Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS®

Theriogenology 64 (2005) 1236–1243 www.journals.elsevierhealth.com/periodicals/the

Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS1 C. Soler a,*, B. Gadea a, A.J. Soler b, M.R. Ferna´ndez-Santos b, M.C. Esteso b, J. Nu´n˜ez a, P.N. Moreira c, M. Nu´n˜ez a, R. Gutie´rrez a, M. Sancho a, J.J. Garde b a

Departament de Biologia Funcional i Antropologı´a Fı´sica, Edifici d’Investigacio´, Universitat de Vale`ncia, C/. Dr. Moliner 50, 46100 Burjassot, Valencia, Spain b Grupo de Biologı´a de la Reproduccio´n, Instituto de Investigacio´n en Recursos Cinege´ticos (IREC), UCLM-CSIC-JCCM, Campus Universitario 02071, Albacete, Spain c Departamento de Reproduccio´n Animal, INIA, Madrid 28040, Spain Received 27 December 2004; received in revised form 11 February 2005; accepted 14 February 2005

Abstract When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2 h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 8C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20 s at 37 8C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris’ Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS1. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS1, and discrimi* Corresponding author. Tel.: +34 9 6354 4387; fax: +34 9 6354 4387. E-mail address: [email protected] (C. Soler). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.02.018

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nates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals. # 2005 Elsevier Inc. All rights reserved. Keywords: Sperm morphology; Morphometry; Stain technique; Red deer; ISAS1

1. Introduction Hunted species present several reproductive problems. One major problem is the difficulty of establishing captive breeding populations of wild animals, and another is the low-genetic stability of these populations owing to the high level of inbreeding in hunting areas [1]. The former increases the difficulty in obtaining sperm from wild animals which could be used to solve the latter (with artificial insemination programs, for example). In order to solve these problems, techniques of epididymal sperm collection from dead hunted animals have been developed and are presently being applied [2]. Cauda epididymal spermatozoa, after a process of sperm maturation in proximal epididymal areas [3], present characteristics similar to those of ejaculated sperm cells [4,5]. The fact that these spermatozoa are fully competent to fertilize makes them useful for many techniques of assisted reproduction in wild and hunted species [2]. At present, since the sperm material of some hunted species is very difficult to obtain and demands quality certification, it is necessary to establish very fine criteria for sperm selection [6]. We believe that the morphometric definition of sperm samples becomes an efficient sperm selection criterion when based on certain parameters of cell size and shape quantified by automatic systems. Such a definition of one sample is, however, a question of technical consistency, efficiency, and repeatability. In previous studies, it has been shown that these definitions vary depending on the species in question [7–10]. Sperm morphometric definitions require techniques of seminal contrast which, especially when automatic detection systems are employed, call for the optimization of experimental protocol for each particular situation prior to the establishment of an efficient work protocol [11–14]. This implies extensive preliminary work. The objective of this study was to define the optimal conditions for the morphometric analysis of epididymal red deer spermatozoa, using the automatic computerized integrated semen analysis system (ISAS1). Here we report a technique of seminal contrast that proved to be useful to morphologically characterize and discriminate between sperm samples of different animals, which is an important step towards the establishment of a correlation between sperm morphology and spermatic quality.

2. Materials and methods 2.1. Epididymal sperm collection, freezing and thawing Epididymal sperm samples were obtained from five mature red deer (Cervus elaphus hispanicus) that were legally culled and hunted in their natural habitat. The hunting of stags

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was performed in accordance with the harvest plan of each game reserve. The harvest plans were made following Spanish Harvest Regulation, Law 2/93 of Castilla-La Mancha, which conforms to European Union Regulations. Game keepers collected the testes and provided the hour of the death of the stags. Scrota containing testes and epididymides were placed in plastic bags and transported to the laboratory at room temperature (approximately 20 8C) within 2 h after males’ death. Samples were processed as soon as they arrived at the laboratory. Spermatozoa were collected from the distal portion of the epididymis according to the method of Soler et al. [2]. Briefly, spermatozoa were recovered from the distal portion of the epididymis by cutting the caudae epididymides with a scalpel, collecting the oozing sperm mass, and placing it in 1 ml of Triladyl1-20% egg yolk medium (Minitu¨b, Tiefenbach, Germany), containing 6% glycerol. The sperm mass was then diluted again at room temperature to a final sperm concentration of 400  106 sperm/ml with the Triladyl1 medium. The diluted sperm was placed in a 15 ml centrifuge tube (Iwaki, Japan) and slowly cooled to 5 8C. In order to do this, the tubes were placed in a beaker with water (75 ml at room temperature) and transferred to a refrigerator at 5 8C. Cooling down to 5 8C took about 1 h, after which extended samples were kept at that temperature for a further 2 h for equilibration. After the equilibration of the diluted sperm samples, the extended sperm was loaded into 0.25 ml plastic straws. The straws were immediately frozen in nitrogen vapors, 4 cm above the surface of the liquid nitrogen, for 10 min and then plunged into liquid nitrogen. Frozen semen was thawed in a water bath (37 8C) for 20 s and the content of the straws poured into a glass tube. The cryoprotective agent was not removed after thawing. The freezing protocol described here is based on that habitually employed by our group for artificial insemination of hind with frozen-thawed semen [2]. Slides of thawed semen were prepared from each sample for morphometric characterization of sperm head. 2.2. Sperm staining Hemacolor (Merck standard kit, Darmstadt, Germany, Cat. No. 11661), Diff-Quik (Baxter DADE AG 3186, Du¨dingen, Switzerland) and Harris’ Hematoxylin (Merck, Darmstadt, Germany, Cat. No 9253) were used to stain two smears from each sperm sample. The first two staining methods were applied following kit recommendations. Hematoxylin staining was performed by a 30 min stain sample immersion and subsequent washing with tap water. All the samples were air dried and permanently mounted on a slide with Eukitt (O. Kindler Gmbh & Co., Freiburg, Germany). 2.3. Sperm morphometric analysis Morphometric analysis of sperm samples was performed using the morphometric module of the ISAS1 (Proiser R + D SL, Bun˜ol, Spain). Slides were examined using an Olympus BH-2 (Tokyo, Japan) microscope equipped with a 100 bright field objective and a 3.3 photo-ocular. The video signal was acquired by a Sony CCD AVC-D7CE video camera (Sony Corporation, Tokyo, Japan) interfaced with a Meteor II (Matrox, Dorval, Canada) frame grabber. The array size was 512  512  8 bits providing digitalized images of 262,144 pixels and 256 grey levels. The resolution of images was 0.083 mm/ pixel in both horizontal and vertical axes.

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Two hundred sperm cells per sample presenting no overlapping with other cells or with background particles were randomly captured using a software function. Several sperm-head parameters of size (length, width, area and perimeter) and shape (elipticity, rugosity, elongation and regularity) were measured (Fig. 1). Inadequately digitalized or badly analyzed cell percentages were recorded to estimate the efficiency of the system. 2.4. Statistic analysis For multiple comparisons between staining methods and animal sperm samples, normality distributions and variance homogeneity were checked by the tests of Kolmogorov–Smirnov and Levene, respectively. For samples that were normally distributed, one-way ANOVA was performed, followed by a Tukey a posteriori test. For non-normally distributed populations, the Kruskal–Wallis one-way ANOVA on ranks was performed. Moreover, considering only 250 randomly selected cells from those correctly analysed after each stain technique, correlation analyses between morphometric

Fig. 1. Morphometric parameters examined in this study. The length (L, along the major axis), width (W, along the shortest axis), area (A) and perimeter (P) of the head are self-evident. Shape parameters are mathematical combinations following the correspondent expressions.

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Fig. 2. Microscopic images of spermatozoa stained with (a) Hemacolor, (b) Harris’ hematoxylin and (c) DiffQuick, bar = 5 mm.

parameters were performed using Pearson’s biponderate statistic. All statistical calculations were performed using the SPSS, Version 11.5 (SPSS Inc., Chicago, IL, USA).

3. Results By microscopic examination, Hemacolor offered the microscopic image with best sperm head/midpiece contrast, while Hematoxylin did not efficiently discriminate sperm midpieces and Diff-Quick presented low-staining intensities (Fig. 2). From the initial number of 1000 spermatozoa (200 cells captured per sample and stain), it was possible to analyse automatically 92% for hemacolor, 91% for Hematoxylin, and 96% for Diff-Quik using ISAS1. Spermatozoa displayed the biggest size when stained with Hemacolor, followed by Hematoxylin and Diff-Quik. Smaller differences between staining techniques were observed in relation to sperm cell shape parameters (Table 1). The parameters with best correlation between data obtained after different staining procedures were those of shape, length and perimeter, while area and width only showed significant correlation between Hemacolor and Diff-Quik (Table 2). The Diff-Quik staining method was the best for differentiating the samples of the animals used here. Significant differences were detected for all of the parameters analyzed

Table 1 Shape and size morphometric values of red deer spermatozoa associated with each staining technique Staining technique

n Length (mm) Width (mm) Area (mm2) Perimeter (mm) Elipticity Rugosity Elongation Regularity

Hemacolor

Hematoxylin

919 8.20  0.44 4.70  0.26 32.57  2.40 22.83  0.90 1.75  0.12 0.78  0.02 0.27  0.03 0.93  0.03

905 8.04  0.45 4.51  0.21 30.67  2.04 22.20  0.83 1.79  0.12 0.78  0.02 0.28  0.03 0.93  0.02

a a a a a a a a

Diff-Quick b b b b b b b a

Values with different letters in the same row are significantly different (P < 0.05).

960 7.67  0.47 4.42  0.25 28.38  2.54 21.11  1.08 1.74  0.10 0.80  0.02 0.27  0.03 0.94  0.03

c c c c a c a b

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Table 2 Correlation values between shape and Size morphometric data of red deer spermatozoa analyzed by ISAS1 after different stain techniques from 250 cells randomly selected from each pool of data

Length (mm) Width (mm) Area (mm2) Perimeter (mm) Elipticity Rugosity Elongation Regularity *

Hemacolor/Diff-Quik

Hemacolor/hematoxylin

0.794* 0.443* 0.641* 0.729* 0.516* 0.405* 0.509* 0.136*

0.395* 0.028 0.119 0.264* 0.294* 0.308* 0.300* 0.192*

Hematoxylin/Diff-Quik 0.278* 0.073 0.068 0.224* 0.195* 0.261* 0.190* 0.087

Significant correlation (P < 0.05).

Table 3 Spermatic morphometric values associated with each red deer male using the Diff-Quik staining technique Male 1 Length (mm) Width (mm) Area (mm2) Perimeter (mm) Elipticity Rugosity Elongation Regularity

7.87  0.41 4.43  0.18 29.07  1.46 21.52  0.62 1.78  0.12 0.79  0.02 0.28  0.03 0.94  0.03

2 a a a a a a a a

7.44  0.28 4.32  0.16 27.16  1.59 20.53  0.64 1.73  0.06 0.81  0.01 0.27  0.02 0.93  0.02

3 b b b b b b, c b b

7.67  0.37 4.65  0.24 29.37  2.19 21.31  0.88 1.65  0.08 0.81  0.02 0.25  0.02 0.95  0.03

4 c c a a c b c c

7.23  0.38 4.18  0.20 25.57  2.08 19.97  0.93 1.73  0.09 0.81  0.02 0.27  0.03 0.93  0.02

5 d d c c b c b b

8.13  0.28 4.51  0.20 30.59  1.76 22.15  0.70 1.81  0.08 0.78  0.02 0.29  0.02 0.94  0.03

e e d d d d d a

Values with different letters in the same row are significantly different (P < 0.05).

among animals, with sperm-head length and width being the most distinguished parameters, and regularity the parameter with the lowest range of diversity (Table 3).

4. Discussion The results obtained in this study highlight the importance of the choice of staining technique for the morphometric characterization of sperm cells, particularly when a computerized system is used [10,14]. In our seminal contrast comparison study, the visual observation of the cells offered the apparent best definition with the Hemacolor technique, since it offered the best sperm head/midpiece/background contrast. Nevertheless, it is important to note that the difference in the percentage of sperm cells that could be analyzed correctly after each staining procedure was due to the ISAS1 analysis algorithm; the specific contrast differences between sperm heads and midpieces and the background generated by each one, and not to sperm sample characteristics. With these considerations, Diff-Quik proved to be the most efficient sperm staining technique tested, indicating that visual appreciations are not trustworthy for the choice of a good technique for automatic image analysis.

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The differences observed between animals highlight the importance of a precise and reliable method of morphometric analysis for the identification of small significant variations in sperm cells, otherwise not detected by ordinary microscopy [9,15]. In general, species can be grouped into those that present different morphologic classes of spermatozoa (human [16], horse [17], alpaca [15]) and those that present an apparent sperm homogeny (ram [9], goat [12], red deer). When differences are observed among individuals, a morphologic definition of what is a ‘‘normal’’ spermatozoon can be established, as has been done in humans [16]. However, in certain domestic species, sperm morphology has been a marginal parameter owing to the insignificant percentage of clear abnormal seminal forms. This work highlights that (at least with red deer epididymal spermatozoa) significant morphometric variations can be observed among animals despite the absence (or lack of detection) of abnormal sperm forms. This observation is of particular importance since it leads to a new morphologic definition of ‘‘normal’’ sperm, which may be the key for the explanation of the differential fertilizing ability and cryopreservation efficiency between sperm samples. Now that the optimal conditions for the epididymal sperm morphometric analysis in red deer have been defined, it will be interesting to analyze, as has already been done with other species (human [18–20]; bovine [21]; horse [17]), whether the morphometric definition of a red deer sperm sample can anticipate its fertilizing ability.

Acknowledgements This study was partially supported by the Ministerio de Ciencia y Tecnologı´a, INIA (RZ01-008). We thank I.V. Costello for linguistic assistance.

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