**5. Materials and methods**

#### **5.1 Presentation of the study areas and breeding structures**

The aim of this work is to identify the most suitable environment for the rearing of *O. niloticus* in Tunisia. To achieve this goal, this study was held investigating two areas with different types of waters: the experimental station of Bechima (geothermal water) and Smati Reservoir (fresh water). In addition to the main infrastructure intended for the preparation of broodstock for reproduction, spawning and the production of larvae, the production of juveniles and young individuals requires appropriate infrastructure, such as cages and enclosures which are efficient and resistant to environmental factors.

#### *5.1.1 Bechima experimental station*

Bechima experimental station is a research unit created in 1999 by the NISTS in Al-Hamma region in the governorate of Gabes in southern Tunisia. It is located in the vicinity of a cooler on a slope of 3% to ensure a continuous circulation of water by simple gravity. Two artesian wells supply the station with geothermal water at a temperature of 70°C. This water is cooled by the atmosphere to reach an average temperature of about 30–40°C. In terms of infrastructure, the station is equipped with 3 greenhouses containing breeding, larval rearing, hatching and rearing tanks [15].

#### *5.1.2 Smati reservoir*

Smati Reservoir covers an area of 121 km<sup>2</sup> and is located in the region of Al-Ala in the governorate of Kairouan, central Tunisia on Smati Wadi. The average salinity and depth are estimated at 2.3 PSU and 1.5–2 m respectively. In addition, according to the General Directorate of Fisheries and Aquaculture [20], this reservoir offers ichthyic habitat for two species namely the mullet *M. cephalus* and the Barbel *Barbus callensis*, with a total production estimated at 17.3 tons in 2015.

#### *5.1.3 Design and assembly of breeding structures*

In order to facilitate handling and transport and to minimize costs, the used cages and enclosures were built with respective volumes of 3 m<sup>3</sup> (with Length = 1.5 m, Width = 1.3 m and Height = 1.5 m), and 2 m<sup>3</sup> (with Length = 1.3 m, Width = 1 m and Height = 1.5 m), using a mesh size of 6 mm of polyamide nets. In total, 31 cages were constructed, of which only five were initially installed in the large tank for the phases *Nile Tilapia "Oreochromis niloticus" Farming in Fresh and Geothermal… DOI: http://dx.doi.org/10.5772/intechopen.106646*

of pre-growing and grow-out in Bechima Station. In addition, six enclosures were built for the rearing of individuals in a semi-extensive system in Smati Reservoir, of which two were used for hatching and two others for rearing at a depth of 1.5-2 m [63].

#### **5.2 Biological material and breeding procedure**

#### *5.2.1 Biological materials*

*O. niloticus*, in particular the strain "Maryout", subject of this zootechnical study was introduced in 1999 from Libya within the framework of research cooperation between the NISTS and the Marine Science Center of Tajoura [64].

The experiments started with the collection of more than 6000 larvae (average weight = 0.01 g) from 13 broodstock (weight between 99 and 190 g) which were well adapted to life in captivity in the geothermal waters of Bechima. These larvae were subdivided into 2 batches, to be prepared for an initial pre-growing phase of 60 days in two larval tanks, located in the larval rearing greenhouse of the station, with the aim to obtain fry of 1 to 2 g. The latter were then transferred to two cages and two enclosures which were installed respectively in Bechima Basin and in Smati Reservoir in order to obtain juveniles of 15–20 g (50 days). Finally, a sorting was ensured based on average weight and sex to produce neo males and to start the last phase of production: the grow-out.

#### *5.2.2 Breeding procedure*

#### *5.2.2.1 Collection of eggs and larvae*

During this study, the broodstocks were maintained under optimal abiotic rearing conditions. In fact, several signs allow the identification of incubating females, such as the appearance of a dark band on the forehead and black spots on the flanks, as well as a fast and discontinuous swimming with a rather aggressive behavior towards other specimens in the tank.

#### *5.2.2.2 Females reproductive parameters*

Once the female identified and isolated, they are weighed and measured. This procedure allows the evaluation of two important parameters: relative and absolute fecundity, which describe the productivity of the females per day according to weight and length. The absolute fecundity refers to the total number of eggs present in a female before fertilization. Relative productivity represents the total number of larvae produced per day in relation to the total biomass of females in g.

#### *5.2.2.3 The broodstock phase*

Larvae were obtained by spitting technique. The handling of the larvae as a count is a delicate operation, requiring a particular follow-up not to stress the unhatched eggs and larvae. The latter were reared for 60 days in cubic tanks with a unit volume of 1m<sup>3</sup> , a density of 800 larvae/m<sup>3</sup> , and a flow rate of 4 to 6 l/min. As for nutrition, larvae were fed 40% of protein levels in order to obtain fry with an average weight of 1 to 2 g.

#### *5.2.2.4 The breeding phase*

The breeding/pre-growing phase started on May 11th, 2017, in the floating cages installed in the rearing tank at Bechima experimental station (average weight: 1 to 2.03 g). One day later, 3009 larvae (average weight of 1.35 g) were transferred to the enclosures. At the end of this phase, a sexual sorting of the fry was required to start the rearing of the mono-sex populations. The density adopted for each structure is 500 individuals/m<sup>3</sup> . Moreover, beyond a size of 20 g, sexual dimorphism can be observed and the distinction between males and females is possible. Indeed, in males, the genital papilla is protuberant in the shape of a cone and carries a urogenital pore at the end. On the other hand, in females, it is small and round, found in the middle of a transverse slit (oviduct) which is located between the anus and the urethral orifice (urinary pore) located at the end.

#### *5.2.2.5 The fattening phase*

From mid-July onwards, we started the fattening/grow-out phase of the reared fry. In general, the sorting was first based on size that allowed the retainment of individuals with an average weight greater than or equal to 20 g. Towards the end of August, males and females were reared separately to later evaluate growth rates for both sexes. Regarding nutrition, the supplemented food for tilapia was the same in both environments with 30% of protein level and was distributed at a rate of 10% on a daily basis.

#### *5.2.2.6 Watching out for pathology*

In order to ensure the success of the different rearing stages, a certain level of cleanliness and hygiene in the experimental tanks had to be maintained on a daily basis. The bottom was siphoned off before feeding, in order to eliminate feces and any kind of deposit of food, thus avoiding the development of pathogenic organisms. In addition, specific measures related to the health of the group, were taken while monitoring the condition of individuals and their behavior.

#### *5.2.3 Nutrition*

The added food was composed of raw materials available on local market and prepared in Bechima pilot station, with variable proportions according to the fish stage development and following the formulas provided by the research of the NISTS (**Table 2**).


#### **Table 2.**

*Composition of tilapia food according to rearing phases [13].*

*Nile Tilapia "Oreochromis niloticus" Farming in Fresh and Geothermal… DOI: http://dx.doi.org/10.5772/intechopen.106646*

This food is rich in protein with a level up to 40%. In addition, vitamins and minerals from the Mineral Vitamin Supplement (MVC) were provided in the food to meet the dietary requirements of the species. The rationing rate, the granulometry and the quantity of the food supplied vary according to the development stages, size and age of the individuals, the energetic value of the food and the variation of the physicochemical properties. The adopted rationing rate ranges from 20 to 10% (from the first day until pre-growth). The amount of food for broodstock (Qg) and for larval rearing (Ql) was determined in accordance with the following formulas:

$$\mathbf{Qg} = (\mathbf{Nm} \ast \mathbf{Mm} + \mathbf{Nf} \ast \mathbf{Mf}) \ast \mathbf{TC} \tag{1}$$

$$\mathbf{Q}\mathbf{l} = (\mathbf{B}\mathbf{f} - \mathbf{B}\mathbf{i}) \ast \mathbf{T}\mathbf{C} \tag{2}$$

**With:** Nm and Nf: numbers of male and female spawners; Mm and Mf: average weights of male and female spawners; TC: conversion rate; Fb and Bi: final and initial biomass during the larval stage.

Additionally, the remaining, not ingested, amount of food was monitored and estimated in order to fully adjust the supplemented amount to the needs of the fish.

#### *5.2.4 Zootechnical parameters*

To estimate the growth of fish during the different rearing phases, a number of indices and zootechnical parameters should be calculated.

#### *5.2.4.1 Body weight gain*

This index is used to evaluate the weight growth of fish during a given time. It is calculated according to the following formula:

$$\text{Gain } in\text{ average weight } (\mathbf{g}) = \text{final weight } (\mathbf{g}) - \text{initial weight } (\mathbf{g}) \tag{3}$$

#### *5.2.4.2 Daily growth rate (DGR)*

This parameter is determined for a short period of time from a fish sample during all rearing phases and is estimated according to the formula:

$$DGR = \frac{\text{Final Mass} - \text{Initial Mass}}{\text{Time}} \ast 100 \tag{4}$$

#### *5.2.4.3 Mortality*

This zootechnical parameter is of crucial importance when evaluating the rearing performance. It is therefore vital to monitor the cumulative mortality rate in the larval, fry and juvenile fish stock. This is the ratio of the number of dead individuals to the total population during a given timespan.

$$\text{Mortality rate} = \left(\frac{\text{Number of dead organisms}}{\text{Total Number}}\right) \ast 100\tag{5}$$

#### *5.2.4.4 Survival rate*

The survival rate is well correlated to the mortality rate. It is calculated from the total number of fish at the end of the experiment and the number of fish at the beginning of the rearing, according to the following relationship:

$$\text{Survival Rate} = \left(\frac{\text{Final specimen number}}{\text{Initial species number}}\right) \ast 100\tag{6}$$

#### *5.2.4.5 Food conversion rate (FCR)*

It is a food conversion index that measures the efficiency of the transformation of nutrition into fish flesh. It represents the ratio between the total amount of the supplied food to the fish and the gain obtained in fish weight.

$$\text{FCR} = \frac{\text{Dry weight of the supplied food}}{\text{Gain in fish weight}} \tag{7}$$

#### *5.2.4.6 Daily feeding ration (DFR)*

It is the ration supplied per day of rearing, and it depends closely on the feeding rate.

$$\text{DFR} = ((\text{Average weight} \* \text{Feeding rate})/100)) \* \text{Nombr of } \text{lavae} \tag{8}$$

#### *5.2.4.7 Length-weight relationship*

In order to properly control the growth parameters of broodstock, pre-grown fry and grown individuals in both environments, it is necessary to establish the relationship between the size and weight of the fish, which is defined according to Le Cren [65] by the following equation:

$$\mathbf{W} = \mathbf{a} \mathbf{L}^{\mathbf{b}} \tag{9}$$

with W: the weight of the fish in grams; L: the length of the fish (Tl, Fl or Sl) in centimeters; a: slope; b: coefficient of allometry defined as the coefficient of relative growth in weight.

Three cases can be distinguished: if b = b theoretical, there is an isometric allometry between the two characters, if b < b theoretical there is a negative allometry, and if b > b theoretical, the allometry is positive [66].

#### *5.2.5 Monitoring of physico-chemical parameters*

Throughout the whole experiment and during all the rearing phases, monitoring the physicochemical parameters was of primary concern. The different parameters, notably temperature, salinity, pH, and dissolved oxygen were measured with a Multi 350i/SET. Nitrites, nitrates and ammonium were also kept track of, because of their impact on water quality. The various physicochemical analyses were made in situ and in the laboratory.

#### *5.2.6 Data analyses*

The study of growth is a very delicate approach in fisheries and it requires a method which best suits the basic data, and the choice of the model that effectively describes the relationship between the variables. For this reason, and in order to have solid understanding of the prior effects of physicochemical and zootechnical parameters on the success of the rearing, it is necessary to carry out some statistical tests based on R, in order to determine the most suitable environment for tilapia farming. Thus, tests were conducted to determine the effect of different physicochemical parameters on larval rearing, pre-growth and grow-out.
