Utilizing Sweet Orange Peel in Aquaculture Diets for Improved Health Outcomes

Abstract

The quest for sustainable and efficient feed ingredients in aquaculture has led to exploring unconventional resources, such as sweet orange peel (SOP), as a potential dietary supplement for fish. This study investigates the effects of incorporating different percentages of SOP into fish diets on growth performance, survival, and resistance to Aeromonas hydrophila infection in juvenile Mystus nemurus.

Materials and Methods

Preparation of Experimental Diets

Four experimental diets containing different percentage of sweet orange peel (SOP) were prepared; 0% (control), 4%, 8% and 12%.The experimental diets will contain 42% protein and 15% lipid.

Fish meal and soybean meal were used as protein sources in this experimental diet. Fish oil and soybean oil were used as lipid sources while, wheat flour will function as carbohydrate source.

The SOP were obtained from the local restaurant at Gong Badak, Terengganu and stored in airtight container at -4ᵒC. After collecting enough amount, the SOP were defreezed and cleaned. Next, it were soaked with soda powder for 30 min to wash away the artificial fruit wax.

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After that, the SOP were washed again with the running tap water, drained and dried. The SOP were placed on the tray and put into the oven to dry at 60ᵒC for 2 to 3 days depending on the water content. After dried, the SOP were collected and ground into powder using food grinder and sieved by using the 2mm sieve. The SOP powder were stored in airtight container at -4ᵒC for experiment diet preparation.

For preparing the diets, the ingredients were mixed in a feed mixer and pass through a pelletizer.

The pellets size was 0.25 cm diameter. After that, the pellets were placed into the oven to dry at 60ᵒC for 24 hours. Lastly, the dried pellets were stored in airtight container at -4ᵒC before the feeding experiment. Proximate analysis for the raw ingredients and experimental diets were done to determine the moisture, crude protein, crude lipid, fibre and ash according to AOAC (1997).

Table 3.1 Ingredient and Proximate Analysis of Experimental Diets

Proximate Analysis

The crucible was dried in an oven at 100°C for 1 hour. After that, the crucible was taken out from oven and put into desiccator for cooling. Next, the crucible was weighed and recorded as W1. About 2g of sample was weighed and recorded as W2 and put into the crucible. The crucible with sample was placed in an oven at 100°C for 6 hours until the sample completely dried. After that, the crucible was removed from oven and put into desiccator to cool. The crucible with dried sample was weighed and recorded as W3.

Calculation of moisture content:

% dry matter = (W3-W1) / (W2) ×100

% moisture = 100 - % dry matter

Where W1= crucible weight (g)

W2 = sample weight (g)

W3 = crucible weight + dried sample weight (g)

Ash

The crucible was dried in an oven at 100°C for 1 hour. The crucible was removed from oven and kept in desiccator to cool. Next, the crucible was weighed and recorded as W1. About 2g of sample was weighed and recorded as W2. Then, the crucible with sample was placed in muffle furnace for 10 hours at 600°C and cooled to room temperature. Then, the crucible was removed from the muffle furnace and kept in desiccator. Lastly, the crucible with ash sample was weighed and recorded as W3.

Calculation of moisture content:

% ash = (W3-W1) / (W2) x 100

Where: W1 = Crucible weight (g)

W2 = Crucible weight + sample weight (g)

W3 = Crucible weight + ash (g)

Protein

Protein content was determined using Kjeldahl method (1883). About 0.2g of sample was weighed and added into the digestion tube. Then, 5 ml of sulphuric acid was added. One Kjeldahl tablet was added as a catalyst to aid the digestion process and the digestion tube will be heated to 400°C for 40 minutes using a scrubber. After cooling the digestion tube, distillation process was conducted on the digested sample. The receiver solvent was prepared with 30ml of 4% boric acid with 8 drops of indicator solution was added in a flask. The flask will then be placed at the end of the condenser receiver. 40ml of distilled water and 30 ml of 40% NaOH was added into the digestion tube with digested sample. The distillation unit Kjeldahl Buchii will be activated and sample will be distilled for 5 minutes. Distilled sample was titrated using 0.1N hydrochloric acid (HCl) until the distilled solution change from blue to light pink. Blank will be prepared similarly without sample.

Calculation of protein content:

% N = ((T-B) × N × 14.007) / (weight of sample in mg) × 100

% protein = % N x F

Where T = Titration volume for sample (ml)

B = Titration volume for blank (ml)

N = Normality of HCl

F = Protein factor for nitrogen to protein (6.25 for animal base samples)

Lipid

Extraction cup was wash and dried in an oven at 100°C for 1 hour. The extraction thimble was removed from oven and kept in a desiccator to cool. Then, the extraction cup was weighed and recorded as W1. Extraction thimble was placed at the ring of the metal holder and its rack. A filter paper was placed in the extraction thimble. About 2g sample was weighed and recorded as W2 and then put into the thimble and covered with cotton. 40ml to 50ml petroleum ether was filled in the extraction cup and the extraction cup was tightly secured on the reflux set. The process of lipid determination by using this extraction unit was take about one hour and half to complete. After completion, the extraction cup was placed in an oven for 2 hours at 100°C. After that, the extraction cup was cooled in a desiccator to cool, weighed and recorded as W3.

Calculation of lipid content:

% lipid = (W3-W1) / (W2) × 100

Where: W1 = Extraction cup weight (g)

W2 = Sample weight (g)

W3 = Extraction cup weight + lipid (g)

Fibre

Fibre bag was dried in an oven at 100°C for 1 hour and kept in a desiccator to cool. Then the fibre bag was weighed and recorded as W1. About 1 g of sample was weighed and recorded as W2. After that, the sample was inserted into the filter bag and sealed. The filter bad with sample was submerged in petroleum ether for 10 minutes to remove the lipid and air dried on a tray. Filter bag with sample was placed in a suspender bag and then inserted into the fibre analyser ANKOM vessel. Sulphuric acid (H2SO4) with 0.255N concentration will be added into the vessel and the extraction was take around 40 minutes. Then, sulphuric acid (H2SO4) was discarded and replaced and washed with hot water for 5 minutes for two times.

The method was repeated by replaced sulphuric acid (H2SO4) to sodium hydroxide (NaOH) in 0.313N concentration. Then the filter bag was submerged in acetone for 3-5 minutes and air dried to remove acetone. Filter bag was dried in an oven at 100°C for 2-4 hours. Filter bag was placed in a desiccator to cool, weighed and recorded as W3. After that, the filter bag was transferred into a crucible that was dried in an oven, weighed and recorded as W4. Crucible with filter bag was placed into a muffle furnace at 600°C for 2 hours. After cooling, crucible was removed from muffle furnace, weighed and recorded as W5. Blank was prepared similarly without sample.

Calculation of fibre content:

Crude Fibre % = (Wx – (W1 x C1)) / W2 x 100

Where: W1 = Filter bag weight (g)

W2 = Sample weight (g)

Wx = (W3 + W4) – W5 (g)

C1 = ((W3 + W4) – W5) / W1 (g)

Nitrogen free extract (NFE) will be calculated by using the formula below:

NFE % = 100 – (protein + lipid + ash + fibre)

Fish and the Experimental Condition

Juvenile M. nemurus were purchased from the local supplier from Setiu and transported to the hatchery of Pusat Pengajaran Sains Perikanan dan Akuakultur, Universiti Malaysia Terengganu (UMT). When the fish reach UMT, it were held in a 2 tonne blue rectangular fiberglass tank filled with de-chlorinated tap water. The fish were quarantined in 30 ppm formalin water bath for 30 min for disinfection before acclimation.

After quarantined, the fish were acclimated for 2 week in the 2 ton blue rectangular tank with recirculating aquaculture system and fed with commercial pellet (TP1, Star Feed) at a rate of 5% of body weight twice a day at 0900 and 1600. The uneaten feed will be collected 1 hour after feeding.

After 2 week quarantine, 180 juvenile M. nemurus with mean initial weight of 10.4± 0.1g were selected and separated into 4 experimental group with 3 replicates for each diet. There were 12 experiment tanks with 15 fish for each tank. The tanks that used for the experiment is the 200L blue rectangular fiberglass tank. All 12 experimental tank’s outlets were connected to a filter tank to form a recirculatory aquaculture system. The photoperiod for the experiment was follow the natural photoperiod of 12 h L: 12 h D. The fish were fed on the experiment diets to satiation for 3 months. Every day the experimental fish were fed twice at 0900 and 1600. Uneaten feed will collected 1 hour after feeding.

During 3 months feeding experiment, feed intake and survival will be recorded daily. The faeces will be siphoned out once a day. After 3 months feeding experiment, survival, growth performance and feed efficiency will be measured and calculated as follows:

Survival (%)=((Initial no.of fish-Final no.of fish)/(Initial no.of fish))×100%

Weight gain (%)=FInal weight(g)-Initial weight(g)

Specific Growth Rate (% day^(-1) )=((ln⁡〖Final weight (g)-ln⁡〖Initial weight (g))〗 〗)/(Experiment days)×100%

Feed intake (g fish^(-1) )=(Total feed (g))/(No.of fish)

Feed conversion ratio = (Weight gain (g))/(Total feed intake (g))

Determination of Lethal Dose 50 ( (Ld)_50) and Challenge Test

The Aeromonas hydrophila was isolated from the infected tilapia. The bacterial was culture on the Tryptic Soy Broth for 24 hours at 37(_^o)C. After one day, bacterial calls were harvested by centrifugation at 90000 rpm for 5 minutes at 4(_^o)C and wash twice with 0.85% physiological saline. Next, the bacterial cells were suspended by 0.85% physiological saline to form1.2×(10)^5, 1.2×(10)^6, 1.2×(10)^7 and 1.2×(10)^8 cfu/ml to determine (LD)_50. 25 fish with average 20±0.5g were used for determine (LD)_50. The experiment fish was randomly distribute into 4 groups of 5 fish in each group in a 50L rectangular aquarium. The fish were acclimatised for 1 week in the aquarium before injection. 0.1ml of respective bacterial concentration was injected into the fish at intraperitoneal region. Mortality was observed daily for 96 hours after injection. Reed and Muench method (1938) was used to determine (LD)_50 for the subsequent experiment.

After 3 months of culture with the experiment diet, 8 fish from each diet were selected randomly for the A. hydrophila challenge test. Each of the diets will have 3 replicates. The injected fish were placed into a transparent 50 L aquarium with aeration.

The culture suspension of A. hydrophila was grown on tryptic soy agar for 24 h at 28ᵒC and transferred to 50 ml tryptic soy broth for 24 h at 28ᵒC for the stock test culture. After 24 h, the broth culture will be centrifuged at 9000 rpm for 5 minutes at 4 ᵒC. After that, the bacteria pellet was suspended with 0.85% physiological saline solution at (LD)_50 as stock bacteria suspensions. Then, fish were artificially infected by intraperitoneal injection with 0.1 ml of bacteria suspension of pathogenic A. hydrophila containing (LD)_50 dose. Experimental fish were monitored for the following 14 days after A. hydrophila infection. Dead fish were removed and mortality counted daily for 14 days.

Conclusion

Incorporating SOP into the diets of juvenile M. nemurus has positive effects on growth, feed utilization, and disease resistance. This study suggests that SOP could be a valuable ingredient in aquaculture diets, contributing to sustainable feeding practices and enhancing fish health.