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74.6±3.39 b


0.98±0.04 b


































Values are means ±S.D. Values with the same superscripts within the same row are not significantly different (P < 0.05). 1See Table 1 for diet abbreviations. 2FCR, food conversion ratio;

3SGR, specific growth rate; 4HIS, Hepatosomatic index;

5VSI, viscerosomatic index; 6CF, Condition factor.


Recent studies on concentrated plant protein inclusion in rainbow

trout diet showed that it can potentially replace whole dietary fish meal with either no reduction or just a slight reduction in growth (Kaushik et al. 1995; Barrows, Gaylord, Stone & Smith 2007), which is in agreement with our results. Results showed that complete substituting fish meal with plant ingredients did not adversely affect fish growth. However this is in accordance with replacing 30 and 35% fish meal with wheat gluten in Atlantic salmon and Atlantic halibut respectively (Helland and Grisdale-helland 2006) and 50% fish meal with corn gluten in Atlantic salmon

(Mente et al. 2003).

There are several explanations for undesirable effects of higher levels of plant derived ingredients in salmonids diet such as higher carbohydrate content which is not generally well digested by salmonids (Singh and Nose 1967). It is not conceivable that carbohydrate could have noticeable effects on fish growth in this study, since all diets had balanced carbohydrate content. We incorporated wheat and corn gluten because of their higher content of protein, lower amounts of fiber and starch and relatively void of any ANFs (Robaina et al. 1997). Moreover, wheat and corn gluten have proved higher digestibility coefficients (99 and 95-96%) in salmonids (Pfeffer et al. 1995).

Essential amino acids are necessary for optimal growth rate and better fish performance (Halver & Hardy 2002). Another problem arisen from higher plant derived ingredients in aquafeed is that many plant meals contain lower protein and essential amino acids compared to fish meal. Gluten based protein contains high protein levels but it is deficient in some essential amino acids such as lysine and methionine (Regost et al. 1999). As lysine and methionine were supplemented to the plant based experimental diets in present study, any amino acids deficiency as growth inhibitor is overruled. Fish fed different diets did not show any significant differences in hepatosomatic index which is in compliance with some existing literature on rainbow trout (Palmegiano et al. 2006; Drew et al. 2007). Similarly there were no significant differences in VSI amongst fish of different dietary groups, which may be attributed to the shorter experimental period in this study. In contrast to our findings, 24 week and 96 days experiments on inclusion of plant protein in rainbow

diet (Francesco et al. 2004, Palmegiano et al. 2006, respectively) resulted in significantly higher VSI.

Replacement of fish oil with vegetable oil at all levels of plant protein inclusion had no significant effect on salmonid fish growth, feed utilization and muscle proximate composition (Torstensen et al. 2005). In the present study, a mixture of linseed, canola, sunflower and safflower oil were used to replace fish oil. These oils were chosen because they are rich in n-3 fatty acid, alpha linolenic acid in linseed and canola oil (53% and 12% respectively; NRC 1993), and n-6 fatty acid, 18:2n-6 in safflower oil and sunflower oil. However, they are devoid of EPA (20:5n-3) and DHA (22:6n-3).

With reference to the main aims of this study and the results obtained, we may conclude that whole dietary fish meal and 80% of fish oil in rainbow trout diet could be replaced with plant sources without significant negative effects on growth indices and feed efficiency.


This study was supported by Artemia and Aquatic Animals Research Institute and Faculty of Natural Resources of Urmia University, Iran.


Barrows F.T., Gaylord T.G., Stone D.A.J. & Smith C.E. (2007) Effect of protein source and nutrient density on growth efficiency, histology and plasma amino acid concentration of rainbow trout (Oncorhynchus mykiss Walbaum). Aquaculture Research 38, 1747-1758.

Drew M.D., Ogunkoya A.E., Janz D.M. & Van Kessel A.G. (2007) Dietary influence of replacing fish meal and oil with canola protein concentrate and vegetable oils on growth performance, fatty acid composition and organochlorine residues in rainbow trout (Oncorhynchus mykiss). Aquaculture 267, 260-268.

Francesco M., Parisi G., Medale F., Kaushik S.J. & Poli B.M. (2004) Effect of long

term feeding with a plant protein mixture based diet on growth and body/fillet quality traits of large rainbow trout (Oncorhynchus mykiss). Aquaculture 263, 413-429.

Francis G., Makkar H.P.S. & Becker K. (2001) Anti-nutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture 199,


Halver J.E. & Hardy R.E. (2002) Nutrient flow and retention In Fish Nutrition, 3rd edn, pp. 768-769. Academic Press, Elsevier Science, San Diego, California, USA.

Helland S.J. & Grisdale-helland B. (2006) Replacement of fish meal with wheat gluten in diets for Atlantic halibut ( Hippoglos sushippoglossus ): Effect on whole-body amino acid concentrations. Aquaculture 261:1363 - 1370.

Kaushik S.J., Cravedi J.P., Lalles J.P., Sumpter J., Fauconneau B. & Laroche M. (1995) Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout (Oncorhynchus mykiss). Aquaculture 133, 257-274.

NRC (1993) Nutrient Requirements of Fish. National Academy Press, Washington, DC.

Oo A.N., Satoh S. & Tsuchida N. (2007) Effect of replacements of fishmeal and fish oil on growth and dioxin contents of rainbow trout. Fisheries Science 750-759.

Palmegiano G.B., Dapra F, Forneris G., Gai F., Gasco L., Guo K., Peiretti P.G., Sicuro B. & Zoccarato I. (2006) Rice protein concentrate meal as a potential ingredient in practical diets for rainbow trout (Oncorhynchus mykiss). Aquaculture


Pfeffer E., Kinzinger S. & Rodenhutscord M. (1995) Influence of the proportion of poultry slaughter by-products and of untreated or hydrothermically treated legume seeds in diets for rainbow trout, Oncorhynchus mykiss Walbaum , on apparent digestibilities of their energy and organic components. Aquaculture Nutrition 1, 111-117.

Regost C., Arzel J. & Kaushik S. J. (1999) Partial or total replacement of fish meal by corn gluten meal in diet for turbot (Psetta maxima). Aquaculture 180:99-117.

Robaina L., Izquierdo M.S., Moyano F.J., Socorro J., Vergara J.M., Montero D. & Fernandez-Palacios H. (1995) Soybean and lupin seed meals as protein sources in diets for gilthead seabream (Sparus aurata): nutritional and histological implications. Aquaculture 130, 219-233.

Robaina L., Moyano F.J., Izquierdo M.S., Socorro J., Vergara J.M. & Montero D. (1997) Corn gluten meal and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): nutritional and histological implications.

Aquaculture 157, 347-359.

Singh R.P. & Nose T. (1967) Digestibility of carbohydrates in young rainbow trout. Bull. Freshwater Fish. Res. Lab 17, 21-25.

Sokal R.R. & Rohlf F.J. (1969) Biometry. Freeman, San Francisco, CA, USA.

Torstensen B.E., Bell J.G., Sargent J.R., Rosenlund G., Henderson R.J., Graff I.E., Lie 0. & Tocher D.R. (2005) Tailoring of Atlantic salmon (Salmo salar L.) flesh lipid composition and sensory quality by replacing fish oil with a vegetable oil blend. J. Agric. Food Chemistry 53, 10166-10178.

Yamamoto T., Sugita T. & Furuita H. (2005) Essential amino acid supplementation to fish meal-based diets with low protein to energy ratios improves the protein utilization in juvenile rainbow trout Oncorhynchus mykiss. Aquaculture 246,


Effect Of Dietary Bovine Lactoferrin On Rainbow Trout (Oncorhynchus Mykiss) Fecundity And Egg


Eslam Ahmadian1, Naser Agh2, Amir Tokmechi2, Reza Jalili1, Sirwe Ghaderpour1

1. Department of Fisheries, Faculty of Natural Resources, Urmia University, Urmia, Iran.

2. Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran.


The primary objective of salmonid brood stock management programme is the production of the maximum number of high quality eggs and fry from available broodstock. It is well known that fecundity and quality of eggs is related not only to the genetics of broods but also to their rearing condition such as feeding and disease(Okumus, 2002). Improvement in broodstock nutrition and feeding has shown to greatly improve the egg and sperm quality, gonadal development and fecundity(Izquierdo et al., 2001). Successful seed production also demands a thorough understanding of the special husbandry and nutritional requirements of broodstock fish, because diet and management procedures can have significant effects on fecundity, egg size and egg and larval quality or survival.( Marimuthu et al., 2009). In the other hand, Fish egg quality can be affected by maternal age, condition factor, the timing of the spawning cycle, genetic factors, overripening processes and Stress. (Kjorsvik et al., 1990; Brooks et al., 1997; Coban et al., 2011). Lactoferrin has been used as an immunostimulant in different fish species(Rahimnejad et al., 2011).

because of high cost of broodstock produce and maintenance, The present study was conducted to determine the effects of Lactoferrin on rainbow trout broodstock fecundity and egg composition.

Materials and methods

Rainbow trout 4 year old broodstock were provided by Artemia and

Aquatic Animals Research Institute farm in Urmia, Iran. fourty eight randomly captured broodstock(24 males and 24 females) were distributed into 4 groups of the 4 rectangular polyethylene tanks of 1000 L capacity containing 800 L of well water supplied at a flow rate of 5 L min1. The experiments were performed in a flow-through system. Commercial trout feed (crude protein: 38%, crude lipid: 12%, ash: 10%, fiber:3.5%) (faradaneh., shahrkord, Iran)) was crushed first and then supplemented with bovine Lf (Biopole SA, Les Isnes, Belgium, Lot No. LODB10) at levels of 0(control), 100, 200 and 300 mg kg1 diet. The diets were repelleted using a lab scale extruder to obtain 8 mm sized pellets. Diets were dried at oven (40°C ), and maintained in a freezer at 4°C until used. The fish were fed the experimental diets to apparent satiation once daily for 90 day.Data were analysed using one-way analysis of variance (ANOVA). The Duncan's test was used to determine significant differences among groups (P < 0.05).


Results of this study showed that total fecundity was significantly higher in fish fed 200 mg kg1 Lf compared to the control group (P < 0.05), but no significant differences among fish fed three different levels of Lf. There was no significant difference in individual weights of the eggs produced by experimental fish compared to the control. Absolute fecundity was significantly higher in fish fed Lf compared to the control group (P < 0.05), but no significant differences among fish fed three different levels of Lf. Relative fecundity was significantly higher in fish fed 200 and 300 mg kg1 Lf compared to the control (P < 0.05). Results of this study also showed that bovine Lf supplementation decreases spawning time in rainbow trout especially in those fed 300 mg kg1 Lf (Table.2).

In this study, the lowest moisture were observed in the group fed the diet supplemented with 100 mg Lf kg1 and the highest were observed in the group fed the diet supplemented with 300 mg Lf kg-1. However, the differences of moisture were not significant (Table.3). mean ash significantly decreased in the groups fed the diet supplemented with 100,

200 and 300 mg Lf kg-1 compared to the control group(Table.3). Crude protein significantly increased in the group fed the diet supplemented with 200 mg Lf kg-1 compared to the control group and group fed the diet supplemented with 100 mg Lf kg-1 (Table.3).Crude lipid significantly increased in the groups fed the diet supplemented with 100, 200 and 300mg Lf kg-1 compared to the control group (Table.3).

Table 1.Mean ± std. Deviation, Measured parameters of rainbow trout broodstock (Duncan test P < 0.05)


Fish early weight(g)

Fish final



fecundity (g)

Eggs weight(g)

Absolute fecundity(N)

Relative fecundity (N/kgbw)



2777 ± 153 a

304.8 ± 57 b

0.067±0.007 a

4562 ± 850 b


100 mg kg1


3128 ± 481 a

397.5 ± 53 ab

0.066±0.008 a

6731 ± 1761 a

2197±660 ab

200 mg kg1


2765 ± 540 a

424 ± 65 a

0.068±0.01 a

6842 ± 669a


300 mg kg1


2760 ± 621 a

391.1± 84 ab

0.069±0.008 a



Table 2. Percent of broodstock spawning in each week


Week 1

Week 2

Week 3

Weeek 4

Week 5









100 mg kg1







200 mg kg1







300 mg kg1







Table 3.Mean ± std. Deviation, Measured parameters of rainbow trout broodstock (Duncan test P < 0.05)




Crude protein(%)

Crude lipid(%)


59.22 ± 2.09 a

13.67 ± 0.76 a

71.24 ± 0.68 b

13.66 ± 0.58 b

100 mg kg1

58.10 ± 0.40 a

11.21 ± 0.84 b

71.76 ± 0.54 b

15.42 ± 0.18 a

200 mg kg1

58.89 ± 0.82 a

10.15 ± 0.53 b

72.73 ± 0.79 a

15.78 ± 1.08 a

300 mg kg1

59.49 ± 1.52 a

11.23 ± 0.97 b

72.04 ± 0.83 ab

15.38 ± 0.74 a

It is clear that reproduction is very sensitive to stress (Okumus, 2002) and stress reduces the quality of gametes produced by rainbow trout (Campbell et al., 1992). Successful seed production also demands a thorough understanding of the special husbandry and nutritional requirements of broodstock, because diet and management procedures can have significant effects on fecundity, egg size and egg and larval quality or survival (Marimuthu et al., 2009).

The overall health status of brood fish can affect breeding performances, quality seed production and protection of offsprings. However, factors such as age, maturation, reproductive behaviour and nutrition (micro and macro-nutrients) may affect the immunity in brood fishes. (Swain and Nayak, 2009).

There was no significant difference in individual weights of the eggs produced by experimental fish compared to the control. These results are in agreement with previous reports on; effects of broodstocks size on egg quality in African catfish (Clarrias gariepinus) (Sule and Adikwu, 2004), Oreochromis niloticus (macintosh,1985) and Pseudopleuronectes americanus(Buckley et al., 1991) and effects of broodstocks age on eggs weight and size in Caspian brown trout (Salmo trutta caspius) (Rahbar et al., 2011). Several reasons have been suggested that influence egg size including depletion of body reserves (Kjesbu et al., 1991; Hsiao et al., 1994; Chambers and Waiwood, 1996) or endogenously influenced hormonal profiles (Kjesbu et al., 1996; Kennedy et al., 2007). However, In the current study bigest egg were seen in groups receiving 100, 200 and 300mg Lf kg-1 compared to the control group. In the current study, relative Fecundity significantly increased in the group receiving 200 and 300 mg Lf kg-1 compared to the control group and absolute Fecundity significantly increased in the group receiving 100, 200 and 300 mg Lf kg-1 compared to the control group. Total Fecundity significantly increased in the group receiving 200mg Lf kg-1 compared to the control group. There is no published documentation on effect of Lf on Fecundity on brood fish. Its positive effect may bedue to immunoregulatory and

stress resistance role of Lf (Tomita et al., 2002; Lonnerdal 2009; Yokoyama et al., 2006).These results are agreement with previous reports on effect of stress on reproduction (Okumus, 2002) and gametes quality and quantity of rainbow trout (Campbell et al., 1992). In the other hand, it could be suggested that effect of Lf on stress reduction (Yokoyama et al., 2006), stimulating immune response (Ringo et al., 2012; Kamilya et al., 2006; Chand et al., 2006; Rahimnejad et al., 2011), and regulation of iron absorption (Welker et al., 2007; Ringo et al., 2012), may have a role in improving Fecundity.


Brooks, S., Tyler, C.R. and Sumpter, J.P. 1997: Egg quality in fish: what makes a good egg? Reviews in Fish Biology and Fisheries, 7: 387—416.

Buckley, J., Smigielski, A.S., Halavik, T.A., Caldarone, E.M., Burns, B.R and Laurence, G.C., 1991: Winter flounder (Pseudopleuronectes americanus) reproductive success. Effects of spawning time and female size on size, composition and viability of eggs and larvae. Marine Ecology Progress Series,

74: 125-135.

Campbell, P.M., Potfinger, T.G and Sumpter, J.P., 1992: Stress Reduces the Quality of Gametes Produced by Rainbow Trout. biology of reproduction, 47: 1140­1150.

Cecchini, S., Caputo, Anna R., 2010: Seminal plasma of brown trout, Salmo trutta fario (L.) contains a factor able to retain iron at acid pH, typical feature of lactoferrin. Fish & Shellfish Immunology 28: 927-930.

Chand, R.K., Sahoo, P.K., Kumari, J., Pillai, B.R and Mishra B.K., 2006: Dietary

administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress. Fish and Shellfish Immunology, 21: 119-129.

Coban, D., Kamaci, H.O., Suzer, C., Yildirim,     Arda, G., Korkut, A.Y., Saka,

Firat, K., 2011: Effect of Some Morphometric Characteristics on Egg Quality in Common Dentex, Dentex dentex (Linnaeus, 1758). Turkish Journal of Fisheries and Aquatic Sciences, 11: 425-431.

Izquierdo, M.S., Fernandez-Palacios, H. and Tacon, A.G.J., 2001: Effect of broodstock nutrition on reproductive performance of fish. Aquaculture, 197: 25—42.

Kamilya D., Ghosh D., Bandyopadhyay S., Mal B.C and Maiti T.K., 2006: In vitro

effects of bovine lactoferrin, mushroom glucan and Abrus agglutinin on Indian major carp, catla (Catla catla) head kidney leukocytes. Aquaculture, 253: 130­139.

Kennedy, J., Geffen, A.J., Nash, R.D.M., 2007: Maternal influences on egg and larval characteristics of plaice (Pleuronectes platessa L.). Journal of Sea Research 58: 65—77.

Kjesbu, O.S., Klungsoyr, J., Kryvi, H.,Witthames, P.R.,Walker,M.G., 1991: Fecundity, atresia and egg size of captive Atlantic cod in relation to proximate body condition. Can. J. Fish. Aquat. Sci. 48: 2333-2343.

Kjorsvik, E., Mangor-Jesen, A., Holmefjord, I., 1990: Egg quality in fishes. Advances in Marine Biology, 26: 71-113.

Lombardo, F., Gioacchini, G and Carnevali, O,. 2011: Probiotic-Based Nutritional Effects on Killifish Reproduction. Fisheries and Aquaculture Journal, Vol. 2011:


Macintosh, D.J and Little, D.C., 1995: Nile tilapia (Oreochromis niloticus), Pages 277­320 In: Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality, Blackwell Science Publication, University Press, Cambridge, UK.. Lonnerdal, B., 2009: Nutritional roles of lactoferrin. Current Opinion in Clinical Nutrition& Metabolic Care, 12: 293-297.

Marimuthu, K., Jesu Arokiaraj, A. and Haniffa, M.A., 2009: Effect of Diet Quality on Seed Production of the Spotted Snakehead (Channa Punctatus) (Bloch) American-Eurasian Journal of Sustainable Agriculture, 3(3): 344-347, ISSN


Okumus, I., 2002: Rainbow Trout broodstock management and seed production in Turkey: present practices, constraints and the future. Turkish journal of fisheries and aquatic scinces, 2: 41-56.

Rahimnejad, S., Agh, N., Kalbassi, M. R and Khosravi, S., 2011: Effect of dietary bovine lactoferrin on growth,haematology and non-specific immune response in rainbow trout (Oncorhynchus mykiss). Aquaculture Research, 2011: 1-9.

Rahbar, M., Nezami, Sh., Khara, H., Rezvani, M., Khodadoust, A., Movahed, R and Eslami S., 2011: Effect of age on reproductive performance in female Caspian brown trout (Salmo trutta caspious, Kessler 1877) .Caspian J. Env. Sci, Vol. 9

No.1 pp. 97-103.

Ren T., Koshio S., Ishikawa M., Yokoyama S., Micheal F.R., Uyan Q and Tung H.T., 2007: Influence of dietary vitamin C and bovine lactoferrin on blood chemistry and non-specific immune responses of Japanese eel, Anguilla japonica. Aquaculture, 267: 31-37.

Ringo, E., Olsen, R.E., Gonzalez Vecino, J. L., Wadsworth, S and Song, S.K., 2012: Use of immunostimulants and nucleotides in aquaculture: a review. Marine Science Research & Development, 2:1.

Sule, O.D and Adikwu, I.A., 2004: Effect of broodstock size on egg and larval size and survival of larvae of the African catfish, Clarias Garipinus, under laboratory conditions. Journal of Aquatic Sciences, 19(1): 1-4.

Swain, P and Nayak S.K., 2009: Role of maternally derived immunity in fish. Fish & Shellfish Immunology, 27: 89-99.

Tomita, M., Wakabayashi, H., Yamauchi, K., Teraguchi, S and Hayasawa, H., 2002: Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochemistry and Cell Biology, 80: 109-112.

Welker, T.L., Lim, C., Yildirim-Aksoy, M and Klesius, P.H., 2007: Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine Lactoferrin. Aquaculture, 262: 156-162.

Yokoyama, S., Koshio, S., Takakura, N., Oshida, K., Ishikawa., Gallardo- Cigarroa, M., Francisco J., Catacutan, M. R and Teshima, S., 2006: Effect of dietary bovine lactoferrin on growth response, tolerance to air exposure and low salinity stress conditions in orange spotted grouper Epinephelus coioides. Aquaculture,

255: 507-513.

Effects of the aromatase inhibitor Letrozole on serum sex steroid levels, egg diameter, gonado-stomatic index (GSI) in adult female rainbow trout (Oncorhynchus mykiss) two months before spawning

Paria Akbari Introduction

In this study we evaluated the effect of an aromarase inhibitor, Letrozoler for blocking estrogen biosynthesis, since inhibition of synthesis of this hormone during oocyte growth may cause retardation in the gonad development (Ankley. et al., 2002).The potential of Aromatase inhibitors (AIs) for blocking estrogen biosynthesis has been demonstrated in both invivo and invitro studies on mammals (Selvaraj. et al., 1994, Bajetta. et al., 2000). Letrozole is a non- steroidal compound and one of the most potent aromatase inhibitors yet developed (Smith 1999). It has potential for use both to prevent the conversion of androgenic steroids to estrogens and prevent or diminish the side effects of androgenic steroid abuse (Haynes. et al., 2003). Aromatase inhibitors can enter aquatic systems and cause ecotoxicological effects .The effects of aromatase inhibitors have been shown in aquaculture to sex differentiation and reproduction of fish (Afonso. et al., 1999, 2000, Ankley, Kahl et al. 2002, LI. et al., 2005). Association between decreased brain aromatase activity ,circulating E2 levels and ovarian somatic index in females of perch (Perca fluviatilis) was reported (Noaksson. et al., 2001). However, our knowledge about the potential of Letrozole to change serum sex steroid levels in females of trout is low Therefore in this paper,we investigated the effects of aromatase inhibitor Letrozole, on serum sex steroid secretion and role of Letrozole in gonad development in females of rainbow trout .

The experiment was conducted using forty eight adult rainbow trout (Oncorhynchus mykiss) female that had not yet undergone maturation fish reared at Dalkhan Fisheries Research Center of Shiraz, Iran, approximately 2 months before spawning (in mid- September 2011) and help outdoors in a 10 m2 concrete pond, with a water depth of 50 cm supplied with through- flowing river water and kept under photoperiod and at ambient temperature (15.8+0.5°C) with oxygen (5.5+0.1 ppm) and pH 7.8.Fish were initially weighted (854+0.1mg) under anesthesia (150 pmm clove oil). All fish were also implanted with a passive integrated transponder (PTT) tag for individual identification. 3The non- steroidal aromatase inhibitor Letrozole (CGS 20267) [1, 2, 6,73H]-4- Androstron-3,17- dione, was obtained as a gift from Iran hormone venture pharmaceutical technology development Co., Ltd., Iran was dissolved in the vehicle dichloromethane (Shilling.- 1et al., 1999). Stock solutions which contained 1.0, 2.5 mg of AI ml-1 were prepared. The fish were injected intraperitonially at the base of the right ventral fin using individual 10 ml syringes fitted with an 18.5 gauge needle. Control -1group was injected with the vehicle dichloromethane only (1.0 ml kg-1body weight).Fish were divided in four groups, (-11) control vehicle injected (n=12), (2) group treated with 1.0 mg AI kg-1 (n=12), (3) group treated with 2.5 mg AI kg-1 (n=12) and (4) group treated weekly with 2.5 mg AI kg-1(3x.5 mg AI kg-1)(n=12) .Blood samples (3ml) were taken by individual 5 ml syringes fitted with 21 gauge needle and collected from the caudal vein at 0 h (just before injection with AI), 6, 24,48, 96, 144,192, 288,384,482,528 h after injection with AI . Serum samples were obtained from six fish per pound (non- lethal bleeding), and allowed to clot at room temperature for 1-2h and then at 4°C overnight. After which the serum was drawn off, frozen in plastic tubes on dry ice and stored at -20°C until assay. After the last blood collection, three fish of each group, were dissected and ovaries were excised and weighted in order to determined the gonado-somatic index [GSI=100x (gonad weight / total body weight)] and oocyte diameter. for analysis. Serum E2, 17a-20B-P and T levels were measured by Enzyme liked imunosorbent assays described by Navas and segner (2000) and Guzman, Norberg, Ramos, Mylonas and Mananos (2008) with slight modification In the steroid ELISA, All data were presented as mean+ standard error of the

mean (S.E.M).Homogeneity of variances and the normal distribution were tested according to the levene and kalmogrov- smirnov tests. ANOVA was also used to detect variation in oocyte diameter and GSI among groups. Group dependent variation in serum concentrations for each hormone were detected by analysis of variance (ANOVA) followed by all pairwise multiple comparison by Duncan test.


Treatment 1715-esradiol (ng ml-1) hours after injection









































0.04 a/c






0.08 d

































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