- Email: [email protected]
The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout
Animal Reproduction Science 55 Ž1999. 279–287
The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout E. Cabrita a , R. Alvarez a , E. Anel b, M.P. Herraez ´
a,)
a
b
Department of Cell Biology, UniÕersity of Leon, ´ 24071 Leon, ´ Spain Department of Animal Pathology, UniÕersity of Leon, ´ 24071 Leon, ´ Spain Accepted 11 February 1999
Abstract The hypoosmotic swelling test ŽHOS. is one of the methods used to evaluate sperm quality in mammals. This test is based on the swelling ability that functional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow trout spermatozoa in such conditions and it is not possible to distinguish between reactive cells Žcells who were capable to increase in volume. and non-reactive cells Ždid not increase in volume. under light microscopy. In our approach we have used the coulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions Ž50, 100, 150, 200, 250 and 320 mosMrkg. and cell volume measured at different times after dilution Ž30 s, 2, 5, 10, 20, and 30 min.. The higher percentage of reactive cells was achieved with the 100 mosMrkg solution and swelling occurred before 30 s. Even with this solution, the small increase in cell size caused the overlapping of volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reactivity, the test was performed in samples containing known rates of liverdead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over a standard volume Žreactive cells.. Results showed a high correlation between the percentages of reactive cells and the known rate of live cells Ž r 2 s 0.65.. This fact suggests that HOS test could be used to analyse the integrity and functionality of rainbow trout fresh sperm. To study the reliability of this test in cryopreserved sperm, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter data. No significant
)
Corresponding author. Tel.: q34-987-291492; fax: q34-987-291487; e-mail: [email protected]
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 1 4 - 7
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E. Cabrita et al.r Animal Reproduction Science 55 (1999) 279–287
correlation was observed in either case, showing that structural and functional integrity do not correlate after freezerthaw. Consistently, the HOS test is not a reliable method to evaluate cryopreserved sperm quality in rainbow trout. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Coulter counter; HOS test; Rainbow trout; Semen assay
1. Introduction Several assays have been developed to evaluate sperm fitness. The standard semen analysis ŽSSA. relies on assessing a number of parameters such as cell concentration, motility and morphology for the assessment of male fertility. It is assumed that these measurements provide enough information about the status of spermatozoa. However, these parameters have limitations and cannot be used as reliable predictors of sperm fertilising capacity ŽMcNiver et al., 1992; Correa and Zavos, 1994.. The study of sperm membrane is of particular importance since it is involved in several processes such as exchanges with the outside or recognition signals and acts as a barrier to maintain the form of the cell. Different methods such as supravital stain, biochemical tests or electron microscopy have been used to assess sperm membrane ŽCorrea and Zavos, 1994., but are sometimes time consuming and provide information about membrane breaking but not about membrane functionality. The cell membrane regulates cell volume in order to reach osmotic equilibrium. It is nevertheless, a highly inelastic structure and therefore cannot increase volume by stretching but only by increasing surface area. However, this swelling capacity will not take place if the cell membrane is ruptured or made highly permeable by damaging stresses ŽArtiga et al., 1991; Hammerstedt et al., 1992.. The viability tests—liverdead stain—measure whether the membrane is intact or not, a sign of cell death. The hypoosmotic swelling test ŽHOS test. evaluates the ability of cells to swell in a hypoosmotic solution, thus indicating whether an intact membrane is also biochemically active or, in other words, determining functional integrity of sperm membranes ŽCorrea and Zavos, 1994; Engel and Petzoldt, 1994.. Biochemically active spermatozoa will increase in volume to establish an equilibrium between the fluid compartment within the spermatozoa and the extracellular environment. Swelling causes changes in cell size and shape, that can be observed using phase contrast microscopy in some mammals Žboar, bull, human, equine and canine spermatozoa.. Different patterns of swelling are due to differences in the sensitivity of plasma membranes to water transport between species ŽCurry and Watson, 1994.. Sperm from teleost fish also show different patterns of swelling after hypoosmotic shock ŽMalejac et al., 1990; Marian ` ` et al., 1992. but sometimes it is difficult to detect under light microscopy. In those cases the use of the coulter counter, an electronic particle counterrsize, can help to determine the changes in sperm volume, allowing a large number of cells to be counted within a short period of time ŽPaulenz et al., 1995.. The aim of this study was to perform the HOS test using a coulter counter and to determine its effectiveness in the evaluation of the functional integrity of trout sperm membrane.
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2. Material and methods 2.1. Milt The milt was obtained by catheterization and the quality of each individual cock was checked using the same methods applied in previous studies Ž12.. Only samples whose quality parameters ranged between the following values were used: pH Ž8.08–8.42., osmolality Ž310–319 mosMrkg., motility Ž80%–100% motile cells., cell concentration Ž48 = 10 9 –114 = 10 9 cellsrml., live cells Žnon-permeable to Hoechst 33285. Ž85– 100%.. All sperm manipulations were carried out at 48C using individual males for every trial. Fertility tests were also made to check semen quality. 2.2. HOS assay technique 2.2.1. Coulter counter A Coulter counter ŽZ1 electronic, Luton, England. equipped with 50 nm aperture tube was used. This apparatus was calibrated according to the user’s manual with latex balls of a known size. All of the analyses were performed by counting 10,000 cellsrsample. 2.2.2. Trial I. Selection of experimental conditions Five solutions of Tris buffered–NaCl, pH 7.4, with different osmolality Ž50, 100, 150, 200, 250 mosMrkg. were used to check their ability to induce sperm swelling. None of these solutions activate sperm motility. An isosmotic solution Ž320 mosMrkg. was the control for normal volume. Semen Ž20 ml. were diluted in 100 ml of every solution and the volume of spermatozoa was assessed at different times Ž30 s, 2, 5, 10, 20 and 30 min. in the coulter counter. The experiment was performed with milt from 4 males and the results from every sample were expressed as mean cell volume. Differences in cell swelling were analysed using a one-way ANOVA Ž p - 0.05.. 2.2.3. Trial II. Analysis of coulter counter data Two parameters obtained from the coulter counter data were analysed: the percentage of reactive cells and the increase in mean cell volume, expressed as a percentage ŽFig. 1a and b.. To define the first parameter, a sample of 100% non-reactive cells was
Fig. 1. Analysis of coulter counter data: Ža. increase of mean volume in the hypoosmotic solution Ž100 mosM. compared to the isosmotic one Ž320 mosM.; Žb. determination of the percent of cells below a standard volume Ž80.13 fl. —reactive cells—.
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Fig. 2. Results obtained from analyses of coulter counter data. Ža. Percentage of reactive cells from fresh sperm samples Ž ns 4. subjected to hypoosmotic solutions Ž50, 100, 150, 200, 250 and 320 mosM.; Žb. percentage of mean cell volume increase subjected to the same conditions Ž ns 4..
obtained by freezing directly in liquid nitrogen and thawing at room temperature. After hypoosmotic dilution cell volumes remained below 80.13 fl in 90% of cells, considering this volume as the threshold for reactive cells ŽFig. 1a.. To study the reliability of the two parameters, nine samples containing known rates of liverdead cells were tested Ž0, 10, 20, 30, 40, 50, 75, 90 and 100% live spermatozoa.. The correlation Žsimple linear regression. between the percentage of live cells and the two parameters analysed from the coulter counter was established using the SPSSq software from Microsoft. 2.2.4. Trial III. Application to sperm samples Before freezing, milt was diluted 1:3 in extender a6 from Erdhal and Graham Ž1980. containing three different cryoprotectants ŽCPTs.: Ž1. Me 2 SO 7%, Ž2. DMA, 7% and Ž3. methanol 5%, Žvrv.. After 30-min equilibration at 48C, 250 ml of diluted semen from all samples was frozen in plastic straws. Freezing was carried out at y208Crmin in a programmable biofreezer until 1008C and then plunged into liquid nitrogen. Thawing was performed in a 258C water bath for 30 s. All experiments were carried out in
Fig. 3. Results obtained from direct analysis of coulter counter data showing volume of sperm cells submitted to different hypoosmotic solutions Ž50, 100, 150, 200, 250.. The 320 mosM solution was used as a control.
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Fig. 4. Results obtained from analyses of coulter counter data. Ža. Percentage of reactive cells and Žb. percentage of mean volume increase from fresh sperm samples Ž ns 4. subjected to a 100 mosM solution over a certain period of time Ž30 s, 2 min, 5 min, 10 min, 20 min, 30 min..
triplicate. The HOS test was performed on frozenrthawed samples Ž n s 9. after a 30 s exposure to a 100 mosMrkg solution. The same method was applied using a 320 mosMrkg solution as a control. Simple linear regressions were used to determine the correlation between the analysis of coulter counter data and the percentage of live cells obtained by Hoechst dye.
3. Results The coulter counter data showed an increase in cell volume after exposure to any hypoosmotic solution. However, no significant differences were observed between the solutions tested ŽFig. 2.. After dilution two populations of cells Žswollen and non-swollen. are not clearly identifiable ŽFig. 3..
Fig. 5. Linear regression between the proportion of liverdead known spermatozoa and Ža. the percentage of reactive cells to the HOS test Ž y s 0.33 x q40.28; r 2 s 0.65. and Žb. the percentage of mean volume increase in a 100 mosM solution Ž y s 0.41 x q1.4; r 2 s 0.48..
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Fig. 6. Linear regression between the percentage of live cells detected by fluorometry stain dye ŽHoechst 33258. and Ža. the percentage of reactive cells to the HOS test cells Ž y s 0.53 x q16.59 r 2 s 0.25. or Žb. the percentage of sperm volume increase in a 100 mosM solution Ž y s 0.28 x q17.68; r 2 s 0.04. in cryopreserved sperm.
Based on these data, a 100 mosMrkg solution was selected for the next experiments, because it promotes cell swelling Ž59% increase in mean cell volume compared to the 320 mosMrkg solution. but does not cause cell lysis in the first 10 min after dilution, as was recorded in other studies ŽCabrita et al., 1997.. No significant differences in volume increase were observed with any of the tested solutions through the time ŽFig. 4a and b.. The analysis of the accuracy of the two parameters obtained from the coulter counter data demonstrate a significant correlation between the known percentage of live spermatozoa and the percentage of reactive cells to the HOS test, with a coefficient of determination Ž r 2 . of 0.65 Ž p - 0.05. ŽFig. 5a.. However a minor correlation was achieved with the increase in cell volume Ž r 2 s 0.48. ŽFig. 5b. The application of the HOS test to frozenrthawed samples showed that sperm frozen with Me 2 SO as a cryoprotectant contains a significantly higher percentage of reactive cells Ž32.4%. than samples frozen with the other CPTs Ž22.04% DMA, 19.04% methanol.. No significant correlation was achieved in cryopreserved sperm when live cells obtained by Hoechst dye were compared either with the percentage of reactive cells Ž r 2 s 0.25. or with the increase in mean cell volume Ž r 2 s 0.04. ŽFig. 6..
4. Discussion The development of simple tests to assess functional activity of spermatozoa should be useful in the diagnosis of sperm quality. At present, standard semen analysis determines sperm parameters such as cell density, motility, cell vitality and morphological characteristics amongst others. Although these are representative of the characteristics of spermatozoa, they give inadequate information about cell viability and fertilisation ability in fishes. Membrane functionality is essential to maintaining motility and
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carrying out all the events related to fertilisation ŽJeyendran et al., 1984.. In this context, one of the most important functions is the regulation of the osmotic equilibrium. Therefore, assessment of this functional aspect is a useful complement to semen analysis. A simple but effective test can be performed to evaluate the physical and functional integrity of the sperm membrane exposed to hypoosmotic conditions: the HOS test. Several authors have reported increases in sperm volume in mammals ŽJeyendran et al., 1984; Engel and Petzoldt, 1994. and in teleost fish ŽBillard, 1978; Malejac et al., 1990; McNiver et al., 1992. when spermatozoa are submitted to hypoosmotic conditions. During the HOS test functional spermatozoa will undergo swelling, and subsequent increase in volume when exposed to hypoosmotic stress, due to the influx of water. In order to identify the hypoosmotic solution that causes maximal swelling of the spermatozoa, the medium should exert an osmotic stress large enough to cause a detectable increase in volume, but small enough to prevent lysis of sperm membrane ŽCorrea and Zavos, 1994.. Results achieved with the saline solutions Ž50, 100, 150, 200, 250 mosMrkg., tested in the present study, showed their ability to induce swelling in rainbow trout spermatozoa. However, no significant differences were observed between the tested solutions. Previous studies ŽCabrita et al., 1997. demonstrate that the 50 mosMrkg medium induce sperm membrane lysis and that 200 and 250 mosMrkg solutions sometimes have an osmolality similar to that of sperm. The results obtained with the coulter counter, together with those of Cabrita et al. Ž1997., showed that the 100 mosMrkg or 150 mosMrkg solution result in a maximal number of clearly identifiable swollen spermatozoa and maintain sperm membrane integrity during the assay in this apparatus. The choice of the 100 mosMrkg solution to perform the test on frozen samples was, therefore, for convenience. The hydraulic conductivity of spermatozoa calculated for several species is very high, leading to the expectation of rapid osmotic equilibration across the membrane within a few seconds ŽCurry and Watson, 1994.. This theory was checked in rainbow trout sperm: no significant differences between data from 30 s to 30 min exposure were observed, suggesting that swelling also occurs in a few seconds in trout sperm. Moreover, the present results suggest that differences in the sensitivity of plasma membrane to water transportrpermeability between species, as well as patterns of swelling, may be important factors that could influence the HOS test. In the present study the data obtained from the coulter counter analysis showed that volume of cells submitted to a 100 mosMrkg solution display a normal distribution which overlaps with the cell volume in isosmotic conditions Ž320 mosMrkg. at some points. In some mammals Ži.e. bull, boar., several authors have demonstrated that a 100 mosMrkg solution causes the appearance of two different populations: the first corresponding to the non-swollen spermatozoa and the second to the cells with biochemically active plasma membrane. These data allow direct identification of reactive and non-reactive spermatozoa. The different results obtained in trout and mammals are explained by the patterns of swelling in those species. Bull spermatozoa, when exposed to hypoosmotic conditions, suffer a spherical expansion of the cell membrane covering the tail, with the flagellum coil inside the membrane causing a very important increase in
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volume. Thus, it is possible to identify swollen spermatozoa using phase contrast microscopy ŽCorrea and Zavos, 1994; Curry and Watson, 1994.. In rainbow trout some authors have identified similar patterns of swelling ŽMalejac et al., 1990., others have noticed just a small expansion in the basal region of the head without any alteration in the structure of the flagellum ŽHammerstedt et al., 1992.. However, in our observations using phase contrast microscopy Žimages not shown. a small increase in volume of the sperm head was detected. In these conditions the coulter counter allows the small volume increase to be detected and a high number of cells to be counted in a short period of time. Nevertheless, the analysis of the obtained data requires the choice of a standard parameter to quantify the population of functional cells. The analysis of two different parameters obtained from the HOS test showed a good correlation between the percentage of live cells and the percentage of reactive cells Ž r 2 s 0.65.. The correlation was less significant when the increase of mean cell volume was analysed Ž r 2 s 0.48.. Spermatozoa could display different shapes after swelling, as stated by Malejac et al. Ž1990., therefore the volume of functional cells in the hypoosmotic medium is more variable than the volume in the isosmotic one. This explains why the analysis of the cell mean volume is not a good indicator for the percentage of swollen cells. These results prove the relationship between the structural integrity and the functionality of the cells in fresh sperm. Yavetz et al. Ž1995. and Esteves et al. Ž1996. obtained similar results with bull and human sperm. However, as in our approach, a lack of correlation was also detected when cryopreserved sperm was analysed Ž r 2 s 0.25 and r 2 s 0.04.. Difference between fresh and thawed sperm could be a result of sublethal damage occurring in the plasma membrane after freezingrthawing ŽCheck et al., 1991; Alvarez and Storey, 1993; Buhr et al., 1994; Cabrita et al., 1997.. The lack of correlation after freezingrthawing reflects the non-functionality of a structurally undamaged population. Freezing and thawing often results in vast structural and functional alterations such as hydration-dependent phase changes and lateral phase separation of membrane components, intramembranous particle aggregation, loss of membrane permeability and fusions. These sublethal cryo-injuries are not detected by the standard procedures of quality analysis of sperm Žlike the Hoechst dye. and make the membrane more prone to early fracture at the stress points ŽAlvarez and Storey, 1993.. As we have seen, a loss in the percentage of HOS reactive cells was detected after cryopreservation with all of the cryoprotectants used. Moreover, we have detected significant differences between CPTs, with Me 2 SO demonstrating better results on sperm membrane protection.
5. Conclusion The results obtained in the present study indicate that fresh rainbow trout spermatozoa react to the hypoosmotic swelling test and that swelling occurs in a few seconds after dilution. The slight detected increase in volume is large enough to identify a population of reactive cells using a coulter counter. The rate of cells over a standard volume Žreactive cells. shows a better correlation than the increase in mean cell volume
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with the rate of structurally intact cells in fresh sperm. However, some cryodamage occurs in cell membrane during cryopreservation and no correlation is observed between structural and functional integrity after freezerthaw.
References Alvarez, J.G., Storey, B.T., 1993. Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamages in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J. Androl. 14, 199–209. Artiga, C.G., Vazquez, I., Nunez, R., Yubero, C.D., 1991. Endosmosis celular en espermatozoides de morueco. Forum Internacional de Reproduccion ´ Animal, 1–5. Billard, R., 1978. Changes in structure and fertilising ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture 14, 187–198. Buhr, M.M., Curtis, E.F., Kakuda, N.S., 1994. Composition and behaviour of head membrane lipids of fresh and cryopreserved boar sperm. Cryobiology 31, 224–238. Cabrita, E., Alvarez, R., Anel, L., Rana, K., Herraez, M.P., 1997. Evidence of sublethal damages during cryopreservation of rainbow trout sperm. Cryobiology 35, 370. Check, M.L., Check, J.H., Long, R., 1991. Detrimental effects of cryopreservation on structural and functional integrity of the sperm membrane. Arch. Androl. 27, 155–160. Correa, J.A., Zavos, P.M., 1994. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine membrane. Theriogenology 42, 351–360. Curry, M.R., Watson, P.F., 1994. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology 31, 39–46. Engel, S., Petzoldt, R., 1994. The spermatozoa volume as indicative of the plasma membrane integrity Žmodification of the hypoosmotic swelling test.: I. Methods. Andrologia 26, 309–313. Erdhal, D.A., Graham, E.F., 1980. Preservation of spermatozoa of brook trout and rainbow trout. Cryo-Letters 1, 203–208. Esteves, S.C., Sharma, R.K., Thomas, A.J., Agarwal, A., 1996. Suitability of the hypoosmotic swelling test for assessing the viability of cryopreserved sperm. Fertil. Steril. 66, 798–804. Hammerstedt, R.H., Crichton, E.G., Watson, P.F., 1992. Comparative approach to sperm cryopreservation: does cell shape and size influence cryosurvival? Theriogenology, Proceeding for annual meeting 8–11. Jeyendran, R.S., Van Der Ven, H.H., Perez-Pelaez, M., Crabo, B.G., Zaneveld, L.J.D., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characterises. J. Reprod. Fertil 70, 219–225. Malejac, M., Loir, M., Maisse, G., 1990. Qualite´ de la membrane des spermatozoides de truite arc-en-ciel Ž Oncorhynchus mykiss .; relation avec l’aptitude du sperm a` la congelation. Aquatic Living Resour. 3 Ž1., ´ 43–53. Marian, ` ` T., Kraznai, Z., Balkay, L., Balazs, ` M., Emri, M., Bene, L., Tron, ´ L., 1992. Hypo-osmotic shock induces an osmolality-dependent permeabilization and structural changes in the membrane of carp sperm. J. Histochemistry and Cytochemistry 41, 291–297. McNiver, M.A., Gallant, R.K., Richardson, G.F., 1992. In vitro methods of assessing the viability of rainbow trout spermatozoa. Theriogenology 38, 679–686. Paulenz, H., Grevle, I.S., Tverdal, A., Hofmo, P.O., Anderson Berg, K., 1995. Precision of the coulter counter for routine assessment of boar-sperm concentration in comparison with haemocymeter and spectrophotometer. Reprod. Dom. Anim. 30, 107–111. Yavetz, H., Hauser, R., Yogev, L., Lessing, J.B., Homonnai, Z.T., Paz, G., 1995. Advanced methods for evaluation of sperm quality. Andrologia 27, 31–35.
The hypoosmotic swelling test performed with coulter counter: a method to assay functional integrity of sperm membrane in rainbow trout E. Cabrita a , R. Alvarez a , E. Anel b, M.P. Herraez ´
a,)
a
b
Department of Cell Biology, UniÕersity of Leon, ´ 24071 Leon, ´ Spain Department of Animal Pathology, UniÕersity of Leon, ´ 24071 Leon, ´ Spain Accepted 11 February 1999
Abstract The hypoosmotic swelling test ŽHOS. is one of the methods used to evaluate sperm quality in mammals. This test is based on the swelling ability that functional spermatozoa have when submitted to hypoosmotic solutions. Only a slight increase in size is caused in rainbow trout spermatozoa in such conditions and it is not possible to distinguish between reactive cells Žcells who were capable to increase in volume. and non-reactive cells Ždid not increase in volume. under light microscopy. In our approach we have used the coulter counter to verify the effectiveness of the HOS test in this species. Semen was diluted in different hypoosmotic solutions Ž50, 100, 150, 200, 250 and 320 mosMrkg. and cell volume measured at different times after dilution Ž30 s, 2, 5, 10, 20, and 30 min.. The higher percentage of reactive cells was achieved with the 100 mosMrkg solution and swelling occurred before 30 s. Even with this solution, the small increase in cell size caused the overlapping of volumes from swollen and non-swollen spermatozoa. In order to analyse the data and to choose a parameter suitable for assessing cell reactivity, the test was performed in samples containing known rates of liverdead cells. Two parameters were analysed after swelling: the increase in volume and the percentage of cells over a standard volume Žreactive cells.. Results showed a high correlation between the percentages of reactive cells and the known rate of live cells Ž r 2 s 0.65.. This fact suggests that HOS test could be used to analyse the integrity and functionality of rainbow trout fresh sperm. To study the reliability of this test in cryopreserved sperm, simple linear regressions were made between cell viability determined by Hoechst 33285 dye and the two parameters obtained from coulter counter data. No significant
)
Corresponding author. Tel.: q34-987-291492; fax: q34-987-291487; e-mail: [email protected]
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 1 4 - 7
280
E. Cabrita et al.r Animal Reproduction Science 55 (1999) 279–287
correlation was observed in either case, showing that structural and functional integrity do not correlate after freezerthaw. Consistently, the HOS test is not a reliable method to evaluate cryopreserved sperm quality in rainbow trout. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Coulter counter; HOS test; Rainbow trout; Semen assay
1. Introduction Several assays have been developed to evaluate sperm fitness. The standard semen analysis ŽSSA. relies on assessing a number of parameters such as cell concentration, motility and morphology for the assessment of male fertility. It is assumed that these measurements provide enough information about the status of spermatozoa. However, these parameters have limitations and cannot be used as reliable predictors of sperm fertilising capacity ŽMcNiver et al., 1992; Correa and Zavos, 1994.. The study of sperm membrane is of particular importance since it is involved in several processes such as exchanges with the outside or recognition signals and acts as a barrier to maintain the form of the cell. Different methods such as supravital stain, biochemical tests or electron microscopy have been used to assess sperm membrane ŽCorrea and Zavos, 1994., but are sometimes time consuming and provide information about membrane breaking but not about membrane functionality. The cell membrane regulates cell volume in order to reach osmotic equilibrium. It is nevertheless, a highly inelastic structure and therefore cannot increase volume by stretching but only by increasing surface area. However, this swelling capacity will not take place if the cell membrane is ruptured or made highly permeable by damaging stresses ŽArtiga et al., 1991; Hammerstedt et al., 1992.. The viability tests—liverdead stain—measure whether the membrane is intact or not, a sign of cell death. The hypoosmotic swelling test ŽHOS test. evaluates the ability of cells to swell in a hypoosmotic solution, thus indicating whether an intact membrane is also biochemically active or, in other words, determining functional integrity of sperm membranes ŽCorrea and Zavos, 1994; Engel and Petzoldt, 1994.. Biochemically active spermatozoa will increase in volume to establish an equilibrium between the fluid compartment within the spermatozoa and the extracellular environment. Swelling causes changes in cell size and shape, that can be observed using phase contrast microscopy in some mammals Žboar, bull, human, equine and canine spermatozoa.. Different patterns of swelling are due to differences in the sensitivity of plasma membranes to water transport between species ŽCurry and Watson, 1994.. Sperm from teleost fish also show different patterns of swelling after hypoosmotic shock ŽMalejac et al., 1990; Marian ` ` et al., 1992. but sometimes it is difficult to detect under light microscopy. In those cases the use of the coulter counter, an electronic particle counterrsize, can help to determine the changes in sperm volume, allowing a large number of cells to be counted within a short period of time ŽPaulenz et al., 1995.. The aim of this study was to perform the HOS test using a coulter counter and to determine its effectiveness in the evaluation of the functional integrity of trout sperm membrane.
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2. Material and methods 2.1. Milt The milt was obtained by catheterization and the quality of each individual cock was checked using the same methods applied in previous studies Ž12.. Only samples whose quality parameters ranged between the following values were used: pH Ž8.08–8.42., osmolality Ž310–319 mosMrkg., motility Ž80%–100% motile cells., cell concentration Ž48 = 10 9 –114 = 10 9 cellsrml., live cells Žnon-permeable to Hoechst 33285. Ž85– 100%.. All sperm manipulations were carried out at 48C using individual males for every trial. Fertility tests were also made to check semen quality. 2.2. HOS assay technique 2.2.1. Coulter counter A Coulter counter ŽZ1 electronic, Luton, England. equipped with 50 nm aperture tube was used. This apparatus was calibrated according to the user’s manual with latex balls of a known size. All of the analyses were performed by counting 10,000 cellsrsample. 2.2.2. Trial I. Selection of experimental conditions Five solutions of Tris buffered–NaCl, pH 7.4, with different osmolality Ž50, 100, 150, 200, 250 mosMrkg. were used to check their ability to induce sperm swelling. None of these solutions activate sperm motility. An isosmotic solution Ž320 mosMrkg. was the control for normal volume. Semen Ž20 ml. were diluted in 100 ml of every solution and the volume of spermatozoa was assessed at different times Ž30 s, 2, 5, 10, 20 and 30 min. in the coulter counter. The experiment was performed with milt from 4 males and the results from every sample were expressed as mean cell volume. Differences in cell swelling were analysed using a one-way ANOVA Ž p - 0.05.. 2.2.3. Trial II. Analysis of coulter counter data Two parameters obtained from the coulter counter data were analysed: the percentage of reactive cells and the increase in mean cell volume, expressed as a percentage ŽFig. 1a and b.. To define the first parameter, a sample of 100% non-reactive cells was
Fig. 1. Analysis of coulter counter data: Ža. increase of mean volume in the hypoosmotic solution Ž100 mosM. compared to the isosmotic one Ž320 mosM.; Žb. determination of the percent of cells below a standard volume Ž80.13 fl. —reactive cells—.
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Fig. 2. Results obtained from analyses of coulter counter data. Ža. Percentage of reactive cells from fresh sperm samples Ž ns 4. subjected to hypoosmotic solutions Ž50, 100, 150, 200, 250 and 320 mosM.; Žb. percentage of mean cell volume increase subjected to the same conditions Ž ns 4..
obtained by freezing directly in liquid nitrogen and thawing at room temperature. After hypoosmotic dilution cell volumes remained below 80.13 fl in 90% of cells, considering this volume as the threshold for reactive cells ŽFig. 1a.. To study the reliability of the two parameters, nine samples containing known rates of liverdead cells were tested Ž0, 10, 20, 30, 40, 50, 75, 90 and 100% live spermatozoa.. The correlation Žsimple linear regression. between the percentage of live cells and the two parameters analysed from the coulter counter was established using the SPSSq software from Microsoft. 2.2.4. Trial III. Application to sperm samples Before freezing, milt was diluted 1:3 in extender a6 from Erdhal and Graham Ž1980. containing three different cryoprotectants ŽCPTs.: Ž1. Me 2 SO 7%, Ž2. DMA, 7% and Ž3. methanol 5%, Žvrv.. After 30-min equilibration at 48C, 250 ml of diluted semen from all samples was frozen in plastic straws. Freezing was carried out at y208Crmin in a programmable biofreezer until 1008C and then plunged into liquid nitrogen. Thawing was performed in a 258C water bath for 30 s. All experiments were carried out in
Fig. 3. Results obtained from direct analysis of coulter counter data showing volume of sperm cells submitted to different hypoosmotic solutions Ž50, 100, 150, 200, 250.. The 320 mosM solution was used as a control.
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Fig. 4. Results obtained from analyses of coulter counter data. Ža. Percentage of reactive cells and Žb. percentage of mean volume increase from fresh sperm samples Ž ns 4. subjected to a 100 mosM solution over a certain period of time Ž30 s, 2 min, 5 min, 10 min, 20 min, 30 min..
triplicate. The HOS test was performed on frozenrthawed samples Ž n s 9. after a 30 s exposure to a 100 mosMrkg solution. The same method was applied using a 320 mosMrkg solution as a control. Simple linear regressions were used to determine the correlation between the analysis of coulter counter data and the percentage of live cells obtained by Hoechst dye.
3. Results The coulter counter data showed an increase in cell volume after exposure to any hypoosmotic solution. However, no significant differences were observed between the solutions tested ŽFig. 2.. After dilution two populations of cells Žswollen and non-swollen. are not clearly identifiable ŽFig. 3..
Fig. 5. Linear regression between the proportion of liverdead known spermatozoa and Ža. the percentage of reactive cells to the HOS test Ž y s 0.33 x q40.28; r 2 s 0.65. and Žb. the percentage of mean volume increase in a 100 mosM solution Ž y s 0.41 x q1.4; r 2 s 0.48..
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Fig. 6. Linear regression between the percentage of live cells detected by fluorometry stain dye ŽHoechst 33258. and Ža. the percentage of reactive cells to the HOS test cells Ž y s 0.53 x q16.59 r 2 s 0.25. or Žb. the percentage of sperm volume increase in a 100 mosM solution Ž y s 0.28 x q17.68; r 2 s 0.04. in cryopreserved sperm.
Based on these data, a 100 mosMrkg solution was selected for the next experiments, because it promotes cell swelling Ž59% increase in mean cell volume compared to the 320 mosMrkg solution. but does not cause cell lysis in the first 10 min after dilution, as was recorded in other studies ŽCabrita et al., 1997.. No significant differences in volume increase were observed with any of the tested solutions through the time ŽFig. 4a and b.. The analysis of the accuracy of the two parameters obtained from the coulter counter data demonstrate a significant correlation between the known percentage of live spermatozoa and the percentage of reactive cells to the HOS test, with a coefficient of determination Ž r 2 . of 0.65 Ž p - 0.05. ŽFig. 5a.. However a minor correlation was achieved with the increase in cell volume Ž r 2 s 0.48. ŽFig. 5b. The application of the HOS test to frozenrthawed samples showed that sperm frozen with Me 2 SO as a cryoprotectant contains a significantly higher percentage of reactive cells Ž32.4%. than samples frozen with the other CPTs Ž22.04% DMA, 19.04% methanol.. No significant correlation was achieved in cryopreserved sperm when live cells obtained by Hoechst dye were compared either with the percentage of reactive cells Ž r 2 s 0.25. or with the increase in mean cell volume Ž r 2 s 0.04. ŽFig. 6..
4. Discussion The development of simple tests to assess functional activity of spermatozoa should be useful in the diagnosis of sperm quality. At present, standard semen analysis determines sperm parameters such as cell density, motility, cell vitality and morphological characteristics amongst others. Although these are representative of the characteristics of spermatozoa, they give inadequate information about cell viability and fertilisation ability in fishes. Membrane functionality is essential to maintaining motility and
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carrying out all the events related to fertilisation ŽJeyendran et al., 1984.. In this context, one of the most important functions is the regulation of the osmotic equilibrium. Therefore, assessment of this functional aspect is a useful complement to semen analysis. A simple but effective test can be performed to evaluate the physical and functional integrity of the sperm membrane exposed to hypoosmotic conditions: the HOS test. Several authors have reported increases in sperm volume in mammals ŽJeyendran et al., 1984; Engel and Petzoldt, 1994. and in teleost fish ŽBillard, 1978; Malejac et al., 1990; McNiver et al., 1992. when spermatozoa are submitted to hypoosmotic conditions. During the HOS test functional spermatozoa will undergo swelling, and subsequent increase in volume when exposed to hypoosmotic stress, due to the influx of water. In order to identify the hypoosmotic solution that causes maximal swelling of the spermatozoa, the medium should exert an osmotic stress large enough to cause a detectable increase in volume, but small enough to prevent lysis of sperm membrane ŽCorrea and Zavos, 1994.. Results achieved with the saline solutions Ž50, 100, 150, 200, 250 mosMrkg., tested in the present study, showed their ability to induce swelling in rainbow trout spermatozoa. However, no significant differences were observed between the tested solutions. Previous studies ŽCabrita et al., 1997. demonstrate that the 50 mosMrkg medium induce sperm membrane lysis and that 200 and 250 mosMrkg solutions sometimes have an osmolality similar to that of sperm. The results obtained with the coulter counter, together with those of Cabrita et al. Ž1997., showed that the 100 mosMrkg or 150 mosMrkg solution result in a maximal number of clearly identifiable swollen spermatozoa and maintain sperm membrane integrity during the assay in this apparatus. The choice of the 100 mosMrkg solution to perform the test on frozen samples was, therefore, for convenience. The hydraulic conductivity of spermatozoa calculated for several species is very high, leading to the expectation of rapid osmotic equilibration across the membrane within a few seconds ŽCurry and Watson, 1994.. This theory was checked in rainbow trout sperm: no significant differences between data from 30 s to 30 min exposure were observed, suggesting that swelling also occurs in a few seconds in trout sperm. Moreover, the present results suggest that differences in the sensitivity of plasma membrane to water transportrpermeability between species, as well as patterns of swelling, may be important factors that could influence the HOS test. In the present study the data obtained from the coulter counter analysis showed that volume of cells submitted to a 100 mosMrkg solution display a normal distribution which overlaps with the cell volume in isosmotic conditions Ž320 mosMrkg. at some points. In some mammals Ži.e. bull, boar., several authors have demonstrated that a 100 mosMrkg solution causes the appearance of two different populations: the first corresponding to the non-swollen spermatozoa and the second to the cells with biochemically active plasma membrane. These data allow direct identification of reactive and non-reactive spermatozoa. The different results obtained in trout and mammals are explained by the patterns of swelling in those species. Bull spermatozoa, when exposed to hypoosmotic conditions, suffer a spherical expansion of the cell membrane covering the tail, with the flagellum coil inside the membrane causing a very important increase in
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volume. Thus, it is possible to identify swollen spermatozoa using phase contrast microscopy ŽCorrea and Zavos, 1994; Curry and Watson, 1994.. In rainbow trout some authors have identified similar patterns of swelling ŽMalejac et al., 1990., others have noticed just a small expansion in the basal region of the head without any alteration in the structure of the flagellum ŽHammerstedt et al., 1992.. However, in our observations using phase contrast microscopy Žimages not shown. a small increase in volume of the sperm head was detected. In these conditions the coulter counter allows the small volume increase to be detected and a high number of cells to be counted in a short period of time. Nevertheless, the analysis of the obtained data requires the choice of a standard parameter to quantify the population of functional cells. The analysis of two different parameters obtained from the HOS test showed a good correlation between the percentage of live cells and the percentage of reactive cells Ž r 2 s 0.65.. The correlation was less significant when the increase of mean cell volume was analysed Ž r 2 s 0.48.. Spermatozoa could display different shapes after swelling, as stated by Malejac et al. Ž1990., therefore the volume of functional cells in the hypoosmotic medium is more variable than the volume in the isosmotic one. This explains why the analysis of the cell mean volume is not a good indicator for the percentage of swollen cells. These results prove the relationship between the structural integrity and the functionality of the cells in fresh sperm. Yavetz et al. Ž1995. and Esteves et al. Ž1996. obtained similar results with bull and human sperm. However, as in our approach, a lack of correlation was also detected when cryopreserved sperm was analysed Ž r 2 s 0.25 and r 2 s 0.04.. Difference between fresh and thawed sperm could be a result of sublethal damage occurring in the plasma membrane after freezingrthawing ŽCheck et al., 1991; Alvarez and Storey, 1993; Buhr et al., 1994; Cabrita et al., 1997.. The lack of correlation after freezingrthawing reflects the non-functionality of a structurally undamaged population. Freezing and thawing often results in vast structural and functional alterations such as hydration-dependent phase changes and lateral phase separation of membrane components, intramembranous particle aggregation, loss of membrane permeability and fusions. These sublethal cryo-injuries are not detected by the standard procedures of quality analysis of sperm Žlike the Hoechst dye. and make the membrane more prone to early fracture at the stress points ŽAlvarez and Storey, 1993.. As we have seen, a loss in the percentage of HOS reactive cells was detected after cryopreservation with all of the cryoprotectants used. Moreover, we have detected significant differences between CPTs, with Me 2 SO demonstrating better results on sperm membrane protection.
5. Conclusion The results obtained in the present study indicate that fresh rainbow trout spermatozoa react to the hypoosmotic swelling test and that swelling occurs in a few seconds after dilution. The slight detected increase in volume is large enough to identify a population of reactive cells using a coulter counter. The rate of cells over a standard volume Žreactive cells. shows a better correlation than the increase in mean cell volume
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with the rate of structurally intact cells in fresh sperm. However, some cryodamage occurs in cell membrane during cryopreservation and no correlation is observed between structural and functional integrity after freezerthaw.
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