Department of Zoology, Arts, Science and Commerce College, Manmad, Nashik-423104 (M.S. India) Affiliated to Savitribai Phule Pune  University, Pune

Corresponding author email: drrahulskale@yahoo.co.in

Article Publishing History

INTRODUCTION

Population explosion, pollution, climate change, unplanned management and other factors increases pressure on the agricultural productivity which reduces access to food quantity and quality. So to fulfill the need of the people, aquaculture now a day’s is a good and reliable source particularly crustaceans; which is an important source of aquatic food protein, ornamental and trade which  are extremely important for any developing countries. It provides both economic development and empowerment in terms of contribution to GDP, consumption, employment, catch value and exports. The crustacean sector generates high value export products which enables producers to buy lower value products in the world market  which is a positive contribution to food security in both producing and exporting countries (Bondad-Reantaso et al.  2012). Enhancement of aquaculture entirely depends upon reproduction which is the most fundamental and essential high energy demanding physiological process in living organisms to continue its race in which gonad maturation plays a vital role; whereas some species require external manipulation for it. The gonadal development can be stimulated or inhibited by affecting the neurosecretory cells by many pharmacological agents and eyestalk ablation (Subramoniam 2000; Lafont and Mathieu  2007; Coccia et al. 2010; Alejandra et al. 2011,Yang Lu et al 2018,Raghavan and Ayanath 2019).

Likewise, steroid hormones are biologically active in crustaceans found in the hepatopancreas, ovary and haemolymph which control vitellogenesis (Stevenson et al. 1979; Quackenbush 2001). Vitellogenesis is an important physiological process associated with female reproduction, because the primary source for the developing crustacean embryo is yolk protein and is a pivotal stage during crustacean reproduction. It is the synthesis of yolk proteins i.e. Vitellin (Vn) and Vitellogenin (Vg) which are the two main yolk proteins, that are important nutritive sources, which are necessary for the proper maturation and development of the oocytes (Tseng et al. 2001; Zapata et al. 2003). In decapod crustaceans, hepatopancreas acts as center for storage and / or synthesis of biochemical material which is transferred to sites of gametogenesis for the purpose of growth, maintenance and reproduction (Adiyodi and Adiyodi 1970). The mobilization and accumulation of protein, lipid and glycogen reserves in several tissues have been documented in several crustacean species (Khayat et al. 1994; Harrison 1997; Tseng et al. 2001; Thomas et al. 2005).

In this regard the researchers have reported the role of some vertebrate-type steroids such as β– estradiol and progesterone in ovarian maturation. Reddy et al. (2006) demonstrated that 17α- hydroxyprogesterone hormone induced ovarian growth and ovarian VTG synthesis in the fresh water crab, Oziotelphusa senex senex. Muhd-Farouk et al. (2014) studied effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea and found enhancement in the ovarian maturation. Sujathamma and Dayakar (2015) observed effect of estradiol and 17α-hydroxyprogesterone on ovarian development of fresh water paddy field crab, Oziotelphusa senex senex and found that both hormones were influencing the gonadal growth.

Kale (2017) found significant ovarian maturation by mobilization of protein from hepatopancreas to ovary under the influence of β– estradiol hormone and eyestalk ablation in fresh water crab, B. cunicularis.  Yang Lu et al. (2018) examined the effect of exogenous estrogen on the ovarian development and gene expression in the female swimming crab, Portunus trituberculatus and found that hormone  injection  probably indirectly stimulate the ovarian development and vitellogenesis by mediating the secretion of hormones and gene expression in the endocrine organs.  Raghavan and Ayanath (2019) investigated the effect of ecdysteroids on oogenesis in the freshwater crab,  Travancoriana schirnerae  and found positive results.

The eyestalk is also one of the critical factors known to control crustacean reproduction by a series of inhibitory neurosecretory factors which effectively targets the ovaries and hepatopancreas (Laufer et al. 1998; Aktas et al. 2003). Hussain et al. (2014) studied the effect of unilateral eyestalk ablation in fresh water prawn, Macrobrachium lamarrei and noticed to induce gonadal development. Samyappan et al. (2015) studied impact of unilateral eyestalk ablation on lipid profiles in fresh water female crab, Oziotelphusa senex senex which showed a marked decrease in the hepatopancreas and a significant increase in ovarian tissue indicating ovarian maturation. Sarojini et al. (2016) studied impact of unilateral eyestalk ablation on protein content in freshwater crab, Spiralothelphusa hydrodroma and found significant increase in ovarian maturation.  Recently, Rana (2018) revealed that eyestalk ablation is the alternative technique in induced breeding for rapid ripening of ovaries to enhance meat yield in aquaculture in freshwater crab, Barytelphusa lugubris. Available literature states that ovarian maturation is the process in which there is intense requirement of biochemical constituents like protein, lipid and glycogen which are transported from the storage organs under controlled hormone action.

Present investigation was undertaken to confirm whether 17α-hydroxy progesterone hormone enhance the ovarian maturation in relation to synthesis and mobilization of protein from hepatopancreas to ovary and the results were compared with eyestalk ablation.

MATERIALS AND METHODS

Barytelphusa cunicularis were collected from Godavari  River near Newasa  and were acclimatized for a week in the laboratory. Healthy well-adapted female crabs of early-reproductive phase of approximately same weight and size of intermoult stage ranging between 35-40g body weights were selected for the experiments. Water in the troughs was changed daily and crabs fed by small pieces of earthworm and bivalve on alternate days. Other parameters like temperature, pH, salinity, photoperiod etc., were maintained constant during experiment.

The crabs were divided into four groups, as base control, experimental control, 17a-hydroxy progesterone injected and eyestalk ablated group, each containing 10 crabs. The final concentration of the hormone preparation was 1ml = 1mg. The hormone was injected into the abdominal musculature of arthropodial membrane through 3^rd walking legs, receiving a dose of 20ml /crab with the help of hypodermic syringe having a 27-guaze needle.

Preparation of 17a- hydroxy progesterone hormone injection

10mg of 17a-hydroxy progesterone hormone was dissolved in 1ml of 1% ethanol and resulting solution diluted to 10ml by adding glass distilled water. The final concentration of the hormone preparation was 1ml = 1mg. From this preparation hormone was injected to crab receiving a dose of 20ml hormone/crab. The used hormone was obtained from Sigma Chem. (USA).

The experiment was conducted for a period of 21 days; base control crabs were sacrificed on o-day of the experiment and remaining grouped crabs were sacrificed on 21^st day of the experiment. Ovaries and hepatopancreas were dissected out for estimation of protein levels by Lowry et al. (1951). Protein levels were calculated on dry weight basis and expressed as % mg.

RESULTS AND DiSCUSSION

Table 1: Shows levels of protein (% mg on dry weight basis) in ovary and hepatopancreas of different groups in freshwater crab, B. cunicularis.

Animal Category	No. of Crabs	Exp. Day	Tissue	Protein (%mg)
Group-I Base control (normal)	10	0-day	Ovary	28.81 ± 0.03
			Hepatopancreas	48.12 ± 0.31
Group-II  Experimental control (1% ethanol injected)	10	21-day	Ovary	31.72 ± 0.34
			Hepatopancreas	47.19 ± 0.23
Group-III 17a-OH -P injected crabs	10	21-day	Ovary	45.12 ± 1.07
			Hepatopancreas	36.47 ± 0.82
Group-IV Eyestalk ablated crabs	10	21-day	Ovary	50.38 ± 0.28
			Hepatopancreas	26.98 ± 0.89

± S. D.: Mean Standard Deviation

17a-OH –P: 17 a- hydroxy progesterone hormone

Figure 1: Comparative levels of protein (% mg on dry weight basis) in ovary and hepatopancreas of different groups in freshwater crab, B. cunicularis

Protein levels in the ovary were found to be 28.81 %, 31.72 % and 45.12 % for base control, experimental control and 17α-hydroxy progesterone injected respectively. Eyestalk ablated crabs showed 50.38 % protein level. While protein content in hepatopancreas were noted to be 48.12 %, 47.19 % and 36.47 % base control, experimental control and 17α-hydroxy progesterone injected crabs respectively. Eyestalk ablated crabs showed 26.98 % of protein level. Increased protein level in eyestalk ablated group was prominent when compared to protein levels obtained in hormone injected.

Biochemical studies are very important from the reproductive as well as the nutritive point of view in any edible crustacean animals particularly protein content. Protein plays very important role in the growth and maintenance of the animals and it is easily digestible too. In the present study (Table 1 and Fig. 1) the protein levels in the ovary were found to be 28.81%, 31.72% and 45.12% for base control, experimental control and 17α-hydroxy progesterone injected respectively. Whereas eyestalk ablated crabs ovary showed 50.38% protein level. While protein content in hepatopancreas were noted to be 48.12%, 47.19% and 36.47% base control, experimental control and 17α-hydroxy progesterone injected crabs respectively. Eyestalk ablated crabs showed 26.98% of protein level.

The present findings indicated that hormone injections and eyestalk ablation showed enhancement in the ovarian maturation by synthesizing or and mobilization of the protein and other  biochemical constituents from hepatopancreas to ovary over the base control and experimental control which seems reasonable that decreases were a consequence of accelerated transport to maturing ovaries in which proteins and lipids are accumulated  and hepatopancreas may be the source for these constituents circulated through the haemolymph (Kulkarni et al. 1979; Teshima et al. 1988; Okumura and Aida 2001). Similarly, the role of steroid hormones in controlling the process of reproduction has been studied by (Yano 1987; Quackenbush 1989a; Subramoniam 2017).

In the same ray of light  Tsukimura (2001) studied the effect of 17 β-estradiol, progesterone and 17 α-hydroxyprogsterone on ovarian maturation of crustaceans and found positive correlation to protein synthesis and its uptake for the maturation in  ridgeback shrimp, Sicyonia ingentis. Zapata et al. (2003) observed ovarian growth in the crab, Chasmagnathus granulata induced by 17a-hydroxyprogesterone and Juvenile hormone III.   Sujathamma and Dayakar (2015) in their studies examined the effect of estradiol and 17α-hydroxyprogesterone on ovarian development of fresh water paddy field crab, Oziotelphusa senex senex  and found that both hormones promotes the gonadal growth and reproduction in female crab. Muhd-Farouk et al. (2016) noticed that the administration of 17α-OH pregnenolone as well as 17α-OH progesterone stimulated ovarian index, oocyte diameter and ovarian maturity in S. olivacea

Kale (2017) studied the influence of β– estradiol hormone and eyestalk ablation on protein metabolism in fresh water crab, Barytelphusa cunicularis and found ovarian maturation due to mobilization of protein from hepatopancreas to ovary.

Recently, Raghavan and Ayanath (2019) investigated effect of ecdysteroids on oogenesis in the freshwater crab, Travancoriana schirnerae study indicated that 20-OH ecdysone can stimulate ovarian growth and maturation in all phases of the oogenic cycle.

Present studies also reveals that decrease in protein level in hepatopancreas and  increased protein level of ovary in eyestalk ablated group was prominent over hormone injected crabs. This may be due to the eyestalk ablation; because neuroendocrine system has long being known to play a significant role in gonadal maturation, and or moulting (Quackenbush 1986; Fingerman 1987).  In the same way Arcos et al. (2003) reported similar results in shrimp, Litopenaeus vannamei suggesting decreases in total protein, cholesterol and triglycerides in haemolymph and ovarian maturation as an effect of eyestalk of eyestalk ablation. Murugesan et al. (2008) noticed eyestalk ablation influenced protein, lipid and carbohydrate content in ovarian maturation of crab, Charybdis lucifera.  Varalakshmi and Reddy (2010) found significant changes in carbohydrate, protein and energy leading to ovarian maturation in Macrobrachium lanchesteri due to eyestalk ablation. Unilateral eyestalk ablation in freshwater female crabs, Oziotelphusa senex senex showed marked decrease in lipid classes in the hepatopancreas but significant increase in ovarian tissue which suggests ovarian maturation (Samyappan et al. 2015).

Lee et al. (2017) and Guan et al.( 2017) have also mentioned that eyestalk ablation is considered as the most effective method to facilitate molting and quick ovarian maturation in captive economic crustacean culture. Rana (2018) studied the effect of eyestalk ablation in freshwater crab, Barytelphusa lugubris  and found that eyestalk ablation not only promoted the process of moulting but it also amplified the process of ovarian maturation. Similarly, Shabnam Banoo et al. (2018) also noticed increased gonadosomatic indices and gonad maturation of freshwater crab, Paratelphusa hydrodromous due to unilateral eyestalk ablation. Mhd Ikhwanuddin et al. (2019) found increase in gonadosomatic index and mean oocyte diameter which indicated ovarian maturation due to eyestalk ablation of blue swimming crab, Portunus pelagicus. But, Nagathinkal et al. (2017) studied molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis. Their results indicate that throughout the annual cycle, both the somatic and the reproductive growth of U . triangularis are under the influence of inhibitory principles from the eyestalks. It is also revealing that mere deprival of the inhibitory principles does not culminate into successful vitellogenesis. Arguably, the inhibitory influence from the eyestalks could be a prerequisite for normal healthy maturation of the oocytes and spawning.

In conclusion, from the available literature and results it seems that transfer of protein from hepatopancreas to ovary for its maturation was due to eyestalk ablation. It showed significant progress over experimental and hormone injected crabs which suggests that hormonal manipulation of crustacean reproduction is limited to eyestalk ablation for the induction of ovarian maturation till date and a new technological advances in such type of hormonal manipulation and eyestalk ablation for understanding of crustacean endocrinology is essential for application of management programs for commercially important species in aquaculture.

ACKNOWLEDGEMENTS

Author is very much thankful to Prof. (Dr.) K.B. Shejule, Head, Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad. My special thanks to the management of Mahatma Gandhi Vidyamandir Nashik and Principal, Vice-Principal of Arts, Science and Commerce College Manmad, Tal. Nandgaon, Dist. Nashik.

CONFLICT of INTEREST

There is no conflict of interest towards this manuscript

REFERENCES

Adiyodi, K. G. and Adiyodi, R. G. (1970) Endocrine control of reproduction in decapod crustacea. Biol. Rev., 46, 121-165.

Aktas M, Kumlu M and Eroldogam OT (2003) Off-season maturation and spawning of Penaeus semisulcatus by eyestalk ablation and/or temperature-photoperiod regimes. Aquaculture, 228, 361-370.

Alejandra, A. V. C., Daniel A. M., Anouk, C. and Enrique, M. R. (2011) In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle. Comparative Biochemistry and Physiology – Part A: Molecular & Integrative Physiology, 158(1), 126-131.

Arcos, G. F., Ibarra, A.  M., Vazquez-Boucard, C., Palacios, E. and   Racotta, I. S. (2003) Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp, Litopenaeus vannamei Boone. Aquaculture Research, 34, 749-755.

Coccia E, De Lisa E, Di Cristo C, Di Cosmo A and Paolucci M. (2010) Effects of estradiol and progesterone on the reproduction of the freshwater Crayfish, Cherax albidus. Biol. Bull., 218, 36–47.

Fingerman, M. (1987) Endocrine mechanisms of crustaceans. J. Crust. Biol., 7(1), 1-24.

Guan, Z. B., Zhao, H. X. H., Shui, Y., Cai, Y. J. and Liao, X. R. (2017) Cell division cycle 2 participates in eyestalk ablation-induced ovarian maturation of Procambarus clarkii. Aquacult, 468, 115-119.

Harrison, K. E. (1997) Broodstock nutrition and maturation diets. In: crustacean nutrition. Advances in world aquaculture Vol. 6 (L. R. D’Abramo, D. E. Conklin and D. M. Akiyama, eds), pp. 390-408. The world aquaculture society, Baton Rouge, Louisiana, USA.

Hussain S, Borana K and Sujad N. (2014) Effect of eyestalk ablation on reproductive organ of Macrobrachium lamarrei lamarrei (H. Milne Edwards, 1837). IAJPR, 4(4), 2247-2250.

Kale, R.S. (2017) Effect of β-estradiol hormone and eyestalk ablation on ovarian maturation in fresh water crab, Barytelphusa cunicularis. Bioscience Discovery, 8(2),140-145.

Khayat, M., Shenker, O., Funkenstein, B., Tom, M., Lubzens, E., Tietz, A. (1994) Fat transport in the penaeid shrimp, Penaeus semisulcatus (De Hann). Isr. J. Aquacult., 46, 22-32.

Kulkarni, G. K., Nagabhushanam, R. and Joshi, P. K. (1979) Effect of progesterone on the ovarian maturation of a marine penaeid prawn, Parapenaeopsis hardwickii. (Miers) (Crustacea, Decapoda, Penaeidae). Ind. J. Expt. Biol., 17, 986-987.

Lafont, R and Mathieu M. (2007) Steroids in aquatic invertebrates. Ecotoxicology ,16:109–130.

Laufer, H., Bigger, W.J. and Ahl J.S.B. (1998) Stimulation of ovarian maturation in the crayfish, Procambarus clarkii by methyl farnesoate. Gen. Comp. Endocrinol., 111, 113-118.

Lee, J. H., Suryaningtyas, I. T., Yoon, T. H., Shim, J. M., Park, H., and Kim, H. W. (2017) Transcriptomic analysis of the hepatopancreas induced by eyestalk ablation in shrimp, Litopenaeus vannamei. Comp. Biochem. Physiol. Part D Genomics Proteomics, 24, 99-110.

Lowry, O. H., Rosebrough, H. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Foline phenol reagent. J. Biol. Chem., 193, 265-275.

Bondad-Reantaso, M. G.,  Rohana P. Submarine, Helga Josupeit, Junning Cai, Xiaowei Zhou. (2012) The role of crustacean fisheries and aquaculture in global food security: Past, present and future. Journal of Invertebrate Pathology, 110, 158–165.

Mhd Ikhwanuddin, Mohd-Fadhil Adnan, Syahnon Mohamad and Ambok Bolong Abol-Munafi (2019) Effect of Eyestalk Ablation on the Ovarian Maturation Stages of  blue swimming crab, Portunus  pelagicus. Asian  J. Biol. Sci., 12(3), 437-441.

Muhd-Farouk H, Abol Munafi AB, Jasmini S and Ikhwanuddin M. (2014) Effect of steroid hormones 17 α – hydroxyprogesterone and 17 α – hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology, 9(1), 23-28.

Muhd-Farouk H, Jasmani S, Ikhwanuddin M. (2016) Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture, 451, 78–86.

Murugesan,  R., P. Soundarapandian and K. Manivannan (2008) Effect of unilateral eyestalk ablation on the biochemical changes of edible portunid crab, Charybdis lucifera  (Fabricus). J. Fish. Aquat. Sci., 3(1), 82-86.

Okumura, T. and Aida K. (2001) Effects of bilateral eyestalk ablation on molting and ovarian development in the giant freshwater prawns, Macrobrachium rosenbergii. Fish. Sci., 67, 1125-1135.

Quackenbush, L.S. (2001) Yolk synthesis in the marine shrimp, Penaeus vannamei. Amer. Zool., 41, 458-464.

Quackenbush, L. S. (1986) Crustacean endocrinology: A review. Can. J. Fish. Aqua. Sci., 43, 2771-2282.

Quackenbush, L. S. (1989a) Vitellogenesis in the shrimp, Penaeus vannamei: In vitro studies of the isolated hepatopancreas and ovary. Comp. Biochem. Physiol., 94B, 253–261.

Rana, S. (2018)  Eyestalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding.  Int. J. Pure Appl. Zool., 6(3), 30-34.

Reddy, P.R, Kiranmayi, P.K, Kumari, T. and Reddy, P.S. (2006) 17β -Hydroxyprogesterone induced ovarian growth and vitellogenesis in the freshwater rice field crab, Oziotelphusa senex senex. Aquaculture, 254,768–775.

Samyappan, K., Saravanan, R., Vijayakanth, T. and Prabakaran, D. (2015) Impact of unilateral eyestalk ablation on lipid profiles in freshwater female crab, Oziotelphusa senex senex. Asian Journal of Science and Technology, 6,1474-1478.

Sarojini, N., Bharathi, A. and Padmaja, M. (2016) Impact of unilateral eyestalk ablation on protein content in freshwater crab, Spiralothelphusa hydrodroma. G.J.B.B., 5 (1), 31-34.

Shabnam Banoo, Showkat Hussain Rather, Mohd Waseem  (2018)  Effect of unilateral eye stalk ablation (ULEA) on gonadosomatic indices and gonad maturation of freshwater crab, Paratelphusa hydrodromous (Henderson 1893)  International Journal of  Zoology Studies,  3(2), 289-292.

Stevenson, J.R., Armstrong, P.W., Cheng, E.S. and O’Conner, J.D. (1979) Ecdysone titers during molt cycle of the crayfish, Orconectes samborni. Gen. Comp. Endocrinol., 39, 20-25.

Subramoniam, T. (2000) Crustacean ecdysteroids in reproduction and embryogenesis. Comparative Biochemistry and Physiology,125,135-156.

Subramoniam, T. (2017) Steroidal Control of Vitellogenesis in Crustacea: A New understanding for improving shrimp hatchery production. Proc Indian Natn. Sci.  Acad., 83(3), 595-610.

Raghavan Sudha Devi Arath and Aswani Ayanath  (2019) Effect of ecdysteroids on oogenesis in the freshwater crab, Travancoriana schirnerae Bott, 1969 (Crustacea: Gecarcinucidae). Brazilian Journal of Biological Sciences, 6(12), 87-101.

Sujathamma, A. and Dayakar, Y. (2015) Effect of estradiol and 17α-hydroxyprogesterone on ovarian development of fresh water paddy field crab, Oziotelphusa senex senex (Fabricius). European Journal of Experimental Biology, 5(9), 27-30.

Supriya Nagathinkal, T., Sudha Kappalli, Krishnakumar Velayudhannair, Anilkumar Gopinathan  (2017) Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chin. J. Oceanol. Limnol., 35(3), 645-657.

Teshima, S. I., Kanazawa, A., Koshio, S. and Horinouchi, K. (1988) Lipid metabolism in destalked prawn, Penaeus japonicus: induced maturation and transfer of lipids reserves to the ovaries. Nippon Suisan Gakkaishi, 54, 1123-1129.

Thomas, S., Eswaralakshmi, R., Jayanthi, J. and Ragunathan, M. G. (2005)  Biochemical mobilization in an eyestalk ablated male freshwater crab, Paratelphusa hydrodromous due to certain neurotransmitters. J. Aqua. Biol., 20(1), 63-66.

Tseng, D., Chen, Y., Kou, G., Lo, C. and Kou, C. (2001) Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon. Comp. Biochem. Physiol., 129A, 909-917.

Tsukimura, B. (2001) Crustacean vitellogenesis: Its role in oocyte development. Amer. Zool., 4, 465–476.

Varalakshmi, K.N. and Reddy, R. (2010) Effects of eyestalk ablation on growth and ovarian maturation of the fresh water prawns, Macrobrachium lanchesteri. Turk. J. Fish. & Aquatic Sci., 10, 403-410.

Yang, Lu., Meimei Liu, Jie Gong, Yongxu Cheng and Xugan, Wu. (2018) Effect of exogenous estrogen on the ovarian development and gene expression in the female swimming crab, Portunus trituberculatus (Miers, 1876) (Decapoda: Brachyura: Portunidae). Journal of Crustacean Biology, 38(3), 367–373.

Yano, I. (1987) Effect of 17a-hydroxyprogesterone on vitellogenin secretion in Kuruma prawn, Penaeus japonicus. Aquaculture, 61, 49–57.

Zapata, V., Lopez Greco, L.S., Medesani, D., Rodriguez, E.M. (2003) Ovarian growth in the crab, Chasmagnathus granulata induced by hormones and neuroregulators throughout the year. In vivo and in vitro studies. Aquaculture, 224(1-4), 339-353.