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Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa
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Theriogenology 69 (2008) 1033–1038 www.theriojournal.com
Technical note
Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa V.M. Tuset a,b,*, G.J. Dietrich b, M. Wojtczak b, M. Słowin´ska b, J. de Monserrat c, A. Ciereszko b a
Departamento de Biologı´a Pesquera, Instituto Canario de Ciencias Marinas, P.O. Box 56, E-35200 Telde (Las Palmas), Canary Islands, Spain b Department of Semen Biology, Institute of Animal Reproduction and Food Research of Polish Academy of Science, ul. Tuwina 10, 10-747 Olsztyn, Poland c Unidad de Ana´lisis por Imagen, Laboratorio de ana´lisis Dr. Echevarne, Barcelona, Spain Received 27 April 2007; received in revised form 20 December 2007; accepted 12 January 2008
Abstract This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93 0.13 mm; head width 2.33 0.15 mm and tail length 34.16 1.66 mm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast. # 2008 Published by Elsevier Inc. Keywords: Staining Techniques; Morphometry; Spermatozoa; Rainbow trout; ISAS1
1. Introduction The rainbow trout Oncorhynchus mykiss (Walbaum, 1792) is the most commonly farmed fish species. Numerous studies have focussed on several of its reproduction features, particularly sperm biochemistry and physiology, as well as the short- and long-term storage of rainbow trout semen [1–6]. The results of * Corresponding author at: Departamento de Biologı´a Pesquera, Instituto Canario de Ciencias Marinas, P.O. Box 56, E-35200 Telde (Las Palmas), Canary Islands, Spain. Tel.: +34 928 132 900; fax: +34 928 132 908. E-mail address: [email protected] (V.M. Tuset). 0093-691X/$ – see front matter # 2008 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2008.01.012
these investigations have improved production efficiency through broodstock selection or milt cryopreservation based on identifying the highest quality sperm in terms of their motility, speed and fertilizing capacity. Despite sperm quality in fishes being also determined by their morphology, data on fish sperm morphology are scarce due to methodological limitations. Sperm morphological variables are usually established by staining sperm samples, and examining the slides under a microscope with the 100 non-phase contrast lens and correctly adjusting field brightness followed by analysis of captured images [7]. Contrast techniques, especially when automatic detection systems are employed, have to be optimized before
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V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
designing an efficient protocol (depending, for example, on the species, whether the semen is fresh or cryopreserved and whether an extender is used [8]). When examining mammalian sperm, this type of preliminary analysis has proved essential [8–10]. However, few studies have tried to assess the efficiency of the staining techniques available for fish sperm. The purpose of this study was to establish the optimal staining technique for morphometric analysis of rainbow trout spermatozoa using an automated computerized integrated semen analysis system (ISAS1). 2. Material and methods 2.1. Sperm collection Milt was obtained from five 3-year-old rainbow trout (spring spawning) reared at the Department of Salmonid Research, Rutki, Poland. Milt was collected by striping the fish anesthetized with propiscin (1 ppm, IRS, Z˙abieniec, Poland). Special care was taken to avoid contamination of milt with urine. Samples were stored in an insulated biocontainer (TMVLN EPS, Termovial, Kern Frio, S.A., Barcelona, Spain) until analysis. The study protocol was approved by the Animal Experiments Committee in Olsztyn, Poland. 2.2. Staining techniques Semen was diluted 1:100 in 3% citrate sodium. The diluted sperm was deposited in an Eppendorf tube and centrifuged for 15 s at 300 g. For each of the five individual milt samples, nine smears were prepared by placing a 5 ml aliquot onto a slide and pulling out into a smear using a second slide followed by air drying for 20–30 s. The staining kits used were: Diff-Quick1 (DQ) (Medion Diagnostics GmbH, Du¨dingen, Germany), Hemacolor1 (HC) (Merck KGaA, Darmstadt, Germany) and Spermac1 (Stain Enterprises Inc., Wellington, RSA). Each kit was used to stain three smears of the nine prepared from each milt sample. The following modifications were made to the procedures recommended by the manufacturers: for Hemacolor, the fixing time was 10 min and staining time was 5 min; for Diff-Quick fixing and staining times were 5 min. In all cases, smears were washed in distilled water to eliminate excess stain, air dried, covered with a coverslip and permanently sealed with Eukitt mounting medium (Kindler & Co., Freiburg, Germany).
2.3. Head morphology In each smear, 100 spermatozoa were randomly captured and subjected to automated sperm morphology analysis (ASMA) using the sperm morphometry module of the ISAS1 (Proiser R+D SL, Bun˜ol, Spain). Slides were viewed under an Olympus BX50 microscope equipped with a 100 bright field objective and images were captured by a digital video camera (Basler A310, Vision Technologies, Basler AG, Germany). The sperm head measurements calculated automatically by ISAS1 included the size variables: length (L, in mm), width (W, in mm), area (A, in mm2), and perimeter (P, in mm); and shape variables: ellipticity (L/W), rugosity (4pA/P2), elongation ((L W)/(L + W)) and regularity (pLW/4A). The best staining technique was determined in terms of the percentage of cells correctly analyzed, variability of parameters and correlations among stains for each variable [8]. 2.4. Tail morphology Using the 100 lens, flagellum length was measured as the distance from its insertion point to the end of the main section (in salmonids the flagellum is comprised of two sections: a main, longer and visible segment, and an end piece, which is narrower, shorter and less visible by light microscopy [11]). Thirty tails were measured per milt sample (10 per smear) [12] and staining technique. Images were analyzed using the Image-Pro Plus version 4.1.0 package (Media Cybernetics L.P., Carlsbad, USA). 2.5. Statistical analysis To determine if the measurements made were influenced by the handling procedures, coefficients of variability (%) were calculated for the sperm head size variables (length, width, area and perimeter) among smears corresponding to each individual milt sample and staining technique. For multiple comparisons among staining techniques, normality distributions and variance homogeneity were checked by the Kolmogorov–Smirnov and Levene tests, respectively. For data showing a normal distribution, one-way ANOVA was performed, followed by a Tukey post hoc test. For non-normally distributed variables, Kruskal–Wallis analysis was performed and the Mann–Whitney test applied for pairwise comparisons with Bonferroni correction (P < 0.017). Finally, a subsample consisting of measurements made on 450 randomly selected sperm heads for each staining
V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
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technique was used to calculate Pearson’s correlation coefficients. 3. Results Sperm heads stained with Diff-Quick were more susceptible to morphometric analysis in that measurements could be made in 98.8% of the sperm heads compared to 90.7% for Hemacolor and 76.1% for Spermac. Spermac was unable to stain all cells with a similar intensity, which hindered morphometric analysis. In contrast, sperm heads stained with Diff-Quick and Hemacolor showed a good intensity of staining and good contrast (Fig. 1). Coefficients of variability revealed that sperm size did not vary among the five smears for each staining technique, indicating the handling procedure did not affect the final results. Similar coefficients were obtained for Hemacolor and Diff-Quick, while Spermac showed greater variability (Table 1). Using a nonparametric test, all the morphometric variables were observed to vary significantly according to the staining method with the exception of the factors sperm head length and regularity provided by Diff-Quick and Spermac. The Hemacolor technique gave rise to the highest head size variable values, while using this stain the shape of the head appeared slightly less elliptical and elongated (Table 2). Our Pearson’s correlation analysis revealed that head parameters determined after staining with Hemacolor were less correlated to values provided by the other techniques, while the data obtained using Diff-Quick and Spermac were strongly correlated (Table 3). In many cases, it was impossible to distinguish between the mid-piece and flagellum. Using Hemacolor and Spermac, contrast for examining the tail was improved over the contrast obtained using Diff-Quick (Fig. 1). However, tail length measurements failed to differ among the three staining techniques (ANOVA, F 2, 447 = 2.957, P > 0.05) (Table 2). Thus, tail lengths ranged from 30.04 to 39.26 mm, with a coefficient of variability of 24.5%.
Fig. 1. Sperm morphology of rainbow trout using the three staining techniques, Hemacolor, Diff-Quick and Spermac.
4. Discussion Our study demonstrates that fish sperm can be stained and morphometrically analyzed using an automated sperm morphology system. To select an appropriate staining procedure, preliminary tests need to consider the effects of different extenders, and drying, fixation and staining times. Moreover the results of staining, such as appropriate grey-level contrast for
accurate morphometric analysis [13–15], need to be assessed. Current knowledge on staining techniques for fish sperm is scarce. Thus, Howell and Butts [16] stained fish spermatozoa using a silver stain with limited success. Wirtz and Steinmann [17] described the use of Diff-Quick on perch sperm, although they only measured sperm tail length under phase contrast.
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Table 1 Coefficient of variation (CV, %) for morphometric measurements of spermatozoa head by staining technique Size parameters
Individual
Hemacolor
Diff-Quick
Length (mm)
1 2 3 4 5
3.050 3.025 3.199 3.562 3.399
3.462 3.525 3.627 3.997 3.455
4.989 5.736 5.775 4.656 3.882
3.245
3.615
5.023
1 2 3 4 5
3.936 5.234 5.141 5.936 4.816
4.287 6.002 5.456 5.000 5.328
5.799 8.511 8.718 7.910 8.134
5.013
5.221
7.795
1 2 3 4 5
4.574 5.501 5.669 6.820 5.986
4.646 6.967 6.025 6.205 5.975
7.174 11.266 11.269 9.773 9.191
5.701
5.987
9.697
2.282 2.705 2.876 3.254 2.914
2.386 3.342 2.978 3.048 2.942
3.753 5.429 5.487 4.810 4.554
2.805
2.944
4.801
Total Width (mm)
Total Area (mm2)
Total Perimeter (mm)
1 2 3 4 5
Total
Spermac
We speculate that the limited success of the procedures used to stain fish spermatozoa is attributable to numerous artefacts arising during the preparation of smears. In this study, we observed that by reducing the
air drying and fixation times recommended by the manufacturers of the staining kits, the morphology of rainbow trout spermatozoa could be assessed. We believe that these steps are critical for the successful staining of fish spermatozoa. Optimizing the air drying and fixation steps for each fish species should be a primary focus of future studies. Another way of performing a morphometric analysis of fish sperm is to fix the spermatozoa in glutaraldehyde and examine the sperm using the contrast phase lens under low magnification (20 or 40) [11,12,18–21]. Thus, phase contrast acts as a ‘stain’, enabling morphometric measurements. The main technical drawbacks of this method are floating cells and the appearance of a phase contrast ring around the sperm head [7]. Marco-Jime´nez et al. [15] recently obtained good results using negative phase contrast at 100 to examine European eel spermatozoa. Accordingly, phase contrast and staining techniques can be used for morphometric studies on teleost fish spermatozoa. However, for lower groups of fish such as sharks, lampreys or sturgeons, the staining method is recommended since these spermatozoa have an acrosome [22–23]. Using Hemacolor and Diff-Quick, the sperm head was evenly colored and its shape was also uniform. The head was accurately delineated such that the measurements made using the ISAS1 showed good precision and reproducibility. Notwithstanding, the Spermac staining kit provided the best contrast for examining the tail and it was even possible to measure the length of the end piece. Although similar head shape variables were provided by all the stains, low correlation was observed among stains. This was due to the effects of
Table 2 Shape and size variables of spermatozoa head and tail lengths by staining technique Variables
Head n Length (mm) Width (mm) Area (mm2) Perimeter (mm2) Ellipticity Rugosity Elongation Regularity Tail n Length (mm)
Staining technique Hemacolor
Diff-Quick
Spermac
1360 3.22 0.13a 2.67 0.17a 7.25 0.59a 9.99 0.39a 1.21 0.07a 0.91 0.01a 0.09 0.03a 0.93 0.03a
1482 2.93 0.13b 2.33 0.15b 5.82 0.49b 8.95 0.37b 1.26 0.07b 0.91 0.01b 0.11 0.03b 0.92 0.03b
1141 2.95 0.20b 2.50 0.24c 6.30 0.82c 9.37 0.64c 1.16 0.09c 0.89 0.03c 0.08 0.04c 0.92 0.04b
150 34.57 1.84a
150 34.16 1.66a
150 34.16 1.54a
Superscript letters (a, b and c) indicate significant differences (P < 0.05).
V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
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Table 3 Correlation values between shape and size morphometric data of different stain techniques from 450 cells randomly selected from each pool of data Shape parameters
Staining technique Hemacolor/Diff-Quick
Length (mm) Width (mm) Area (mm2) Perimeter (mm2) Ellipticity Rugosity Elongation Regularity Significant differences *P < 0.05;
0.045 0.008 0.079 0.075 0.054 0.153** 0.049 0.024 **
Hemacolor/Spermac 0.034 0.002 0.007 0.035 0.055 0.177** 0.045 0.056
Diff-Quick/Spermac 0.478** 0.095* 0.224** 0.210** 0.029 0.078 0.026 0.181**
P < 0.01.
the fixative and dyes during the staining protocol. Hence, Hemacolor produced clear morphologic changes including rounding of the sperm head, which led to low correlation with the measurements obtained using the other two stains. Spermac provided similar means to Diff-Quick but variability was high. In effect, only the head size variables and not the shape parameters were significantly correlated with those recorded using Diff-Quick. In conclusion, our findings indicate that Diff-Quick staining is suitable for the morphometric analysis of rainbow trout sperm. The sperm shapes obtained using this staining kit are similar to those previously described for scanning and transmission electron micrographs [24]. The method exhibits good reproducibility and requires a short drying time (20 s). Notwithstanding, because of the improved tail contrast obtained using Spermac, we recommend this technique for morphometric analysis of the rainbow trout sperm tail. Acknowledgments We thank Halina Karol and Wieslaw Demianowicz for their excellent technical assistance. Also Mrs. Ana Burton for proof reading and the reviewers for their suggestions. This project was funded by grant number PR2006-0354 from the Education and Science Ministry of the Spanish Government. References [1] Lahnsteiner F, Patzner RA, Weismann T. Physiological and biochemical determination of Rainbow Trout, Oncorhynchus mykiss, semen quality for cryopreservation. J Appl Aquat 1996;6:47–73. [2] Lahnsteiner F, Berger B, Weismann T, Patzner RA. Determination of semen quality of rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoa metabolism. Aquaculture 1998;163:163–81.
[3] Glogowski J, Kwasnik M, Piros B, Dabrowski K, Goryczko K, Dobosz S, Kuzminski H, Ciereszko A. Characterization of rainbow trout milt collected with a catheter: semen parameters and cryopreservation success. Aqua Res 2000;31(3):289. ´ lvarez R, Herra´ez MP. Cryopreservation [4] Cabrita E, Robles V, A of rainbow trout sperm in large volume straws: application to large scale fertilization. Aquaculture 2001;201(3–4): 301–14. [5] Gage MJG, Macfarlane CP, Yeates S, Ward RG, Searle JB, Parker GA. Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. Curr Biol 2004;14:44–7. [6] Dietrich GJ, Kowalski R, Wojtczak M, Dobosz S, Goryczko K, Ciereszko A. Motility parameters of rainbow trout (Oncorhynchus mykiss) spermatozoa in relation to sequential collection of milt, time of post-mortem storage and anesthesia. Fish Phys Biochem 2005;31(1):1–9. [7] NAFA and ESHRE-SIGA. Manual on basic semen analysis; 2002. p. 1–34. [8] Soler C, Gadea B, Soler AJ, Fernandez-Santos MR, Esteso MC, Nun˜ez J, Moreira PN, Nun˜ez M, Gutierrez R, Sancho M, Garde JJ. Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS1. Theriogenology 2005;64(5):1236–43. [9] Sancho M, Perez-Sanchez P, Tablado L, de Monserrat JJ, Soler C. Computer-assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques. Theriogenology 1998;50:27–37. [10] Iguer-Ouada M, Verstegen JP. Validation of the sperm quality analyzer (SQA) for dog sperm analysis. Theriogenology 2001;55:1143–58. [11] Vladic´ TV, Afzelius BA, Bronnikov GE. Sperm quality as reflected through morphology in salmon alternative life histories. Biol Reprod 2002;66:98–105. [12] Burness G, Schulte-Hostedde AI, Casselman SJ, Moyes CD, Montgomerie R. Sperm swimming speed and energetics vary with sperm competition risk in bluegill (Lepomis macrochirus). Behav Ecol Sociobiol 2004;56:65–70. [13] Gravance CG, Liu IK, Davis RO, Hughes JP, Casey PJ. Quantification of normal head morphometry of stallion spermatozoa. J Reprod Fertil 1996;108:41–6. [14] Verstegen J, Iguer-Ouada M, Onclin K. Computer-assisted semen analysers in andrology research and veterinary practice. Theriogenology 2002;57:149–79.
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[15] Marco-Jime´nez F, Pe´rez L, Viudes de Castro MP, Garzo´n DL, Pen˜aranda DS, Vicente JS, Jover M, Asturiano JF. Morphometry characterisation of European eel spermatozoa with computerassisted spermatozoa analysis and scanning electron microscopy. Theriogenology 2006;65:1302–10. [16] Howell WM, Butts D. Silver staining of spermatozoa from living and preserved museum fishes: a new taxonomic approach. Copeia 1983;4:974–8. [17] Wirtz S, Steinmann P. Sperm characteristics in perch Perca fluviatilis L.. J Fish Biol 2006;68:1896–902. [18] Gage MJG, Stockley P, Parker GA. Sperm morphometry in the Atlantic salmon. J Fish Biol 1998;53:835–40. [19] Gage MJG, MacFarlane C, Yeates S, Shackelton R, Parker GA. Relationship between sperm morphometry and sperm motility in the Atlantic salmon. J Fish Biol 2002;61:1528–39.
[20] Balshine S, Leach BJ, Neat F, Werner NY, Montgomerie R. Sperm size of African cichlids in relation to sperm competition. Behav Ecol 2001;12(6):726–31. [21] Van Look KJW, Kime DE. Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm. J Fish Biol 2003;63:1020–33. [22] Jaana H, Yamamoto TS. The ultrastructure of spermatozoa with a note on the formation of the acrosomal filament in the maprey, Lampetra japonica. Jpn J Ichthyol 1981;28:135–47. [23] DiLauro MN, Kaboord W, Walsh RA. Sperm-cell ultrastructure of North American sturgeons. III. The lake sturgeon (Acipenser fulvescens Rafinesque, 1817). Can J Zool 1998;78(3): 438–47. [24] Jamieson BGM. Fish evolution and systematics: evidence from spermatozoa. Cambridge: University Press; 1991.
Theriogenology 69 (2008) 1033–1038 www.theriojournal.com
Technical note
Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa V.M. Tuset a,b,*, G.J. Dietrich b, M. Wojtczak b, M. Słowin´ska b, J. de Monserrat c, A. Ciereszko b a
Departamento de Biologı´a Pesquera, Instituto Canario de Ciencias Marinas, P.O. Box 56, E-35200 Telde (Las Palmas), Canary Islands, Spain b Department of Semen Biology, Institute of Animal Reproduction and Food Research of Polish Academy of Science, ul. Tuwina 10, 10-747 Olsztyn, Poland c Unidad de Ana´lisis por Imagen, Laboratorio de ana´lisis Dr. Echevarne, Barcelona, Spain Received 27 April 2007; received in revised form 20 December 2007; accepted 12 January 2008
Abstract This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93 0.13 mm; head width 2.33 0.15 mm and tail length 34.16 1.66 mm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast. # 2008 Published by Elsevier Inc. Keywords: Staining Techniques; Morphometry; Spermatozoa; Rainbow trout; ISAS1
1. Introduction The rainbow trout Oncorhynchus mykiss (Walbaum, 1792) is the most commonly farmed fish species. Numerous studies have focussed on several of its reproduction features, particularly sperm biochemistry and physiology, as well as the short- and long-term storage of rainbow trout semen [1–6]. The results of * Corresponding author at: Departamento de Biologı´a Pesquera, Instituto Canario de Ciencias Marinas, P.O. Box 56, E-35200 Telde (Las Palmas), Canary Islands, Spain. Tel.: +34 928 132 900; fax: +34 928 132 908. E-mail address: [email protected] (V.M. Tuset). 0093-691X/$ – see front matter # 2008 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2008.01.012
these investigations have improved production efficiency through broodstock selection or milt cryopreservation based on identifying the highest quality sperm in terms of their motility, speed and fertilizing capacity. Despite sperm quality in fishes being also determined by their morphology, data on fish sperm morphology are scarce due to methodological limitations. Sperm morphological variables are usually established by staining sperm samples, and examining the slides under a microscope with the 100 non-phase contrast lens and correctly adjusting field brightness followed by analysis of captured images [7]. Contrast techniques, especially when automatic detection systems are employed, have to be optimized before
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V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
designing an efficient protocol (depending, for example, on the species, whether the semen is fresh or cryopreserved and whether an extender is used [8]). When examining mammalian sperm, this type of preliminary analysis has proved essential [8–10]. However, few studies have tried to assess the efficiency of the staining techniques available for fish sperm. The purpose of this study was to establish the optimal staining technique for morphometric analysis of rainbow trout spermatozoa using an automated computerized integrated semen analysis system (ISAS1). 2. Material and methods 2.1. Sperm collection Milt was obtained from five 3-year-old rainbow trout (spring spawning) reared at the Department of Salmonid Research, Rutki, Poland. Milt was collected by striping the fish anesthetized with propiscin (1 ppm, IRS, Z˙abieniec, Poland). Special care was taken to avoid contamination of milt with urine. Samples were stored in an insulated biocontainer (TMVLN EPS, Termovial, Kern Frio, S.A., Barcelona, Spain) until analysis. The study protocol was approved by the Animal Experiments Committee in Olsztyn, Poland. 2.2. Staining techniques Semen was diluted 1:100 in 3% citrate sodium. The diluted sperm was deposited in an Eppendorf tube and centrifuged for 15 s at 300 g. For each of the five individual milt samples, nine smears were prepared by placing a 5 ml aliquot onto a slide and pulling out into a smear using a second slide followed by air drying for 20–30 s. The staining kits used were: Diff-Quick1 (DQ) (Medion Diagnostics GmbH, Du¨dingen, Germany), Hemacolor1 (HC) (Merck KGaA, Darmstadt, Germany) and Spermac1 (Stain Enterprises Inc., Wellington, RSA). Each kit was used to stain three smears of the nine prepared from each milt sample. The following modifications were made to the procedures recommended by the manufacturers: for Hemacolor, the fixing time was 10 min and staining time was 5 min; for Diff-Quick fixing and staining times were 5 min. In all cases, smears were washed in distilled water to eliminate excess stain, air dried, covered with a coverslip and permanently sealed with Eukitt mounting medium (Kindler & Co., Freiburg, Germany).
2.3. Head morphology In each smear, 100 spermatozoa were randomly captured and subjected to automated sperm morphology analysis (ASMA) using the sperm morphometry module of the ISAS1 (Proiser R+D SL, Bun˜ol, Spain). Slides were viewed under an Olympus BX50 microscope equipped with a 100 bright field objective and images were captured by a digital video camera (Basler A310, Vision Technologies, Basler AG, Germany). The sperm head measurements calculated automatically by ISAS1 included the size variables: length (L, in mm), width (W, in mm), area (A, in mm2), and perimeter (P, in mm); and shape variables: ellipticity (L/W), rugosity (4pA/P2), elongation ((L W)/(L + W)) and regularity (pLW/4A). The best staining technique was determined in terms of the percentage of cells correctly analyzed, variability of parameters and correlations among stains for each variable [8]. 2.4. Tail morphology Using the 100 lens, flagellum length was measured as the distance from its insertion point to the end of the main section (in salmonids the flagellum is comprised of two sections: a main, longer and visible segment, and an end piece, which is narrower, shorter and less visible by light microscopy [11]). Thirty tails were measured per milt sample (10 per smear) [12] and staining technique. Images were analyzed using the Image-Pro Plus version 4.1.0 package (Media Cybernetics L.P., Carlsbad, USA). 2.5. Statistical analysis To determine if the measurements made were influenced by the handling procedures, coefficients of variability (%) were calculated for the sperm head size variables (length, width, area and perimeter) among smears corresponding to each individual milt sample and staining technique. For multiple comparisons among staining techniques, normality distributions and variance homogeneity were checked by the Kolmogorov–Smirnov and Levene tests, respectively. For data showing a normal distribution, one-way ANOVA was performed, followed by a Tukey post hoc test. For non-normally distributed variables, Kruskal–Wallis analysis was performed and the Mann–Whitney test applied for pairwise comparisons with Bonferroni correction (P < 0.017). Finally, a subsample consisting of measurements made on 450 randomly selected sperm heads for each staining
V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
1035
technique was used to calculate Pearson’s correlation coefficients. 3. Results Sperm heads stained with Diff-Quick were more susceptible to morphometric analysis in that measurements could be made in 98.8% of the sperm heads compared to 90.7% for Hemacolor and 76.1% for Spermac. Spermac was unable to stain all cells with a similar intensity, which hindered morphometric analysis. In contrast, sperm heads stained with Diff-Quick and Hemacolor showed a good intensity of staining and good contrast (Fig. 1). Coefficients of variability revealed that sperm size did not vary among the five smears for each staining technique, indicating the handling procedure did not affect the final results. Similar coefficients were obtained for Hemacolor and Diff-Quick, while Spermac showed greater variability (Table 1). Using a nonparametric test, all the morphometric variables were observed to vary significantly according to the staining method with the exception of the factors sperm head length and regularity provided by Diff-Quick and Spermac. The Hemacolor technique gave rise to the highest head size variable values, while using this stain the shape of the head appeared slightly less elliptical and elongated (Table 2). Our Pearson’s correlation analysis revealed that head parameters determined after staining with Hemacolor were less correlated to values provided by the other techniques, while the data obtained using Diff-Quick and Spermac were strongly correlated (Table 3). In many cases, it was impossible to distinguish between the mid-piece and flagellum. Using Hemacolor and Spermac, contrast for examining the tail was improved over the contrast obtained using Diff-Quick (Fig. 1). However, tail length measurements failed to differ among the three staining techniques (ANOVA, F 2, 447 = 2.957, P > 0.05) (Table 2). Thus, tail lengths ranged from 30.04 to 39.26 mm, with a coefficient of variability of 24.5%.
Fig. 1. Sperm morphology of rainbow trout using the three staining techniques, Hemacolor, Diff-Quick and Spermac.
4. Discussion Our study demonstrates that fish sperm can be stained and morphometrically analyzed using an automated sperm morphology system. To select an appropriate staining procedure, preliminary tests need to consider the effects of different extenders, and drying, fixation and staining times. Moreover the results of staining, such as appropriate grey-level contrast for
accurate morphometric analysis [13–15], need to be assessed. Current knowledge on staining techniques for fish sperm is scarce. Thus, Howell and Butts [16] stained fish spermatozoa using a silver stain with limited success. Wirtz and Steinmann [17] described the use of Diff-Quick on perch sperm, although they only measured sperm tail length under phase contrast.
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V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
Table 1 Coefficient of variation (CV, %) for morphometric measurements of spermatozoa head by staining technique Size parameters
Individual
Hemacolor
Diff-Quick
Length (mm)
1 2 3 4 5
3.050 3.025 3.199 3.562 3.399
3.462 3.525 3.627 3.997 3.455
4.989 5.736 5.775 4.656 3.882
3.245
3.615
5.023
1 2 3 4 5
3.936 5.234 5.141 5.936 4.816
4.287 6.002 5.456 5.000 5.328
5.799 8.511 8.718 7.910 8.134
5.013
5.221
7.795
1 2 3 4 5
4.574 5.501 5.669 6.820 5.986
4.646 6.967 6.025 6.205 5.975
7.174 11.266 11.269 9.773 9.191
5.701
5.987
9.697
2.282 2.705 2.876 3.254 2.914
2.386 3.342 2.978 3.048 2.942
3.753 5.429 5.487 4.810 4.554
2.805
2.944
4.801
Total Width (mm)
Total Area (mm2)
Total Perimeter (mm)
1 2 3 4 5
Total
Spermac
We speculate that the limited success of the procedures used to stain fish spermatozoa is attributable to numerous artefacts arising during the preparation of smears. In this study, we observed that by reducing the
air drying and fixation times recommended by the manufacturers of the staining kits, the morphology of rainbow trout spermatozoa could be assessed. We believe that these steps are critical for the successful staining of fish spermatozoa. Optimizing the air drying and fixation steps for each fish species should be a primary focus of future studies. Another way of performing a morphometric analysis of fish sperm is to fix the spermatozoa in glutaraldehyde and examine the sperm using the contrast phase lens under low magnification (20 or 40) [11,12,18–21]. Thus, phase contrast acts as a ‘stain’, enabling morphometric measurements. The main technical drawbacks of this method are floating cells and the appearance of a phase contrast ring around the sperm head [7]. Marco-Jime´nez et al. [15] recently obtained good results using negative phase contrast at 100 to examine European eel spermatozoa. Accordingly, phase contrast and staining techniques can be used for morphometric studies on teleost fish spermatozoa. However, for lower groups of fish such as sharks, lampreys or sturgeons, the staining method is recommended since these spermatozoa have an acrosome [22–23]. Using Hemacolor and Diff-Quick, the sperm head was evenly colored and its shape was also uniform. The head was accurately delineated such that the measurements made using the ISAS1 showed good precision and reproducibility. Notwithstanding, the Spermac staining kit provided the best contrast for examining the tail and it was even possible to measure the length of the end piece. Although similar head shape variables were provided by all the stains, low correlation was observed among stains. This was due to the effects of
Table 2 Shape and size variables of spermatozoa head and tail lengths by staining technique Variables
Head n Length (mm) Width (mm) Area (mm2) Perimeter (mm2) Ellipticity Rugosity Elongation Regularity Tail n Length (mm)
Staining technique Hemacolor
Diff-Quick
Spermac
1360 3.22 0.13a 2.67 0.17a 7.25 0.59a 9.99 0.39a 1.21 0.07a 0.91 0.01a 0.09 0.03a 0.93 0.03a
1482 2.93 0.13b 2.33 0.15b 5.82 0.49b 8.95 0.37b 1.26 0.07b 0.91 0.01b 0.11 0.03b 0.92 0.03b
1141 2.95 0.20b 2.50 0.24c 6.30 0.82c 9.37 0.64c 1.16 0.09c 0.89 0.03c 0.08 0.04c 0.92 0.04b
150 34.57 1.84a
150 34.16 1.66a
150 34.16 1.54a
Superscript letters (a, b and c) indicate significant differences (P < 0.05).
V.M. Tuset et al. / Theriogenology 69 (2008) 1033–1038
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Table 3 Correlation values between shape and size morphometric data of different stain techniques from 450 cells randomly selected from each pool of data Shape parameters
Staining technique Hemacolor/Diff-Quick
Length (mm) Width (mm) Area (mm2) Perimeter (mm2) Ellipticity Rugosity Elongation Regularity Significant differences *P < 0.05;
0.045 0.008 0.079 0.075 0.054 0.153** 0.049 0.024 **
Hemacolor/Spermac 0.034 0.002 0.007 0.035 0.055 0.177** 0.045 0.056
Diff-Quick/Spermac 0.478** 0.095* 0.224** 0.210** 0.029 0.078 0.026 0.181**
P < 0.01.
the fixative and dyes during the staining protocol. Hence, Hemacolor produced clear morphologic changes including rounding of the sperm head, which led to low correlation with the measurements obtained using the other two stains. Spermac provided similar means to Diff-Quick but variability was high. In effect, only the head size variables and not the shape parameters were significantly correlated with those recorded using Diff-Quick. In conclusion, our findings indicate that Diff-Quick staining is suitable for the morphometric analysis of rainbow trout sperm. The sperm shapes obtained using this staining kit are similar to those previously described for scanning and transmission electron micrographs [24]. The method exhibits good reproducibility and requires a short drying time (20 s). Notwithstanding, because of the improved tail contrast obtained using Spermac, we recommend this technique for morphometric analysis of the rainbow trout sperm tail. Acknowledgments We thank Halina Karol and Wieslaw Demianowicz for their excellent technical assistance. Also Mrs. Ana Burton for proof reading and the reviewers for their suggestions. This project was funded by grant number PR2006-0354 from the Education and Science Ministry of the Spanish Government. References [1] Lahnsteiner F, Patzner RA, Weismann T. Physiological and biochemical determination of Rainbow Trout, Oncorhynchus mykiss, semen quality for cryopreservation. J Appl Aquat 1996;6:47–73. [2] Lahnsteiner F, Berger B, Weismann T, Patzner RA. Determination of semen quality of rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoa metabolism. Aquaculture 1998;163:163–81.
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