Effect of the Incorporation Level of Ripe Avocado Pulp Powder Persea americana on the Zootechnical Performances of Clarias jaensis Juveniles (Boulanger, 1909)

: In order to contribute to the valorization of agricultural by-products in fish feed, the effect of the incorporation level of the ripe avocado pulp powder persea americana on the zootechnical performance of Clarias jaensis Juveniles (Boulanger, 1909), was studied between February and July 2019 in the highland zone of West Cameroon. To attend the aims, 156 juvenile Clarias jaensis ( 25.82 ± 7.43g) settled in triplicates in floating tanks were fed four iso-protein rations (40% crude protein) corresponding to 0, 8, 12 and 16% incorporation of ripe avocado . Feed were distributed twice daily (8 am and 6 pm) at 5% of the fish biomass for 63 days. The following results were as follows: Survival rate, final mean weight, daily mean weight gain, specific growth rate and condition


Introduction
Fish, whether from the fisheries or aquaculture sector, plays a crucial role in improving and securing household food and nutrition in developing countries (Béné et al., 2015).Indeed, fish is an excellent source of protein, micronutrients and polyunsaturated fatty acids (FAO, 2018).Its global consumption person has increased from an average of 9.9 kg in the 1960s to 19.7 kg in 2013, and initial estimates for 2015 point to the crossing of the 20 kg mark (FAO, 2016).In Cameroon, this consumption is estimated at nearly 17.9 kg per person per year, which is an annual demand in tons of nearly 400,000 t/year, mostly met by massive imports of frozen fish of 220,000 t/year, a total national production of fish estimated at nearly 180,000 tons of fish of which less than 0,1% comes from aquaculture (Temegne and Momo, 2019).To overcome this problem, the Cameroonian government has decided to revive aquaculture to meet demand and reduce imports of frozen fish.However, the emergence of the aquaculture sector in Cameroon is confronted with many constraints, of which feeding is one of the main ones, influenced by the low availability and high cost of quality feed inputs to combine optimal growth, fish health and product quality, especially flesh for human consumption (FAO, 2016).
It is well known that in aquaculture, feed represents a large part of the production cost (Gandaho, 2007.However, the use of an inadequate feed can lead to reduced growth but more seriously to nutritional deficiencies or mortality due to increased susceptibility to infectious diseases.Therefore, it is important to assess the nutritional value of alternative feeds for fish based on locally available fruit byproducts and waste.Mabuhay in 2018 evaluated the nutrient contribution of plant and fruit wastes as feed additives in aquaculture.The use of plants as an input to fish feed have already been studied extensively, mainly on Chromolaena odorata (Boukila & al., 2009) and Desmodium uncinatum (Efole & al.,2016).However, not all plant components are generally used as feed within the fruit, while some of them, notably avocado (Persea americana), end up as waste in post-sale and post-harvest losses to the environment, leading to organic pollution.This waste can potentially be recycled and returned to the food chain by converting it into aquaculture feed.In Cameroon, nearly 30 to 40% of this avocado production is lost post-harvest and post-sale due to the lack of processing and storage units (Ludovic T., 2015), estimated at 200 tons / month by RECOSAF.However, based on the observation that avocado pulp is degraded by catfish and the fact that no study has been published on the use of avocado as an ingredient in the intensive feeding of Clarias jaensis, it seemed important and interesting to carry out this work.

Geographic Location
The study took place between April and June 2019 in the Kanhe-Moyo Integrated Fisheries and Aquaculture Research and Application Farm in Baham,West Region -Cameroon, the average altitude is 1700 m.
The objective was to evaluate the influence of the incorporation rate of avocado pulp meal (Table 1) on the zootechnical performances, a total of 156 juveniles of Clarias jaensis with an average weight of 25.82g ± 7.43g and a size of 15.06±0.4cm were randomly distributed in the 12 tanks of 40l each, placed in a pond of 80 m2 .The fish were fed for 63 days on four (04) iso-protein 219 feeds with 40% protein (tab.2) incorporated with 0, 8, 12 and 16% of ripe avocado pulp flour.15 kg of fresh pulp from post-harvest losses on the farm and from post-sale losses at the various avocado depots in Bafoussam were used.12 basins of 0.035m3 perforated with holes of 10cm diameter on both sides were used as infrastructure, mosquito nets to cover the basins, string to hold them together, a Secchi disc of 50cm length, a landing net for control fishing, an ichthyometer, a multiparameter of the brand HANNA Hi 9813-5, an oximeter of the brand MILWAUKEE, an electronic balance of 1g precision was used to take the weight of the juveniles.

Formulation and Preparation of the Feed Used
04 experimental feeds were formulated at 40% crude protein and separated into different treatments according to the incorporation levels (Tab.2)

Practical Feeding
The raw ingredients were finely ground and sieved through 400 micrometer mesh.In order to determine the amount of fresh pulp to be incorporated in each ration, a traditional process (Fig. 1) was used to condition the fresh pulp and determine its dry matter.After determining the dry matter of the avocado pupa, the pulp harvested from the spoiled ones was then added to the mixture of ingredients in the same proportions as the different incorporations, to which palm oil was added.Water was previously boiled to 2/3 of the amount of food taken, considering that a good part of it would evaporate during boiling, and was then added to form a malleable paste to be transformed into spaghetti through the die of a meat grinder (TC 22SL), it gave filaments of 3 mm in diameter.These filaments were then dried in a storfish electric dryer, broken down to the desired size, bagged and stored until distribution to juveniles.
Collection of spoiled avocados at the market and around the farm.

Experimental Design
12 basins of 35 L of useful volume were installed in triplicates in a random manner in a pond of 80m 2, equipped with a tarpaulin in its base, to prevent infiltration, and each corresponding to a treatment so as to allow water circulation and oxygen diffusion.They are held in place by a string attached to stakes at each end of the pond, posts at each end and floats attached to the basins to allow them to float.They were covered with mosquito netting and above them were suspended half bottles to serve as feeding points.
Subsequently, a total of 156 fish were introduced at a rate of 13 individuals per tank and acclimatised for one week.

Feed Distribution, Growth and Environmental Monitoring
The distribution of the different treatments was done at a frequency of twice a day (06h, 18h) at a rationing rate of 5% of the fishy biomass (photo a).In order to monitor growth and adjust the quantities of feed distributed per period, control fisheries were carried out every 21 days, at the cool hours of the day (6:30 am).All individuals were caught, weighed and then measured individually on a 1g precision scale and an ichthyometer.To monitor the physicochemical parameters of the environment, the temperature, dissolved oxygen and ph of the water were taken in situ every day (photo c) between 7 and 8 am using a Hanna multiparameter and a Milwaukee oximeter.The percentage of dry matter is determined by drying the feed and dry fish samples in an oven at 103°C overnight (12 hours) and calculated using the following formula: x 100 where Pf is Weight of crucible + sample after ovun, Pc is Weight of empty crucible, Po is Weight of sample.
After determination of DM, th e sample is placed in a muffle furnace at 500°C for 6 hours for the ash content and the following calculation is made: Where: Pf = Weight of crucible + sample after 6 hours in the oven; Pc = Weight of empty crucible

Lipids or Fats
The lipid content is determined by the Soxhlet method (AOAC, 1999).This method is based on the solubilisation of lipids in apolar organic solvents (petroleum ether, chloroform, benzene).In the sample, it is determined by the following relationship: Where m0 = 1g = weight of the sample; P0= weight of sample + cellulose weight of cartridge.

Crude protein
The total nitrogen content is determined by the Kjeldhal method (AOAC, 1990).The nitrogen (N) content is then calculated according to the following formula:

Economic Characteristics
The economic evaluation of the different treatments was carried out on the basis of the cost of food production calculated from the unit price of the ingredients obtained on the local market, with the exception of Persea americana pulp, the price of which was estimated from the transport from the market where they were collected to the study farm.The only parameter considered was the consumption index.Thus, the characteristics evaluated were the following: • Cost of a kg of food (Cf)  = Σ [quantiy of ingrédient (g) x cost of one kg (FCFA)x 1000] 100 • Food cost per kg of fish produced (FCf)

Statistical Analysis
Growth and biochemical parameters were expressed as mean ± standard deviation and as percentages.
An analysis of variance (ANOVA) was used to compare the different treatments and Duncan's test at a 5% threshold to separate the mean in case of significant difference using SPSS 20.0.The curves were made with Microsoft Office Excel 2016.

Clarias jaensis Juveniles as a Function of the Persea americana Ripe pulp Meal
The highest values of survival rate (SR) were recorded in treatments receiving 8 and 12% incorporation of Persea américana but statistical analysis showed no significant difference (P ˃ 0.05).The final average weight (pmf), and weight gain (GP) were significantly higher with the batches receiving 8% compared to the batches receiving 0% and 16%.On the other hand, the condition factor K did not vary much with the level of incorporation.However, the CI recorded a higher value with 8% incorporation.Incorporation levels (%) of ripe avocado pulp meal Persea americana in the feed

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The weight increased at all levels of incorporation steadily throughout the trial.This increase was more rapid, first at 8, then at 12 and 0 and finally at 16% incorporation.These results suggest that growth remained lower in the batch receiving 16% incorporation of Persea americana pulp flour.

Evolution of the Average Daily Weight Gain in Juveniles of Clarias jaensis
Figure 3 shows the evolution of the average daily gain as a function of the level of incorporation of ripe Persea americana pulp meal in the feed.

Figure 3. Evolution of the Average Daily Gain in Juvenile Clarias jaensis as a Function of the Level of Incorporation of the Ripe Avocado Pulp Meal Persea americana in the Feed
It was found that the average daily gain decreased throughout the trial regardless of the level of incorporation.At the end of the experiment, the values obtained were comparable, although the highest without significant difference (p>0.05) was observed in the batches fed at 8% incorporation.

Clarias jaensis Juveniles
Figure 4 opposite shows that the evolution of the specific growth rate of juveniles varied with the level of incorporation.
The evolution varied with the levels of incorporation, as shown in figure 4. Between the 1st catch ( 21) and the 2nd catch (42), there was a slight increase with all levels of incorporation, followed by a slight decrease until the end of the trial.However, the lowest values without significant difference with the other incorporation levels (p>0.05) were obtained with 16% incorporation of Persea americana pulp flour in the feed.

Juveniles of Clarias jaensis
Figure 5 shows the evolution of the K-factor as a function of the level of incorporation.

Biochemical analysis of juveniles
They show that the relative water content of the fish at the end of the experiment decreased with increasing lipid in all diets compared to the initial sample.While, protein and ash content increased but no significant difference (p>0.05) was found.Regarding lipid content, the results show that fish receiving 0 and 8% incorporation had the lowest levels compared to those receiving 12 and 16% incorporation of pulp meal which appeared to be the fattest at the end of the experiment than at the beginning.Note: Means on the same line with letter a are not significantly different (P<0.05).

Nutrient retention in juvenile Clarias jaensis in relation to incorporation levels
Nutrient retention is illustrated in Figure 6 below.It was found that protein retention was highest with 8 and 16% incorporation of avocado pulp flour, and lowest with 0 and 12% incorporation.
The highest value of lipid retention was obtained with 16% pulp flour incorporation followed by that with 12%.The ash retentions varied little, although the highest was obtained with 8% incorporation.

Clarias jaensis juveniles according to the level of incorporation
This table 5 summarises the feed cost of one kg of feed and one kg of Clarias jaensis as a function of the level of incorporation of Persea américana avocado pulp meal.The results show that the cost of feed was lower at 8% incorporation

Discussion
The highest survival rate of Clarias jaensis juveniles was obtained with 8% incorporation.
The values obtained at the end of the trial are comparable to those (94.87 to 100%) reported by Ekoué (2013) for Clarias gariepinus juveniles fed with Néré almond meal and soybean meal.and 12% pulp incorporation.The differences in growth observed at the end of the trial between these batches could be related to a difference in digestibility and assimilation which are a function of the nature of the ingredients used as pointed out by Burel et al (2000).
The specific growth rates (SGR) obtained at the end of the trial remain higher than those obtained by Hoffman et al. 1997  The results of the analysis of the protein content in the fish flesh varied between treatments.These values were lower than 20.29% (Francis & al., 2001) obtained with C. gariepinus and 34.16% (Efole & al., 2016) on juveniles of the same species.This could be due to the high fiber content of the experimental feed.Because Adelakun & al. (2014) showed that the high fiber content of the feed interferes with the processing of the feed in the fish gut, thus reducing the digestibility of the feed and contributing to protein loss.In contrast to the water content which decreased with the increase in fat.
The prices of the ingredients were those on the market at the time of the test, while the price of the avocado was determined by reference to the cost of transport.Feed costs varied with the levels of incorporation, however, they were lower than those obtained by Efole et al. in 2012 with desmodium incorporations (422.56 -430.5 FCFA), and comparable to those obtained by Kemegne, 2004 with incorporation rates (R0, R10, R15) of Chromolaena odorata whose values were 356.7; 227 381.26and 375 FCFA.Similarly, the cost of producing 1 kg of fish was low for the 8% incorporation and high for the 12% incorporation.The results obtained in this trial show that, under the experimental conditions, the incorporation of 8% of Persea americana pulp has the lowest feed cost, which indicates that it is the most economically efficient.

Conclusion
At the end of this trial, which focused on the effect of the level of incorporation of avocado pulp flour Persea americana on the zootechnical performance of Clarias jaensis juveniles, and in view of the economic profitability analyses, the results obtained showed that the incorporation of this agricultural by-product at a rate of 8% leads to a reduction in the financial charges linked to the cost of production of a kilogram of fish without any prejudice to the growth of fish.Therefore, the incorporation of avocado pulp flour is possible and advantageous and allows for the valorisation of this locally available by-product.

Note:
Figure 2 illustrates the evolution of the average individual weight of Clarias jaensis

Figure 2 .
Figure 2. Daily Evolution Mean Weight of Clarias jaensis Juveniles in Function of the Incorporation Level of the Ripe Avocado Pulp Meal Persea americana in the Feed

Figure 4 .
Figure 4. Evolution of the Specific Growth Rate in Clarias jaensis Juveniles as a Function of the Level of Incorporation of the Ripe Avocado Pulp Flour Persea americana in the Feed avocado pulp meal Persea americana in the feed 225

Figure 6 .
Figure 6.Variation in Nutrient Retention in Juveniles of Clarias jaensis Depending on the Level of Incorporation of the Ripe Persea Pulp Powder Americana in Feed (%)

Table 3 . Survival Rate and Growth Characteristics of Clarias jaensis Juveniles as a Function of the Level of Incorporation of Ripe Persea Americana Pulp Meal in the Feed Survival Rate and Growth Characteristics Incorporation levels of avocado ripe pulp meal
Note:Values are means ± standard deviation of three replicates of basins in each treatment; SR=Survival Rate; MWf=final Mean Weight; ADWG=Average Daily Weight Gain; SGR=Specific Growth Rate; CI= Consumption Index and K= condition factor.