reproductive biology of nile tilapia Brood Stock Fish

A field study was conducted on brood stock Nile tilapia to increase the propagation. 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 (0.5, 1.0 and 2.0 g Therigon^®; 1.0, 2.0, and 3.0 g Nuvisol Hatch P^®; 20, 40 and 60 mg Gibberllic acid and 700, 900 and 1100 mg L - carnitine / Kg diet). The obtained results were evaluated and the best treatment for each sex was chosen for mating. Results indicated that all pretreatments for males and females brood stocks of Nile tilapia positively affected the total count of the offspring produced. Yet, the Haba, in which the females were pretreated with 0.5 g Therigon^® / Kg diet and the males pretreated with 700 mg L - carnitine / Kg diet, gave the highest total count of the offspring comparing with the other  Habas. But, because of the high feed cost due to the additives cost, 0.5 g Therigon^® / kg diet as pretreatment for ? only (3^rd Haba), 2 g Nuvisol Hatch P^® / Kg diet as pretreatment for ? only (5^th Haba), followed by  0.5 g Therigon^® and 700 mg L - carnitine / Kg diet for ? and ?, respectively (4^th Haba), respectively were the best economically.                                                                                      

Key words: Nile tilapia - Brood stock - Feed additives - Reproductive performance. 

 Tilapia aquaculture is and will continue to be an important fish, particularly for the lesser-developed countries in the tropics [1]. 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 [2] , where tilapia culture is believed to have originated some 4000 years ago. Tilapia consist 36% of the Egyptian production from fish culture [3] and occupy the 10^th order concerning the world production from aquaculture [4]. Hence, Egypt produces 20% of the world tilapia capture and 12% of the world farmed tilapia [5]. 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 [6]. 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 the complex reproductive biology and asynchronously spawning with 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 [7]. The objective of the present research was to study the possibility of improving reproductive performance of Nile tilapia fish using some feed additives.  

The present study was carried out during Nile tilapia hatching season of 2008 (June and July) in  two phases, the first was 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 characteristics and some reproductive traits. The second phase was to select treatments of both 0.5 g Therigon^® and 2 g Nuvisol Hatch P^® / Kg diet (for females ) and  700 mg L - carnitine / Kg diet (for males )  to apply in a mating trial to be evaluated via hatchability.                                                                                       

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 [8] and moistened to be pelleted via a hand mincer. The feeding continued for males and females before mating. Water of fish rearing in each individual Haba was tested daily for some water quality parameters including water temperature, pH value, and dissolved oxygen concentration.                                                                                                                                                                                                                               

 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, three fish from each experimental Haba were catched to collect blood, seeds (eggs), and milt for different measurements and determinations. Tri sodium citrate was used as an anticoagulant for blood collection. Blood determinations for follicle stimulating hormone (FSH), luteinizing hormone (LH) and progesterone hormone were done using commercial colorimetric kits (Diamond, Diagnostic, Egypt), and milt analyses (count, motility, forward, sluggish and dead percents) were done too.

Six Habas (similar to those used in phase one, in the same earthen pond, at the same private farm) were used in the second phase (beginning from the 9^th of July) of this study. The Habas were stocked with 9 females and 3 males each (sex ratio 3 ?: 1 ?) to test the best treatments from phase one as following in Table 2:

The level 0.5 g Therigon^® / Kg diet was chosen for its high value of FSH (Table 7), 2 g Nuvisol Hatch P^® / Kg diet was chosen for its high value of progesterone (Table 7). On the 22^nd July, the fry were collected and counted. Throughout this phase also, water quality parameters were measured daily as in phase one, at 9 - 11 am.

Data collected were statistically analyzed using SAS [9], when ANOVA-test was significant (P ≤ 0.05), least significant difference was calculated [10] to differentiate between means.                            

As shown from Table 3, there were no significant differences among all Habas concerning the tested water quality parameters. Therefore, the data were presented as overall means ± standard errors (SE). The tested parameters showed very suitable water conditions for rearing tilapia.

 

Data of the studied females' reproductive traits are illustrated in Table 4. Except GSI, the other tested parameters showed significant (P ≤ 0.05) differences among treatments. The significantly heavier body weight (288.9 ± 23.8 g) after the 19 day-study was realized by the fish in the 8^th Haba (20 mg gibberllic acid / Kg diet) followed by Haba No. 6 (2 g Nuvisol Hatch P^® / Kg diet) without significant difference  between both Habas (6 & 8). The best (P ≤ 0.05) ovaries weight (11.5 ± 0.07 g) was recorded in fish of the 4^th Haba (2 g Therigon^® / Kg diet); yet, there were no significant differences among all treatments and the control. Egg number per fish was the highest (935.5 ± 120.2 and 926.0 ± 12.7) significantly (P ≤ 0.05) by the 5^th  and 3^rd Habas' fish (treated with 1 g Nuvisol Hatch P^® / Kg diet and 1 g Therigon^® / kg diet), respectively. Yet, the egg number / Kg fish weight (fecundity) of these fish groups in Habas No. 5 and 3 did not differ significantly (P ≥ 0.05) with the control, which was better (P ≤ 0.05) than all the other treatments. Moreover, the lowest (P ≤ 0.05) egg diameter was reflected by the fish groups of Habas No. 6 and 5, being 0.96 ± 0.06 and 1.25± 0.00 mm. Otherwise, all other treatments were significantly similar to the control.

* Means with the same letter within the same column don't differ significantly (P ≥ 0.05).

**: BW=body weight.

***: GSI = gonado - somatic index = gonads weight (g) × 100 / fish weight (g). 

Tables 5 and 6 present data of males reproductive traits tested including testes weight and GSI as well as milt quality parameters. Although there were no significant differences among treatments and the control; yet, the control was more pronounced in fish weight, testes weight, GSI, and sperms count than the treatments. But the motility and dead percentages were better in fish group of Haba No. 12 (700 mg L - carnitine / Kg diet) followed by Haba No. 13 (900 mg L - carnitine / Kg diet) concerning motility, forward, sluggish and dead percentages.

*:Means don't differ significantly (P ≥ 0.05).

**: GSI = gonado - somatic index = gonads weight (g) × 100 / fish weight (g). 

Data of serum sexual hormones of the experimental fish are presented in Table 7. Females fish of the 3^rd and 5^th Habas reflected lower concentrations of (FSH) but higher concentrations of either (LH) or progesterone hormone, comparing with the other Habas' fish. This is in good accordance with the fecundity which was given in Table 4. Also, male fish of the 12^th and 13^th Habas (Table 7) had sera with higher levels of FSH, LH, and testosterone comparing with the other treatments. This also confirms the previous results of the milt analysis for its quality parameters (Table 6).               

The following Table 8 shows that all pretreatments for males and females brood stocks of Nile tilapia positively affected propagation (2^nd phase of the experiment, mating), i. e total count of the offspring produced. Yet, the 4^th Haba  (in which the females were pretreated with 0.5 g Therigon^® / Kg diet and the males were pretreated with 700 mg L - carnitine / Kg diet, for 19 days before mating or fertilization) gave the highest total count of the offspring comparing with the other  Habas. But, the 3^rd, 5^th, and 4^th Habas were the best economically, since they were responsible for 43.5, 31.7, and 25.3 % superiority than the control (1^st Haba).                                                                                                                    

* Economic efficiency = Income from buying the produced fry in LE / feed costs of  the brood stock during the pretreatment in LE, where the local  price of 1000 fry was 35 LE and for 1 Kg diet without additives was 2.2 LE.                                                   

The quality of fish rearing water did not influence by the experimental treatments, and was suitable for rearing Nile tilapia brood stock according to Abdelhamid [11].                                                                                                                                                                                                                

Reproductive performance of fish is influenced by many factors, e.g. feeding regime including dietary protein [12-14] and vitamin [15, 16] levels, feeding rate [17], and hatchery management [18], as well as endocrine regulation [19-24]. Other environmental conditions may also affect, including photoperiod and water temperature [25-27] as well as water depth [28]  

The fish farming industry has been more focused on the quality of eggs or larvae rather than that of sperm, even though the quality of both gametes may affect fertilization success and larval survival. Sperm quality in farmed fish may be affected by different components of brood stock husbandry, during collection and storage of sperm prior to fertilization or the fertilization procedure. Motility is most commonly used since high motility is a prerequisite for fertilization and correlates strongly with fertilization success [29].

Gibberellins (GAs) are involved in a wide range of plant developmental processes. Of all the plant hormones the GAs represent perhaps the most diverse group, with currently 126 different structures known [30]. Gibberellins are tetracyclic diterpenes that are found in plants and fungi. A few of the identified to date are known to be active hormones that are involved in seed germination, seedling emergence, stem elongation, fertility, and flower and fruit development. The gibberellin receptor has not yet been conclusively identified. GAs act in stem growth via an enhancement of both cell division and cell elongation. Gibberellins get their unusual name from the fungus Gibberella fujikuroi, from which they were first isolated [31]. GA₃ is naturally widespread than the other gibberellins which have sexual influences [32]. GA₃ has a pesticide effect [33], and also carcinogenic effect on rectal protozoon [34] and Egyptian toads' liver [35]. But, [36] clearly demonstrated that it was essentially nontoxic by various routs of applications for different animal species.                                                                                                   

Yet, it promotes growth of rats, poultry, pigs, and calves [37, 38] as well as it improves laying hens' production, concerning egg production, egg mass and hatchability [39, 40]. Gibberellins have an estrogenic effect on animals [41]. It increased blood protein significantly, but affected different organs weight and their histology in chickens [42]. In fish, GA₃ at low levels improved Nile tilapia growth and gonado-somatic index [43], since it is a nitrogenous compound [37] with estrogenic effect; where it increased the percent of egg production, hatchability and ovary and oviduct relative weight significantly [44]. So using natural GA₃ instead of the synthetic estrogen is safer and environmentally friend therefore should be considered.                                                                                                                   

L-carnitine is a naturally occurring amino acid derivative (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 [45]. It is an essential cofactor of fatty acid metabolism (it provides energy by transporting long and medium chain fatty acids to mitochondria to act as fuel). Deficiency in carnitine is associated with male infertility. 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. Carnitine lowers triglycerides and raises the high density lipoprotein levels. L-isomer of carnitine is more effective than DL - isomer [46].        

Cellular parameters of the seminogram have been previously shown to correlate with L-carnitine concentration in the seminal fluid. Carnitine is involved in maintaining an active oxidative phosphorylation (OXPHOS). Therefore, it was strongly suggested that relationship between carnitine secretions, seminal quality and OXPHOS activities could be because of a parallel response to the same regulatory event [47, 48]. L-carnitine improved semen quality and histological characteristics of the testes [49]. Generally, a low level of L-carnitine enrichment provides several protective effects in fish reared under intensive pond-culture conditions [50-52].

Pituitary homogenate induced artificially maturing and increased both serum testosterone and estradiol [53]. Gonadotropin-releasing hormones (GnRHs) bind to the specific receptor on the gonadotrophs to activate the synthesis and release of gonadotropins (follicle stimulating hormone or FSH and luteinizing hormone or LH), which in turn control gonadal maturation, gametogenesis and gamete release [54, 55]. Not only do teleosts exhibit the highest variety of GnRH variants, but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptor [56]. Synthetic analog of gonadotropin-releasing hormone was used for inducing ovulation and enhancing spermiation in brood fish [57]. Moreover, territorial males of African cichlid, Haplochromis burtoni, characterized by aggressive and reproductive activity, have significantly larger hypothalamic gonadotropin-releasing hormone (GnRH)-containing neurons and larger testes than nonterritorial males [58].

Dietary inclusion of 1 g Nuvisol Hatch P^® / Kg diet and 1 g Therigon^® / kg diet before mating realized good females reproduction performance. Also, dietary supplementation with 700 and 900 mg L-carnitine / Kg diet before mating gave better results of males' reproductive performance. But, because of the high feed cost due to the additives cost, 0.5 g Therigon^® / kg diet as pretreatment for ? only (3^rd Haba of the 2^nd phase, mating), 2 g Nuvisol Hatch P^® / Kg diet as pretreatment for ? only (5^th Haba of the 2^nd phase, mating), followed by  0.5 g Therigon^® and 700 mg L-carnitine / Kg diet for ? and ?, respectively (4^th Haba of the 2^nd phase, mating), respectively were the best economically. 

It could be recommended to use such commercial feed additives for improving reproductive performance of Nile tilapia brood stocks to offer enough seeds for fish farms .It is recommend also to make other trials on different other additives at economical levels.                                                                                                                 

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