SHORT COMMUNICATION Immersion bath treatment of tilapia fry with myostatin-1 prodomain does not affect tilapia growth at market size

Transcription

1 Aquaculture Research, 2012, 1 6 doi: /j x SHORT COMMUNICATION Immersion bath treatment of tilapia fry with myostatin-1 prodomain does not affect tilapia growth at market size Yong-Soo Kim 1, Bradley Fox 1, Kyung Ho Kim 1, Sang Beum Lee 2, Hyung Joo Jin 2 & Clyde S Tamaru 1 1 Department of Molecular Bioscience and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, USA 2 Department of Marine Molecular Biotechnology, Gangneung-Wonju National University, Gangneung, Korea Correspondence: Y-S Kim, Department of Molecular Bioscience and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, HI 96822, USA. ykim@hawaii.edu Introduction Myostatin (MSTN), a member of the transforming growth factor-b (TGF-b) superfamily, is a potent negative regulator of skeletal muscle growth in mammalian species (Lee 2004). Recent studies have also indicated that fish MSTN negatively regulates muscle growth in fish (Xu, Wu, Zohar & Du 2003; Amali, Lin, Chen, Wang, Gong, Lee, Ko, Lu, Her, Chen & Wu 2004; Acosta, Carpio, Borroto, Gonzalez & Estrada 2005; Lee, Hu, Gong, Chen, Lu & Wu 2009; Medeiros, Phelps, Fuentes & Bradley 2009; Lee et al. 2010; Lee, Cho, Kim, Kim & Jin 2011). Because of the prominent role of MSTN in regulating muscle mass, there has been much interest in developing MSTN-based strategies to improve skeletal muscle growth in agriculturally important animal species, including fish (Tsuchida 2008).The MSTN prodomain (MSTNpro), the N- terminal part of MSTN precursor molecule, has been shown to be a potent suppressor of MSTN activity in mammalian species (Lee & McPherron 1999; Yang, Ratovitski, Brady, Solomon, Wells & Wall 2001; Matsakas, Foster, Otto, Macharia, Elashry, Feist, Graham, Foster, Yaworsky, Walsh, Dickson & Patel 2009; Hu, Chen, Sheng, Sun, Cao & Qiao 2010; Li, Zhao, Kim, Hu & Yang 2010). Fish MSTNpro also appears to suppress MSTN activity. The MSTN-1 prodomain (MSTN1pro) of the marine fish Sparus aurata suppressed MSTN activity in vitro (Rebhan & Funkenstein 2008). An increase in fibre number was observed in transgenic zebrafish overexpressing MSTN1pro (Xu et al. 2003). Recently, it was also observed that immersion bath treatment with flatfish (Paralichthys olivaceus) MSTN1pro (pomstn1pro) expressed in an Escherichia coli system improved the growth of young juvenile rainbow trout (Lee et al. 2010), indicating the potential of fish MSTN-1 prodomain as an agent to improve the growth of commercially important aquaculture species. Similarly, immersion bath treatment of African catfish, goldfish and tilapia larvae with a soluble form of MSTN receptor (activin type IIB receptor) improved body mass growth of those fish (Carpio, Acosta, Morales, Santisteban, Sanchez & Estrada 2009). These studies, however, examined the growth response only during the days treatment period, and did not follow the growth response to market size. Thus, it is not known whether the enhanced weight gain induced by treatment with MSTN inhibitor at the early stage of fish growth would last to later growth stages, resulting in heavier market weight when harvested at a fixed growth period. The current study was designed to address this question. Materials and methods Six hundred tilapia (Oreochromis spp.) fry weighing about 110 mg were obtained from the same cohort of brood stock breeding tanks at the 2012 Blackwell Publishing Ltd 1

2 Effect of myostatin-1 prodomain treatment on tilapia growth Y-S Kim et al. Aquaculture Research, 2012, 1 6 CTAHR Freshwater Aquaculture and Aquaponics Research and Extension Facility at Windward Community College, Oahu, Hawaii, randomly divided into four treatment groups, and each group was allocated into three equal sized glass tanks holding 50 L of fresh water; such that each treatment group was represented with triplicated rearing tanks. Tanks were located indoor with overhead fluorescent lamps set to a 12 h on, 12-h off photoperiod, supplied with aerated flowthrough municipal drinking water (22 24 C). The first group experienced no immersion bath treatment, and the three remaining groups were subjected to immersion bath treatment by dipping tilapia for 2 h in water containing 0, 0.05 or 0.2 mg L 1 pomstn1pro respectively. The triplicates were immersed separately. The production of pomstn1pro was reported previously (Lee et al. 2010). The flatfish myostatin prodomain-1 (pom- STN1pro) shares about 90% homology in amino acid sequence with that of tilapia. The immersion bath treatment was performed twice a week for 4 weeks. At the time of sampling, all the fish were caught with a hand net, and transferred to a perforated 1 L container that was submerged for 2 h in the appropriate bath treatment. Body weight and standard length were measured weekly just prior to the immersion bath treatment. Fish were fed live Moina spp. ad libitum twice daily for the initial 6 weeks of the experiment, after which they were fed a high protein commercial starter diet in a similar manner (Silver Cup Steelhead; Nelson & Sons, Murray, HT, USA). The survival rate was not affected by the immersion bath treatment, and was around 93% for all groups at the end of treatment. At 6 weeks after the last immersion bath treatment when fish weight reached about 2.5 g, fish were transferred into bigger identical outdoor tanks (500 L) supplied with aerated flow-through municipal drinking water (22 24 C) under natural photoperiod, and fed a larger commercial pellet ad libitum twice daily for 13 weeks (Silver Cup Trout Chow 3.5 mm; Nelson & Sons). Body weight and standard length were measured monthly. At 23 weeks of age when fish reached about 70 g body weight, 15 fish were randomly selected from each group, surgically implanted with passive integrated transponder tags (PIT tags; Destron IDI, Boulder, CO, USA), and transferred to a L outdoor tank supplied with aerated flow-through municipal drinking water (22 24 C) under natural photoperiod, and fed a commercial pellet ad libitum twice daily (Silver Cup Trout Chow 3.5 mm; Nelson & Sons) until fish were sacrificed at 45 weeks of age. Body weight and standard length were monitored monthly. At sacrifice, head and visceral content were removed to measure empty carcass weight, and fillets were collected from both sides of carcass and weighed. Results and discussion Table 1 summarizes the effect of immersion bath treatment with pomstn1pro on tilapia growth. At 6 weeks (2 week after the last immersion bath treatment), fish body weights of both immersion bath treatment groups (0.05 and 0.2 mg L 1 ) were significantly (P < 0.05) heavier by about 9% without dose effect than the non-immersion bath group or the group treated with 0 mg L 1 (Table 1). This improvement of body weight gain of tilapia during the early period of immersion bath treatment with pomstn1pro is in agreement with a previous study which demonstrated that the growth of young juvenile rainbow trout was improved during the 4 weeks of immersion bath treatment with the same pomstn1pro (Lee et al. 2010), even though the extent of improvement in tilapia (9%) is less than that in rainbow trout (27 42%). However, the increased weight gain observed in the pomstn1pro treated groups disappeared at 10 weeks and afterwards, resulting in no difference in body weights among treatment groups when tilapia reached close to market size of 450 g at 45 weeks. Body lengths of both the immersion bath treatment groups were significantly (P < 0.05) longer than the non-immersion bath group at 6 weeks, but were not different from the group treated with 0 mg L 1. Similar to the results observed in body weight gain, the increase in body length disappeared at 10 weeks and afterwards, resulted in no difference in body length at 45 weeks. No significant differences were observed in the weights of empty carcass, liver, heart and fillet among treatment groups when sacrificed at 45 weeks (Table 2). The current results, thus, indicate that the improved tilapia growth induced by the pomstnb1pro treatment at fry stage was not sustained during the later stages of growth following the immersion bath treatment, eventually resulting in no treatment effect on the weights of body and fillet at market size. In mammals, the post-natal increase in skeletal muscle mass is achieved mostly by an increase in Blackwell Publishing Ltd, Aquaculture Research, 1 6

3 Aquaculture Research, 2012, 1 6 Effect of myostatin-1 prodomain treatment on tilapia growth Y-S Kim et al. Table 1 Effects of immersion bath with myostatin prodomain on tilapia growth Weeks Treatment groups * P value after immersion bath Treatment groups * No bath Bath 1, 0 mg L 1 Bath 2, 0.05 mg L 1 Bath 3, 0.2 mg L 1 P value Phase 1 (n = 150) (n = 150) (n = 150) (n = 150) 0 day, mg ± ± ± ± week, mg ± ± ± ± week Wt, mg a ± a ± b ± b ± Length, cm 2.34 a ± b ± b ± b ± week Wt, g 2.41 ± ± ± ± Length, cm 3.95 a ± b ± ab ± ab ± week Wt, g ab ± ab ± b ± a ± Length, cm ± ± ± ± Phase 2 (n = 13) (n = 18) (n = 16) (n = 18) 23 week Wt, g 73.1 a ± a ± a ± b ± Length, cm a ± a ± a ± b ± week Wt, g ± ± ± ± Length, cm ± ± ± ± week Wt, g ± ± ± ± Length, cm ± ± ± ± Data are least square mean ± (SEM); SEMs of 0 day weight are from 3 tanks and SEMs of the rest are from individual weights. * Mean difference was analysed using Fisher s pairwise comparisons. Means in the same row not sharing the same superscript differ at P < In phase 2 grow-out experiment, fish were randomly selected and tagged and raised in a same outdoor tank to remove tank effect on tilapia growth Blackwell Publishing Ltd, Aquaculture Research, 1 6 3

4 Effect of myostatin-1 prodomain treatment on tilapia growth Y-S Kim et al. Aquaculture Research, 2012, 1 6 Table 2 Effects of immersion bath with myostatin prodomain on body weight, length, and organ weights of tilapia Treatment groups Parameters No bath (n = 13) Bath 1, 0 mg L 1 (n = 17) Bath 2, 0.05 mg/l (n = 16) Bath 3, 0.2 mg L 1 (n = 15) P value Wt, g ± ± ± ± Empty body wt, g ± ± ± ± % empty body wt 91.3 ± ± ± ± Liver wt, g 5.26 ± ± ± ± % liver wt 1.78 ± ± ± ± Heart wt, g 0.24 ± ± ± ± % heart wt ± ± ± ± Fillet wt, g 93.6 ± ± ± ± % fillet wt 34.9 ± ± ± ± Data are least square means ± (SEM). % of organ wt = (organ wt/body wt) fibre size and not by an increase in fibre numbers, thus animals born with more muscle fibre have a greater potential to have a greater muscle mass in later growth stages (Byrne, Hooper & McCarthy 1973; Hanrahan, Hooper & McCarthy 1973; Hegarty & Allen 1978; Powell & Aberle 1981; Brown & Stickland 1994). However, unlike mammals, many fish species which exhibit indeterminate growth, such as important finfish aquaculture species like the tilapia, increase muscle mass by recruitment of new muscle fibres throughout life (Stickland 1983; Brooks & Johnston 1993). Although it has been shown that fish with more muscle fibre at hatching grow faster in the initial post-hatching period than fish with fewer muscle fibres (Nathanailides, Lopez-Albors & Stickland 1995), little is known about whether the muscle fibre number and/or body mass in the larval or early juvenile stages are/is associated with the late muscle growth or mass. Continual recruitment of new fibre is the major mechanism of muscle growth in fast-growing fish that can easily achieve a 1000-fold increase in muscle mass from the larval or young juvenile stages to later mature stage. Thus, a temporary increase in muscle fibre number or mass caused by extrinsic factors during early growth stages may not have lasting effects on its muscle mass in later stages of growth unless the extrinsic factors induce an imprinting influence on the pathways regulating the recruitment of new muscle fibres or muscle hypertrophy. Recently, studies have demonstrated that the growth of larval or early juvenile fish can be improved by nutritional supplements or growth factors in various aquaculture species, including the tilapia (Chang & Lin 1995; Acosta, Morales, Morales, Alonso & Estrada 2007; Acosta, Carpio, Besada, Morales, Sanchez, Curbelo, Ayala & Estrada 2008; Apun-Molina, Santamaria-Miranda, Luna-Gonzalez, Martinez-Diaz & Rojas-Contreras 2009; Meurer, da Costa, de Barros, de Oliveira & da Paixao 2009; Lee et al. 2010; Zhou, Tian, Wang & Li 2010). However, if these improvements in growth rates during early growth stages have no lasting effects on the later stages of fish growth, the strategies of improving skeletal muscle growth during these early life stages of fish may not be feasible to improve the growth rate of aquaculture species, especially with respect to decreasing rearing time to harvest weight. The current results demonstrate that tilapia growth in the adult stage (near harvest weight) is not necessarily influenced by improved growth rate during the early fry stage induced by MSTN1pro treatment, raising a question regarding the utility of treating young fish with MSTN-inhibiting agents to improve fish growth. Future studies therefore should examine whether long-term administration of MSTNinhibiting agents using probably different mode of administration in later growth stages improve the growth of fish. In addition, it needs to be investigated how the earlier growth rate of skeletal muscle influences the eventual growth of skeletal muscle at later stages, or whether the genetic potential of skeletal muscle growth of fast-growing fish, such as tilapia and other aquaculture species, can be modulated by intervention of the growth process during earlier growth stages Blackwell Publishing Ltd, Aquaculture Research, 1 6

5 Aquaculture Research, 2012, 1 6 Effect of myostatin-1 prodomain treatment on tilapia growth Y-S Kim et al. References Acosta J., Carpio Y., Borroto I., Gonzalez O. & Estrada M. P. (2005) Myostatin gene silenced by RNAi show a zebrafish giant phenotype. Journal of Biotechnology, 119, Acosta J., Morales R., Morales A., Alonso M. & Estrada M.P. (2007) Pichia pastoris expressing recombinant tilapia growth hormone accelerates the growth of tilapia. Biotechnology Letters, 29, Acosta J., Carpio Y., Besada V., Morales R., Sanchez A., Curbelo Y., Ayala J. & Estrada M.P. (2008) Recombinant truncated tilapia growth hormone enhances growth and innate immunity in tilapia fry (Oreochromis sp.). General and Comparative Endocrinology, 157, Amali A.A., Lin C.J., Chen Y.H., Wang W.L., Gong H.Y., Lee C.Y., Ko Y.L., Lu J.K., Her G.M., Chen T.T. & Wu J.L. (2004) Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1. Developmental Dynamics, 229, Apun-Molina J.P., Santamaria-Miranda A., Luna- Gonzalez A., Martinez-Diaz S.F. & Rojas-Contreras M. (2009) Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature. Aquaculture Research, 40, Brooks S. & Johnston I.A. (1993) Influence of development and rearing temperature on the distribution, ultrastructure and myosin subunit composition of myotomal muscle-fiber types in the plaice pleuronectes-platessa. Marine Biology, 117, Brown S.C. & Stickland N.C. (1994) Muscle at birth in mice selected for large and small body size. Journal of Anatomy, 184 (Pt2), Byrne I., Hooper J.C. & McCarthy J.C. (1973) Effects of selection for body size on the weight and cellular structure of seven mouse muscles. Animal Production, 17, Carpio Y., Acosta J., Morales R., Santisteban Y., Sanchez A. & Estrada M.P. (2009) Regulation of body mass growth through activin type IIB receptor in teleost fish. General and Comparative Endocrinology, 160, Chang C.F. & Lin S.J. (1995) Immersion in Bovine Insulin Stimulates Growth of Tilapia. Reproduction, Nutrition, Development, 35, Hanrahan J.P., Hooper A.C. & McCarthy J.C. (1973) Effects of divergent selection for body weight on fiber number and diameterin two mouse muscles. Animal Production, 16, Hegarty P.V.J. & Allen C.E. (1978) Effect of pre-natal runting on the postnatal development of skeletal muscles in swine and rats. Journal of Animal Science, 46, Hu S., Chen C., Sheng J., Sun Y., Cao X. & Qiao J. (2010) Enhanced muscle growth by plasmid-mediated delivery of myostatin propeptide. Journal of Biomedicine and Biotechnology, 2010, Lee S.J. (2004) Regulation of muscle mass by myostatin. Annual Review of Cell and Developmental Biology, 20, Lee S.J. & McPherron A.C. (1999) Myostatin and the control of skeletal muscle mass. Current Opinion in Genetics & Development, 9, Lee C.Y., Hu S.Y., Gong H.Y., Chen M.H., Lu J.K. & Wu J.L. (2009) Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish. Biochemical and Biophysical Research Communications, 387, Lee S.B., Kim Y.S., Oh M.Y., Jeong I.H., Seong K.B. & Jin H.J. (2010) Improving rainbow trout (Oncorhynchus mykiss) growth by treatment with a fish (Paralichthys olivaceus) myostatin prodomain expressed in soluble forms in E. coli. Aquaculture, 302, Lee S.B., Cho M.J., Kim J.H., Kim Y.S. & Jin H.J. (2011) Production of bioactive rockfish (Sebastes schlegeli) myostatin-1 prodomain in an Escherichia coli system. Protein Journal, 30, Li Z., Zhao B., Kim Y.S., Hu C.Y. & Yang J. (2010) Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth. Molecular Reproduction and Development, 77, Matsakas A., Foster K., Otto A., Macharia R., Elashry M. I., Feist S., Graham I., Foster H., Yaworsky P., Walsh F., Dickson G. & Patel K. (2009) Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice. Neuromuscular Disorders, 19, Medeiros E.F., Phelps M.P., Fuentes F.D. & Bradley T.M. (2009) Overexpression of follistatin in trout stimulates increased muscling. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 297, R235 R242. Meurer F., da Costa M.M., de Barros D.A.D., de Oliveira S.T. & da Paixao P.S. (2009) Brown propolis extract in feed as a growth promoter of Nile tilapia (Oreochromis niloticus, Linnaeus 1758) fingerlings. Aquaculture Research, 40, Nathanailides C., Lopez-Albors O. & Stickland N.C. (1995) Influence of prehatch temperature on the development of muscle cellularity in posthatch Atlantic salmon (Salmo salar). Canadian Journal of Fisheries and Aquatic Sciences, 52, Powell S.E. & Aberle E.D. (1981) Skeletal muscle and adipose tissue cellularity in runt and normal birth weight swine. Journal of Animal Science, 52, Rebhan Y. & Funkenstein B. (2008) Inhibition of fish myostatin activity by recombinant fish follistatin and myostatin prodomain: potential implications for enhancing muscle growth in farmed fish. Aquaculture, 284, Blackwell Publishing Ltd, Aquaculture Research, 1 6 5

6 Effect of myostatin-1 prodomain treatment on tilapia growth Y-S Kim et al. Aquaculture Research, 2012, 1 6 Stickland N.C. (1983) Growth and Development of Muscle-Fibers in the Rainbow-Trout (Salmo-Gairdneri). Journal of Anatomy, 137, Tsuchida K. (2008) Targeting myostatin for therapies against muscle-wasting disorders. Current Opinion in Drug Discovery & Development, 11, Xu C., Wu G., Zohar Y. & Du S.J. (2003) Analysis of myostatin gene structure, expression and function in zebrafish. Journal of Experimental Biology, 206, Yang J., Ratovitski T., Brady J.P., Solomon M.B., Wells K. D. & Wall R.J. (2001) Expression of myostatin pro domain results in muscular transgenic mice. Molecular Reproduction and Development, 60, Zhou X., Tian Z., Wang Y. & Li W. (2010) Effect of treatment with probiotics as water additives on tilapia (Oreochromis niloticus) growth performance and immune response. Fish Physiology and Biochemistry, 36, Keywords: Myostatin-1, prodomain, growth, tilapia, immersion bath Blackwell Publishing Ltd, Aquaculture Research, 1 6