DOI: 10.1111/jpn.12199
ORIGINAL ARTICLE
Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation M. S. Aramli Young Researchers and Elites Club, Ardabil Branch, Islamic Azad University, Ardabil, Iran
Summary Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing-thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 0.026 g/l, and the TP after cryopreservation was 1.86 0.6 g/l. The activities of acid phosphatase (0.82 0.042 U/l), lactate dehydrogenase (234.4 19.4 U/l), arylsulfatase (143.1 32.5 U/l) and b-N-acetylglucosaminidase (58.39 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen-thawed semen samples (7.43 0.64, 3224.6 167.2, 422.6 21.3 and 90.2 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing-thawing. Keywords enzyme activities, protein concentration, fresh semen, Frozen-thaw cycle, Huso huso Correspondence Mohammad Sadegh Aramli, Young Researchers and Elites Club, Ardabil Branch, Islamic Azad University, Ardabil 31567-56157, Iran. Tel: +98 914 754 1626; E-mails: [email protected]; [email protected] Received: 22 March 2014; accepted: 31 March 2014
Introduction The sturgeon is among the world’s most valuable wildlife resources. Most of the world’s sturgeon populations have experienced significant declines in recent decades, primarily because of over-fishing, habitat destruction and pollution (Pikitch et al., 2005). The beluga (Huso huso) is one of 27 chondrostean fish species that are included in the family, Acipenseridae. The beluga appeared about 200 million years BP (Bemis et al., 1997; Ludwig, 2008) and inhabits the Northern Hemisphere. Belugas have a high growth rate, making them highly suitable for aquaculture (Aramli et al., 2013a). Previous studies of biochemical indicators of semen quality have generally focused on the activities of enzymes in the plasma of fresh semen or the supernatants of semen samples following long- or short-term cryostorage (Ciereszko, 2008). The assessment of the biochemical characteristics of semen, particularly in terms of enzyme characteristics, can provide important information regarding spermatozoa motility and viability. Studies of the techniques for assessing semen quality might facilitate improvements in artificial reproduction and germplasm resource conservation procedures (Li et al., 2008).
The enzymology of fish gametes is not well understood, compared with the wealth of available knowledge regarding that of mammals. The activity levels of aspartate aminotransferase (AST) (Ciereszko and Dabrowski, 1994), lactate dehydrogenase (LDH) (McNiven et al., 1992), acid phosphatase (AcP) (Schemehl et al., 1987), b-N-acetylglucosaminidase (bNAGase) and arylsulfatase (AS) (Piros et al., 2002; Aramli et al., 2013b, 2014a) in the semen of certain fish species have been previously described. However, to our knowledge, no data are available on the activity of these enzymes in beluga semen. The objective of our study was to measure the enzymatic activity of LDH, AcP, bNAGase, AS and the total protein concentration (TP) in beluga semen before and after cryopreservation. Materials and methods Broodstock handling and collection of semen
Ten male belugas (30–45 kg, 1–2 m) were collected from the south-western region of the Caspian Sea and transported to the Rajaee Sturgeon Hatchery Center in Sari, Mazandaran, Iran (latitude 36°370 N, longitude 53°050 E), during March and April in 2012. The fish were maintained in tanks with a water temperature of 15.5°C 0.5°C, an oxygen content of >5 mg/l and a
Journal of Animal Physiology and Animal Nutrition 99 (2015) 13–16 © 2014 Blackwell Verlag GmbH
13
Enzyme activities and protein content of Huso huso semen
pH of 7.6–7.9. Fish were feed using a commercial diet (Biomars, no. 1.9, Nersac, France; 50% crude protein, 18% crude fat, 10% ash, 1.3% fibre) during the experimental period. To induce spermiation, the fish were given an intramuscular injection of 5–10 lg/kg of luteinizing hormone-releasing hormone agonist between the dorsal and lateral scutes, as previously described (Nazari et al., 2010; Aramli et al., 2014b). Spermiation occurred at 14–18 h post-injection. Semen was collected from the urogenital opening using rigid tubing and a syringe. Special care was taken to avoid contamination with urine, mucus, faeces or water. Three males did not mature during the experiment. Semen samples were collected from seven of the male fish collected.
M. S. Aramli
0.5 mM p-nitrophenyl-b-N-glucosoaminide as a substrate in 0.1 M citrate buffer, pH 5.0. After a 60-min incubation at 37°C, the reaction was stopped by the addition of 0.1 M NaOH, and the absorbance at 410 nm was measured (Jauhiainen and Vanha-Perttula, 1986). The level of TP was measured using a folin-phenol reagent (Lowry et al., 1951) at 37°C, and the absorbance was measured at 546 nm. Data analysis
The data were analysed using a one-way analysis of variance and the Duncan multiple-range test. A value p < 0.05 was considered to indicate a statistically significant difference between data sets. All data are expressed as the mean standard error.
Sample preparation
The semen samples were stored in glass tubes on ice for up to 3 h during transportation to the laboratory. Seminal plasma was collected immediately by centrifugation at 2500 g for 10 min in a Labnet Spectrafuge 16 M (Woodbridge, NJ, USA). An aliquot of each semen sample was transferred to a 1.5-ml microcentrifuge tube and frozen at 79°C for 1 day to evaluate enzyme release from the spermatozoa damaged during freezing and thawing. The semen samples were thawed at room temperature and centrifuged. The supernatant was collected by centrifugation and used for the enzyme analysis.
Results and Discussion
The TP and the activities of the various enzymes in the seminal plasma samples and the supernatant of the frozen-thawed samples were assessed. The activity of AcP was determined using a solution containing 5 mM p-nitrophenyl phosphate and 20 mM citrate, pH 5 (Glogowski et al., 1996). After incubation at 37°C for 30 min, the reaction was stopped by the addition of 0.1 M NaOH, and the absorbance at 410 nm was measured (Glogowski et al., 1996). The LDH activity was measured in a solution containing 1.6 mM sodium pyruvate, 0.2 mM NADH, 200 mM NaCl and 80 mM Tris-HCl, pH 7.3, and the absorbance at 339 nm was measured following a 5-min incubation at 30°C (Vassault, 1983). The activity of AS was determined using 0.2 ml of 20 mM p-nitrocatechol sulphate as a substrate in 0.5 M sodium acetate buffer, pH 4.9. After a 30-min incubation at 37°C, the reaction was stopped by the addition of 2.5 ml 1 M NaOH, and the absorbance at 515 nm was measured (Yang and Srivastava, 1974). The activity of bNAGase was measured using
Table 1 lists the TP and enzyme activities detected in the beluga semen. Almost all of the parameters analysed were statistically higher in the supernatants of the frozen-thawed samples than in the seminal plasma of fresh semen. Seminal plasma proteins play a key role in prolonging sperm viability in teleost fish (Lahnsteiner et al., 2004). Thus, a better understanding of the enzyme activities in seminal plasma and spermatozoa is necessary to identify indicators of freeze-thaw damage to sturgeon semen. Currently, little is known about the proteins present in the seminal plasma of sturgeons. However, certain proteins in the seminal plasma may protect the spermatozoa. Previous studies have reported the TP in the seminal plasma of various sturgeon species (Table 1). Most sturgeons, including beluga, have a much lower TP in the seminal plasma than that typically observed in higher vertebrates. The AS and bNAGase are key enzymes found in the mammalian acrosome, which play a critical role in the penetration of the oocyte (Nikolajczyk and O’Rand, 1992; Brandon et al., 1997). In our current study, these two enzymes were present in the beluga semen. Their presence was expected because of the active acrosome in sturgeon semen. Previous studies of various sturgeon species have shown that the activities of AS and bNAGase in fresh seminal plasma were lower than those of the supernatants of frozen-thawed semen (Table 1; Piros et al., 2002; Sarosiek et al., 2004; Aramli et al., 2014a). The level of AcP activity was much lower in the seminal plasma of fresh semen than that in the supernatant of frozen-thawed semen (Table 1). Sarosiek et al. (2006) reported that two isoforms of AcP (39 and 19 kDa) were present in the semen of Russian
14
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Analytical procedures
Enzyme activities and protein content of Huso huso semen
M. S. Aramli
Table 1 Comparison of enzyme activities and protein content and of fresh and frozen-thawed semen from different sturgeon species Species
Semen type
TP (g/dl)
AS (U/l)
bNAGase (U/l)
AcP (U/l)
LDH (U/l)
Authors
Sterlet
Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh* Frozen-thawed*
0.5 1.33 0.24 1.6 0.58 4.33 0.57 4.40 0.3
207.0 760.2 54.1 937.1 684.3 2720.5 638.5 2993.9
53.68 79.09 23.65 44.59 16.39 61.59 20.49 67.28
1.85 7.89 0.9 5.82 1 20.6 1.21 23.81
761.7 8803.0 71 9493.0 278.4 4420.0 400.9 7217.3
Piros et al. (2002)†
Siberian sturgeon
Stellate Russian sturgeon Persian sturgeon Beluga
Piros et al. (2002)‡ Piros et al. (2002)§ Piros et al. (2002)¶ Li et al. (2011)
0.6 0.28 1.68 0.47 1.86
Li et al. (2011)
0.031 0.1 0.026 0.6
169.1 699.6 143.1 422.6
44.4 25.1 32.5 21.3
36.31 2.25 70.7 2.24 58.39 4.14 90.2 5.37
0.59 9.89 0.82 7.43
0.035 0.81 0.042 0.64
484.4 6262.7 234.4 3224.6
27.3 263.2 19.4 167.2
Aramli et al. (2014a) Present study
TP, total protein; AS, arylsulfatase; bNAGase, b-N-acetylglucosaminidase; AcP, acid phosphatase; LDH, lactate dehydrogenase. †Fish captured from the Danube River. ‡Fish captured from the Dolna Odra Power Plant. §1st semen collection. ¶2nd semen collection. *Significant different between seminal plasma of fresh and freeze–thawed semen (p < 0.05; n = 7).
sturgeon. The role of AcP in sturgeon reproduction is unknown. In mammals, AcP functions in phosphorylation and dephosphorylation reactions during sperm capacitation, hyperactivation, acrosome formation and sperm–egg fusion (Urner and Sakkas, 2003). The formation of lactate allows glycolysis to proceed in the absence of oxygen. LDH functions in energy production by converting lactate to pyruvate, the end product of glycolysis pathway (Miki, 2007). The level of LDH activity was higher in the supernatant of frozen-thawed semen samples than that in seminal plasma of fresh semen samples. Previous studies have shown that LDH activity was also higher in the seminal plasma of semen of the Siberian sturgeon, sterlet and Persian sturgeon following cryopreservation (Table 1; Piros et al., 2002; Aramli et al., 2014a). Piros et al. (2002) suggested that the increased activity of LDH likely resulted from the disruption of the energy supply during cryopreservation, which subsequently affects sperm motility. According to the data presented in Table 1, the activities of all of the enzymes evaluated were significantly higher in the supernatant of the frozen-thawed References Aramli, M. S.; Nazari, R. M.; Kalbassi, M. R.; Aramli, S., 2013a: Semen of beluga, Huso huso: ionic content and osmolality of seminal plasma and their physiologi-
semen samples. Therefore, the activities of these enzymes may represent potential biomarkers of cryoinjury, which is consistent with previous studies of teleost fish (Glogowski et al., 1996; Lahnsteiner et al., 1996; Babiak et al., 1997). The activities of LDH and AcP may be indicators of damage to the plasmalemma and the midpiece area of the sperm structure, whereas the activities of AS and bNAGase may be useful indices of damage to the acrosome. However, future studies are warranted to explore the roles of these enzymes in the viability of beluga spermatozoa. In conclusion, our results contribute to the available data regarding the enzyme properties of beluga semen. These data are critical to the development of optimal diluents for the long- and short-term storage of sperm collected from this endangered species. Acknowledgements The authors wish to thank the technical staff of Rajaee Sturgeon Hatchery Center in Sari, Iran, for their help and support during this study.
cal correlation with sperm motility indices. Journal of Fisheries and Aquaculture 4, 2. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2013b: Spermatozoa and seminal plasma enzyme activity in Persian stur-
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
geon, Acipenser persicus, in relation to short-term storage. Aquaculture Research. doi:10.1111/are.12349. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2014a: Protein concentration and enzyme activities of fresh and
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Enzyme activities and protein content of Huso huso semen
frozen-thawed Persian sturgeon, Acipenser persicus (Borodin, 1897) semen. International Aquatic Research 6, 54. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2014b: Sex steroid levels of Persian sturgeon, Acipenser persicus, Borodin, 1897, males in negative and positive responding to LH-RH-analogue. Journal of Applied Ichthyology 30, 18–19. Babiak, I.; Glogowski, J.; Luczynski, M. J.; Luczynski, M., 1997: Effect of individual male variability on cryopreservation of northern pike, Esox lucius L., sperm. Aquaculture Research 28, 191–197. Bemis, E.; Findeis, E.; Grande, L., 1997: An overview of Acipenseriformes. In V. J. Birstein, J. R. Waldman, W. Bemis, E. Kluwer (eds) Sturgeon Biodiversity and Conservation. Academic Publishing, Dordrecht, pp. 25–71. Brandon, C. I.; Srivastava, P. N.; Heusner, G. L.; Fayer-Hosken, R. A., 1997: Extraction and quantification of acrosin, b Nacetylglucosaminidase, and arylsulfatase-A from equine ejaculated spermatozoa. Journal of Experimental Zoology 279, 301–308. Ciereszko, A., 2008: Chemical composition of seminal plasma and its physiological relationship with sperm motility, fertilizing capacity and cryopreservation success in fish. In: S. M. H. Alavi, J. Cosson, K. Coward, G. R. Rafiee (eds), Fish Spermatology. Alpha Science Ltd, Oxford, pp. 215–240. Ciereszko, A.; Dabrowski, K., 1994: Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage. Fish Physiology and Biochemistry 12, 357–367. Glogowski, J.; Babiak, I.; Goryczko, K.; Dobosz, S., 1996: Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout semen. Reproduction, Fertility and Development 8, 1179–1184. Jauhiainen, A.; Vanha-Perttula, T., 1986: b -N-acetylglucosaminidase in the reproductive organs and seminal plasma
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of the bull. Journal of Reproduction and Fertility 76, 239–250. Lahnsteiner, F.; Berger, B.; Weismann, T.; Patzner, R., 1996: The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation. Journal of Applied Ichthyology 12, 99–106. Lahnsteiner, F.; Mansour, N.; Berger, B., 2004: Seminal Plasma Prolong the Viability of Rainbow trout (Oncorhynchus mykiss) Spermatozoa. Theriogenology 62, 801–808. Li, P.; Hulak, M.; Linhart, O., 2008: Sperm proteins in teleostean and chondrostean (sturgeon) fishes. Fish Physiology and Biochemistry 35, 567–581. Li, P.; Rodina, M.; Hulak, M.; Li, Z. H.; Linhart, O., 2011: Spermatozoa concentration, seminal plasma composition and their physiological relationship in the endangered stellate sturgeon (Acipenser stellatus) and Russian sturgeon (Acipenser gueldenstaedtii). Reproduction in Domestic Animals 46, 247–252. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Raudall, R. J., 1951: Protein measurement with folin-phenol reagent. Journal of Biological Chemistry 193, 265–275. Ludwig, A., 2008: Identification of Acipenseriformes species in trade. Journal of Applied Ichthyology 24, 2–19. McNiven, M. A.; Gallant, R. K.; Richardson, G. F., 1992: In vitro methods of assessing the viability of rainbow trout spermatozoa. Theriogenology 38, 679–686. Miki, K., 2007: Energy metabolism and sperm function. Society of Reproduction and Fertility supplement 65, 309–325. Nazari, R. M.; Modanloo, M.; Ghomi, M. R.; Ovissipor, M. R., 2010: Application of synthetic hormone LHRH-A2 on the artificial propagation of Persian sturgeon Acipenser persicus. Aquaculture International 18, 837–841. Nikolajczyk, B. S.; O’Rand, M. G., 1992: Characterization of rabbit testis b-galactosidase and arylsulfatase purification
and localization in spermatozoa during the acrosome reaction. Biology of Reproduction 46, 366–378. Pikitch, E. K.; Doukais, P.; Lauck, L.; Chakrabarty, P.; Erickson, D. L. 2005: Status trends and management of sturgeon and paddlefish fisheries. Fish Fisher 6, 233–265. Piros, B.; Glogowski, J.; Kolman, R.; Rzemieniecki, A.; Domagala, J.; Horvath, A.; Urbanyi, B.; Ciereszko, A., 2002: Biochemical characterization of Siberian sturgeon, Acipenser baeri and sterlet, Acipenser ruthenus, seminal plasma and spermatozoa. Fish Physiology and Biochemistry 26, 289–295. Sarosiek, B.; Ciereszko, A.; Kolman, R.; Glogowski, J., 2004: Characteristics of arysulfatase in Russian sturgeon (Acipenser gueldenstaedtii) semen. Comparative Biochemistry and Physiology 139B, 517–579. Sarosiek, B.; Wysocka, J.; Wysocki, P.; Kowalski, R.; Glogowski, J., 2006: Characteristics of acid phosphatase from Russian sturgeon (Acipenser gueldenstaedtii) spermatozoa. Journal of Applied Ichthyology 22, 375–379. Schemehl, M. K.; Graham, E. F.; Erdhal, D. A., 1987: Chemical constituents of trout seminal plasma and spermiduct tissue from rainbow trout Salmo gairdneri (Richardson). Fish Physiology and Biochemistry 1, 55–61. Urner, F.; Sakkas, D., 2003: Protein phosphorylation in mammalian spermatozoa. Reproduction 125, 17– 26. Vassault, A. 1983: Lactate dehydrogenase. UV-method with pyruvate and NADH. In: H. U. Bergmeyer (ed), Methods in Enzymatic Analysis Vol 3. Verlag Chemie, Weinheim, pp. 118–126. Yang, C. H.; Srivastava, P. N., 1974: Purification and properties of arylsulfatases from rabbit sperm acrosomes. Proceedings of the Society for Experimental Biology and Medicine 145, 721–725.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
ORIGINAL ARTICLE
Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation M. S. Aramli Young Researchers and Elites Club, Ardabil Branch, Islamic Azad University, Ardabil, Iran
Summary Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing-thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 0.026 g/l, and the TP after cryopreservation was 1.86 0.6 g/l. The activities of acid phosphatase (0.82 0.042 U/l), lactate dehydrogenase (234.4 19.4 U/l), arylsulfatase (143.1 32.5 U/l) and b-N-acetylglucosaminidase (58.39 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen-thawed semen samples (7.43 0.64, 3224.6 167.2, 422.6 21.3 and 90.2 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing-thawing. Keywords enzyme activities, protein concentration, fresh semen, Frozen-thaw cycle, Huso huso Correspondence Mohammad Sadegh Aramli, Young Researchers and Elites Club, Ardabil Branch, Islamic Azad University, Ardabil 31567-56157, Iran. Tel: +98 914 754 1626; E-mails: [email protected]; [email protected] Received: 22 March 2014; accepted: 31 March 2014
Introduction The sturgeon is among the world’s most valuable wildlife resources. Most of the world’s sturgeon populations have experienced significant declines in recent decades, primarily because of over-fishing, habitat destruction and pollution (Pikitch et al., 2005). The beluga (Huso huso) is one of 27 chondrostean fish species that are included in the family, Acipenseridae. The beluga appeared about 200 million years BP (Bemis et al., 1997; Ludwig, 2008) and inhabits the Northern Hemisphere. Belugas have a high growth rate, making them highly suitable for aquaculture (Aramli et al., 2013a). Previous studies of biochemical indicators of semen quality have generally focused on the activities of enzymes in the plasma of fresh semen or the supernatants of semen samples following long- or short-term cryostorage (Ciereszko, 2008). The assessment of the biochemical characteristics of semen, particularly in terms of enzyme characteristics, can provide important information regarding spermatozoa motility and viability. Studies of the techniques for assessing semen quality might facilitate improvements in artificial reproduction and germplasm resource conservation procedures (Li et al., 2008).
The enzymology of fish gametes is not well understood, compared with the wealth of available knowledge regarding that of mammals. The activity levels of aspartate aminotransferase (AST) (Ciereszko and Dabrowski, 1994), lactate dehydrogenase (LDH) (McNiven et al., 1992), acid phosphatase (AcP) (Schemehl et al., 1987), b-N-acetylglucosaminidase (bNAGase) and arylsulfatase (AS) (Piros et al., 2002; Aramli et al., 2013b, 2014a) in the semen of certain fish species have been previously described. However, to our knowledge, no data are available on the activity of these enzymes in beluga semen. The objective of our study was to measure the enzymatic activity of LDH, AcP, bNAGase, AS and the total protein concentration (TP) in beluga semen before and after cryopreservation. Materials and methods Broodstock handling and collection of semen
Ten male belugas (30–45 kg, 1–2 m) were collected from the south-western region of the Caspian Sea and transported to the Rajaee Sturgeon Hatchery Center in Sari, Mazandaran, Iran (latitude 36°370 N, longitude 53°050 E), during March and April in 2012. The fish were maintained in tanks with a water temperature of 15.5°C 0.5°C, an oxygen content of >5 mg/l and a
Journal of Animal Physiology and Animal Nutrition 99 (2015) 13–16 © 2014 Blackwell Verlag GmbH
13
Enzyme activities and protein content of Huso huso semen
pH of 7.6–7.9. Fish were feed using a commercial diet (Biomars, no. 1.9, Nersac, France; 50% crude protein, 18% crude fat, 10% ash, 1.3% fibre) during the experimental period. To induce spermiation, the fish were given an intramuscular injection of 5–10 lg/kg of luteinizing hormone-releasing hormone agonist between the dorsal and lateral scutes, as previously described (Nazari et al., 2010; Aramli et al., 2014b). Spermiation occurred at 14–18 h post-injection. Semen was collected from the urogenital opening using rigid tubing and a syringe. Special care was taken to avoid contamination with urine, mucus, faeces or water. Three males did not mature during the experiment. Semen samples were collected from seven of the male fish collected.
M. S. Aramli
0.5 mM p-nitrophenyl-b-N-glucosoaminide as a substrate in 0.1 M citrate buffer, pH 5.0. After a 60-min incubation at 37°C, the reaction was stopped by the addition of 0.1 M NaOH, and the absorbance at 410 nm was measured (Jauhiainen and Vanha-Perttula, 1986). The level of TP was measured using a folin-phenol reagent (Lowry et al., 1951) at 37°C, and the absorbance was measured at 546 nm. Data analysis
The data were analysed using a one-way analysis of variance and the Duncan multiple-range test. A value p < 0.05 was considered to indicate a statistically significant difference between data sets. All data are expressed as the mean standard error.
Sample preparation
The semen samples were stored in glass tubes on ice for up to 3 h during transportation to the laboratory. Seminal plasma was collected immediately by centrifugation at 2500 g for 10 min in a Labnet Spectrafuge 16 M (Woodbridge, NJ, USA). An aliquot of each semen sample was transferred to a 1.5-ml microcentrifuge tube and frozen at 79°C for 1 day to evaluate enzyme release from the spermatozoa damaged during freezing and thawing. The semen samples were thawed at room temperature and centrifuged. The supernatant was collected by centrifugation and used for the enzyme analysis.
Results and Discussion
The TP and the activities of the various enzymes in the seminal plasma samples and the supernatant of the frozen-thawed samples were assessed. The activity of AcP was determined using a solution containing 5 mM p-nitrophenyl phosphate and 20 mM citrate, pH 5 (Glogowski et al., 1996). After incubation at 37°C for 30 min, the reaction was stopped by the addition of 0.1 M NaOH, and the absorbance at 410 nm was measured (Glogowski et al., 1996). The LDH activity was measured in a solution containing 1.6 mM sodium pyruvate, 0.2 mM NADH, 200 mM NaCl and 80 mM Tris-HCl, pH 7.3, and the absorbance at 339 nm was measured following a 5-min incubation at 30°C (Vassault, 1983). The activity of AS was determined using 0.2 ml of 20 mM p-nitrocatechol sulphate as a substrate in 0.5 M sodium acetate buffer, pH 4.9. After a 30-min incubation at 37°C, the reaction was stopped by the addition of 2.5 ml 1 M NaOH, and the absorbance at 515 nm was measured (Yang and Srivastava, 1974). The activity of bNAGase was measured using
Table 1 lists the TP and enzyme activities detected in the beluga semen. Almost all of the parameters analysed were statistically higher in the supernatants of the frozen-thawed samples than in the seminal plasma of fresh semen. Seminal plasma proteins play a key role in prolonging sperm viability in teleost fish (Lahnsteiner et al., 2004). Thus, a better understanding of the enzyme activities in seminal plasma and spermatozoa is necessary to identify indicators of freeze-thaw damage to sturgeon semen. Currently, little is known about the proteins present in the seminal plasma of sturgeons. However, certain proteins in the seminal plasma may protect the spermatozoa. Previous studies have reported the TP in the seminal plasma of various sturgeon species (Table 1). Most sturgeons, including beluga, have a much lower TP in the seminal plasma than that typically observed in higher vertebrates. The AS and bNAGase are key enzymes found in the mammalian acrosome, which play a critical role in the penetration of the oocyte (Nikolajczyk and O’Rand, 1992; Brandon et al., 1997). In our current study, these two enzymes were present in the beluga semen. Their presence was expected because of the active acrosome in sturgeon semen. Previous studies of various sturgeon species have shown that the activities of AS and bNAGase in fresh seminal plasma were lower than those of the supernatants of frozen-thawed semen (Table 1; Piros et al., 2002; Sarosiek et al., 2004; Aramli et al., 2014a). The level of AcP activity was much lower in the seminal plasma of fresh semen than that in the supernatant of frozen-thawed semen (Table 1). Sarosiek et al. (2006) reported that two isoforms of AcP (39 and 19 kDa) were present in the semen of Russian
14
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Analytical procedures
Enzyme activities and protein content of Huso huso semen
M. S. Aramli
Table 1 Comparison of enzyme activities and protein content and of fresh and frozen-thawed semen from different sturgeon species Species
Semen type
TP (g/dl)
AS (U/l)
bNAGase (U/l)
AcP (U/l)
LDH (U/l)
Authors
Sterlet
Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh Frozen-thawed Fresh* Frozen-thawed*
0.5 1.33 0.24 1.6 0.58 4.33 0.57 4.40 0.3
207.0 760.2 54.1 937.1 684.3 2720.5 638.5 2993.9
53.68 79.09 23.65 44.59 16.39 61.59 20.49 67.28
1.85 7.89 0.9 5.82 1 20.6 1.21 23.81
761.7 8803.0 71 9493.0 278.4 4420.0 400.9 7217.3
Piros et al. (2002)†
Siberian sturgeon
Stellate Russian sturgeon Persian sturgeon Beluga
Piros et al. (2002)‡ Piros et al. (2002)§ Piros et al. (2002)¶ Li et al. (2011)
0.6 0.28 1.68 0.47 1.86
Li et al. (2011)
0.031 0.1 0.026 0.6
169.1 699.6 143.1 422.6
44.4 25.1 32.5 21.3
36.31 2.25 70.7 2.24 58.39 4.14 90.2 5.37
0.59 9.89 0.82 7.43
0.035 0.81 0.042 0.64
484.4 6262.7 234.4 3224.6
27.3 263.2 19.4 167.2
Aramli et al. (2014a) Present study
TP, total protein; AS, arylsulfatase; bNAGase, b-N-acetylglucosaminidase; AcP, acid phosphatase; LDH, lactate dehydrogenase. †Fish captured from the Danube River. ‡Fish captured from the Dolna Odra Power Plant. §1st semen collection. ¶2nd semen collection. *Significant different between seminal plasma of fresh and freeze–thawed semen (p < 0.05; n = 7).
sturgeon. The role of AcP in sturgeon reproduction is unknown. In mammals, AcP functions in phosphorylation and dephosphorylation reactions during sperm capacitation, hyperactivation, acrosome formation and sperm–egg fusion (Urner and Sakkas, 2003). The formation of lactate allows glycolysis to proceed in the absence of oxygen. LDH functions in energy production by converting lactate to pyruvate, the end product of glycolysis pathway (Miki, 2007). The level of LDH activity was higher in the supernatant of frozen-thawed semen samples than that in seminal plasma of fresh semen samples. Previous studies have shown that LDH activity was also higher in the seminal plasma of semen of the Siberian sturgeon, sterlet and Persian sturgeon following cryopreservation (Table 1; Piros et al., 2002; Aramli et al., 2014a). Piros et al. (2002) suggested that the increased activity of LDH likely resulted from the disruption of the energy supply during cryopreservation, which subsequently affects sperm motility. According to the data presented in Table 1, the activities of all of the enzymes evaluated were significantly higher in the supernatant of the frozen-thawed References Aramli, M. S.; Nazari, R. M.; Kalbassi, M. R.; Aramli, S., 2013a: Semen of beluga, Huso huso: ionic content and osmolality of seminal plasma and their physiologi-
semen samples. Therefore, the activities of these enzymes may represent potential biomarkers of cryoinjury, which is consistent with previous studies of teleost fish (Glogowski et al., 1996; Lahnsteiner et al., 1996; Babiak et al., 1997). The activities of LDH and AcP may be indicators of damage to the plasmalemma and the midpiece area of the sperm structure, whereas the activities of AS and bNAGase may be useful indices of damage to the acrosome. However, future studies are warranted to explore the roles of these enzymes in the viability of beluga spermatozoa. In conclusion, our results contribute to the available data regarding the enzyme properties of beluga semen. These data are critical to the development of optimal diluents for the long- and short-term storage of sperm collected from this endangered species. Acknowledgements The authors wish to thank the technical staff of Rajaee Sturgeon Hatchery Center in Sari, Iran, for their help and support during this study.
cal correlation with sperm motility indices. Journal of Fisheries and Aquaculture 4, 2. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2013b: Spermatozoa and seminal plasma enzyme activity in Persian stur-
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
geon, Acipenser persicus, in relation to short-term storage. Aquaculture Research. doi:10.1111/are.12349. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2014a: Protein concentration and enzyme activities of fresh and
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Enzyme activities and protein content of Huso huso semen
frozen-thawed Persian sturgeon, Acipenser persicus (Borodin, 1897) semen. International Aquatic Research 6, 54. Aramli, M. S.; Kalbassi, M. R.; Nazari, R. M., 2014b: Sex steroid levels of Persian sturgeon, Acipenser persicus, Borodin, 1897, males in negative and positive responding to LH-RH-analogue. Journal of Applied Ichthyology 30, 18–19. Babiak, I.; Glogowski, J.; Luczynski, M. J.; Luczynski, M., 1997: Effect of individual male variability on cryopreservation of northern pike, Esox lucius L., sperm. Aquaculture Research 28, 191–197. Bemis, E.; Findeis, E.; Grande, L., 1997: An overview of Acipenseriformes. In V. J. Birstein, J. R. Waldman, W. Bemis, E. Kluwer (eds) Sturgeon Biodiversity and Conservation. Academic Publishing, Dordrecht, pp. 25–71. Brandon, C. I.; Srivastava, P. N.; Heusner, G. L.; Fayer-Hosken, R. A., 1997: Extraction and quantification of acrosin, b Nacetylglucosaminidase, and arylsulfatase-A from equine ejaculated spermatozoa. Journal of Experimental Zoology 279, 301–308. Ciereszko, A., 2008: Chemical composition of seminal plasma and its physiological relationship with sperm motility, fertilizing capacity and cryopreservation success in fish. In: S. M. H. Alavi, J. Cosson, K. Coward, G. R. Rafiee (eds), Fish Spermatology. Alpha Science Ltd, Oxford, pp. 215–240. Ciereszko, A.; Dabrowski, K., 1994: Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage. Fish Physiology and Biochemistry 12, 357–367. Glogowski, J.; Babiak, I.; Goryczko, K.; Dobosz, S., 1996: Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout semen. Reproduction, Fertility and Development 8, 1179–1184. Jauhiainen, A.; Vanha-Perttula, T., 1986: b -N-acetylglucosaminidase in the reproductive organs and seminal plasma
16
M. S. Aramli
of the bull. Journal of Reproduction and Fertility 76, 239–250. Lahnsteiner, F.; Berger, B.; Weismann, T.; Patzner, R., 1996: The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation. Journal of Applied Ichthyology 12, 99–106. Lahnsteiner, F.; Mansour, N.; Berger, B., 2004: Seminal Plasma Prolong the Viability of Rainbow trout (Oncorhynchus mykiss) Spermatozoa. Theriogenology 62, 801–808. Li, P.; Hulak, M.; Linhart, O., 2008: Sperm proteins in teleostean and chondrostean (sturgeon) fishes. Fish Physiology and Biochemistry 35, 567–581. Li, P.; Rodina, M.; Hulak, M.; Li, Z. H.; Linhart, O., 2011: Spermatozoa concentration, seminal plasma composition and their physiological relationship in the endangered stellate sturgeon (Acipenser stellatus) and Russian sturgeon (Acipenser gueldenstaedtii). Reproduction in Domestic Animals 46, 247–252. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Raudall, R. J., 1951: Protein measurement with folin-phenol reagent. Journal of Biological Chemistry 193, 265–275. Ludwig, A., 2008: Identification of Acipenseriformes species in trade. Journal of Applied Ichthyology 24, 2–19. McNiven, M. A.; Gallant, R. K.; Richardson, G. F., 1992: In vitro methods of assessing the viability of rainbow trout spermatozoa. Theriogenology 38, 679–686. Miki, K., 2007: Energy metabolism and sperm function. Society of Reproduction and Fertility supplement 65, 309–325. Nazari, R. M.; Modanloo, M.; Ghomi, M. R.; Ovissipor, M. R., 2010: Application of synthetic hormone LHRH-A2 on the artificial propagation of Persian sturgeon Acipenser persicus. Aquaculture International 18, 837–841. Nikolajczyk, B. S.; O’Rand, M. G., 1992: Characterization of rabbit testis b-galactosidase and arylsulfatase purification
and localization in spermatozoa during the acrosome reaction. Biology of Reproduction 46, 366–378. Pikitch, E. K.; Doukais, P.; Lauck, L.; Chakrabarty, P.; Erickson, D. L. 2005: Status trends and management of sturgeon and paddlefish fisheries. Fish Fisher 6, 233–265. Piros, B.; Glogowski, J.; Kolman, R.; Rzemieniecki, A.; Domagala, J.; Horvath, A.; Urbanyi, B.; Ciereszko, A., 2002: Biochemical characterization of Siberian sturgeon, Acipenser baeri and sterlet, Acipenser ruthenus, seminal plasma and spermatozoa. Fish Physiology and Biochemistry 26, 289–295. Sarosiek, B.; Ciereszko, A.; Kolman, R.; Glogowski, J., 2004: Characteristics of arysulfatase in Russian sturgeon (Acipenser gueldenstaedtii) semen. Comparative Biochemistry and Physiology 139B, 517–579. Sarosiek, B.; Wysocka, J.; Wysocki, P.; Kowalski, R.; Glogowski, J., 2006: Characteristics of acid phosphatase from Russian sturgeon (Acipenser gueldenstaedtii) spermatozoa. Journal of Applied Ichthyology 22, 375–379. Schemehl, M. K.; Graham, E. F.; Erdhal, D. A., 1987: Chemical constituents of trout seminal plasma and spermiduct tissue from rainbow trout Salmo gairdneri (Richardson). Fish Physiology and Biochemistry 1, 55–61. Urner, F.; Sakkas, D., 2003: Protein phosphorylation in mammalian spermatozoa. Reproduction 125, 17– 26. Vassault, A. 1983: Lactate dehydrogenase. UV-method with pyruvate and NADH. In: H. U. Bergmeyer (ed), Methods in Enzymatic Analysis Vol 3. Verlag Chemie, Weinheim, pp. 118–126. Yang, C. H.; Srivastava, P. N., 1974: Purification and properties of arylsulfatases from rabbit sperm acrosomes. Proceedings of the Society for Experimental Biology and Medicine 145, 721–725.
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