Effect of Lactobacillus plantarum and Pediococcus pentosaceus on the Growth Performance and Morphometry of the Genetically Improved Farmed Tilapia (Oreochromis niloticus)

Zahir Muhammad1, Muhammad Zubair Anjum1*, Shamim Akhter1, Muhammad Irfan1, Saira Amin1, Yousaf Jamal1, Sharjeel khalid2 and Shakira Ghazanfar2 1Department of Zoology, Wildlife and Fisheries, PMAS-Arid Agriculture University Rawalpindi, 46300, Pakistan 2National Institute for Genomics, Advanced Biotechnology (NIGAB), National Agriculture Research Center, Park Road, Islamabad-44500, Pakistan Article Information Received 03 July 2022 Revised 20 July 2022 Accepted 05 August 2022 Available online 14 October 2022 (early access)


INTRODUCTION
T ilapia is the second most commercial farmed fish worldwide after carp (Xia et al., 2020). In 2018, tilapia production was approximately 6.882 million tonnes (FAO, 2020) anticipated it could be reached 7.3 million O n l i n e

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the world (Dey and Gupta, 2000). GIFT strain plays a significant role in improving aquaculture outcomes and establishes farmers' income in Asia (Tran et al., 2021). The main issue in fish farming is low fish production, control of communicable diseases and cost-effectiveness. Antibiotics have been used for the prevention and treatment of diseases in aquatic animals to improve aquaculture production as a result antibiotics used produced antibiotics resistance bacteria, disturbed microbiota of the host, that destroy the host, aquatic environment, and reminders in the flesh are hazards for the consumer (Kuebutornye et al., 2020). Currently, synbiotics, probiotics and prebiotics are used in aquaculture as fed additives instead of antibiotics to enhance the growth rate and immunity of the host (Hoseinifar et al., 2017;Sayes et al., 2018). The microorganisms either live, dead, or their components provide health benefits to their host when used for a specific duration and optimum concentrations are termed probiotics (Salminen et al., 2021). Probiotics act as immune modulators, manipulate gut microbial community towards beneficial microbiota, have no side effects, ability to remove pathogens, and improve the growth of culture species. Probiotics are also defined as substances or microbes that can renovate microbial balance define by parker (Hill, 1993). According to World Health Organization (WHO) probiotics as live microorganisms either used as single strain or consortia forms of strains that provide health benefits to organisms by taking in recommended amounts (Rehaiem et al., 2014). Probiotics are eco-friendly feed additives that increased fish production (Chowdhury et al., 2020).
The most commonly used probiotics belong to Lactic acid bacteria Lactobacillus (acidophilus, fermentum, plantarum), Lactococcus, Pediococcus, Enterococcus, Bifidobacterium, and yeast such as Saccharomyces boulardii (El-Saadony et al., 2021;Sehrawat et al., 2021). Due to unique physiological, morphological, and metabolic characteristics lactic acid bacteria as well as secreting different enzymes (i.e. amylases, lipases, proteases) and a variety of health-promoting organic acid and aromatic compounds, make them effective probiotics. They secrete antimicrobial peptides, which are harmful to pathogens without any harm to the host (Siripornadulsil et al., 2014). Probiotics tolerate gastrointestinal (GI) tract harsh environmental barriers such as acidic secretions, pH, enzymes, and bile acids because they ferment carbohydrates into short-chain fatty acids, which lower celiac pH (Levy et al., 2017).
The fish digestive tract also grants a site for attachment and multiplication of various bacteria (probiotics) that compete with pathogenic bacteria for the attachment site and nutrients to improve the fish immune system that provokes lysozyme and burst respiratory function, and stimulate a cellular immune response against pathogens. Probiotics also inhibit pathogens proliferation; by producing different substances such as bacteriocins, hydrogen peroxide, antibiotics, siderophores and lysozymes (Akhter et al., 2015;Xia et al., 2018) There is no publication on probiotics bacteria Lactobacillus plantarum and Pediococcus pentosaceus on the growth performance and morphometric traits of the GIFT tilapia. Therefore, the purpose of this study was to assess the impacts of probiotics bacteria strains L. plantarum and P. pentosaceus on growth, and morphometry of the GIFT tilapia.

Ethical statement
All experiments were carried out following the rules and regulations adopted by the ethical committee of PMAS-Arid Agriculture University Rawalpindi, Pakistan.

Experimental site and fish collection
The research experiment was conducted at Aquaculture and Fisheries Laboratory, Department of Zoology, Pir Mehr Ali Shah, Arid Agriculture University Rawalpindi, Pakistan.

Acclimatization of fish
The fingerlings (n=120) were randomly distributed to 12 aquaria having the size of 1×1×1 foot each, equipped with air stones for the supply of oxygen. Fish were acclimatized to the laboratory environment for 7 days and fed with basal diet, i.e., 30% crude protein (1.5mm) commercial feed (Marine Grow Fish Feed; Hi-Tech Feeds Private Limited, Pakistan).

Experimental setup
Four treatment groups (control, T1, T2 and T3) of

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aquaria in triplicate manner were established randomly containing 30 fingerlings in each group. The control group was treated with the T0-basal diet; T1 was treated with the basal diet + L. plantarum; T2 was treated with the-basal diet + P. pentosaceus and T3 was treated with the basal diet + L. plantarum + P. pentosaceus. For the preparation of diet, the required amount of fluid suspended probiotics were taken when needed and dried by using a fan, and stored in airtight plastic jars at 4 o C. The probiotics were sprayed on the stored basal diet after every 7 days to maintain the original 1×10 8 cfu. Fish were fed two times per day at the rate of 5% body weight for 60 days. A total of 50% water was exchange every day to maintain the water quality.

Assessment of fish growth performance and feed utilization
For growth performance and morphometry measurements, 5 fish samples were randomly collected from each aquarium fortnightly. Fish were weighed with an electronic balance. After the measurement, fish were put to their corresponded aquarium. For evaluation of growth performance final body weight (FBW), average daily weight gain, weight gain, specific growth rate, percent weight gain and feed conversion ratio were taken. Growth performance calculations were carried out by using the following formulae; described by Chowdhury et al. (2020) and Panase and Mengumphan (2015).

Morphometric traits measurements
Morphometric parameters were measured by standard protocol described by (Apparao, 1961). Five fish samples were randomly collected from each aquarium for morphometric measurements. Morphometric traits were measured in centimeter, using a measuring board and transparent ruler. A total of nine morphometric parameters of GIFT were measured including; total length (TL) was measured from the tip of snout to the end the caudal fin, and standard length (SL) from the tip of snout to the start of caudal fin. Similarly, head length (HL) also measured the tip of snout to the most posterior part of the operculum. Dorsal fin length (DFL), pectoral fin length (PFL), pelvic fin length (PvFL), fin length (AFL) and caudal fin length (CFL)were measured from the base of fin to the most anterior tip of fin. The eye diameter (ED) was measured as space joining the front and lateral edges of eye in the longitudinal position.

Statistical analysis
The mean growth and morphometry parameters were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) to identify the significant differences among the treatment groups in Statistical Package for the Social Sciences (SPSS) software. The level of significance was at P < 0.05. The results were presented as means ± standard deviation.

Fortnightly growth performance
Fortnightly weight gains of T1, T2, and T3 groups were significantly better (P < 0.05) than the control (Fig.  1). However, weight gain was increased in T3 as compared to the control, T1 and T2 groups (P < 0.05).

Fortnightly growth performance
Fortnightly weight gains of T1, T2, and T3 groups were better significantly (P < 0.05) than the control (Fig.  1). However, weight gain was increased in T3 as compared to the control, T1 and T2 groups (P < 0.05).

Morphometrics traits
The morphometric parameters length values are presented in the Table II. Weight gain and morphometry  are correlated Table III. In T3, TL, SL, DFL, HL, ED, PFL, PvFL, AFL and CFL lengths were increased significantly (P < 0.05) than the control and 2 other treatments. T1 and T2    Table I. TL, total length; SL, standard length; DFL, dorsal fin length; HL, head length; ED= eye diameter; PFL, pectoral fin length; PvFL, pelvic fin length; AFL, anal fin length; CFL, caudal fin length; cm, centimetre. differ non-significantly (P > 0.05) to each other in terms of TL, SL, OL, and PFL. There was no significant difference (P > 0.05) among T0, T1, and T2 in terms of ED, PvFL, AFL, and CFL. In T1, HL length increased significantly (P < 0.05) as compared to T2 and T0.  For statistical details and abbreviations, see Table II. r, coefficient correlation; W, weight.

DISCUSSION
Probiotics are successfully implemented in aquaculture due to their potential effects on aquatic animals  and also used a bioremediation tools (Eissa et al., 2022). For the sustainable aquaculture industry, probiotics were suggested to use for improving growth performance and well-being of aquatic animals (Ringo et al., 2020). According to author knowledge there is no data available on the effect of consortium (L. plantarum O n l i n e

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Effect of Lactobacillus plantarum and Pediococcus pentosaceus on the Growth Performance 5 + P. pentosaceus) on the growth rate, feed utilization, and morphometrics traits of GIFT. In current study, significantly improved growth rate and feed utilization were observed in all probiotics treatment groups as compared to control. Significantly higher growth performance, lowest FCR, improved morphometric traits and more positive allometric weight-length correlation were examined in T3. The current study is in order with previous studies confirmed that tilapia fed with probiotics diet showed improved growth performance and feed consumption (Dawood et al., 2019;Elsabagh et al., 2018;Gobi et al., 2018;Mirzakhani et al., 2019), and Labeo rohita (Ahmad et al., 2016). Possible reasons for improved growth rate and feed utilization in probiotics tested groups could be (1) by producing growth factors such as vitamins, co-factors, fatty acids, amino acids (Balami et al., 2022) and essential amino acids (i.e. isoleucine, lysine, tryptophan, leucine and histidine) and non-essential) amino acids (i.e. glutamate, tyrosine and alanine) are released during fermentation process (Ndagijimana et al., 2009;Rodrigues et al., 2011), and vitamins (i.e. vitamin C, vitamin B12, and vitamin B9) (Rodrigues et al., 2011;Rossi et al., 2005). These biologically active compounds might play vital role in food absorption, assimilation and growth of aquatic animals.
(2) P. pentosaceus releasing extracellular enzymes such as amylases, proteases, and lipases reported in shrimp (Adel et al., 2017;Wanna et al., 2021). Similar mechanism showen by P. pentosaceus fed to Cyprinus carpio (Ahmadifar et al., 2020). Likewise results of L. plantarum when fed to Nile Tilapia (Van Doan et al., 2018) and also reported in other species (Dawood and Koshio, 2016;Jannathulla et al., 2019) which enhanced nutrients breakdown such protein, starch, and lipid, thereby improved growth rate and feed utilization of GIFT.
(3) L. plantarum producing exopolysaccharides that increase intestinal adhesion and colonization of probiotics which turn to improve intestinal health (Zhao et al., 2021). The intestinal surface area of GIFT was increased by increasing height, width of villi . (4) L. plantarum upregulated growth related genes expression such as glucose-6-phosphate dehydrogenase (G6PD), insulin-like growth factor (IGF-1) and down regulated fatty acid synthase (FAS) gene expression in the muscle and liver tissues of GIFT. The enhanced level of cellular respiration is the indication of high level of G6PD expression is responsible to maintains energy supplies needed for fish growth . Broiler chicken fed with L. plantarum also shows higher expression of IGF-1 and growth hormone receptor (GHR) (Humam et al., 2019). (5) probiotics suppressing pathogenic bacterial activities and modulated beneficial gut microbiota (Terpou et al., 2019).

CONCLUSIONS
The mixture of probiotics (L. plantarum +P. pentosaceus) caused improved growth performance, feed utilization and morphometry in GIFT as compared to L. plantarum or P. pentosaceus alone and control. Therefore, these mixture probiotics could be a better option for culturing GIFT.

Statement of conflict of interest
The authors have declared no conflict of interest.