena Czubaszek b , Barbara Biesiada–Drzazga a a

Department of Breeding Methods and Poultry and Small Ruminant Breeding, University of Natural Sciences and Humanities, 14 Prusa Str., 08-110 Siedlce, Poland b Department of Animal Genetics and Horse Breeding, Institute of Bioengineering and Animal Breeding, University of Natural Sciences and Humanities, 14 Prusa Str., 08-110 Siedlce, Poland

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a r t i c l e

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i n f o

a b s t r a c t

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Article history: Received 2 April 2015 Received in revised form 24 June 2015 Accepted 25 June 2015 Available online xxx

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Keywords: Bull Sperm Spermatozoon Morphometry Tygerberg criteria

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Contents

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1. 2. 3. 4.

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Q3

Sperm morphometry has some value as an indicator of reproductive capacity in males. In laboratory practice a variety of slide-staining methods are used during morphological evaluation of semen to predict male fertility. The aim of this study was to determine the effect of staining of semen using four different techniques on the morphometry of the bull sperm cell. The material for the study consisted of semen collected from test bulls of the Black-and-White variety of Holstein-Friesians. The results obtained in the study indicate differences in the dimensions of bull sperm heads when different slide staining techniques were used. The most similar results for sperm head dimensions were obtained in the case of SpermBlue® and eosin + gentian violet complex, although statistically significant differences were found between all the staining techniques. Extreme values were noted for the other staining techniques – lowest for the Papanicolaou and highest for silver nitrate, which may indicate more interference in the cell by the reagents used in the staining process. However, silver nitrate staining was best at identifying the structures of the sperm cell. Hence it is difficult to determine which of the staining methods most faithfully reveals the dimensions and shape of the bull sperm. © 2015 Elsevier B.V. All rights reserved.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Uncited reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. Tel.: +48 442 56431212. E-mail address: [email protected] (K. Andraszek). http://dx.doi.org/10.1016/j.anireprosci.2015.06.019 0378-4320/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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1. Introduction

Q4

Sperm morphology continues to be an important 35 parameter in predicting fertility in both humans and ani36 mals (Keel et al., 2002; Maree et al., 2010; Lasiene et al., 37 2013). One solution in evaluation of sperm morphology is 38 computer-assisted sperm analysis (CASA), which reduces 39 the subjectivism of morphological analysis (Gago et al., 40 1998), but these systems are relatively expensive and also 41 not free of errors resulting from automation of the analy42 sis process. The concept of morphology is closely linked 43 to sperm cell dimensions falling within the norms for a 44 given species. Hence many authors have observed rela45Q5 tionships between sperm morphometry and male fertility 46 (Casey et al., 1997; Hirai et al., 2001; Esteso et al., 2006; ˜ 47 Núnez-Martínez et al., 2007). 48 Sperm morphometry is so an important indicator of 49 reproductive capacity in the male (Gosz et al., 2010). 50 According to a study by Katz et al. (1986), the sperm cells of 51 infertile men were larger in terms of the long and short axis 52 of the sperm head than in fertile men, and the length-to53 width ratio of the head was greater as well. This has also 54 been confirmed by other studies (Klimowicz et al., 2005; ˙ nski ´ 55 Niza and Klimowicz, 2005). The results of experiments 56 on human sperm correspond to data obtained in veteri57 nary medicine. A pronounced difference in sperm head 58 size in fertile and infertile males has been discovered in ˜ 59 stallions, boars and dogs (Gravance et al., 1996; Núnez60 Martínez et al., 2007; Banaszewska et al., 2011). Animals 61 with smaller sperm head dimensions have been found to 62 be more fertile. Research has shown that not only the size of 63 the sperm affects fertilization capacity, but also the size of 64 the tail and midpiece. Sperm cells with longer tails have 65 greater fertilization capacity due to their greater motil66 ity. Data concerning morphometric dimensions enhance 67 knowledge of the actual in vitro and in vivo fertilization 68 capability of sperm, and also make it possible to determine 69 the suitability of sperm for freezing prior to fertilization 70 (Hirano et al., 2001), while morphological evaluation can 71 to a certain extent indicate the functional capabilities of 72 sperm in terms of acrosome function. Based on evaluation 73 of the state of the acrosome during morphological analy74 sis of the sperm cell we can draw conclusions about the 75 acrosome reaction and the release of proteolytic enzymes 76 during penetration of the zona pellucida (Nikolettos et al., 77 1999), which in consequence allows us to better predict 78 fertilization capacity (McAlister, 2010; Menkveld et al., 79 2011). 80 A problem in evaluating the morphology and mor81 phometry of sperm is the lack of standardization of 82 staining techniques. The method of staining and evaluating 83 specimens can significantly affect the results of morpho84 metric measurements. In laboratory practice a variety 85 of slide-staining methods are used during morphological 86 evaluation of semen to predict male fertility. SpermBlue® 87 is a stain that is often preferred for evaluation of human 88 and animal sperm (Van der Horst and Maree, 2009). The 89 Papanicolaou method is used for analysis of human semen 90 (Menkveld et al., 1990; WHO, 1999). For evaluation specif91 ically of bull semen, a staining technique using a complex 92 of eosin and gentian violet is recommended (Blom, 1981; 34

Kondracki et al., 2012). As an experimental method, staining with silver nitrate, which enables more precise analysis of individual structures of the sperm cell, may be used as well (Andraszek and Smalec, 2011; Andraszek et al., 2014a,b). Each of these methods, due to the chemical composition of the reagents used during the staining process, has a different effect on the stained cells. Hence the aim of this study was to determine the effect of staining of semen using four different techniques on the morphometry of the bull sperm cell. 2. Material and methods The material for the study consisted of semen collected from test bulls of the Black-and-White variety of HolsteinFriesians. A total of 20 individuals at the age of one and a half years were selected for the study. At least one ejaculate from each bull were collected and assessed. The ejaculates were taken by means of the artificial vagina technique. The semen was maintained at room temperature until needed for slide preparation for morphology and morphometry. Slides were prepared within 15 min of collection. Sperm morphology was evaluated using: Papanicolau, SpermBlue® , eosin + gentian violet complex and silver nitrate. Firstly, the routine sperm smear was made and allowed to air dry. For Papanicolau staining method the air-dried slides were placed into 96% ethanol for fixation for 15 min and then stained using the routine protocol recommended by WHO (WHO, 2010) (reagents from Sigma Chemical Co., St. Luis, MO, USA). At the end of the procedure the slides were dehydrated with equal parts of absolute ethanol and xylene, then cleared with xylene alone for 1 min and mounted with DPX medium. The SpermBlue® staining method was carried out as previously described using commercially available kit (Microptic SL, Barcelona, Spain) (Van der Horst and Maree, 2009). Briefly, the slides were placed horizontally on a staining tray and covered with 1 ml of SpermBlue® fixative for 10 min. Then the fixative was gently removed. Immediately, without washing or drying the slides, 0.5 ml of SpermBlue® stain was put onto each fixed sperm smear for 12–15 min. The care was taken to displace stain equally across the smear surface. After removal of the stain by gently running off, the slides were slowly dipped into distilled water (only one or two dips lasting for 3 s). Then the slides were left in an upright position to air dry. Finally, the slides were mounted with DPX medium. All chemicals in this study were purchased from Sigma Chemical Company. For complex dye Q6 eosin and gentian staining method smears were prepared by carefully dragging a drop of the fresh sperm across a degreased microscopic slide heated to 37 ◦ C (Kondracki et al., 2005). The slides were allowed to air dry for a minimum of 2 h, and were then prepared and preserved in a 96% ethanol solution during a 5-min exposure. After 30 min, the preserved slides were washed in distilled water, and then coloured with a 10% aqueous solution of eosin during a 20- to 60-s exposure. The coloured slides were washed in distilled water and coloured with gentian pigment during a 3- to 5-min exposure. After colouring, the slides were washed and dried. The slides were gently rinsed with distilled water for 2 min to remove debris. This procedure led

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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Table 1 Formulas used to calculate sperm head morphometry measurements.

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Variable

Formula

Head length (␮m) Head width (␮m) Head perimeter (␮m) Head area (␮m2 ) Head elipticity Head elongation Head roughness Head regularity

L W P A L/W (L − W)/(L + W) 4␲(A/P2 ) ␲(L*W/4*A)

to a clean background, and thus a good contrast against the stained spermatozoa. All the reagents used were purchased from Sigma Chemical (Germany). A 50% AgNO3 solution and a gelatin colloid solution were applied to the specimens in a proportion of 1:2. The smears were covered with a cover slip and incubated for 15–20 min at 60 ◦ C in saturated humidity (Andraszek and Smalec, 2011). When the samples took on a brown colour the chemical reaction was stopped by washing the specimen several times with distilled water. The specimens were dried at room temperature without access to light. The slides were prepared and assessed at the same time, and by the same person using a microscope. The sperm cells were evaluated with an Olympus BX50 fluorescence microscope and the MultiScan image analysis system and measurement software from Computer Scanning system. The Olympus BX50 microscope is a high quality research microscope with phase objective lenses and the capability of analysing fluorescence slides. The microscope works in conjunction with the MultiScan image analysis system by Computer Scanning Systems. The MultiScan system has an option for brightness correction enabling optimal adjustment of brightness for maximum contrast. The system is intended for the visualization, acquisition, management, storage, processing and analysis of images, with particular focus on measurement functions. The source of the image for the MultiScan software is the subprogram Multiplier, which uses a frame grabber card. An image from a video camera is displayed by the card in real time on the monitor. MultiScan is an image analysis programme and is not used to improve the quality of photographs of slides but to extract important information from them. In each ejaculate, morphometric measurements were made of the heads of 100 randomly selected sperm that were clearly visible in the field of view of the microscope. A total of 4000 sperm heads were evaluated. The length, width, circumference and surface area of the sperm head were measured according to the

Fig. 1. Boar sperm head: Papanicolaou staining.

method described by Kondracki et al. (2005). In addition, the frequency of nuclear vacuoles in the sperm head was determined. Tygerberg’s strict criteria (Kruger et al., 2004), which most precisely characterize the sperm head, were used in the morphological evaluation of the sperm (Table 1). The data for the morphometric measurements of the spermatozoa were stored in a database and exported for further statistical analysis. Statistical differences between the samples were tested using Tukey’s test and ANOVA (STATISTICA version 10.0, StatSoft Inc., PL). The level of significance was set at P ≤ 0.01. 3. Results

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Table 2 presents data comparing the morphometric characteristics of the heads of bull sperm stained using four different slide-staining techniques (Figs. 1–4). The data show that the staining method affects the dimensions of the bull sperm heads. The smallest sperm head dimensions were observed in the case of the Papanicolaou staining. The heads of sperm cells stained with Papanicolaou stain were 0.50–0.72 ␮m shorter and 0.19–0.43 ␮m narrower than in the case of sperm stained by other techniques (P ≤ 0.05). These dimensions also resulted in a smaller circumference and surface area for the heads of these sperm cells. The largest sperm head dimensions by far were noted in the case of silver nitrate staining. The morphometric dimensions of the sperm cells stained with eosin + gentian violet complex, recommended for bull semen, were closest to those of the cells stained with SpermBlue® , and in most cases the standard deviation was lower than in the sperm

Table 2 Morphometric variables of the bull sperm head measured manually with Multiscan software. Morphometric parameter

Papanicolaou

SpermBlue®

Eosin + gentian

Silver nitrate

Number of sperm cells Head length (␮m) Head width (␮m) Head perimeter (␮m) Head area (␮m2 )

1.000 9.78a ± 0.51 5.03a ± 0.27 30.48a ± 1.64 37.25a ± 3.04

1.000 10.08b ± 0.45 5.22b ± 0.24 31.53b ± 1.51 39.51b ± 2.47

1.000 10.17c ± 0.43 5.31c ± 0.26 31.83c ± 1.45 40.61c ± 2.31

1.000 10.50d ± 0.30 5.46d ± 0.23 32.50d ± 1.82 41.98d ± 2.76

Different superscripts designate significant differences between means within rows; upper-case letters: P ≤ 0.05.

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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Table 3 Sperm morphology according to Tygerberg’s strict criteria and the frequency of nuclear vacuoles in the bull sperm. Morphometric parameter

Papanicolaou

SpermBlue®

Eosin + gentian

Silver nitrate

Number of sperm cells Elipticity Elongation Roughness Regularity Nuclear vacuoles (%)

1.000 1.95a ± 0.13 0.32a ± 0.03 0.50a ± 0.04 1.04a ± 0.03 0.04a ± 0.23

1.000 1.94a,c ± 0.12 0.32a ± 0.03 0.50a ± 0.04 1.05b ± 0.03 0.11b ± 0.45

1.000 1.92b ± 0.13 0.31b ± 0.03 0.51b ± 0.04 1.05b ± 0.03 0.06a ± 0.28

1.000 1.93b,c ± 0.09 0.32a ± 0.02 0.50a ± 0.03 1.07c ± 0.09 0.00 ± 0.00*

Different superscripts designate significant differences between means within rows; upper-case letters: P ≤ 0.05. * Vacuoles not visible in silver nitrate staining.

Fig. 2. Boar sperm head: SpermBlue® staining.

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stained using other methods. Statistically significant differences at P ≤ 0.05 were found between all of the dimensions of sperm stained using the four techniques. Table 3 presents parameters taking into account standard sperm head measurements (length, width, circumference and surface area) in the form of Tygerberg’s strict criteria, which enable more precise analysis of the shape of the sperm head. These data show that the sperm cells stained with Papanicolaou stain had the highest ellipticity index in comparison with the cells stained by the other techniques. Ellipticity indicates the degree to which the sperm heads are oval, narrow or conical. Elongation

Fig. 3. Boar sperm head: eosin + gentian complex staining.

indicates rounding of the sperm head. In this case the sperm stained with eosin + gentian violet complex stood apart, as this index was 0.01 lower than in the sperm stained using other methods (P ≤ 0.05). A similar tendency was observed in the case of roughness, which indicates an amorphous shape. In this case as well the sperm cells stained with eosin + gentian violet complex stood out, as the roughness indicator was 0.01 greater than in the sperm stained using other methods (P ≤ 0.05). Regularity indicates whether the sperm head has a symmetrical shape, and the degree to which it is pyriform. Greater regularity values mean that the sperm heads are more symmetrical. In the present study the most symmetrical sperm heads were observed in the semen stained with silver nitrate, as the index was highest in this case (1.07) and differed significantly from the other staining techniques (P ≤ 0.05). As an additional parameter in the morphological evaluation of the sperm heads the percentage of sperm heads with nuclear vacuoles was determined, and was found to range from 0.04% to 0.11%. Vacuoles were best identified by SpermBlue® , as they were found in the highest numbers where this stain was used. Staining of semen with silver nitrate does not identify vacuoles in the sperm head, hence the zero percentage of vacuoles in Table 3. It does, however, clearly identify the acrosome, which in the case of the other techniques is very faintly visible or not at all. Correlations between the morphometric traits of the sperm cells according to each staining technique are presented in Tables 4–7. The data show significant positive correlations mainly between the length and circumference

Fig. 4. Boar sperm head: silver nitrate staining.

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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D. Banaszewska et al. / Animal Reproduction Science xxx (2015) xxx–xxx Table 4 Phenotypic correlation (Pearson) coefficients between the morphometric traits of the spermatozoa of the bull staining with Papanicolaou. Morphometric traits

Head length

Head width

Head perimeter

Head area

Elipticity Elongation Roughness Regularity

0.62* 0.62* −0.22* −0.12*

−0.65* −0.66* 0.24* 0.31*

0.31* 0.31* −0.64* −0.06

0.08* 0.07* 0.07* −0.22*

*

P ≤ 0.05.

Table 5 Phenotypic correlation (Pearson) coefficients between the morphometric traits of the spermatozoa of the bull staining with SpermBlue® . Morphometric traits

Head length

Head width

Head perimeter

Head area

Elipticity Elongation Roughness Regularity

0.66* 0.66* −0.30 0.12*

−0.68* −0.69* 0.25* 0.39*

0.35* 0.34* −0.76* 0.02

0.05 0.05 −0.01 −0.03

*

P ≤ 0.05.

Table 6 Phenotypic correlation (Pearson) coefficients between the morphometric traits of the spermatozoa of the bull staining with Eosin + gentian. Morphometric traits

Head length

Head width

Head perimeter

Head area

Elipticity Elongation Roughness Regularity

0.69* 0.70* −0.35* 0.14*

−0.78* −0.78* 0.35* 0.32*

0.34* 0.35* −0.78* 0.10*

−0.11* −0.10* 0.13* −0.08*

*

P ≤ 0.05.

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of the sperm head and its ellipticity and elongation. They indicate that as the length of the sperm head increases, the heads are more oval and rounded. Moreover, a negative correlation was shown in the case of the length of the sperm head and roughness, which means that the longer the sperm head, the less regular and more amorphous it is.

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4. Discussion

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Microscopic examination of the sperm showed that the morphology of sperm is markedly heterogeneous. Within one ejaculate there are sperm with different shapes, mainly of the head, and of different sizes and forms. This has led researchers to attempt to identify and define normal sperm traits (Gago et al., 1998; Gage, 1998). An additional element of the morphological evaluation of sperm heads is the presence of vacuoles, which are regarded as a defect in sperm structure (Vanderzwalmen Table 7 Phenotypic correlation (Pearson) coefficients between the morphometric traits of the spermatozoa of the bull staining with silver nitrate. Morphometric traits

Head length

Head width

Head perimeter

Head area

Elipticity Elongation Roughness Regularity

0.50* 0.51* −0.10* 0.29*

−0.81* −0.80* 0.11* 0.59*

0.17* 0.17* −0.88* −0.73*

0.15* 0.16* −0.59* −0.79*

*

P ≤ 0.05.

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et al., 2008). Vacuoles in the sperm head may be associated with DNA fragmentation (Oliveira et al., 2010; De Almeida Ferreira Braga et al., 2011) or abnormal chromatin condensation (Boitrelle et al., 2011; Perdrix et al., 2011; Franco et al., 2012). However, the origin and exact causes of the formation of vacuoles are still the subject of much research (Oliveira et al., 2010; Perdrix et al., 2011; Boitrelle et al., 2011; Franco et al., 2012; Gatimel et al., 2013). Vacuoles are believed to appear during spermatogenesis, and may already be present in the spermatids (Johnson and Truitt-Gibert, 1982). It is also thought that the presence of vacuoles in human sperm heads can have a negative effect on fertilization, and in consequence on the quality of embryos (Berkovitz et al., 2006; Leandri et al., 2013). Although the presence of vacuoles in the sperm head is linked to abnormal semen morphology, in a study by Park et al. (2014) in human semen with better morphology the number of vacuoles in the sperm heads was greater than in semen with a higher percentage of abnormal sperm cells. This suggests that the presence of vacuoles in the sperm head may be a normal characteristic of its morphological structure (Park et al., 2014). For identification of vacuoles a modification of Papanicolaou staining is recommended (Park et al., 2014), which enables observation of pale blue spots in the acrosomal region of the sperm head and dark blue ones in the post-acrosomal region (WHO, 2010). The results of the present study show that the highest percentage of nuclear vacuoles was observed in the case of staining with SpermBlue® . Thus it can be surmised that in the case of bull semen this is the most objective staining method for identifying these sperm structures. The present study was carried out in order to analyze sperm cells morphologically and morphometrically following staining by different methods. Silver nitrate is a stain with basic pH used to identify acidic chromatin proteins. However, for routine evaluation of sperm morphology in Poland, conducted at animal breeding and insemination centres, the most commonly used stain is the acidic eosin + gentian violet complex, which is recommended for bull semen (Blom, 1981; Kondracki et al., 2012). The use of stains with basic and acidic pH enabled more precise characterization of individual elements of sperm structure. Silver nitrate in comparison with routine staining more accurately differentiates the sperm head and the tail, with a clearly visible midpiece. Most proteins of the sperm head have basic pH and after application of silver nitrate the acrosome is stained less intensively than the post-acrosomal region. These differences are shown in photographs included with the description of the results of the present study (Figs. 1–4). Different staining techniques use different chemical reagents. In many cases fixation of semen on the microscope slide alone can change the structure of the sperm cell. Alcohol is commonly used and in various concentrations may cause dehydration and thus shrinkage of the sperm head, which may result in smaller dimensions of the sperm head in the case of Papanicolaou staining. Initial incubation of specimens in saline solution may act as a hypotonic solution and cause swelling of the head, midpiece and tail. The results obtained in the present study confirm this hypothesis.

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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Sperm stained with silver nitrate had longer, wider heads with greater circumference and head area, while the smallest dimensions of sperm heads were noted in the case of the Papanicolaou staining. Comparison of the indices characterizing the sperm head in the case of Papanicolaou, SpermBlue® and silver nitrate staining with the results of staining with eosin + gentian violet complex shows that the sperm heads stained by this method have a less oval and rounded shape, as indicated by the lower ellipticity and elongation values. The sperm heads stained with eosin and gentian violet also had a fairly high regularity value. According to the literature data, the morphometry of the sperm cell may also be influenced by osmotic pressure, staining duration, freezing, and thawing. The changes may affect not only the dimensions of the sperm cell, which may lead to an erroneous false evaluation, but also chromatin structure, which translates directly into reduced fertility or even infertility (Aziz et al., 1998; Andraszek et al., 2014a). Preservation of semen in low temperatures has been shown to decrease sperm head dimensions in bull semen (RubioGuillén et al., 2007). Osmotic pressure in human semen has been determined to range from 330 to 370 mOsm/kg (Rossato et al., 2002). The osmotic water permeability coefficient in the membranes of human sperm is very high, which indicates the presence of numerous pores in the cytoplasmic membrane. In a hypoosmotic environment water penetrates into the sperm cell in order to achieve osmotic balance. This influx of water to the inside of the cell causes the dimensions of the head to increase and a bulge may appear on the cell membrane, disturbing the surface-area-to-volume ratio. The reverse takes place when the sperm are placed in hyperosmotic conditions. Water is then lost and the sperm head shrinks (Abraham-Peskir et al., 2002; Esteso et al., 2006; Maree et al., 2010). In the case of human sperm, research has been carried out to compare two staining techniques commonly used for morphological evaluation: Rapidiff® (RD) and Papanicolaou (PAP) (Maree et al., 2010). Osmotic pressure was calculated for the components of RD and all three components were found to be hyperosmotic in relation to the semen. The differences between the osmotic pressure in the solution and the sperm head can lead to the detection of a substantial number of swollen heads during RD staining. PAP staining is a multi-stage procedure involving the use of ethanol and xylene, which act hyperosmotically and cause the sperm head to shrink (Maree et al., 2010). No negative impact on the dimensions of human sperm was observed in the case of staining with SpermBlue® (SB), probably due to substances having an isosmotic effect on the semen. Unlike RD and PAP, SB staining also had a positive impact on the effectiveness of staining of the acrosome. In general SB revealed morphometric values that were most similar to the results obtained on fresh, unstained semen (McAlister, 2010). Thurston et al. (1999) conclude that the shape of the sperm head is genetically determined. Studies by some authors indicate that the shape of the sperm head depends on certain factors which may arise during spermatogenesis. It has been determined that morphologically varied gametes may appear as early as during the spermatogenesis process when a genetic factor significantly influences

˜ the structure and size of the cell (Thurston et al., 1999; Pena et al., 2005). Scientists explain that an abnormally shaped sperm head linked to poor chromatin condensation may result in the presence in the semen of sperm cells with an elongated, narrow head. Sperm cells with such a shape may result in functional disturbances in the form of immature chromatin and fragmented DNA, causing a potential disadvantage for embryo development (Gandini et al., 2000; Prisant et al., 2007; Auger, 2010). Abnormal sperm chromatin structure is correlated with reduced fertility in the individual (Kazerooni et al., 2009). Research carried out on bull semen confirms this correlation (Ostermeier et al., 2001a). Differences in the dimensions of the sperm head may also be determined by the structure and arrangement of microfibres present in the sperm head. The cytoskeleton of the sperm head consists of proteins of the nucleus and the nuclear membrane, which are partly responsible for shaping of the nucleus. Depending on how the specimens are fixed and on the stain applied, changes may occur in the arrangement of actin fibres in the sperm head (Dvorakova et al., 2005). The relationship between sperm morphometry and fertility is studied mainly in research on human fertility. The results of experiments on human sperm correspond to data obtained in veterinary medicine. Few studies discuss this type of relationship in horses (Casey et al., 1997; Henkel et al., 2008; Hidalgo et al., 2008; Phetudomsinsuk et al., 2008), in which fertility disorders are positively correlated with enlarged sperm heads (Gravance et al., 1996). The subject has also been taken up in regard to fertility in boars ˜ et al., 2005; Saravia et al., 2007; Banaszewska et al., (Pena 2011), bulls (Boersma et al., 1999; Kondracki et al., 2012) ˙ nski ´ ˜ and dogs (Niza and Klimowicz, 2005; Núnez-Martínez ˙ nski ´ et al., 2007; Niza et al., 2009). Animals with smaller sperm head dimensions have been found to be more fertile. Ostermeier et al. (2001a) confirm that bulls with high fertility rates have more elongated sperm than bulls with lower fertilization capacity. Ostermeier et al. (2001b) suggest that Fourier harmonic amplitudes may indicate subtle changes in the shape of the heads of normal sperm. This suggests that even small differences in the shape of the head are significant in diagnosing fertility and may be an indicator of abnormal sperm chromatin structure. The shape of the sperm head is not an unambiguous parameter. Bulls whose semen contained a high percentage of sperm with seemingly abnormal, conical heads were found to be more fertile than bulls whose sperm was assessed as normal (Barth et al., 1992). Research has shown that not only the size of the sperm affects fertilization capacity, but also the size of the tail and midpiece (Ciftci and Zülkadir, 2010). Sperm cells with longer tails have greater fertilization capacity due to their greater motility (Gomendio and Roldan, 2008). Sperm cells with longer heads and shorter midpieces have also been found to swim faster than sperm cells with longer midpieces and shorter heads (Malom et al., 2006). Furthermore, many studies have shown the existence of relationships between the head and the midpiece (Gage, 1998; Piasecka and Kawiak, 2003; Malom et al., 2006). A negative correlation between midpiece length and head length has been reported (Humphries et al., 2008).

Please cite this article in press as: Banaszewska, D., et al., The effect of selected staining techniques on bull sperm morphometry. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.06.019

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Sperm morphometry is also influenced by the age of the individual (Kondracki et al., 2005). This has been confirmed in research on boars. Boar age has been shown to be associated with sperm dimensions and shape. As boars grew older, sperm length and width and head area increased. However, young boars under 11 months old had slightly longer spermatozoa than older boars this was true of both the length of the entire spermatozoon and that of the flagellum. Indicators of the morphological structure of sperm show that the sperm cells of boars aged about 16 months are characterized by elongated heads. This assertion is based on the proportion between the width and length of the head which was smaller than in the sperm of boars over the age of 17 months (Banaszewska et al., 2011). These observations suggest that it is crucial to determine the natural size of the sperm head for each staining technique so that accurate assessment and classification can be made in the diagnosis of male fertility. It is also essential to select an appropriate staining technique for a given animal species, as research by many authors indicates that some methods that work well for one species are not suitable for analysis of another species.

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