Oyster mushroom meal as partial replacement of maize meal in diets of Nile Tilapia (Oreochromis niloticus)

Samuel Segun Ashley-Dejo, Musa Alhaji Musa¹, Ijabo Samuel Ogah¹, Mohammed Sabo¹ and Ogo Princes Mercy¹

Department of Forestry, Wildlife and Fisheries, Olabisi Onabajo University Ago-Iwoye, Ogun State, Nigeria
ashleydejosamuel@gmail.com
¹ Department of Fisheries and Aquaculture, Federal University Gashua, Yobe State, Nigeria

Abstract

Performance of Oreochromis niloticus fed with Oyster mushroom was evaluated as maize meal was supplemented in part by Oyster mushroom at 0%, 25%, 50%, 75% and 100%. Fish samples with initial average weight 2.84 g were stocked randomly into fifteen tanks (M0%, M25%, M50%, M75% and M100%) at a density of 20fish/tank and were fed twice daily for 56 days. M50% had best mean values for weight gain (10.1±0.05), SGR (2.81±0.07) and MGR (8.91±0.15). Haematological parameters revealed high mean values of PCV (41.1) and Hb (12.5) for M50% and M100% respectively. Also, M50% had the best mean value of MCV (150±5.66) and MCH (42.9±5.66). It was further observed that high density lipoprotein cholesterol (HDLc) was highest in M0% (59.1±0.49) while M50% (37.6±0.49) recorded the lowest HDLc. M50% had the highest LDLc (34.1±1.27) while M25% (15.3±1.73) recorded the lowest LDLc. Best performance of O.niloticus after 56days was observed in 50% inclusion level of Oyster mushroom.

Keywords: fish, growth, haematology, serum

Introduction

Fish is important as a valuable source of animal protein in human diets. Its demand in developing countries is higher than the supply (Ashley-Dejo and Adelaja 2022). Fish has continued to be the most easily affordable source of animal protein to millions of people in both developed and developing nation (FAO 2020). It is a good source of thiamine, riboflavin, vitamins A and D phosphorus, calcium and iron (FAO 2020). In meeting the supply of fish most especially in developing countries there is need to venture into more productive fish production enterprise (Ashley-Dejo and Adelaja 2022). Fish production in Nigeria is adversely affected by spontaneous rise in cost of production especially fish feed which account for over 65% of cost of production (Ashley-Dejo et al 2017). In order to intensify fish production through aquaculture, adequate nutrition would be required for attaining high fish yields. Okoye and Sule (2001) stated that the rapid expansion and success of commercial fish culture depends largely on availability of good quality and cheap feed. To fish farmers, in order to minimize running cost, it is important to use cheaper alternative feed ingredients that are locally available to produce good quality, suitable and palatable fish feeds (Omojowo et al 2010; Ashley-Dejo et al 2014). Harnessing non-conventional feedstuffs and by-products for aqua feeds could reduce unit cost of fish production (Ashley-Dejo et al 2020). Akinrotimi et al (2007) and Jamiu and Ayinla (2003) opined that feed management determines the viability of fish farming which accounts for at least 65% of the cost of production.

There is need to intensify the culture of fish, so as to meet the ever-increasing demand, also to develop suitable diets either in supplementary forms for ponds or as complete feed in tanks (Olukunle, 2006). For the purpose of nutritional and economic benefits, researchers have made attempts at increasing the use of non-conventional plant and animal materials to replace conventional feed ingredients such as maize and fish meal in fish feed (Falaye, 1988; Fagbenro, 1992; Olatunde, 1996; Baruah et al 2003; Eyo 2004). The use of cereal products, especially maize in fish feeds is becoming increasingly unjustified in economic terms, because of its ever-increasing cost. There is therefore need to exploit cheaper energy sources to replace expensive cereals. Also, to relieve the food competition between man and animal and for-profit maximization.

Both protein and energy are important components in fish feed maintenance, growth and reproduction. Their adequate supply in both quality and quantity enables fish to realize their full growth potential in aquaculture. Conventionally, fishmeal and cereals have been used as protein and energy sources respectively (El-Sayed, 1999; Gatlin et al 2007).

Mushrooms are the fruiting body of micro-organisms, which is known as fungi. Like other plants, mushrooms lack in chlorophyll and do not produce photosynthesis. It has medicinal values, aside from being good source of carbohydrate, vitamins, minerals, antimicrobial and therapeutic qualities (Batool et al 2020). This support its potential to replace traditional feeds (Batool et al 2020). The wellbeing of fish is reflected significantly on its blood profile, this have made haematology major monitoring indicator in fish culture (Jan and Ahmed 2021). This research established the performance and blood indices of Oreochromis niloticus fed with Mushroom

Materials and methods

Study Area

This research was carried out in the Department of Fisheries and Aquaculture, Federal University Gashua, Yobe State, Nigeria.

Source of ingredients/ preparation of experimental diets

Oyster mushroom (Pleurotus ostreatus) was sources from department of Forestry and wildlife, Federal University Gashua, Yobe State, it was identified by an agronomist in the department, while other ingredients such as soyabeans, groundnut cake, maize, vitamin premix, lysine, methionine, salt and binder were procured from local market in Gashua. Oyster mushroom were air-dried after harvesting for eight days. This was introduced into the feed at various inclusion levels (0%, 25%, 50%, 75% and 100%) respectively and pelleted into 2 mm and 3 mm sized feeds using a locally fabricated flat-die pelletizer. The pelleted diets were dried at room temperature for five days and stored in air tight plastic container.

Oyster mushrooms

Oyster mushrooms are widely distributed throughout the world. It grows in the wild on dead organic matter in tropical and temperate regions. Belonging to the genus Pleurotus and species ostreatus. The cap is normally shell-like about 1.9 – 7.8 in diameter, fleshy, with eccentric or lateral stipe. Its cultivation dated 1917 in Germany, using natural spawn for inoculation of wood logs and stumps. The first large-scale cultivation on logs was achieved in Hungary in 1969 and is now grown commercially around the world for food.

Nutritional value of oyster mushroom (Pleurotus ostreatus)

On the average the protein content of oyster mushroom ranged between 10,5-30.4% on dry weight basis, with essential amino acids between 33.4-46 g/100g of corrected crude protein showing significant amounts of lysine, leucine, and methionine. Fat content ranged between 1.1 - 2.2% on a dry weight basis, having a high proportion of unsaturated fatty acid (79.3%). The carbohydrate content varies from 46.6 - 81.8% on a dry weight basis. Main vitamins present in 100 g dry weight of oyster mushrooms are thiamine (1.16 - 4.8mg), niacin (46 - 108.7mg), and ascorbic acid (7.4). Fiber (7.4 - 27.6% on a dry weight basis) and minerals (potassium, phosphorus, iron, copper, zinc) are also present in good proportion. (Martinez-Carrera et al 1999).

Photo 1. Growing oyster mushroom	Photo 2. Harvested oyster mushroom	Photo 3. Processed oyster mushroom

Experimental Fish and Experimental Set Up

Three hundred O. niloticus juvenile were procured from a notable fish farm in Kano and transported to the Fisheries Laboratory, Federal University Gashua, where the initial body weight was measured with triple bean balance. The juveniles measured an average weight of 2.84 g. At the laboratory, fish samples were disinfected with potassium per manganate (2 ppm) for 20 minutes. Thereafter, stocked in a tank of (2 m x 2 m x 0.4) and fed twice daily, in the morning at 08.00 hour and evening at 20.00 hour with 2 mm Coppens feed at 5% body weight and proper management routine was carried out. This process lasted for two weeks prior to the transfer of samples to experimental tanks.

The experimental fish Nile tilapia samples were separated into fifteen tanks of 3.2 m² (2 x 2 x 0.8) water holding capacity and each tank was stocked with twenty (20) O. niloticus juveniles. The tanks M0% had no mushroom meal in the experimental diet, while M0%, M25%, M50%, M75% and M100% were fed with the experimental diets respectively.

Duration of study

For a total of 8 weeks this study was performed, across July to August, 2020.

Nutrient digestibility

The digestibility trial was conducted in white rectangular plastic tanks, 15 Nile tilapia fingerlings were stocked per tank (three tanks per treatment) and fed the experimental diets at 5% body weight / day, twice daily for 15 days. Feces from fish fed each diet were collected at the bottom of settling column in 150ml conical flask on 15 separate days and pooled for each treatment. Pooled feces were oven-dried to a constant weight at 105ºC. The chromic oxide content of the diets and the feces was determined in triplicate 50-100mg. Portions of moisture – free samples using Furukawa and Tsukahara (1966) methods. The proximate composition and gross energy content of the feces were determined according to AOAC (1990) methods. Apparent digestibility coefficients (ADC) for protein and energy were calculated according to Austreng and Refstie (1979) and Page and Andrews (1973) as follows;

ADC protein = 10 x (a – b) / a;

ADC energy = Ed – (Ef x Id / If)

Where a: a = protein in diet / Cr₂O₃ in diet; b = protein in feces / Cr₂O₃ in feces; Ed = gross energy of diet; Ef = gross energy of feces; Id = Cr₂O₃ in diet; If = Cr₂O₃ in feces.

Laboratory analyses

The experiment was terminated after 8 weeks and the fish were killed. At the end of the experiment, fish were anaesthetized with tricaine methanesulfonate (MS222) at 250ppm in water. Blood was drawn near caudal peduncle from one fish from each group and one part of blood was transferred into a heparinized tube for hematological study and another part of blood was centrifuged at 1500*g for 5 mins at room temperature (24ºC) to obtain plasma, which was then stored at -20ºC for chemical composition analysis. Prior to determination to the determination of proximate composition, the fish were autoclaved at 121ºC for 20 mins, thoroughly homogenized using an Ultra-Turrax M25%5, frozen and freeze-dried.

The proximate composition of diet ingredients, diets and whole body of fish was determined using the standard methods of the Association of Official Analytical Chemists (AOAC 1990). Gross energy (GE) determination was done using bomb calorimeter (IKA C7000) and benzoic acid was used as a standard. Detailed analytic methods are as follows.

Dry matter

Samples of approximately 0.5 g were weighed into pre-weighed crucibles, placed into drying oven at 120ºC overnight. The crucibles were then transferred into a dessicator for about 1hour to cool off. Finally, the crucibles were weighed at room temperature (25 ºC). Prior to the weighing, all corresponding crucible weight was noted. The dried matter was calculated as follows. DM %= [(crucible weight with sample – empty crucible weight)/sample weight] *100.

Ash

This was a follow up from dry matter sample. The dry sample from dry matter determination were placed into muffle furnace (Naberherm) at 500ºC for 6hr. Then, the crucible with the sample from dry matter determination were transferred to a dessicator for approximately one hour and weighed at room temperature (25 ºC). Ash was calculated as follows. Ash % = [(crucible weight with ash – empty crucible)/sample weight]*100.

Crude protein

Samples (approximately) 0.2g were weighed into crucibles in replicates. The samples were placed in the C/N analyzer to determine the nitrogen content. Crude Protein was then determined by multiplying nitrogen values with the factor 6.25.

Crude lipid

Smedes methods was used to determine the crude lipids content of the samples. About 0.2g of the samples was weighed into 12ml plastic tube, thereafter 2ml of isopropanol and 2.5ml of cyclohexane was added to the sample. The tube with the content was then vortexed for 30seconds and shook strongly by hands for 2mins. This tube containing mixture was hanged on the ultrasonic bath for 15mins and 2.75ml of nanopor-H2o was added. Vortex was repeated for another 30seconds and the mixture was shook again for 2mins and then centrifuged for 8 minutes at 3000*g. The supernatant was transferred using Pasteur pipettes into a glass vial that was earlier weighed. The residue was again suspended with cyclohexane/isopropanol mixture, vortexed for 30seconds and shook strongly for 2mins. The supernatant was also collected with pipettes and added into one earlier collected. This content was then allowed to evaporate at 37ºC under compressed air for approximately 30min. The dried fat was transferred into an oven at 50ºC for 2hr and later placed in a dessicator to cool down before weighing. Fat level was calculated as follows.

Lipids (%) = {[(Vial with lipid (g) – empty vial (g)] /sample weight}*100

Gross energy

Samples (0.25g) were weighed in triplicates and placed in a bomb calorimeter (IKA-Calorimeter C7000 Isoperibolic) to determine the gross energy (GE) content using benzoic acid as a standard.

Evaluation of Growth parameters and nutrient utilization

Growth performance and diet utilization for the experiment was determined by measuring the following. Weight gain (WG), Specific growth rate (SGR), Metabolic growth rate (MGR), Feed conversion ratio (FCR), Protein efficiency ratio (PER), Apparent lipid conversion (ALC), and Energy retention (ER). The underlisted formular were used.

WG = Final body mass – Initial body mass

WG% = [(Final body mass – Initial body mass) / Initial body mass]*100

SGR % = [(In final body mass in g) – In initial body mass in g) / number of trial days]*100

MGR = (Weight gain, g) / [(initial body mass in g / 1000)^^0.8 + (final body mass,g /1000^^0.8} / 2] / duration of trial days (Gatlin et al1996)

FCR = dry feed fed (g) / weight gain

PER = fresh body weight gain (g) / crude protein fed

PPV = [(final fish body protein, g – initial fish body protein, g) / total protein consumed,g]*100

ALC (%) = [(final fish body lipid, g – initial fish body lipid, g)/ total crude lipid consumed, g]*100

ER (%) = [(final fish body energy – initial fish body energy) / (gross energy intake)]*100

Hemato-immunological parameter evaluation

A comprehensive diagnostic of the blood composition was done on experimental set up.

Blood collection

Tricaine methane sulfonate (MS222, sigma chemical Co., USA) at 250 ppm in water was used to anaesthesized the fish, after which blood sample were taken with 2ml heparinized syringe and needles from the caudal vein of five O. niloticus from each treatment and put separately in 2ml heparinized tube. This was later taken to the Veterinary Clinic Laboratory in our university for haematological and immunological analysis.

Total erythrocyte (RBC) and total leucocyte (WBC) count

RBC and WBC were counted by Neubauers counting chamber of haemocytometer. Trapping of air bubbles was avoided. The RBC lying inside the five small squares were counted under high power (40X) of light microscope. Formula used to calculate the number of RBC per mm³ of the blood sample was.

Number of RBC/mm³ = ( N x dilution)/area counted x depth of fluid

Haemoglobin and Packed Cell Volume (PCV) content

Haemoglobin content of the blood was analysed using Reflotron Haemoglobin test (REF 10744964, Roche diagnostic GmbH, Mannheim Germany). PCV was determined on the basis of sedimentation of blood. Heparinised blood (50µl) from the hematocrit capillary (Na-heparinised) and centrifuged in the hematocrit 210 Hettich Centrifuge (Tuttlingen Germany) to obtain PCV value.

Following parameters were calculated from analyses of PCV, Hb and RBC.

Mean cell volume [MCV (Fl)] = (PCV [in L/L] x1000)/(RBC count [in millions/µL]);

Mean corpuscular haemoglobin, [MCH (pg)] = (Hemoglobin [g/dL] x 10 / (RBC count [in millions/µL]), and

Mean cell haemoglobin concentration, MCHC [in g/dL ] = Haemoglobin [in g/dL ] / PCV [in L/L]

Cholesterol and triglyceride estimation

Nobiflow cholesterin (kit lot number 60041889; Hitado Diagnostic system) and Nobiflow triglyceride-GPO (kit lot number 60040710) were used to determine plasma cholesterol and triglycerides respectively. The color intensity was measured spectro-photometrically and it was directly proportional to the concentration of cholesterol and triglycerides in the plasma sample.

Cholesterol concentration in the sample (mg/dl)

Where 200 is the concentration of triglycerides in blood sample

Triglyceride concentration (mg/dl) = Absorbance of sample x standard concentration (mg/dl)/Absorbance of standard

The concentrations of very low-density lipoprotein cholesterol (VLDLc) and low-density lipoprotein cholesterol (LDLc) were calculated by;

While low density lipoprotein cholesterol LDLc was calculated by the equation below:

LDLc = Total serum cholesterol – (HDLc + TG/2.2) mmol/l

Water quality monitoring

Water quality parameters in each tank were recorded once during the experimental period. The pH was recorded by a pH meter (Schott Greate, Florida state, USA) and dissolved oxygen (DO mg/L) by Winkler titration (Kyle et al 2019). Temperature (ºC) was recorded daily with mercury in glass thermometer at 8.00 h and 14.00 h.

Data Analysis

Data collected were analyzed using Analysis of Variance (ANOVA) in an entirely randomized strategy. Comparisons among diets means was carried out by Duncan Multiple Range Test at p < 0.05. All computation was performed using statistical package SPSS version 23.

Results

The proximate composition of Oyster mushroom was examined and presented in Table 2.

The proximate composition of feed with varying inclusion level of mushroom was presented in Table 3. It was revealed that highest value of crude protein (36.5%) and NFE (40.1%) was found in M75%, while lowest was recorded in M100% (35.3%) and (35.6%) respectively. While highest value of crude lipid (8.92%), crude fibre (4.84%) and ash (7.86%) was found in M100%

Table 4 shows the carcass composition of O. niloticus fingerlings before and after the feeding trial. The crude protein content of fish fed M0% was greater (p > 0.05) than protein content of M25%, M50%, M75% and M100%. Same trend of results was observed for dry matter and fat content.

The growth indices of O. niloticus at M50% revealed that the mean weight gain (10.1±0.05), SGR (2.81±0.07) and MGR (8.91±0.15) showed better performance compared to the other treatments. The regression result in Figure 1 where R² = 0.89 showed that 89% of the weight gain trend can be said to have resulted from the variation in diets inclusion level.

Figure 1. Relationship between weight gain and inclusion level of mushroom meals in diets of O. niloticus

The apparent digestibility coefficient (ADC) of fish was highest in M50% and lowest in M100%. The regression result in Figure 2 where R² = 0.67 showed that 67% of the feed conversion ratio trend can be said to have resulted from the variation in diets inclusion level.

Figure 2. Relationship between feed conversion ratio and inclusion level of mushroom meals in diets of O. niloticus

Hematological parameters of O. niloticus fed experimental diet was presented in Table 7. PCV of fish feed M50% (41.1±0.51) was the highest (p > 0.05) compared to PCV of fish feed M75% and M100% respectively. Experimental fish fed M0% (32.4) recorded the lowest PCV. The Haemoglobin (Hb) of fish fed M100% (12.5) was the highest while fish in M0% (11.4) recorded lowest Hb. Same trends of results as observed for Hb was RBC, WBC and MCHC.

Cholesterol and triglyceride levels of O. niloticus fed experimental diets was presented in table 8. Triglyceride content was greater in M50% (120±1.59) (p < 0.05) compared to other treatments, nevertheless, M25% (101±0.19) recorded the least triglyceride content. Cholesterol content of fish in M50% (98.3±1.05) was greater (p > 0.05) compared to cholesterol of M0% and M100% treatments. M75% (87.0±0.99) recorded the lowest cholesterol content. High density lipoprotein cholesterol (HDLc) was greater (59.1±0.49) (p > 0.05) while M50% (37.6±0.49) recorded the lowest HDLc. Similar trends were observed in triglyceride level was recorded for very low-density lipoprotein cholesterol (VLDLc). M50% had the greatest LDLc (34.1±1.27) (p < 0.05) from other treatments, while M25% (15.3±1.73) recorded the lowest LDLc.

TRIG-Triglyceride CHOL- Cholesterol, HDLc – High Density Lipoprotein cholesterol, VLDLc – Very Low-Density Lipoprotein cholesterol LDLc- Low Density Lipoprotein cholesterol

Summary of water quality parameters of experimental water was presented in Table 9. The parameters measures were temperature, pH, total dissolved solid and dissolved oxygen which were similar in all treatment.

Discussion

The growth response and nutrient utilization of experimental fish ( O. niloticus) fed experimental diet (mushroom) did not follow a particular trend. Fish fed with diet M50% had the best MWG, SGR, FCG. ADC protein value were also similar among treatments but ADC energy was lower in M100%. This result was similar to the findings of Moreau et al (2003) who substituted maize with coffee pulp in O. niloticus. The results also corroborate with the findings of El-Sayed and Gaber (2004) who replacement yellow corn torpedo grass. Possible explanation that could be given to the decrease in growth performance and nutrient utilization at higher replacement level as observed in M75% and M100% respectively might be due to lower palatability of the experimental diet. The body protein composition of O. niloticus does not varies this indicate optimum protein utilization. Lovel (1989) stated that for protein and other nutrients to be optimally utilized, non-protein energy is important and must be adequately provided for. This indicated that all the treatments have satisfied the energy requirements of O. niloticus.

The haematological parameters observed in this study was similar to that reported by Yue and Zhou (2008) and El- Saidy and Gaber (2004) who fed cotton seed meal to juvenile hybrid Tilapia and Nile Tilapia respectively. Also, the result corroborates with the study of Osuigwe et al (2005) and were all in the range of normal haematological parameter of a healthy fish (Clark et. al. 1979; Erondu 1993; Osigwe et al 2005).

No haematological effect was observed in O. niloticus fed experimental diets suggests that fish growth was not affected by the health of the fish. The decrease in plasma cholesterol concentrations in fish fed diets with plant proteins is in accordance with the results of Yamamoto et al (2007), Kumar et al (2008) and Makkar et al (2009). The fish hypocholesterolemia in response to dietary plant protein supply could be either due to an increased in the excretion of bile salt, to an inhibition of cholesterol intestinal absorbtion, or just to the withdrawal of fish meal rather than to the direct effects of plant protein (Kakwi and Olusegun, 2020). Serum triglycerides act as a short-term indicator of feeding or nutritional status (Bucolo and David 1973). In our study 50% oyster mushroom inclusion levels exhibited higher cholesterol and triglyceride level in plasma than other diets.

Water quality parameters observed in this study falls within an acceptable range and with limits of national standard (Dinesh et al 2020). It was observed that at high range of dissolved oxygen (7.67), growth performance was high. This finding agrees with Dinesh et al (2020) they establish that growth performance decrease with decrease level in dissolved oxygen and also Batool et al (2000) established that as dissolved oxygen declined from 100% to 30% there was a progressive reduction in growth performance.

Conclusion

• Maize is one of the most important fish feed ingredients. It is also an expensive ingredient due to its nutritional value and can be very scarce in some seasons.
  
  
• Oyster mushroom is one of the cheapest close substitutes, being a plant feed stuff. It has a nutrient profile that can substitute adequately to what is obtained in maize and it can also be made available all year round to support the aquaculture industry.
  
  
• This study has shown that 50% substitution level of oyster mushroom meal in O. niloticus diet produces better result than without substitution.

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