Length-weight relationship and condition factor of Nile tilapia ( Oreochromis niloticus) fed diets supplemented with guava and star gooseberry leaf extract

Background Nile tilapia ( Oreochromis niloticus) is predominant cultured species in aquaculture. However, there is a scarcity of literature regarding relationship between guava and star gooseberry leaf extract and the condition factor. Thus, the present study aims to investigate the effect of guava and star gooseberry leaf extract-supplemented diets on the specific growth rate, length-weight relationship, and condition factor of Nile tilapia. Methods Six hundred and thirty Nile tilapia (8.7±0.4 g) were randomly distributed among twenty-one tanks (30 fish per tank) within a recirculation system. Over a 60-day period, the fish were fed diets supplemented with 5g/Kg and 10g/Kg of guava leaf extract (GLE-5, GLE-10), star gooseberry leaf extract (SGLE-5, SGLE-10), and a mixture of both (MxLE-5, MXLE-10). Subsequently, specific growth rate, length-weight relationship, and condition factor were determined. Results After 60 days, the specific growth rate was significantly higher in all the GLE, SGLE, and MxLE groups compared to the control group. The final lengths and weights differed significantly both in the control group and all the GLE, SGLE, and MxLE groups. The analysis of the regression equation indicated a positive correlation (r = 0.970, 0.977, 0.976, 0.974, 0.974, 0.974, and 0.969) between the length and weight of Nile tilapia in the control group and in all the GLE, SGLE, and MxLE groups. The regression exponent " b" values in all the GLE, SGLE, and MxLE groups were >3, indicating a positive allometric growth pattern in Nile tilapia compared to the control ( b=2.866), which exhibited a negative allometry. The final condition factor values did not differ significantly in either the control group or any of the plant extract groups. Conclusions Nile tilapia exhibited positive allometric growth patterns and maintained good health when fed with GLE, SGLE, and MxLE groups. Therefore, these plant extracts demonstrate suitability for commercial Nile tilapia production.


Introduction
2][3][4] In 2020, the global production of Nile tilapia nearly reached 4.9 million metric tons (Mmt), and the continuous growth is attributed to the robust worldwide demand. 5Furthermore, technological advancements with high stocking densities play a significant role in the occurrence of disease outbreaks, resulting in substantial economic losses and impeding the sustainable progression of tilapia culture globally. 6e incorporation of plant extracts into aquafeed is significant in finfish aquaculture 7 because the indiscriminate use of antibiotics has led to the development of drug-resistant bacteria. 8,9Plant extracts can serve as cost-effective and environmentally sustainable prophylactic or antimicrobial agents.Guava (Psidium guajava) leaf (GLE) are rich in various phenolic and flavonoid compounds 10 and possess antimicrobial, 11 antiviral, 12 and antioxidant 13 activities.5][16][17][18][19][20] Star gooseberry (Phyllanthus acidus) leaf (SGLE) contain flavonoids, kaempferol, hypogallic acid, caffeic acid, and phenolic compounds. 21,22SGLE exhibited antioxidant, anti-inflammatory and antimicrobial activities. 23,24It is also employed as a dietary supplement to augment the immune response of fish 9,25 and chickens. 26e length-weight relationship (LWR) plays an essential role in estimating the biomass of diverse fish populations. 27dditionally, LWR is valuable for assessing fish condition and growth patterns. 28The condition factor (CF) contributes to comprehending the development and adaptability of fish. 29A heightened condition factor indicates the robust health and positive growth of the fish. 30Hence, the condition factor can be employed to assess the feeding activity of a species, indicating its effective utilization of available feeding sources. 271][32][33] Unfortunately, there is a scarcity of literature regarding relationship between guava and star gooseberry leaf extract and the condition factor.Thus, the present study aims to investigate the effect of guava and star gooseberry leaf extract-supplemented diets on the specific growth rate, length-weight relationship, and condition factor of Nile tilapia.

Preparation of guava and star gooseberry leaf aqueous extracts
The guava (P.guajava) and star gooseberry (P.acidus) leaf powder were received from All-Season Herbs Pvt. Ltd., Bangalore, India and the aqueous extract was prepared. 35Briefly, distilled water (100 mL) was mixed with leaf powder (10 g) and homogenized using an orbital shaker (Hsiangtai D500) at 100 rpm for 20 h at room temperature (RT).The resultant mixture underwent centrifugation at 8,000 rpm for 15 min at RT.A rotavapor (BÜCHI R-200/205) at 35 °C used to evaporate the supernatant and continue drying for 48 h in a hot air oven at 50 °C.The dried samples obtained were placed in bottles and kept refrigerated at 4°C until further use.

Ethical statement
This study was conducted under the project entitled 'Application of Phytobiotic Supplementation to Improve Disease Resistance of Nile Tilapia (Oreochromis niloticus)' and funded by the Social Justice and Special Assistance Department, Government of Maharashtra, India, and Second Century Fund (C2F) Postdoctoral Fellowship, Chulalongkorn University, Bangkok, Thailand.This study was carried out in strict accordance with the recommendations of the Asian Institute of Technology (AIT), Thailand, and the protocol was approved by the Local Ethical Committee for Experiments on Animals of the AIT, Thailand (Project Number: AIT-AQ-18-01, May 2018-April 2019; April 29, 2018).Additionally, according to Thailand Legislation, the Nile tilapia were not categorized as a protected species.The research was carried out at the Laboratory of the Aquaculture and Aquatic Resources Management (AARM, Asian Institute of Technology (AIT), Thailand, over 60 days from February to March, 2019.This study is reported in line with the ARRIVE guidelines.A total of 630 fish were used in the experiment, with 90 fish allocated to each treatment.Each treatment comprised 30 fish per replicate, totaling three replicates.Prior to sampling, the fish underwent a 12-hour fasting period to empty their digestive tracts.In this study, to reduce stress on the experimental animals, the animals housed in the containers were gently anesthetized using 1 mL of clove oil per 10 L of water for a period of 2-3 min, after which their weights were measured.Following this, the fish were returned to their designated glass tanks according to their treatment and replication groups.

Fish
Nile tilapia monosex fingerlings (8.7 AE 0.4 g and 8.0 AE 1.0 cm) were obtained from a GMP Certified fish farm, Ayutthaya Province, Thailand.The research was conducted at the laboratory of Aquaculture and Aquatic Resources Management (AARM), Asian Institute of Technology (AIT), Thailand.For a period of 15 days, the fish were acclimatized in three fiberglass tanks (500 L) before the commencement of the experiment.Fish were fed pelleted feed twice daily at 4% of their body weight.Following the acclimatization period, the fish were randomly distributed among twenty-one 150-L glass tanks (30 fish per tank), all integrated into a recirculation system.
Preparation of experimental diets supplemented with guava and star gooseberry leaf aqueous extracts Plant extracts (10 mg), dissolved in 1 mL distilled water, were mixed with commercial feed ((Charoen Pokphand (CP)-7710), and spaghetti-like strands were prepared using a mincer (MITSUYAMA YC80B-4).Pellets approximately 5 mm in length were formed and dried for 24 h at 50 °C in a hot air oven.The dried pellets were then stored at 4 °C until the end of the experiment.The proximate composition of the commercial diet, including moisture, ash, crude lipid, and crude protein, was 7.6%, 8.2%, 6.5%, and 30.5%, respectively.

Experimental design
In this study, a completely randomized design was employed, comprising seven experimental treatments: 5g/kg and 10g/kg of guava leaf extract (GLE-5, GLE-10), star gooseberry leaf extract (SGLE-5, SGLE-10), and a mixture of both (MxLE-5, MXLE-10).The mixed diets (MxLE) were prepared with an equal proportion (1:1) of GLE and SGLE extracts.The control group, on the other hand, was fed a diet without plant extract supplementation.Fish were fed twice daily at 4% of their body weight.Prior to sampling, the fish underwent a 12-hour fasting period to empty their digestive tracts.In this study, to reduce stress on the experimental animals, the animals housed in the containers were gently anesthetized using 1 mL of clove oil per 10 L of water for a period of 2-3 min, after which their weights were measured.Following this, the fish were returned to their designated glass tanks according to their treatment and replication groups.The specific growth rate, length-weight relationship, and condition factor were determined after 60 days of feeding.

Water quality parameters
During the experiment, a Eutech Cyberscan PC300 multi-parameter apparatus was employed to measure daily water quality parameters, including temperature, pH, and dissolved oxygen (DO).The weekly concentrations of ammonianitrogen (NH 3 -N) were assessed using Phenate or indophenol method, 36 nitrite-nitrogen (NO 2 -N) concentrations were quantified through direct spectrophotometric assay, 36 and nitrate-nitrogen (NO 3 -N) levels were determined employing cadmium reduction method. 36

Specific growth rate of Nile tilapia fed diets supplemented with GLE, SGLE and MxLE
The specific growth rate 37 was evaluated after 60 days of feeding trials using the following equation Length-weight relationship (LWR) of Nile tilapia fed diets supplemented with GLE, SGLE and MxLE Length-weight relationship (LWR) 38 between the total length and body weight was estimated using the following formula Where W is body weight of fish (g), L is the total length (cm), a is the exponent denotes the rate of weight change with length (constant), and b is the slope representing the weight of one unit length.These values are estimated through the linear regression equation, which is transformed by applying the natural logarithm (Log) to both sides.
Where b = 3, growth pattern is isometric.However, when b is greater or less than 3, an allometric pattern emerges.This allometric pattern can be positive, indicating increase in length relative to body thickness, or negative, signifying an increase in length relative to body thinness. 28lative condition factor (K n ) of Nile tilapia fed diets supplemented with GLE, SGLE and MxLE The health condition of the tilapia was assessed by evaluating relative condition factor (K n ) in all the treatments.
where W o = observed weight, and W c = calculated weight. 39When K n is greater than 1, it indicates favorable growth conditions for the fish.Conversely, when K n is less than 1, it suggests that the organism is in poorer growth condition compared to an average individual of the same length.

Data analysis
The results were presented as means AE SE, and statistical analysis was conducted using IBM SPSS Statistics software (SPSS, Inc., Version 29).The homogeneity of data was determined using Levine's test.The impact of plant extract treatments on specific growth rate was assessed through one-way analysis of variance (ANOVA), followed by Tukey post-hoc tests for multiple comparisons.An independent t-test was used to determine the significant difference between the initial and final length and weight of Nile tilapia with plant extract treatments.Linear regression analysis was employed to evaluate the length-weight relationship.A significance level of p < 0.05 was considered for statistical significance.

Discussion
Aquafeed supplementation with herbal or plant extracts has proven to be an effective means of enhancing the growth performance, feed utilization, and immune response in farmed fish species. 40,41The supplementation of natural bioactive rich-plant extracts may boost natural immunity and improve disease resistance, 40 resulting in higher production and profitability in the aqua industry.The length-weight relationship is used to predict the weight of fish in proportion to their length over a certain period. 38Therefore, the present study investigated the effects of plant extract-supplemented diets on the length-weight relationship and condition factor of Nile tilapia.
The water quality parameters in all treatments were within the acceptable range, consistent with the findings of previous studies on spotted snakehead (Channa punctata) under various feeding regimes involving agro-industrial by-products, 42 and on silver barb (Barbonymus gonionotus) fingerlings fed with dietary blanched moringa leaf meal. 43The specific growth rate showed a significant increase in all plant extract treatments.Similarly, the specific growth rate of spotted snakehead (C.punctata) and silver barb (B.gonionotus) was considerably improved under various feeding regimes involving agro-industrial by-products 42 and dietary blanched moringa leaf meal. 43ua farmers and consumers prefer fish with isometric growth or body structure. 27,44Furthermore, the length-weight relationship provides insights into the growth and health of a fish species. 45,46In the present study, plant extractsupplemented diets observed significant correlation between length and weight of Nile tilapia.Our results are corroborated with previous study involving different maltose levels fed to Nile tilapia (O.niloticus). 27Importantly, Nile tilapia fed diets supplemented with GLE-5, GLE-10, SGLE-5, SGLE-10, MxLE-5, and MxLE-10 groups exhibited positive allometric growth pattern compared to the control.Similarly, silver barb (B.gonionotus) and zebrafish (Danio rerio) fed with dietary blanched moringa leaf meal 43 and plant-protein based diets 47 found positive allometric growth.On the contrary, gold fish (Carassius auratus) fed with different concentrations of carrot 48 and gurami sago (Osphronemus goramy) stocked in concrete ponds, floating net cages, and earthen freshwater ponds, 49 found negative allometric growth.
In the present study, plant extracts diets revealed a significant improvement in specific growth rate and positive allometric growth patterns of Nile tilapia.This can be ascribed to the presence of bioactive compounds in GLE and SGLE, including gallic acid, p-coumaric acid, quercetin, and kaempferol.These compounds have been demonstrated to enhance the growth of various fish species, including common carp Cyprinus carpio, 50 snakehead fish Channa argus, 51 grass carp Ctenopharyngodon idellus, 52 as well as certain animals. 53,54Further research should be conducted in pond or cage culture systems to substantiate the positive allometric growth patterns exhibited by Nile tilapia when fed diets supplemented with GLE and SGLE.
The mean K n values for both the plant extract diets and control were higher than one, indicating that the Nile tilapias were in good health condition during the experimental period.Similarly, condition factor values were more than one when Sargassum meal fed to Nile tilapia O. niloticus, 31 Mansoa alliacea hydroalcoholic extracts fed to pirarucu Arapaima gigas, 55 different maltose levels fed to Nile tilapia O. niloticus. 27

Conclusion
The specific growth rate was significantly higher in GLE, SGLE, and MxLE groups compared to the control group.The analysis of the regression equation indicated a positive correlation between the length and weight of Nile tilapia in the control group and the GLE, SGLE, and MxLE groups.Furthermore, the GLE, SGLE, and MxLE groups exhibited a positive allometric growth pattern in Nile tilapia compared to the control.The final condition factor values did not differ significantly between the control group and any of the GLE, SGLE, and MxLE groups.Consequently, these plant extracts demonstrate suitability for commercial Nile tilapia production.
-Table 8: Initial and final length (cm), weight (g), and condition factor of Nile tilapia (N=75) fed diet supplemented with control for 60 days.
-Table 15: Summary of the chemicals and reagents used in the experiment.
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Agumassie Tesfahun
Debre Tabor University, Debre Tabor, Ethiopia I advice the authors to include correlation matrix or principal component analysis (PCA) regarding the water quality parameter and fish biometric relationships (length and weight).To this end, please refer other published works in the same title.The paper is sound, however, it should be more sound if the authors include more statistical analysis such as correlation matrix or principal component analysis (PCA) to know the relationship between environmental variables and fish biometric data.Except for this issue, the paper is well presented and innovative for the scientific community.
Capture fishery is now declining due to human induced impacts on the water bodies.Aquaculture is the alternative option to feed this fast-growing population.Therefore, this work is very important for the fish farmers and the community as well for food security.Reviewer Expertise: fisheries, wildlife I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Is
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Figure 1 .
Figure 1.Specific growth rate of Nile tilapia fed diets supplemented with plant extracts.Results with the means of three replicates AE SE; Different superscripts in the bar indicate significant difference (p < 0.05).

Figure 2 .
Figure 2. Final logarithmic length-weight relationship with regression equation for Nile tilapia fed plant extracts.
the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesIs the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?NoAre the conclusions drawn adequately supported by the results?PartlyCompeting Interests: No competing interests were disclosed.

Table 1 .
Water quality parameters of Nile tilapia fed diets supplemented with plant extracts.

Table 2 .
Length-weight relationship, growth pattern, and condition factor of Nile tilapia fed diets supplemented with plant extracts.