Commercial feed additives

ABSTRACT 

A field study was conducted on brood stock Nile tilapia to increase the reproduction performance. Both sexes were individually stocked into habas (enclosures) in an earthen pond and fed for 19 days on a basal diet supplemented with different additives at graded levels of each ( 0.0, 0.5, 1.0 and 2.0 g Therigon^®  (T₁-T₄; T₁ as a control treatment respectively ); 1.0, 2.0, and 3.0 g Nuvisol Hatch P^®; (T₅-T₇); 20, 40 and 60 mg Gibberllic acid (T₈-T₁₀) for females fish and 700, 900 and 1100 mg L - carnitine / Kg diet for fish meals (T₁₁-T₁₄) respectively. Ovaries in all treatments of O. niloticus (T_1- T₁₀) contained more advanced oocytes at final maturation stage (i.e. yolk vesicle and ripe stage) with some clear histological alterations of fish ovaries fed on the highest levels of these feed additives, especially with Gibberllic acid treatments. Fish treated with dietary supplementation of 40 and 60 mg Gibberllic acid /Kg diet (T₉-T₁₀) showed the worst effects on fish ovary among all treatments, where T9 showed depletion with atresia of oocytes with abnormal shape of oocyte in Vitellogenic yolk stage, also, T₁₀ showed erosion in margin of some wall of oocytes, coagulative necrosis and liquefaction of the yolk sphere with large vacuoles of ripe oocyte of O. niloticus, and irregular wall of oocytes. Dietary supplementation of L-carnitine at levels 700 & 900 mg caused histological maturation of testis better than the control treatment (T₁₁) which showed normal structure of somniferous tubules filed with spermatozoa and spermatids. Meanwhile, fish fed on basal ration plus 1100 mg L-carnitine / Kg diet (T₁₄) showed degeneration and sever hemolysis in somniferous tubules, which were free from spermatocytes or spermatids. These drastic histological alterations in testes were confirmed by the macroscopic findings, where clear atrophy of testes was observed. The low bacterial loads of all tested organs (gills, liver, intestine and ovary / testes) at the high level of Nuvisol Hatch (on NA and SSA media). Data proved that the high bacterial load was detected in fish treated with L-carnitine in both tested media and fish organs. The results reported that the low bacterial load was observed in both of liver and ovary /testes in all media comparing with the other organs (gills and intestine) in addition to the total count of bacterial obtained on SSA and BP media was lower in all tested organs than the other media (NA, BR and TSA). The statistical comparison of bacterial load of tested fish organs among different treatments showed some variations. Significant differences were found between gills and the other tested organs of all treatments.

Key words: Tilapia brood stock - Gonads - Anatomy - Histology - Microbiology - Therigon^® -  Nuvisol Hatch P^® -  Gibberllic acid - L - carnitine.

Tilapia aquaculture is important, particularly for the lesser-developed countries in the tropics (FAO, 2001).  Nile tilapia Oreochromis niloticus are considered as the most common and popular fish in Egypt.  Egypt, a country where, arguably, the farming of tilapia has its roots (Stickney, 2006). Tilapia consist 36% of the Egyptian production from fish culture (Sadek, 2000) and occupy the 10^th order concerning the world production from aquaculture (Van Hauwaert et al., 2000).  Hence, Egypt produces 20% of the world tilapia capture and 12% of the world farmed tilapia (EL-Sayed, 2006). The culture of O. niloticus in Egypt has recently developed into a major industry. This industry, however, is still growing in a remarkable way with apparent intend towards intensification that pressurizing the need of enormous number of seeds. Many limitations associated tilapia fry production under the prevailing Egyptian conditions were described by El-Gamal (2002). Also, brood stock husbandry and spawning techniques are constantly upgraded as Egyptian hatcheries require a high number of good quality eggs to satisfy the needs for aquaculture, so rigorous management of large numbers of brood stock are necessary for mass production of eggs and fry due to relatively small number of eggs produced per spawning. Accordingly, the development of more elaborated forms of brood stock management is crucial to improve fry yield and system efficiency.  Today, it is widely accepted that effective seed production demands a thorough understanding of the special husbandry and particular nutritional requirements of brood stock fish which significantly affect fecundity, survival, egg size and egg and larval quality (Bromage, 1998). Therefore, the present research aimed to study the effects of using some commercial synthetic feed additives for improving brood stock fish reproduction, from the view point of gonads morphology and histology besides the microbiological examination.

The present study was carried out during Nile tilapia hatching season of 2008 (June and July) to study the effects of three commercial feed additives on females (Therigon^®, Nuvisol Hatch P^® and Gibberllic acid) and fourth one (L - carnitine) on males brood stock, concerning their gonads' anatomical and histopathological characteristics and some microbiological traits.                                                                                    

A field study was conducted in a private earthen pond fish farm located at Alabhar belonging to Alhamol, Kafr Alshiekh governorate. Fourteen habas (each 3 m width × 6 m length × 0.5 m water depth and 1 m total depth) were constructed in a two feddans earthen pond. The first ten habas were stocked with females' brood stock of one year (yearlings) Nile tilapia fish (average body weight of 150 g) from the same farm. The other four habas were stocked with males' brood stock of (the same age as the females, but of an average body weight of 200 g) Nile tilapia fish from the same farm also. Each haba was stocked with twenty fish. This study was a feeding trial to test the effects of some commercial dietary supplements on Nile tilapia (O. niloticus) propagation. The experimental feeding began on the 20^th June till the 8^th of July, where the feed was offered to fish twice daily, at a daily feeding rate of 3% of the fish biomass in each haba. The feed additives were purchased from the local market and added directly to a mash diet, which was purchased also from the local market (contained 90.31% dry matter, 80.88 % organic matter, 23.81% crude protein, 5.47% ether extract, and 9.43% ash) after the proximate analysis according to AOAC (2000) and moistened to be pelleted via a hand mincer. The feeding continued for males and females before mating.                                                                                                                                                                           

 Fish were fed on a basal ration (BR) with or without (control) the tested feed additives (as illustrated in the following Table (1) which were:

1- Therigon^® powder for veterinary use, manufactured by Adwia Co., S. A. E. 10^th of Ramadan city, Egypt. Each 1 g contains Alpha - Amino - p - hydroxyhydrocynnamic acid, 1000 g package as GnRH stimulant (Batch No. 0601116).                                  

2- Nuvisol Hatch P^®, imported by Khirat Alnile Co., 27 Alferdos Buildings, Flat 43, Nasr city, Egypt from Newtrix Co., Belgium, in 500 g package. Each 1 Kg contains the following vitamins (in mg): B₁ 4000, B₂ 5000, B₃ 4000, B₆ 2000, B₉ 1000, B₁₂ 20, PP 10000, Biotin 50, and L - carnitine 30000.                                                        

3- Gibberllic acid (C₁₉ H₂₂ O₆), type analysis, Art. 3930,  M. W. 346.38, M. P. 225 °C,  GA₃ content 90 %, 1 g  package, Batch No. 43124, imported from Lobal Chemie, Pvt. LTD, 2042 Bombay, India.                                                                       

 4- L - carnitine powder from Mebaco, Egypt.

After the 19 days feeding trial of the separate sexes of brood stock, fish samples from each experimental haba were catched for the anatomical, histological and microbiological examinations of the gonads. Microbiological tests were carried out too for gills, livers, and intestine as well as for rearing water and feeding diet. 

        At the end of the experiment, fish were sacrificed and the target organs (gonads) were sampled.  Samples were fixed in 10% neutralized formalin solution followed by washing with tab water, then dehydrated by different grades of alcohol (70, 85, 96 and 99%).  Samples were cleared by xylene and embedded in paraffin wax.  The wax blocks were sectioned to six micron. The sections were stained by hematoxyline and eosin and then subjected to a histological examination according to Pearse (1968) and Roberts (2004).

             Bacteria were identified by general and specific media and biochemical testes, the following were the cultivation media used for different purposes.

1-Nutrient agar, NA (general media). 2-Salmonella Shigella agar, SSA. 3- Brucella agar, BR. 4-Baird- parker agar, BP. 5- Trypticas soy agar, TSA. 6- R-S agar (Shotts and Rimler, 1973). 7- Potato dextrose agar (PDA). For every sampling, 3 fish (in each group) from each hapa were used for bacterial counts in fish organs (gills, liver, intestine and ovary/ testes). The fish were killed by physical destruction of the brain and the number of incidental organisms was reduced by washing the fish skin with 70% ethanol before taking the gills and opening the ventral surface with sterile scissors to expose the body cavity. From each fish, 1 g of each tested organs was removed and suspended in 10 ml of sterile saline (0.85% (w/v) NaCl). The suspension was serially diluted to 10³, and 1 ml of the solution was spread in each media plates. The inoculated plates were cultured as described above and the number of colonies was counted. Water samples were collected in sterile glass bottles. Appropriate serial dilutions of fish and water samples were then plated onto plate count agar.

Aerobic plate count (APC): For total heterotrophic aerobic bacterial counts of water, diets, gills, liver, intestine and ovary/ testes of tilapia, all the inoculated plates were incubated at 30^oC for 48 h and colony forming units (cfu) were counted. The temperature and incubation time used were found to be suitable for the growth of the investigated bacteria. Readings obtained with 30 to 300 colonies on a plate were used to calculate bacterial population numbers, recorded as cfu per unit of sample. The bacterial colonies were divided into different types according to the colony characteristics of shape, size, color and opacity, and the number of colonies of each recognizable type was counted. Three to five representatives of each colony type were then streaked on additional TSA plates repeatedly until pure cultures were obtained.

Identification of bacteria: All the purified isolates were observed for cell shape, spores, and Gram staining. The isolates were then subjected to biochemical tests following the criteria described in the Bergey's Manual of Systematic Bacteriology (Holt et al., 1994) for identification to genus or species level.

All numerous data collected were statistically analyzed using SAS (2001), when ANOVA-test was significant (P ≤ 0.05), least significant difference was calculated (Duncan, 1955) to differentiate between means. Data of the microbiology were statistically analyzed also using coefficient of variance, cross tab, and Chi-Square test. 

Gonad maturation and gonadosomatic index (GSI): 

In the present study the macroscopic examination of the ovaries of O. niloticus brood stock in all treatments showed maturation (Fig. 1 a) and some ovaries showed few atretical oocytes (Fig. 1b)   with different levels of the tested fed additives, in general the ripe females had swollen green ovaries. While testes of O. niloticus brood stock males fed on basal ration only (T₁₁, as a control male) and basal ration plus 700 and 900 mg L-carnitine / Kg diet (T₁₂ and T₁₃) appeared swollen milky white and well vascularized and readily released milt under gentle pressure of the abdomen (Fig 1c). While testes of brood stock fish fed on basal ration plus 1100 mg L-carnitine / Kg diet (T₁₄) showed an atrophy case in testes among all treatments (Fig. 1d).  These results of control treatments were agreement with Latif and Saady (1973). In addition to,  some symptoms had been registered in ovaries during the anatomical examination, e.g. hemorrhage, air pupils, fatty ovaries (T₄); gall bladder enlargement, atrophy of liver (T ₈),   and ovaries, discoloration, of fatty ovaries and liver (T ₇), friable liver (T₅  and T₆); over ripeness of ovaries (T ₇), and hepatic destruction (T ₉ and T ₁₀).

Fig 1 (a-d): Shows ovaries and testes in Nile tilapia brood stock with normal structure (Figs.a &c), (Fig 1d): shows start of atretic of oocytes in ovary (Fig b). (Fig 1d): shows testes with an atrophy case.

 In complementary study of this work (Abdelhamid et al. 2010), it is clear that, females have higher values of GSI than males where GSI values were determined in 8^th of July.  That is agreement with, (EL-Sayed et al., 2007), who reported that GSI showed higher values in females than males during the period from June to September with a peak in July. Also, O. niloticus has prolongedspawning activities with a peak in June; these results are in accordance with the results of Payne and Collinson (1983), Bayoumi and khalil (1988) and El Shazly (1993). Female reproductive maturity was commonly quantified by the GSI. Determination by sizing is low accurate because the size to which the fish grow, and at which they mature, varied greatly (Lowe-McConnell, 1982). In general, correlated with the results of the present study, the GSI of female tilapia O. niloticus was different in all treatments. However, determination of reproductive maturity using only the GSI is not enough because the structures within the ovary, such as oocytes at different stages, interstitial tissue with accumulation of yolk materials, can not be interpreted by weight. Direct observation of histological architecture is the most accurate method to let usknow exactly the stage of maturation at which the ovary is undergoing.

The histological characteristics of O. niloticus ovarian tissues fed on basal ration (BR) with or without the tested feed additives (T₂-T₁₀, and T₁ respectively), were investigated and showed in Figs.1& 2. It was observed that, three phases of developmental stages of oocytes (Oocyte is the building unit in ovary of any species of fish, it undergoes sequential changes named Oogenesis) were appeared; Cortical alveoli formation (yolk vesicle stage), Vitellogenic (yolk) and Ripe (mature) stage, but few oocytes appeared at early

Basal ration (control treatment) showing normal structure of ovarian tissues; ovary of O.niloticus showed ripe stage of oocyte with migratory nucleus and start of yolk liquefaction of cytoplasm of oocyte and large connected vacuoles of some oocyte (Fig 1a). Ovary in O. niloticus fed on BR plus 0.5g Therigon/ Kg diet (T₂) showed ripe stage of oocyte stage (Fig 1b).While the ovaries of  O. niloticus fed on BR plus 1.0g Therigon/ Kg diet (T₃) showed oocyte in yolk vesicle stage (in normal structure) beside many oocytes in  ripe stage, also, clear histological lesions were detected in ovary as coagulation necrosis in yolk granules, separation of follicular layers, degeneration in oocyte (atresia) and focal area of necrosis in ovarian interstitial tissues where the Ovarian interstitial tissues were found to consist of interstitial cells, adipose cells, yolk granules and blood capillaries (Fig.1c). Degenerative changes (atretic state) were observed in the ripe oocytes in ovary of O. niloticus fed on BR plus 2.0g Therigon/ Kg diet (T₄) with depletion of the yolk granules( Fig 1d). So, the ovaries in fish fed with diets supplemented with 0.5g Therigon/ Kg diet were protected to be atretic comparing with the high levels of Therigon (1.0 &2.0g / Kg diet).

O. niloticus fed on BR plus 1.0g Nuvisol Hatch P® / Kg diet (T5) showed ovary become more developed and reached to the final maturation ( ripe oocytes stage) with some atretic state in ripe (Fig 1e).  The level 2.0g Nuvisol Hatch P^® / Kg diet (T₆) showed different development stages of oocytes of O. niloticus,  Chromatin nucleolar stage, Perinucleolar stage, Cortical alveoli formation stage, Vitellogenic (yolk) stage, Ripe (mature) stage, with irregular wall of oocytes (Fig 1f). Whereas the high level of Nuvisol Hatch P^® 3.0 / Kg diet (T₇) showed ovary in ripe oocytes stage with migratory nucleus which loses its circularity besides hemolysis in ovarian interstitial tissues (Fig 2a). Concerning the ovaries histological of O. niloticus treatments fed on Gibberllic acid at the 3 levels 20, 40 and 60 mg /Kg diet (T_8- T₁₀, respectively) were showed in Figs. 2c-d. T₈ showed ovary in ripe stage with slight histological changes of oocytes; liquefaction of cytoplasm of oocyte with nucleus loses its circularity and degeneration of wall of oocyte (Fig 2b). While T₉ showed severe histological alterations of ovary; as lysis or depletion with atresia of oocytes (Fig 2c) with abnormal shape of oocyte in Vitellogenic (yolk) stage (Fig 2d). T₁₀ fed on BR plus 60 mg /Kg diet Gibberllic acid showed ovary in ripe stage with histological alterations of ovary as erosion in margin of some wall of oocytes, coagulative necrosis and liquefaction of the yolk sphere with large vacuoles of ripe oocyte of O. niloticus, and irregular wall of oocytes (Figs 2e & f) in addition to, the Vitellogenic oocyte showing the start of the nucleus migration to the animal pole (Fig 2e).

Ovaries of O. niloticus which were fed on the two higher levels of tested feed additives showed pathological alterations. Furthermore, fish treated with dietary supplementation of gibberllic acid showed the worst effects on fish ovary among all treatments,  In contrast the low level of these feed additives showed no clear histopathological changes of ovary especially, 0.5g Therigon/ Kg diet (T₂) which reflected the high level of FSH (91 u/ml) among all treatments (Abdelhamid et al. 2010; In complementary study of this work), according to Nagahama et al. (1995 a, b), vitellogenesis and oocyte maturation are regulated primarily by FSH and LH, respectively. Also Abdelhamid et al. (2010) confirmed that dietary inclusion of 1 g Nuvisol Hatch P^® / Kg diet (T₅) realized good female's reproduction performance.

The histological changes of ovaries of O. niloticus observed in some treatments  may be  attributed to many causes, such as a mechanism to regulate fecundity may be due to the tested feed additives or to environmental stress (as pollution), where the different pollutants such as industrial and agriculture wastes, pesticides and also different types of bacteria have histopathological effects on the reproductive tissues of fish gonads (Jhonson, et al., 1991 and  Lye,et al., 1998), these effects may disturb the development of germ cells andmay reduce the ability of fish to reproduce. Johnson et al. (1997) reported that, artesiais thought to be an uncommon event in healthy femalesand it has been linked to poor nutrition, environmental stress and starvation. Also, atretic oocytes were also recorded in the same gonads, a sign that some oocytes failed to mature normally. This indicates insufficient stimuli for normal gonads development (Msiska, 2002).

Testes of Nile tilapia brood stock fed on basal ration only (T₁₁, as a control) showed normal structure of semniferous tubules filed with spermatocytes and spermatids (Fig. 4 a & b). However, dietary supplementation of L-carnitine caused histological maturation of testis of treated fish especially at level of 700 mg (T₁₂), which showed normal structure of semniferous tubules filed with spermatozoa (Fig. 4c) or 900 mg L-carnitine / Kg diet, which showed normal structure of semniferous tubules filed with spermatids and spermatozoa (Fig. 4d). All of these histological developments in testis of experimental fish due to dietary supplementations and also are confirmable with those of the anatomical findings in the present study (Fig. 1c).

Meanwhile, fish fed on basal ration plus 1100 mg L-carnitine / Kg diet (T₁₄) showed degeneration in semniferous tubules, which were free from spermatocytes or spermatids among all treatments (Fig. 4e &f). These drastic histological alterations in testes may be related with increasing level of L-carnitine in fish diet and also associated with those of the atrophy case in testes of anatomical findings among all treatments (Fig. 1 d).

Several stages of spermatogenesis (spermatocytes, spermatid and spermatozoa) in the present study are similar to those reported by Msiska (2002). In addition, secondary spermatocytes were illustrated by darkly staining chromatin as in other teleost fish. Meanwhile, spermatozoa were concentrated in the lumen (Tyler and Sumpter, 1996). Furthermore, Abdelhamid et al. (2010) confirmed that dietary supplementation with 700 (T₁₂) and 900 (T₁₃) mg L-carnitine / Kg diet gave better results of males' reproductive performance. L-carnitine is a naturally occurring amino acidderivative (dipeptide amino acid), synthesized from methionine and lysine. L-carmitin, a betaine derivative of β - hydroxybutyrate, could be biosynthesized in plant and animal cells via lysine, methionine, and some vitamins like B₆, C, nicotinic acid and folate (Zeyner and Hameyer, 1999).

Fig.2 (a-f): Sections of ovaries of O. niloticus fish stained with H &E(a) ovary of control fish showing normal ovary tissue (x250); (b-d) ovaries of O. niloticus fed on BR plus 0.5,1.0 &2.0g Therigon/ Kg diet (T₂-T₄ respectively) (b) showing normal structure of ripe stage of oocyte (x200),(c) showed coagulation necrosis in yolk granules, and atresia of ripe oocytes (x200), (d) showed depletion in yolk granules, and atresia of oocytes (x200), (e & f) ovaries of O. niloticus fed on BR plus 1.0& 2.0g Nuvisol Hatch P® / Kg diet (T₅&T₆);(e) showed ovary in ripe oocytes stage with some atretic state (x200).(f) different development stages of oocytes with irregular wall of oocytes (x 300).   

Fig.3 (a): Sections of ovary of O. niloticus fish fed on BR + 3.0g Nuvisol Hatch P® / Kg diet (T₇) showed ovary in ripe oocytes stage with migratory nucleus which loses its circularity besides hemolysis in ovarian interstitial tissues (x250). Fig 2 (b, c&d, e&f): Sections of ovaries of O. niloticus fed on BR + 20, 40 and 60 mg /Kg diet (T8- T10, respectively). (b); T8 showed ovary in ripe stage with slight histological changes of oocytes as liquefaction of cytoplasm of oocyte with nucleus loses its circularity and degeneration in wall of oocyte (x200). (c); T9 showed severe of pathological alterations ovary; as lysis with atresia of oocytes (x200), (d); T9 showed abnormal shape of oocyte in Vitellogenic stage(x300). (e&f); T₁₀ showed coagulative necrosis and liquefaction of the yolk sphere with large vacuoles of ripe stage  (x200) and irregular wall of oocytes (x 350) respectively.

                                                                                                                       

Fig.4 (a-f): Sections of testes of O. niloticus fish stained with H &E (a &b) testes of control fish (T₁₁) showing normal structure of semniferous tubules filed with spermatocytes and spermatids (x150,200); (c) testes of O. niloticus fed on BR plus 700 mg L - carnitine / Kg diet (T₁₂) showed normal structure of semniferous tubules filed with spermatozoa (x 300 ). (d) testes of O. niloticus fed on BR plus 900 mg L - carnitine / Kg diet(T13) showed normal structure of semniferous tubules filed with spermatids and spermatozoa (x200), (e & f) ) testes of O. niloticus fed on BR plus 1100 mg L - carnitine / Kg diet(T₁₄);(e) testes showed  showed degeneration in semniferous tubules, which were free from spermatocytes or spermatids (x 60 ), (f); showed severe hemolysis testes tissue (x 200) 

Since L-carnitine provides an energetic substrate for the spermatozoa in the epidydimis, contributes directly to sperm motility and may be involved in the successful maturation of the sperm (Chatzifotis et al., 1995). L-carnitine improved semen quality and histological characteristics of the testes (El-Damrawy, 2007). Generally, a low level of L-carnitine enrichment provides several protective effects in fish reared under intensive pond-culture conditions (Harpaz, 2005).Yet, Jayaprakas et al. (1996) reported that supplementation with 900 mg l-carnitine / kg diet had a positive effect on the reproductive performance of male Mossambique tilapia, significantly increasing both testis weight and sperm cell concentration per ml compared with the control. Moreover, administration of GA₃   induced many histopathological changes in the liver revealed marked reduction in total carbohydrates and total protein contentsin the hepatocytes. GOT, GPT and alkaline phosphatase in serum were significantly decreased. So, GA₃ affected the structure and function of the rat liver (Sakr et al., 2003).

Furthermore, Abdelhamid et al. (2010) confirmed that dietary inclusion of 1 g Nuvisol Hatch P^® / Kg diet (T₅) and 1 g Therigon^®(as GnRH stimulant) / kg diet (T₃) realized good female's reproduction performance.

Gonadotropin releasing hormone (GnRH) is a hypothalamic neuronal secretory decapeptide that plays a pivotal role in reproduction. In addition, it is also believed that GnRH may have a role as a modulator of the activity of diverse systems in the brain and many peripheral organs (Emons and Schally, 1994).The development of potent GnRH analogues by modification of mammalian, avian and piscine GnRHs has had a significant impact on the development of practica1 means for the induction of final maturation, ovulation and spermiation in farmed teleosts. GnRH analogue stimulates gonadotropin secretion (Van Der Kraak et al., 1983) steroidogenesis (Van Der Kraak et al., 1984) and ovulation (Haraldsson and Sveinsson, 1993) in salmon and in many other species. Another GnRH analogue that has been widely used in salmonids (Wei1 et al., 1991) and in carps (Peter et al., 1988) is sGnRH ethylamide. Most successful protocols for induction of ovulation involve intramuscular or intraperitoneal injection of an aqueous solution of GnRH analogue; other forms of administration have been tested (Myolonas et al., 1995). Other studies have demonstrated that GnRH can be administered orally to fish (McLean et al., 1991) and this technique has been utilized to induce ovulation in both warm water (Spotted seatrout, Cynoscion nebulosus, Thomas and Boyd, 1989) and cold water marine species (sablefish, AnopZopoma~mbriu, Solar et al., 1990). Also, Sukumasavin et al. (1992) demonstrated the oral induction of ovulation in the Thai catfish when GnRHA is coadministered with domperidone. In fish, Gibberllic acid (GA₃)at low levels improved Nile tilapia growth and gonado-somatic index (Abdelhamid et al., 1998), since it is a nitrogenous compound (Alkhiat et al., 1981) with estrogenic effect; where it increased the percent of egg production, hatchability and ovary and oviduct relative weight significantly (El-Sebai et al., 2003). So using natural GA₃ instead of the synthetic estrogen is safer and environmentally friend therefore should be considered.

The results of the total count of bacterial isolated on different media from gonads of O. niloticuss among different treatments are shown in Table (2). Data exhibited absentce of bacterial loads of ovaries in fish fed on diets supplemented with the three different levels of Nuvisol Hatch, among different tested media, So, Nuvisol Hatch may have a protective effect against bacterial infection, in contrast, in testes the high bacterial load were appeared on BR and BP media with the two high levels of L- carnitine (900 & 1100 mg / Kg diet; T₁₃ &T₁₄). Only T₁₃ showed that SSA appeared total count 60 x 10³ CFU/g, this media has given an appreciable count of Salmonella spp., Shigella spp., Proteus spp. and Escherichia coli. All these genera are belonging to the family Enterobacteriaceae which are capable of inducing gut wrenching gastroenteritis as reported by Anon. (2005). The biochemical tests (table  ) showed that the isolated bacteria from testes were  Bacillus sp., Salmonella. Sp and Brucella sp. Which were found in T₁₃ more than T_14, SO, the histological alterations of testes in T₁₄ attributed to the treatment with high level of L- carnitine (1100 mg/Kg diet) not to the bacterial pollution  

The typical colonial morphology on SSA medium is as follows: both Salmonella and Proteus spp. produce colorless colonies with black center, Shigella colony is colorless (Biolife, 1996), while E. coli colony is pink to red. Baird- parker agar (BP) culture medium was specially used for isolation and identification of Staphylococcus spp., Micrococcus (coccid bacteria) and Bacillus (Bacillus is belonged to family Bacillaceae). Staphylococcus produces black colony on BP medium while Micrococcus and Bacillus species produced brown colony on the same medium. Brusella agar (BR) medium was used for the isolation and cultivation of Brucella that is Coccobacilli and belonging to the family of Brucellaceae. Trypticas soy agar (TSA) medium was specially used for isolation of Streptococcus. R-S agar prepared after Shotts and Rimler (1973), with dehydrate ampicillin supplement, this medium was used for isolation of Aeromonas spp., from water, but not from diets. Yellow colonies from this medium was selected and identified to the genus Aeromonas with panel tests according to Popoff (1984). Some biochemical testes were carried for classification and identification of these isolated colonies of bacteria as shown in Table (4).                                                                                                                                                               

 Table 3: Statistical comparison of bacterial count obtained on different media in fish organs from different experimental treatments (means ± SE).  

 

 

Chi-Square value = 12.32 for gills and P value = 0.015; Chi-Square value = 0 for liver and P value = 1 (not significant); Chi-Square value = 3.000 for intestine and P value = 0.558; Chi-Square value = 8.500 for gonads and P value = 0.386. 

a-c: Treatments, organs or media have the same letter (in the same column) are not significantly different (P ≤ 0.05).

 The results reported that the high bacterial load was observed in both of gills and intestine in all media comparing with the other organs (liver and ovary /testes), that is agree with Austin (1982) who reported that most of the microorganisms in fish tissue are thought to result from surface, gills, or intestinal contamination, where microorganisms are adsorbed on the surfaces of the fish and found in their intestinal contents. So the initial microbial flora on the caught fish is dependent upon the contamination of the water, bottom sediment from the area of catch and the food entering the digestive tract which may contain microorganisms. In addition to the total count of bacteria obtained on SSA and BP media was lower in all tested organs than the other media (NA, BR and TSA). The statistical comparison of bacterial load of fish tested organs among different treatments showed some observation (Table 3).  Non Significant differences were found between tested organs (except gills) and treatments. Generally, tabulated data proved that the high bacterial load was detected in fish treated with both of Gibberllic acid and L-carnitine in both tested media and fish organs. The present of Enterobacteriaceae in some fish organs in this study may be attributed to the rearing water of fish (Sewage), Austin and Austin 1987 reported that fish harvested from water polluted with human and animal wastes can contain Salmonella, Shigella, Proteus and Escherichia coli which are usually found among the most prevalent bacteria on the most rearing water of fish. E. coli is still the most widely used indicator for faecal pollution (Snieszko and Axelrod, 1976).   Also,   Streptococcus spp. are opportunistic pathogens in aquaculture and their pathogenicity depends on environmental stresses as low dissolved oxygen levels, high nitrite concentration (Bunch and Bejeramo, 1997), water hardness, and crowding with water temperatures exceed 20ºC (Ohnishi and Jo., 1981). All vital organs of fish stock infected with streptococci become heavily infected and mortality becomes massive (50-60%; Hubber, 1989).  Streptococcus has been associated with serious economic losses among cultured freshwater and saltwater fish in many parts of the world, especially among tilapia fish. El-Gawady (2002) counted up to 1.9 x 10⁶ - 2.4 x 10⁶ CFU/ml total aerobic bacterial in water of fish farm in winter and summer, respectively. However, the gut and skin of O. niloticus microbial count reached 4.3 x 10⁸ CFU/g. Ampofo and Clerk (2003a) identified 25 species of bacteria as associated with the fish culture systems. They (2003b) also added that manuring causes organic enrichment; it may also hasten the deterioration of the water quality making the aquatic environment favorable for the growth and multiplication of human pathogenic bacteria. Moreover, Abdelhamid et al. (2006 and 2007) registered the presence of pathogenic bacteria (1.3 - 2.0 x 10⁵) in samples of water, feed, sediments and fish, mainly in summer season. There was no difference between fish of natural resources and those of aquaculture concerning bacterial contamination. Also, Abdelhamid et al. (2008) found pathogenic bacteria (X 10⁶ CFU) in samples of water, feed, intestinal, and liver of Nile tilapia up to 113.0, 38.7, , 23.33, and 25.00, respectively. However, Abdelhamid et al.(2009) did not find critical counts of bacteria, whether in water, diet or fish tissues.  Also, there were no relations among the counts in water, diets, muscles and/or liver of the tested tilapia fish. Yet, Abd El-Shahid et al. (2009) found that all fish (including Nile tilapia) samples (100) collected from Alexandria fish markets were contaminated with Enterobacteriaceae (5.14 X 10³) and Coliform (1.o2 X 10³).Moreover, Shaltout et al. (2009) counted the total microbial count including fungi, yeasts and highest total bacterial counts (TBCs) as indicators for faecal pollution in Kafr El-Zayat industrial area.

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