Determination of optimum dietary protein requirement, Ammonia tolerance and Biodiversity of potential aquaculture species Clarias batrachus in Pakistan

Abstract

Clarias batrachus is a commercially important food fish of Pakistan. In the present study, the effect of varying levels of protein was evaluated on the survival, growth parameters, and proximate composition of C. batrachus. Diets comprising 25%, 30%, 35%, 40%, 45%, and 50% crude protein were supplied to fish in T1, T2, T3, T4, T5, and T6, respectively, @ 5% of fish body weight for the entire 90 days, twice daily. Size of each stocked C. batrachus was recorded after 15 days. Results revealed a 100% survival rate of C. batrachus in all treatments. Highest significant (P<0.001) mean value of weight gain (g/fish), percent weight gain, daily growth rate, specific growth rate, and protein efficiency ratio (PER) in C. batrachus were recorded that were reared in T4 by feeding 40% CP in diet. The best FCR value (1.90±0.02) for C. batrachus was obtained in T4 by feeding 40% crude protein in the diet. Mean values of water, ash, fat, and protein contents (% wet mass) were ranged 74.10-79.23, 3.12-4.68, 3.90-4.43, and 13.09- 16.79 for C. batrachus in the studied treatment groups. Water content (%) was found significantly (P<0.05) higher in the body of C. batrachus for T1, T2, T3, and T6 than in T4 and T5. Ash was found significantly (P<0.05) higher in the fish reared in T4 and T5. Fat content in the wet body mass of C. batrachus was found significantly higher in T4 and T1. While significantly higher value of mean protein was noted in C. batrachus reared in T4 and T5. Body composition of C. batrachus was also categorically affected by body size, however, condition factor showed an insignificant correlation in most of the relationships in the present study. 180 specimens of Clarias batrachus, reared upon six experimental diets comprising 25% CP (T1), 30% CP (T2), 35% CP (T3), 40% CP (T4), 45% CP (T5), and 50% CP (T6), were collected for the study of concentrations of twelve important metals and to evaluate the effect of body size and condition factor on the concentration of these metals. Total length (TL) and body weight (BW) of each fish specimen were recorded and mean values were ranged 6.59-6.72 cm and 3.10-6.96 g, in various studied treatment groups. Concentrations (μg/g) of Fe, Cu, Zn, Cr, Co, Mn, Na, K, Ca, Mg, Cd, and Pb were analyzed in whole carcasses of the C. batrachus by atomic absorption spectrophotometer. Concentrations of Fe, Cu, Zn and Mg were found significantly xxi different (p<0.05) among treatments with varying dietary protein levels, with range of mean concentrations as 21.47-12.93 μg/g, 1.00-2.37, 11.37-18.76 and 173.31-302.56 μg/g in the carcasses of C. batrachus, comprising the highest concentrations in T4, in which the fish were fed upon a diet containing 40% of crude protein. Concentrations of Cr, Mn, Na, K, and Ca were not significantly different (p>0.05) among different treated groups and mean values were ranged 9.14-11.47 μg/g, 3.45-5.09 μg/g, 268.02-381.37 μg/g, 1265.57-1368.11 μg/g and 1331.58-1706.20 μg/g, respectively, in the body of C. batrachus. Mean concentration of Co remained at 0.01 μg/g in the fish body reared in all studied treatments. Whereas, two toxic heavy metals, Cd and Pb were not quantified in this study. Regression analyses represented significant correlation (on either p<0.05, p<0.01, or p<0.001) for Cu in T1 and T3; Co, Mn, and Na in T1; while Ca and Mg concentration (μg/g) in T4, with the BW of C. batrachus showing a definite impact of BW on metal concentrations in C. batrachus. TL of the fish also showed significant relationships for Cu and Nain T1, Zn in T3, and Co concentration (μg/g) in T4. Condition factor (Kn) of C. batrachus also represented correlations for Zn in T1, Co in T4 and T5, and for K concentration (μg/g) in T1. Overall findings of the study show that the fish reared in T4 by feeding 40% of CP contained high nutritional values concerning essential metals (Fe, Cu, Zn, and Mg), as these minerals are essential to our everyday lives. Ammonia nitrogen is the major oxygen-consuming pollutant in aquatic environments. The present study was designed to analyze the effects of ammonia exposure on growth performance, hematological parameters, and proximate composition of C. batrachus. 150 fish samples (70 in each treatment) with a mean initial total length of 13.33 ± 1.08, 13.46±1.80, 13.53±0.94, 13.40±1.94, and 16.31±1.57 cm, while body weight 22.36±5.84, 24.36±9.93, 23.38±5.17, 23.11±9.36, and 42.66±11.68 g were exposed for 3 months with different levels of ammonia concentrations (0, 0.25, 0.50, 0.75 and 1.00 mg/L), in T-o, T-I, T-II, T-III, and T-IV respectively. At the end of the experimental trial, 50 samples were randomly selected (10 from each treatment) for hematological study. While another 50 samples of C. batrachus (10 from each treatment group) were taken to analyze the body composition. It was observed that ammonia exposure induced a significant decrease in mean values of growth, condition factor and various hematological parameters such as White Blood Cell (WBC), Hemoglobin (Hb), xxii Red Blood Cell (RBC), Hematocrit (HCT), Platelets (PLT), Monocyte (Mon), Lymphocyte (LYM), and Mean Platelet Volume (MPV), while other haematological parameters including Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Volume (MCV), and Red Cell Distribution Width Standard Deviation (RDW-SD) were significantly increased (P < 0.05) as determined by Duncan’s multiple range test. Mean (±SE) values of water, ash, fat, and protein contents in % wet body weight of C. batrachus were found 77.07±0.80% to 82.23±0.55%, 2.98±0.03%to 3.34±0.17%, 3.90±0.19% to 4.58±0.30% and 10.53±0.40 to 15.36±0.56 in the different studied treatment groups. Water and protein contents were significantly (P<0.05) affected by the exposure of ammonia to C. batrachus. Significant highest level of protein content was found in the fish group which was not treated with ammonia exposure (T-0). Whereas, ash and fat contents showed no significant (P>0.05) difference between ammonia treated groups and the control group. Regression analysis showed a clear effect of fish size on water and protein contents in C. batrachus reared in different treatment groups. While, ash was found correlated only in T-0 (p<0.001; r=0.939), and fat in T-0 (p<0.001; r=0.929) and T-III (p<0.05; r=0.636). However, condition factor remained constant with the proximate composition of C.batrachus. In the research work, seven different DNA extraction methods (GeneJET kit, Phenol chloroform, TNES, Urea, High Salt, SNET, and Rapid MT) were used to compare the quality of DNA from the muscle tissue of C. batrachus. The highest DNA concentration range (973 to 1371 ng/μl) was observed in GeneJET kit, while the lowest (121 to 473 ng/μl) was in SNET extraction method. Trend of DNA purity A260/A280 and A260/A230 among all studied methods is following: GeneJET Kit > Phenol Chloroform >High Salt >Rapid MT >Urea> TNES >SNET and GeneJET Kit extraction > Phenol chloroform >Urea >High salt >Rapid MT > TNES > SNET respectively. Post hoc multiple comparison analysis of the seven different DNA extraction methods showed a significant difference between GeneJet Kit, phenol-chloroform with TNES, SNET, UREA, Rapid MT, and HS methods (Tukey, p<0.01). The present study will be effective for future researchers for successful and timely isolation of valuable DNA for further analysis of PCR amplification and sequencing process. xxiii To analyze Quality DNA isolate from different organs (Muscle, Caudal Fins, Dorsal Fin, Pelvic Fin, Pectoral Fin, Heart, liver, Intestine Gill) of C. batrachus by using Nanophotometer. Ninety samples were used, 10 from each organ, for DNA extraction using the Phenol chloroform DNA extraction method. It was found muscle showed the best yield based on concentration and absorbance value at A260/A280 and A260/A230 ratios with Nanophotometer, then other organs of C. batrachus. For molecular identification 15 (3 from each River) fish specimens of C. batrachus were collected from five different rivers in Punjab, Pakistan. DNA was isolated from muscle samples through the Phenol Chloroform protocol. The concentration purity of samples was assessed by determining their absorbance at A260/A280nm. Best samples of DNA were used for PCR amplification. Samples were sequenced from Lab Sdn Bhd, Malaysia. After sequencing, the size of C. batrachus sequences was 600 bp from River Jhelum, 560 bp from River Chenab, 492 base pairs from River Ravi, 594 bp from River Sutlej, and 601 bp from River Indus. Data on GenBank showed a maximum similarity index match with C. batrachus. Interspecific pairwise K2P minimum genetic distance was observed at 0.0000, and maximum genetic distance was observed at 0.1378%. Phylogenetic trees indicated that C. batrachus species identified in the current study showed a common ancestor reported from India. This study concludes that fish muscle is best for DNA extraction and molecular identification of C. batrachus based on CO1 gene sequence can be adopted for accurate identification of unknown specimens as this is a novel attempt in Pakistan to the best of our knowledge. To study the genetic diversity of C. batrachus in Pakistan, therefore, we tested C. batrachus samples from five river populations collected from River Jhelum (Rasul Barrage), River Chenab (Merala Headworks), River Ravi (Baluki Barrage), River Satluj (Head Sulemanki), and River Indus (Taunsa Barrage) in Punjab, Pakistan. Five RAPD primers (OPA-01 to OPA-05) were used to analyze the genetic diversity of C. batrachus. Results showed a total of 35 polymorphic bands out of total bands (331) were detected with five primers, associated with genetic diversity. Highest genetic diversity was found in River Chenab with 09 polymorphic bands (25.7%), and then River Satluj with 08 polymorphic bands (22.9%), River Ravi and River Indus both showed 07 polymorphic bands (20%), while lowest genetic diversity was found in River Jehlum with 04 xxiv polymorphic bands (11.4%). The current results conclude that a varied level of genetic diversity was detected among five river populations of C. batrachus using a RAPD technique in Pakistan. The physiological responses of Clarias batrachus (Linnaeus 1758), (body weight 1.30-1.37 g and length 5.25-5.30 cm) were observed. Fish were acclimated at 27 °C and critical thermal tolerance showed the highest CTmax (41.03 ±0.20) and CTmin value (11.06 ±0.15). At CTmax Total protein and SOD showed lowest while Respiratory brust, LPO, CAT, and POD showed higher values. Additionally, at CTmin Lysozyme, Respiratory burst and CAT showed lower while Total Protein, LPO, POD and SOD showed higher value which illustrated strong physiological mechanism of Clarius batrachus.