Mehdi Nikoo a & Bahram Falahatkar b a Department of Environmental Science and. To link to this article:

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1 This article was downloaded by: [Dr Kenneth Shapiro] On: 09 June 2015, At: 10:44 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Applied Animal Welfare Science Publication details, including instructions for authors and subscription information: Physiological Responses in Wild Broodstocks of the Caspian Kutum (Rutilus frisii kutum) Subjected to Transportation Stress Mehdi Nikoo a & Bahram Falahatkar b a Department of Environmental Science and Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran b Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran Published online: 25 Sep To cite this article: Mehdi Nikoo & Bahram Falahatkar (2012) Physiological Responses in Wild Broodstocks of the Caspian Kutum (Rutilus frisii kutum) Subjected to Transportation Stress, Journal of Applied Animal Welfare Science, 15:4, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and

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3 JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 15: , 2012 Copyright Taylor & Francis Group, LLC ISSN: print/ online DOI: / RESEARCH REPORTS Physiological Responses in Wild Broodstocks of the Caspian Kutum (Rutilus frisii kutum) Subjected to Transportation Stress Mehdi Nikoo 1 and Bahram Falahatkar 2 1 Department of Environmental Science and Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran 2 Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran Caspian Kutum (Rutilus frisii kutum) broodstocks in the wild were subjected to 2 different densities (100 and 300 kg m 3 ) for 2 hr of transport, and their physiological responses were examined. Fifteen fish were placed into the plastic container for each replicate and blood was taken at 0, 10, 30, 60, and 120 min after transportation in different densities. Mean levels of cortisol and glucose as primary and secondary responses to the stress were significantly different between densities. Significant differences in both densities were observed in cortisol for all times and glucose levels at 30 and 120 min after transportation, and the highest values were found in both densities at 120 min. Testosterone concentrations declined considerably in the first 10 min, and then they showed a gradual decrease with a significant difference between 2 densities at 10 and 30 min after transportation. Serum estradiol reached the lowest level at 120 min after transportation, and differences were significant between the 2 densities after 30 min. Based on the results, it was concluded that Kutum broodstocks are sensitive to transport, especially at high density. Therefore, welfare during transport should be considered. Mehdi Nikoo is now at the School of Food Science and Technology, Jiangnan University, China. Correspondence should be sent to Bahram Falahatkar, Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, 1144, Guilan, Iran. falahatkar@guilan.ac.ir 372

4 TRANSPORTATION STRESS 373 Common practices in aquaculture, including capture of fish in the wild for gametes collection, high stocking density, handling, and transportation can be stressful to the fish and thus affect reproduction (Pankhurst & Van Der Kraak, 1997). Several studies in teleosts showed that both corticosteroids and sex steroids can be affected by stressors (Greenburg & Wingfield, 1987; Schreck, 2009). This feature of fishes may result in an interaction between corticosteroids and sex steroid values under stressful conditions. Cortisol and glucose are regarded as reliable indicators of stress response in fish as their values are elevated under stressful conditions (Barton, 2002; Martinez-Porchas, Martinez-Cordova, & Ramos-Enriquez, 2009). Reproductive hormones in fishes are affected by stressors, where the stressed fish have elevated circulatory adrenocorticotropic hormone and cortisol and decreased sex steroids such as testosterone, 11-keto testosterone, and 17ˇ-estradiol (See Schreck, 2009). Acute stressful conditions in mature fish resulted in sex steroid suppression; fewer oocytes could be stripped and the quality of eggs was reduced (Soso, Barcellos, & Ranzani-Paiva, 2008). The propagation and rearing of Caspian Kutum (Rutilus frisii kutum) using wild broodstocks is aimed at restocking the natural environment in southern parts of the Caspian Sea, and every year up to 200 million of 1-g juveniles are released to their natural environments (Abdoli & Naderi, 2009). During reproduction season, hundreds of broodstocks are captured from the sea and estuaries and are transported to the hatcheries for gamete collection. It is crucial to examine the response of broodstocks to transport and determine optimal densities for transportation. Understanding the stress responses and managing the stress is necessary for optimal performance in fish transport (Carneiro & Urbinati, 2002). The first step of the restocking program is supplying the brooders from natural habitats. Catching, handling, and transporting methods may affect the physiology of fish in terms of their different responses to the stress. Many physiological parameters are altered during stressful conditions, and they may have an effect on gamete quality and reproductive success (Barton, 2002; Schreck, 2009). Modifying transportation procedures and capture techniques are recommended for the success of hatchery restocking programs. Additionally, assessments of physiological responses of fish to transportation-related stressors provide essential information for the development of stock programs. Therefore, the objective of the current study was to examine several physiological indices, including cortisol, glucose, testosterone, and 17ˇ-estradiol levels during the transportation of Caspian Kutum wild broodstocks. This information will provide a better understanding of the fish and can lead to improvements in transportation and handling of Kutum broodstocks.

5 374 NIKOO AND FALAHATKAR TABLE 1 Weight and Length (M SD) of Caspian Kutum Broodstocks for Each Experimental Density Density (kg m 3 ) n Body Weight (g) Total Length (cm) , , Broodstocks METHODS Experimental wild broodstocks were caught on March 25, 2009, from the coastal waters of the southern Caspian Sea by a cast net with a 2-cm mesh size. Weights and lengths of fish are presented in Table 1. Ovary conditions of the captured fish were recorded as either ripe (ovulated eggs free within the body cavity or loose) or gravid (oocytes still attached or tight) according to Patterson, Macdonald, Hinch, Healy, and Farrell (2004). There were 60 gravid broodstocks used in this study, who were divided into four groups of 15 fish each. After capture, fish were placed in four 300-L plastic containers (each group in one tank) containing Caspian Sea water at a low density of 100 and a high density of 300 kg m 3 (two tanks for each density as replicates). Water was well aerated manually during transportation. Broodstocks were then transported for 2 hr using a boat for shipping fish to the hatchery for gamete collection. The water temperature and dissolved oxygen were 15.9 ı C and 7.3 mg L 1, respectively. Blood Sampling Blood samples were collected immediately after capture at resting time and then at 10, 30, 60, and 120 min during transportation from the sea to breeding place, which was located near the Goharbaran River in Mazandaran, Iran. At resting time, 10 fish were sampled. For each sampling time, 4 fish of each treatment were selected from holding containers. At 120 min, the 3 remaining fish were sampled. To minimize the effect of handling stress, fish were subjected to 200 mg L 1 clove powder as anesthesia and blood samples were taken in less than 1 min from the caudal vein with a nonheparinized 5-mL plastic syringe. After sampling, fish were tagged then returned to the same tank for holding exactly the designed loading density. Samples were then centrifuged at 4,000 g for 10 min to obtain the serum and stored at 20 ı C until analysis (Mingist, Kitani, Koide, & Udea, 2007).

6 TRANSPORTATION STRESS 375 Biochemical Measurements Serum cortisol, testosterone, and 17ˇ-estradiol concentrations were measured using the radioimmunoassay method (Rottlant, Balm, Perez-Sanchez, Wendelaar- Bonga, & Tort, 2001; Suresh, Baileand, & Prasada Rao, 2008). Serum glucose levels were determined by colorimetric method using a commercial kit (Greiner, Bahlingen, Germany) according to Bayunova, Barannikova, and Semenkova (2002). Statistical Analysis Data were analyzed using the general linear model and univariate model with densities and sampling times as the factors. When a significant difference was observed (p <.05), Tukey s post hoc test was used to compare the means. Independent sample t tests were used to find differences between high and low densities at each time. All analyses were performed by SPSS The levels of significance were taken as p <.05. All values are presented as M SD. RESULTS Initial mean levels of serum cortisol in both low and high densities were high ( and ng ml 1, respectively). After 30 min of transportation, cortisol increased significantly in both densities. The mean cortisol content was significantly different between low and high densities after catching (p <.000). Cortisol concentrations reached their peak at the end of the transportation (Figure 1). The initial amount of glucose at the two densities was and mg dl 1, respectively, and it did not show considerable elevation in the first 10 min (p D.650). After 30 min, the glucose dramatically rose (p <.000) and a significant difference was observed in both densities at 30 min (p D.003) and 120 min (p D.001) after transportation. The highest glucose concentrations were found in both low and high densities at 120 min (Figure 2). Testosterone at low and high densities was measured at and ng ml 1, respectively, before transport and showed considerable decline in the first 10 min (p <.000). Thereafter, until the end of the broodstocks transportation, they showed a gradual decrease with a significant difference between two densities at 10 min (p D.001) and 30 min (p D.000) after transport began (Figure 3). Serum estradiol levels in both treatments at the beginning of the experiment were and ng ml 1, respectively, and reached the lowest levels ( for low and ng ml 1 for high density) at 120 min

7 376 NIKOO AND FALAHATKAR FIGURE 1 Changes in plasma cortisol (ng ml 1 ) in wild Caspian Kutum, Rutilus frisii kutum, during transportation at low (100 kg m 3 ) or high (300 kg m 3 ) density. Values are M SD from two replicates with n D 2 per replicate. Bars accompanied by different letters are significantly different (p <.05) from others within each treatment. Asterisks show differences between two densities at each time. LD D low density; HD D high density. FIGURE 2 Changes in plasma glucose (mg dl 1 ) in wild Caspian Kutum, Rutilus frisii kutum, during transportation at low (100 kg m 3 ) or high (300 kg m 3 ) density. Values are M SD from two replicates with n D 2 per replicate. Bars accompanied by different letters are significantly different (p <.05) from others within each treatment. Asterisks show significant differences between two densities at each time. LD D low density; HD D high density.

8 TRANSPORTATION STRESS 377 FIGURE 3 Changes in plasma testosterone (ng ml 1 ) in wild Caspian Kutum, Rutilus frisii kutum, during transportation at low (100 kg m 3 ) or high (300 kg m 3 ) density. Values are M SD from two replicates with n D 2 per replicate. Bars accompanied by different letters are significantly different (p <.05) from others within each treatment. Asterisks show significant differences between two densities at each time. LD D low density; HD D high density. after transportation (p <.000). The trend of decline was significant during transportation and differences were significant between the two densities after 30 min (Figure 4). DISCUSSION Transport density and duration had a significant effect on the physiological response of the Caspian Kutum broodstocks as evidenced by the increase in blood cortisol and glucose and decrease in sex steroid levels. Initial values for cortisol were high in this study compared with our previous study on Kutum broodstocks, which showed that the cortisol concentration at resting time was 308 ng ml 1 (Nikoo et al., 2010). High resting values of blood cortisol were also reported in sockeye salmon, Oncorhynchus nerka (Patterson et al., 2004) and rohu, Labeo rohita fingerlings (Hasan & Bart, 2007). High resting levels are attributed to stressful conditions before transport, perhaps as a result of capture and handling (Hasan & Bart, 2007). The levels may also be attributed to the fact that spawning season was close, which has a large impact on the physiological condition of fish. Serum cortisol has been reported to increase with increasing transportation time (Robertson, Thomas, & Arnold, 1987). In adult Brycon cephalus, cortisol levels increased two-fold compared with basal values after 4- hr transport (Carneiro & Urbinati, 2001), and a similar increase was observed in

9 378 NIKOO AND FALAHATKAR FIGURE 4 Changes in plasma estradiol (ng ml 1 ) in wild Caspian Kutum, Rutilus frisii kutum, during transportation at low (100 kg m 3 ) or high (300 kg m 3 ) density. Values are M SD from two replicates with n D 2 per replicate. Bars accompanied by different letters are significantly different (p <.05) from others within each treatment. Asterisks show significant differences between two densities at each time. LD D low density; HD D high density. juveniles of this species following transport stress (Urbinati, Abreu, Camargo, & Parra, 2004). A two-fold increase in serum cortisol concentrations was observed in Eurasian perch, Perca fluviatilis, after transport (Acerete, Balasch, Espinosa, Josa, & Tort, 2004) and following handling; its levels increased significantly after 30 min passed. High values of cortisol were found in golden perch, Macquaria ambigue, after 30 min of netting and confinement (Carragher & Rees, 1994). Although the level of cortisol was a little bit high before transport, this procedure can affect the hypothalamus-pituitary-interrenal cells axis and result in the excretion of cortisol in blood for both densities. If it s sustained, elevated plasma cortisol can lead to decreases in body size, the gonadosomatic index, egg size, gamete quality, and fertilization rates (Bobe & Labbé, 2010; Campbell, Pottinger, & Sumpter, 1992, 1994; Foo & Lam, 1993; Kime & Nash, 1999; Kubokawa, Watanabe, Yoshioka, & Iwata, 1999). The effects of transportation, especially when it occurs at high densities, on the gamete quality and embryo survival should be examined in future studies. Stress is an energy-demanding process, and the production of glucose provides energy to cope with that demand (Gamperl, Vijayan, & Boutilier, 1994; Iwama, Afonso, & Vijayan, 2006; Wendelaar Bonga, 1997). Glucose commonly changes after subjecting fish to stressors such as capture, handling, or transport (Barton & Iwama, 1991; Braley & Andersson, 1992; Falahatkar, Poursaeid, Shakoorian, & Barton, 2009; Rottlant et al., 2001). During stressful events, catecholamines act directly on the liver to stimulate glycogenolysis, which results

10 TRANSPORTATION STRESS 379 in the mobilization of glucose (Axelrod & Reisine, 1984). It has also been reported that the glucose levels increase with the corticosteroid levels under acute stress (Rottlant & Tort, 1997; Vijayan, Pereira, Grau, & Iwama, 1997; Vijayan, Pereira, & Moon, 1994). In the present study, the mean glucose level gradually increased with the duration of the exposure to the stress in Caspian Kutum. Glucose in the experimental fish was less than 50 mg dl 1 in two densities after the first 30 min but increased at the end of transportation. The highest blood glucose levels in Rhamdia quelen (mean weight 1 kg) were observed in fish subjected to high transport density (Carneiro & Urbinati, 2002), and higher glucose values at the end of transportation with the highest density were found with juveniles of Jundiá (Rhamdia quelen; Carneiro, Kaiseler, Swarofsky, & Baldisserotto, 2009). In this study, serum testosterone concentrations declined in the first 10 min of confinement, and this reduction continued until the end of transportation. The same trend was observed for estradiol, but its significant decrease happened after 60 min. Serum testosterone and estradiol levels of snapper, Pagrus auratus, also declined following confinement stress (Cleary, Pankhurst, & Battaglen, 2007). A lower level of estradiol was observed in mature Jundiá when they were subjected to stressful handling (Soso et al., 2008). In brown trout, Salmo trutta, circulating testosterone concentrations decreased with crowding (Pickering, Pottinger, Carragher, & Sumpter, 1987). Testosterone concentrations in both sexes, 11-ketotestosterone levels in males, and plasma 17a-progesterone levels in females decreased with the confinement time in sockeye salmon, Oncorhynchus nerka (Kubokawa et al., 1999). Also, in stellate sturgeon, Acipenser stellatus, the testosterone value decreased in females when they were subjected to the stress of forced swimming (Bayunova et al., 2002). Caspian Kutum responded by decreasing sex steroids levels in the acute stress transport. This result indicates that Caspian Kutum are refractory to acute stress during the breeding season and can respond to the stress by decreasing androgen secretion or increasing androgen clearance. However, the results were related to the females, and perhaps different changes in response to stress would be observed in cortisol secretion and other steroid hormone responses to stress in both male and female Caspian Kutum, which were previously demonstrated in salmonids (Kubokawa et al., 1999). CONCLUSION In conclusion, the present data demonstrate that the high transportation densities of broodstocks lead to the elevation of serum cortisol and glucose and the decrease of sex steroid hormones, which are indicative of stress responses during transportation. Also, the results show that after 120 min, the main metabolite response of cortisol will not decrease and both low- and high-density fish

11 380 NIKOO AND FALAHATKAR suffer, which may decrease the quantity of sex steroids and consequently the quality of reproduction. Therefore, lower density packing is proposed for the transportation of Caspian Kutum broodstocks from the catching area to the hatcheries, and special care during transport will help the welfare of the brooders. In this study, transporting the fish caused stress and affected serum hormones and glucose concentrations. Consequently, these findings suggest that potential stressors should be avoided when possible and transportation procedures and capture techniques should be modified to reduce the degree of stress (Harmon, 2009) to achieve the highest quality of hatcheries during restocking programs for this species. The results regarding changes in the plasma concentrations of cortisol, glucose, and sex steroids in wild Caspian Kutum provide interesting information on the relationship between endocrine function and offer evidence for examining this stressful condition in post-reproductive fish and success in fertilization. ACKNOWLEDGMENTS We thank Shahid Rajaee Hatchery for assisting with fish sampling and Mr. Maleki for helping us with the laboratory assay. REFERENCES Abdoli, A., & Naderi, M. (2009). Biodiversity of fishes of the southern basin of the Caspian Sea. Tehran, Iran: Abzian Scientific. Acerete, L., Balasch, J. C., Espinosa, E., Josa, A., & Tort, L. (2004). Physiological responses in Eurasian perch (Perca fluviayilis L.) subjected to stress by transport and handling. Aquaculture, 237, Axelrod, J., & Reisine, T. D. (1984). Stress hormones: Their interaction and regulation. Science, 224, Barton, B. A. (2002). Stress in fishes: A diversity of response with particular reference to changes in circulating corticosteroids. Integrative Comparative Biology, 42, Barton, B. A., & Iwama, G. K. (1991). Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annual Reviews in Fish Diseases, 1, Bayunova, L., Barannikova, I., & Semenkova, T. (2002). Sturgeon stress reaction in aquaculture. Journal of Applied Ichthyology, 18, Bobe, J., & Labbé, C. (2010). Egg and sperm quality in fish. General and Comparative Endocrinology, 165, Braley, H., & Andersson, T. A. (1992). Changes in blood metabolite concentrations in response to repeated capture, anesthesia and blood sampling in the golden perch, Macquaria ambigua. Comparative Biochemistry and Physiology, 103A, Campbell, P. M., Pottinger, T. G., & Sumpter, J. P. (1992). Stress reduces the quality of gametes produced by rainbow trout. Biology of Reproduction, 47,

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