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Stocking Density Induced Stress on Plasma Cortisol and Whole Blood Glucose Concentration in Nile Tilapia Fish (Oreochromis niloticus) of Lake Victoria, Kenya

Stocking Density Induced Stress on Plasma Cortisol and Whole Blood Glucose Concentration in Nile... Hindawi International Journal of Zoology Volume 2020, Article ID 9395268, 8 pages https://doi.org/10.1155/2020/9395268 Research Article Stocking Density Induced Stress on Plasma Cortisol and Whole Blood Glucose Concentration in Nile Tilapia Fish (Oreochromis niloticus) of Lake Victoria, Kenya 1 1 2 1 Elija Odhiambo , Paul O. Angienda, Patrick Okoth , and David Onyango Department of Zoology, Maseno University, Kisumu, Kenya Department of Biological Sciences, School of Natural Sciences, Masinde Muliro University of Science and Technology, Kakamega, Kenya Correspondence should be addressed to Elija Odhiambo; elija.odhiambo@gmail.com Received 16 November 2019; Revised 16 April 2020; Accepted 19 June 2020; Published 17 July 2020 Academic Editor: Jo o Pedro Barreiros Copyright © 2020 Elija Odhiambo et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Effects of high stocking densities (HSDs) were evaluated for Nile tilapia fish (Oreochromis niloticus) under culture to determine its influence on plasma cortisol and whole blood glucose concentration. Plasma cortisol levels (ng/ml) were assayed by Enzyme- Linked Immunosorbent Assay (ELISA). Whole blood glucose levels were determined using a hand-held one touch ultraglucose meter (MD-300) and test strips. Plasma cortisol and whole blood glucose level determinations were replicated three times for O. niloticus reared under both low stocking densities (LSD) and HSD. One way Analysis of Variance (ANOVA) was performed on the data collected, and comparison of significant differences in means was carried out between LSD and HSD at 0.01%. Plasma cortisol levels revealed statistically (P≤ 0.01) significant values of HSD at 6.32 ± 1.06 ng/ml than in LSD at 4.62± 1.58 ng/ml for the O. niloticus groups studied. Whole blood glucose analysis revealed a statistical (P< 0.05) difference in the means in HSD and LSD O. niloticus groups (F � 7.946> F � 4.414; P � 0.01). Mean plasma glucose concentration was statistically (P≤ 0.01) (df,1; 8) crit higher for HSD than LSD O. niloticus groups at mean± SD, 96.84± 5.28 and 76.82± 5.92, respectively. (e findings of this study demonstrate that high stocking densities increase both cortisol and whole blood glucose concentration in tilapia fish, indicating a marked increase in stress levels. Elevated plasma cortisol and whole blood glucose concentration can be used as biomarkers for acute stress in O. niloticus produced under aquaculture systems. (e findings of this study can help inform policy on the management of stress caused by overstocking of O. niloticus and other related Cichlids under industrial aquaculture production. fish [8] and the most desired by Lake Victoria Community 1. Introduction [9]. It is also an important fish model for studies on social Fish and fisheries play an important role in addressing stress due to overcrowding [10]. nutritional and livelihood food security, especially of the Stocking density is considered a key factor in deter- poor in the developing countries [1]. Globally over 2 billion mining the productivity of fish aquaculture systems [11]. people get at least 20% of their animal protein intake from Crowding is judged as one of the most influential stressors fish [2, 3]. Nile tilapia (Oreochromis niloticus)are among the affecting fish physiology and, thus, the status of well-being in most important warm water fishes used for aquaculture aquaculture [12], and it is a common husbandry practice in production [4] and only the second most popular farmed aquaculture [13, 14]. High stocking density negatively affects fishes after Carps [3, 5, 6]. (ey are recognized as one of the both fish growth and feed utilization [1, 15]. High stocking most important species in tropical and subtropical aqua- densities have been shown to reduce feeding activity and culture [2, 5, 7]. Nile tilapia have a mild white flesh that growth rates in Coho Salmon (Oncorhynchus kisutch) and appeals to customers, making them economically important rainbow trout (Oncorhynchus mykiss) but have a positive 2 International Journal of Zoology effect on these parameters in Arctic charr (Salvelinus alpi- aquarium were fed on a carbohydrate-based feed of chick nus) [16]. It also increases the level of metabolites such as mash (18% protein) (Sigma Feeds Ltd., Nairobi, Kenya) urine and faeces in cultured Chrysichthys nigrodigitatus [17]. supplemented with crushed silver cyprinid (Rastrineobola (is stress response changes water quality [16], thus further argentea) to 25% protein at a feed portion of 10 g/kg of life subjecting fish to chemical stressors [18]. High stocking body weight, and the feeding was terminated 24 hours to density subjects fish to chronic stress [19, 20]. It is reported sampling [13]. by [21] that stocking density and social interactions between specific fish have a significant influence on stress. Stress is 2.3. Sample Collection and Storage. 21-day-old fish were considered to be a generated response, but it can be mod- anaesthetized with 2-phenoxyethanol (chemical formula: ulated by specific stressor conditions. One of the most C H O ; active substance: ethylene glycol monophenyl 8 10 2 frequent causes of chronic stress is social interaction among ether) obtained from BDH Laboratory Reagents, England at members of the same species. −1 a concentration of 0.30 ml l of water for 10 min before Stressors cause distinct stress responses in O. niloticus samples were extracted [31] to minimize suffering. A large [22] leading to elevated plasma cortisol [16, 23], the main plastic sieve was then used to draw fish (n � 5) samples from hormone that activates glucose [24], therefore elevating each of the six aquaria. Blood was then drawn through glucose levels [19, 23, 25]. Fish respond to stress by in- cardiac puncture using EDTA- (ethylene diamine tetra- creasing circulating cortisol [16, 23] and glucose [23]. acetic acid-) coated (obtained from BDH Laboratory Cortisol is released in response to chronic stressors. Elevated Reagents, England) hypodermic syringes [32] as an anti- plasma cortisol [19, 26] and glucose levels [16, 21, 26, 27] are coagulant. (is took not more than 1 min for each fish so as used as indicators of stress. (is study investigated the effect to avoid rise in blood cortisol levels due to handling stress of high stocking density-induced stress on the concentration [13, 22]. Some of the drawn blood samples (n � 20, i.e., three of cortisol and whole blood glucose in Oreochromis niloticus samples from each of the six aquaria and a further one of Lake Victoria under culture. sample from each of the second aquaria of the LSD and HSD groups) were then transferred into labelled Eppendorf tubes, 2. Materials and Methods each containing 1 ml EDTA solution, placed onto crushed ice (0 C) in an icebox, and transported to the laboratory for 2.1. Study Area. Nile tilapia fingerlings were obtained from 2 storage under refrigerated conditions. the Kenyan part of Lake Victoria (4, 100 km ), a fresh water lake. (ey were then transferred to the fish rearing facility at o o Maseno University (0 22′ 11.0″ S, 35 55′ 58.0″ E/Latitude; 2.4. Sample Analyses 0.369734, Longitude; 35.932779) located 400 km west of Nairobi where this study was conducted. Tropical climatic 2.4.1. Plasma Cortisol. Plasma cortisol levels (ng/ml) were conditions characterize this region. assayed for by Enzyme-Linked Immunosorbent Assay (ELISA) using a Neogen Corporation ELISA kit (Lansing, Ml, USA, 2010) [33] and the company’s Cortisol ELISA 2.2. Fish and Experimental Procedures. (e study protocol protocol. (is quantitative analysis of cortisol levels in the was reviewed and optimised by experts from the Department biological fluid (O. niloticus blood plasma) was performed at of Zoology, Maseno University, Kenya, and the Department the Kenya Medical Research Institute (KEMRI), Kisumu, of Biological Sciences, Masinde Muliro University of Science Kenya. An antibody-coated 96-well microplate was used. and Technology, Kenya. Healthy Nile tilapia of Lake Victoria (e standard solutions and the diluted samples were first were obtained from the fish rearing facility of the Depart- added to the microplate in duplicates. Diluted enzyme ment of Zoology, Maseno University. (ey were first conjugate was then added, and the mixture was shaken and acclimatised to the laboratory conditions (temperature, incubated at room temperature for 1 hour to allow com- dissolved oxygen, water quality, water pH, nutritional status, petition to take place between the enzyme conjugate and photoperiod, size, weight, colour, and shape of experimental cortisol in the samples for the limited number of binding aquaria) and progenesis (i.e., fish obtained from the same sites on the antibody-coated plate. (e plate was then place) as reported in [13, 26, 28]. (e fish were then reared washed with a wash buffer to remove all the unbound under natural environmental conditions (natural photoperiod material. (e bound enzyme conjugate was detected by the 12L :12D, tropical temperature, and standard water quality) by action of substrate which generated an optimal colour after the seining technique [29]. Fish of mixed sex [30] were ran- 30 minutes. Quantitative test results were obtained by domly distributed into 2 indoor glass aquaria (0.050 m ) in measuring and comparing the absorbance reading of wells of triplicate at low and high stocking densities of 150 g and 300 g, the samples against the standards with a microplate reader respectively. All fish were matched for body weight (standard set at 650 nm using SoftMax Pro Microplate Data Acqui- mass, mean± SD 15± 1 g) [13] and age [28]. sition & Analysis Software. (e samples were each diluted (e aquaria were fitted with aerator pumps (Lp Low ten (10) times before being assayed. Noise Air pumps) manufactured by Resun , China, and mercury thermometers. A sand-fine gravel filter system was also put into each aquarium. (e aquaria were filled with 2.4.2. Whole Blood Glucose. Blood glucose levels were de- equal volumes (40 litres) of rain water. (e fish in each termined from whole blood using a hand-held one touch International Journal of Zoology 3 ultraglucose meter (MD-300) and test strips manufactured (e data on cortisol concentration levels (Table 2) by TaiDoc. Technologies Corporation and supplied by MD were then organised in an a ascending order for both HSD instruments Inc., as was established by [30, 34, 35] at the and LSD and, then, used to establish a graph of cortisol sampling site. Whole blood was applied onto the test strips concentration in ng/ml against individual samples of O. fixed in the hand-held glucose meter. Glucose concentra- niloticus assayed and plotted so as to show trends (Fig- −1 ∗ tions were read in mmol. ure 2). Note that the data marked with an asterisk ( ) were obtained from samples extracted from the second aquaria for HSD and LSD groups to bring the data to 10 a piece for 2.5. Statistical Analyses. Means, standard deviations (SD), better graphing. (is was due to the fact that the second and Standard Error of Means (SEM) have been used to aquaria positions in between the first and third aquaria describe the data. (ey were determined using One-Way ensured homogeneity in experimental conditions for the ANOVA. Microsoft Office Professional plus Excel 2013 HSD and LSD groups. software was used to present the data graphically for easy Note that the sample number marked with an asterisk interpretation and understanding. (e accuracy with which did not have its cortisol values used in computing the LSD the distributions of the extracted blood samples for LSD and and HSD group means. HSD groups represents the expression of stress related to One-way ANOVA analysis of the triplicate O. niloticus stocking densities in O. niloticus under aquaculture systems control groups gave no statistical (P> 0.05) difference in is measured using SD at 2 standard deviation of the means at their means (F � 0.26< F . � 5.14; P � 0.78) at (df,2; 6) crit 95% level of confidence [36]. (e SEM shows the precision of 4.80± 0.53, 4.80± 1.12, and 3.87± 2.83 mg/dl for LSD 1, the sample means of LSD and HSD groups to the true LSD , and LSD , respectively. Similarly, no statistical 2 3 control and experimental groups’ means. One-Way (P> 0.05) difference (F � 0.43< F . � 5.14; P � 0.67) (df, 2; 6) crit ANOVA was used to test the hypotheses; H0: x̄ � x̄ was revealed between the means of the triplicate O. niloticus LSD1 LSD2 � x̄ , x̄ � x̄ � x̄ , H : not H at F experimental groups at 4.8± 0.53, 4.77± 1.12, and LSD3 HSD1 SHD2 HSD3 A 0 crit (df � 2, df � 6, α � 0.05) � 5.14 for individual 3.87± 2.83 mg/dl for HSD , HSD , and HSD , respectively, AMONG WITHIN 1 2 3 samples and H0: x̄ � x̄ H not H for the true using one-way ANOVA analysis. A comparison of the LSD1,2,3 HSD1,2,3, A: 0 sample means at F (df � 1, df � 4, α � 0.05) � statistical (P< 0.05) difference (F � 20.32> F . � 5.14; crit AMONG WITHIN (df,2; 6) crit 7.71 for cortisol. Similar statistical analyses were conducted P � 0.01) between the means 4.49± 0.54 and 6.33± 0.46 for on whole blood glucose concentrations at F (df � 2, LSD and HSD groups, respectively, revealed a sig- crit AMONG 1,2,3 1,2,3 df � 12, α � 0.05) � 3.18 for LSD , LSD , LSD , HSD , nificant difference between the two means. (e small values WITHIN 1 2 3 1 HSD , and HSD individual sample groups and at F of SEM (0.31 ; 0.27 ) for the true means (4.49 ; 2 3 crit LSD HSD LSD (df � 1, df � 8, α � 0.05) � 5.32 for LSD ; 6.33 ) confirm the fact that the true means vary negligibly AMONG WITHIN 1,2,3 HSD HSD true sample means. from the control and experimental groups’ means and are, 1,2,3 therefore, reliable (Table 3). 3. Results 3.2. Whole Blood Glucose. One-way ANOVA analysis of the 3.1. Plasma Cortisol. (e percent of maximal binding (%B/ triplicate O. niloticus control groups gave no statistical (P< 0.05) B value) was determined by dividing the averages of each difference in their means (F � 0.161< F . � 3.885; (df,2;12) crit standard absorbance value (B . . . B ) by the B absorbance 1 7 0 P≥ 0.01) at 77.76± 12.03, 74.52± 11.64, and 78.12± 9.23 mg/dl value and, then, multiplied by 100 to achieve the percentages for LSD LSD , and LSD , respectively. Similarly, no statistical 1, 2 3 (Table 1). (P< 0.05) difference (F � 0.674< F . � 3.885; (df,2; 12) crit (e data from Table 1 was used to graph the standard P≥ 0.01) was revealed between the means of the triplicate curve (Figure 1) by plotting the %B/B for each standard O. niloticus experimental groups at 100.8± 14.40, concentration on the ordinate (y) axis against concentration 94.68± 5.78, and 95.04± 5.28 mg/dl for HSD , HSD , and 1 2 on the abscissa (x) axis a using a curve-fitting routine. HSD , respectively, using one-way ANOVA analysis (e standard curve was used to determine the con- (Table 3). One-way ANOVA analysis revealed a statistical centration of the samples from their respective %B/B and, (P< 0.05) difference in the means of whole blood glucose then, multiplied with a dilution factor of 10 (Table 2). concentrations in HSD and LSD O. niloticus groups One-way ANOVA analysis revealed a statistical (F � 31.845> F . � 5.318; P � 0.01). Mean plasma (df,1;8) crit (P< 0.05) difference in the means of plasma cortisol con- glucose concentration was statistically (P≤ 0.01) higher centrations in HSD and LSD O. niloticus groups (F � for HSD than LSD O. niloticus groups at mean± SD, (df,1;18) 7.946> F . � 4.414; P � 0.01). Mean plasma cortisol con- 96.84± 5.28 mg/dl and 76.80± 5.92 mg/dl, respectively. crit centration was statistically (P≤ 0.01) higher for HSD than Like in cortisol, the small values of SEM (2.65 ; 2.36 ) LSD HSD LSD O. niloticus groups at mean± SD, 6.32± 1.06 ng/ml and for the true means (76.80± 5.92 ; 96.84± 5.28 ) LSD HSD 4.62± 1.58 ng/ml, respectively (Table 2). It is evident from similarly confirm the fact that the true means vary neg- the small values of standard error, SE, (0.33 ; 0.50 ) that ligibly from the control and experimental groups’ means LSD HSD the sample means (6.32 ; 4.62 ) are reliable indications and are, therefore, reliable (Table 4). LSD HSD of cortisol levels in O. niloticus reared under the two stocking Finally, whole blood glucose concentration levels were densities in aquaculture systems. organised in an ascending order for both LSD and HSD and, 4 International Journal of Zoology Table 1: Standard concentration (ng/ml), optical density (absorbance value), and %B/B . Standard Optical density Standard %B/B Concentration (ng/ml) (Absorbance value) S (B ) 0.0 0.764 100 o o S (B ) 0.04 0.655 86 1 1 S (B ) 0.10 0.627 82 2 2 S (B ) 0.2 0.439 57 3 3 S (B ) 0.4 0.427 56 4 4 S (B ) 1.0 0.290 38 5 5 S (B ) 2.0 0.252 33 6 6 S (B ) 10.0 0.226 30 7 7 Cortisol in EIA buffer –2 0 2 4 6 8 10 12 Concentration (ng/ml) Figure 1: Standard curve. Table 2: Plasma cortisol levels and means (P< 0.05) for HSD and LSD O. niloticus. HSD LSD Samples Cortisol, ×10 ng/ml Sample Cortisol, ×10 ng/ml Un 7.2 Un 0.6 1 11 Un 6.0 Un 5.0 2 12 Un 6.7 Un 5.4 3 13 Un 4.28 Un 3.5 4 14 Un 6.5 Un 5.2 5 15 Un 5.9 Un 5.6 6 16 Un 6.7 Un 5.2 7 17 Un 5.6 Un 5.6 8 18 Un 8.3 Un 4.2 9 19 ∗ ∗ Un 6.0 Un 5.9 10 20 Mean 6.32 Mean 4.62 SD 1.06 SD 1.58 SE 0.33 SE 0.50 Un –Un represent HSD, while Un –Un represent LSD. Un: extra samples drawn from the second aquaria from both HSD and LSD groups. 1 10 11 20 then, used to establish a graph of glucose concentration in 47.97± 9.37 ng/ml (n � 6) obtained on day 0 in an experi- −1 mgdl against individual samples of O. niloticus, as shown ment on conditioning of stress in Nile tilapia [28]. Other in (Figure 3). studies cited in [37] show the mean basal plasma cortisol ranges of 5–15 ng/ml, 16.43–39.22 ng/ml, and 5–50 ng/ml for O. niloticus and 20–60 ng/ml for a related Cichlid, O. 4. Discussion mossambicus. Studies involving other related fish families such as Cyprinid Cyprinus carpio, Salmonids Oncorhynchus 4.1. Plasma Cortisol. (e plasma cortisol concentration level clarkii, and Oncorhynchus mykiss showed similar ranges of was significantly high in HSD than in LSD for the O. basal plasma cortisol levels. niloticus groups. (e mean ± SD (i.e., 6.32± 1.06 ng/ml and (e slightly lower than normal basal corticosteroid 4.62± 1.58 ng/ml) plasma cortisol values obtained from stress response mean± SD value in LSD in this study is HSD and LSD groups (Table 2) compared favourably with not a universal phenomenon in this fish group [12]. the reported normal mean basal cortisol range of 5–60 ng/ml However, the relatively low intensities of cortisol in both for O. niloticus [22,37]. (is range also accommodates the experimental and control groups O. niloticus may the mean± SD basal value range of 31.08± 4.94 to % B/Bo International Journal of Zoology 5 Plasma cortisol 02468 10 12 Samples HSD LSD Linear (HSD) Figure 2: Plasma cortisol concentrations in High Stocking Density- (HSD-) and Low Stocking Density- (LSD-) reared O. niloticus after 21 days. Table 3: Cortisol concentrations and group means for LSD and HSD triplicates, at P< 0.05. −1 Cortisol concentrations, ×10 in ngml Sample number LSD LSD LSD Mean HSD HSD HSD Mean 1 2 3 1 2 3 1 5.2 3.5 5.6 4.8 5.6 6.7 4.28 5.5 2 4.2 5.6 0.6 3.5 8.3 7.2 6.7 7.4 3 5.0 5.2 5.4 5.2 6.0 5.9 6.5 6.1 4 — 5.9 — — — 6.0 — — Mean 4.80 4.80 3.87 4.50 6.60 6.60 5.80 6.30 SD 0.53 1.12 2.44 0.89 2.12 0.64 1.34 1.08 SE 0.31 0.65 1.41 — 1.22 0.37 0.77 — SEM — — — 0.51 — — — 0.62 Table 4: Whole blood glucose concentrations and group means for LSD and HSD triplicates, at P< 0.05. . −1 −1 Concentrations (mmol l ×18) in mgdl Sample number LSD LSD LSD Mean HSD HSD HSD Mean 1 2 3 1 2 3 1 84.6 79.2 66.6 76.8 99.0 90.0 99.0 96.0 2 57.6 77.4 88.2 74.4 126.0 95.4 93.6 105.0 3 84.6 79.2 72.0 78.6 91.8 102.6 90.0 94.8 4 86.4 82.8 86.4 85.2 91.8 88.2 91.8 90.6 5 75.6 54.0 77.4 69.0 95.4 97.2 100.8 97.8 Mean 77.76 74.52 78.12 76.80 100.80 94.68 95.04 96.84 SD 12.03 11.64 9.23 5.92 14.40 5.78 4.67 5.28 SE 5.38 5.21 4.13 — 6.44 2.58 2.09 — SEM — — — 2.65 — — — 2.36 have been as a result of extrinsic nature where response is secretion [26]. Different hormones such as alpha-melano- affected by external factors, i.e., season, time of the day, cyte-stimulating hormone (MSH), endorphin from the pars and from the intrinsic nature dependent on the genotype intermedia (PI), and some sympathetic nerve fibres [38] or phenotype of the fish such as rapid conversion of have been implicated in cortisol release during the chronic cortisol into less immunoreactive cortisone [26]. It phase in fishes, functioning as an emergency system. should also be noted that differences in corticosteroid However, if the suboptimal condition persists, this system stress responses exist among stocks of the same fish, may be deleted [16], leading to impaired cortisol release in hence the low cortisol levels recorded [12]. fish subjected to stressors. It should, however, be noted that Effects of extrinsic stress factor(s) or prolonged sub- the net effect of these apparent unknown stress factors had optimal conditions unknown to the researcher may also have no bearing on cortisol levels of the control and experimental led to the relatively low cortisol intensities in both control fish because of the standardized experimental conditions. and experimental O. niloticus groups because the interrenal However, if the effect was there, then it must have been uniform due to randomization and replication of the tissues may have become less sensitive to the action of ACTH or other pituitary hormones leading to low cortisol experiments. Cortisol concentration (ng/ml) 6 International Journal of Zoology Whole blood glucose level –5 0 5 10 15 20 Samples HSD LSD Linear (HSD) Figure 3: Glucose concentrations in Low Stocking Density- (LSD-) and High Stocking Density- (HSD-) reared O. niloticus. Both physiological and biological status of the fish auratus) [26]. During stress episodes, catecholamine acts used in this study were standardized prior to the ex- directly on the liver to stimulate glycogenolysis, which re- periment leading to a basal cortisol mean value of sults in the mobilization of glucose [43]. Catecholamines 4.62± 1.58 ng/ml in LSD-reared fish against which the promote the phosphorylation of the enzyme glycogen rise in plasma cortisol in HSD-reared O. niloticus is phosphorylase which results in increased glycogenolysis. compared. Enhanced glycogenolysis or a decreased clearance of glucose from the blood is the source for increased plasma glucose In the current study, the high plasma cortisol con- centration level in the HSD group than in the LSD group concentrations in stressed tilapia [44]. was stress-induced [39], with chronic stress (i.e., high Barreto and Volpato [22] report a mean basal glucose stocking density), producing a sustained elevation of concentration of 39.6 and 34.2 mg/dl for electroshock and cortisol in fish [40]. Elevated plasma cortisol, therefore, social stressors, respectively, both of which induced acute indicates that chronic stress occurred in HSD-reared O. stress in O. niloticus. (ese concentrations compare niloticus [16, 26]. Plasma cortisol is actually a good acute favourably with a whole blood glucose mean concentration stress marker [41], with adrenaline considered as the of 76.82± 5.92 mg/dl obtained from the triplicate LSD stress hormone and cortisol, the adaptive hormone [42]. control O. niloticus groups. In addition, the triplicate O. Figure 2 clearly demonstrates variation in the trend of niloticus groups’ whole blood concentration means for LSD , the cortisol concentrations with subsequent HSD samples LSD , and LSD , respectively, showed no statistical differ- 2 3 against lower but increasing cortisol concentrations in ence. Similarly, no statistical difference was established subsequent LSD samples. (e linear (HSD) trend line shows between the means of the triplicate O. niloticus experimental that all but one sample had cortisol concentration below the groups, HSD , HSD , and HSD , respectively (Table 4). (is 1 2 3 highest cortisol concentration recorded from LSD samples. could be attributed to the standardized experimental con- (is reinforces the fact that high stocking density induced ditions. In the present study, mean± SD for the blood stress in O. niloticus. glucose concentration level was statistically high in HSD at 96.84± 5.28 mg/dl than in LSD at 76.80± 5.92 mg/dl for the O. niloticus groups. (is is supported by strong evidence that 4.2. Whole Blood Glucose. Since glucose is an innate immune the blood glucose concentration means for both experi- parameter mediated by stress, an increase in glucose con- mental and control groups were statistically (P< 0.05) dif- centration is a secondary response to stress, and the level of ferent. Stress hormones, adrenaline, and noradrenaline in the increase is a measure of stress [21]. Glucose concen- conjunction with cortisol mobilized and elevated glucose to tration has been widely used in a variety of fish species as an cope with the energy demand in response to high stocking indicator of stress in genetic studies [26, 27]. Glucose is an density-induced stress and, hence, an increase in the blood indicator of sympathetic activation during stress [38]. plasma glucose level in HSD O. niloticus. Like in cortisol, Figure 3, a clear demonstration of (e high blood glucose concentration recorded for HSD in this study can be associated with the single independent variation in the trend of whole blood concentrations with subsequent HSD samples against lower but also increasing variable introduced, high stocking density, thus indicating the occurrence of chronic stress in the experimental O. whole blood glucose concentrations in subsequent LSD samples (Figure 3) is observed. (e linear (HSD) trend line niloticus group. (is is consistent with previous studies which have reported statistically increased blood glucose shows that only the highest whole blood concentration (88.2 mg/dl) in LSD matched the lowest whole blood con- levels in stressed fish. A three- (3-) week period of crowding stress elevated glucose in gilthead sea bream (Sparus centration recorded in HSD samples. (e linear trend line Glucose concentration (mg/dl) International Journal of Zoology 7 [3] B. O. Acosta and M. V. Gupta, “(e genetic improvement of further shows a steady rise in whole blood concentrations in farmed tilapias project: impact and lessons learned,” Success HSD samples compared to LSD samples. (is proves that Stories In Asian Aquaculture, Springer, Berlin, Germany, high stocking density induced stress in O. niloticus. [4] H. Charo-Karisa, M. A. Rezk, H. Bovenhuis, and H. Komen, 5. Conclusions “Heritability of cold tolerance in Nile tilapia, Oreochromis (e results of this study demonstrate the fact that high niloticus, juveniles,” Aquaculture, vol. 249, no. 1–4, pp. 115–123, stocking densities have a significant effect on plasma cortisol [5] A.-F. M. El-Sayed, “Effects of stocking density and feeding and whole blood glucose concentration in tilapia fish in levels on growth and feed efficiency of Nile tilapia aquaculture systems. (e elevated level of plasma cortisol (Oreochromis niloticus L.) fry,” Aquaculture Research, and whole blood glucose indicated that chronic stress in- vol. 33, no. 8, pp. 621–626, 2002. duced by high stocking density occurred in the experimental [6] G. T. Getinet, “Effects of maternal age on fecundity, spawning fish. It can, therefore, be argued that the fish suffered in- interval, and egg quality of nile tilapia, Oreochromis niloticus creased glycogenolysis or a decreased clearance of glucose (L.),” Journal of World Aquaculture Society, vol. 39, no. 5, from the blood, thus raising plasma glucose concentration in pp. 671-672, 2008. the tilapia. [7] S. F. Li, J. L. Zhao, M. Dey, and R. Dunham, “Isozyme var- It was evident that cortisol concentration in plasma iation of nile tilapia (Oreochromis niloticus) in China,” Journal elevated in response to stress and aided in carbohydrate Asian Fisheries Science, vol. 14, pp. 411–416, 2001. metabolism and promoted glucogenesis. Stress can even- [8] J. Masterson, Mosambique Tilapia, Oreochromis mossambicus, tually affect physiological activities such as feeding and the Smithsonian Marine Station at Fort Pierce, Fort Pierce, FL, immunity of the fish leading to exposure to pathogenic USA, 2007. microbes that may lead to impaired growth thus a reduction [9] M. Njiru, J. E. Ojuok, J. B. Okeyo-Owuor, M. Muchiri, in yields in tilapia reared under aquaculture systems. M. J. Ntiba, and I. G. Cowx, “Some biological aspects and (e findings of this study can help inform the policy on life history strategies of Nile tilapia Oreochromis niloticus the management of stress caused by overpopulation of O. (L.) in Lake Victoria, Kenya,” African Journal of Ecology, niloticus and other related Cichlids under industrial aqua- vol. 44, no. 1, pp. 30–37, 2006. [10] G. L. Volpato, A. C. Luchiari, C. R. A. Duarte, R. E. Barreto, culture production. and G. C. Ramanzini, “Eye color as an indicator of social rank in the fish Nile tilapia,” Brazilian Journal of Medical and Data Availability Biological Research, vol. 36, no. 12, pp. 1659–1663, 2003. (e primary data used to support the findings of this study [11] A. Rebl, M. Zebunke, A. Borchel, R. Bochert, M. Verleih, and T. Goldammer, “Microarray-predicted marker genes and are available from the corresponding author upon request. molecular pathways indicating crowding stress in rainbow Table 1 contains part of the primary data. trout (Oncorhynchus mykiss),” Aquaculture, vol. 473, pp. 355–365, 2017. Conflicts of Interest [12] B. A. Barton, “Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids,” (e authors declare no conflicts of interest. Integrative and Comparative Biology, vol. 42, no. 3, pp. 517– 525, 2002. Acknowledgments [13] M. Abdel – Tawwab, M. A. A. Mousa, S. M. Sharaf, and (e Department of Zoology at Maseno University and the M. H. Ahmad, “Effects of crowding stress on some physi- Kenya Medical Research Institute (KEMRI) are acknowl- ologial functions of nile tilapia, Oreochromis niloticus (L) fed different dietary protein levels,” International Journal Zoo- edged for providing laboratory space, equipment, and logical Research, vol. 1, no. 1, pp. 41–47, 2005. technical expertise that enabled the success of this study. Dr [14] A. O. Ochieng, A. O. Paul, R. John, and W. N. Eliud, “Effect of Cyrus Ayieko of the Department of Zoology, Maseno Stocking density on the expression of glucose transporter University, is highly appreciated for providing valuable protein 1 and other physiological factors in the Lake Victoria scientific and technical assistance during sample analysis. Nile tilapia, Oreochromis. niloticus (L.),” International (is study was funded by the authors’ own resources with Aquatic Research, vol. 6, no. 2, pp. 1–8, 2014. some support from the School of Graduate Studies of [15] Y. Abou, E. D. Fiogbe, and J. Micha, “Effects of stocking Maseno University. density on growth, yield and profitability of farming Nile tilapia, Oreochromis niloticus L., fed Azolla diet, in earthen References ponds,” Aquaculture Research, vol. 38, no. 6, pp. 595–604, [1] M. T. Ridha, “Evaluation of growth performance of non- [16] S. E. Wendelaar Bonga, “(e stress response in fish,” Physi- improved and improved strains of the nile Tilapia (L.), ological Reviews, vol. 77, no. 3, pp. 591–625, 1997. Oreochromis niloticus,” Journal of the World Aquaculture [17] K. Pangni, B. C. Atse, and N. J Kovassi, “Effect of Stocking Society, vol. 37, no. 2, pp. 218–223, 2006. Density on Growth and Survival of the African catfish [2] H. A. Hassanien and J. 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Stocking Density Induced Stress on Plasma Cortisol and Whole Blood Glucose Concentration in Nile Tilapia Fish (Oreochromis niloticus) of Lake Victoria, Kenya

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Copyright © 2020 Elija Odhiambo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2020/9395268
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Hindawi International Journal of Zoology Volume 2020, Article ID 9395268, 8 pages https://doi.org/10.1155/2020/9395268 Research Article Stocking Density Induced Stress on Plasma Cortisol and Whole Blood Glucose Concentration in Nile Tilapia Fish (Oreochromis niloticus) of Lake Victoria, Kenya 1 1 2 1 Elija Odhiambo , Paul O. Angienda, Patrick Okoth , and David Onyango Department of Zoology, Maseno University, Kisumu, Kenya Department of Biological Sciences, School of Natural Sciences, Masinde Muliro University of Science and Technology, Kakamega, Kenya Correspondence should be addressed to Elija Odhiambo; elija.odhiambo@gmail.com Received 16 November 2019; Revised 16 April 2020; Accepted 19 June 2020; Published 17 July 2020 Academic Editor: Jo o Pedro Barreiros Copyright © 2020 Elija Odhiambo et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Effects of high stocking densities (HSDs) were evaluated for Nile tilapia fish (Oreochromis niloticus) under culture to determine its influence on plasma cortisol and whole blood glucose concentration. Plasma cortisol levels (ng/ml) were assayed by Enzyme- Linked Immunosorbent Assay (ELISA). Whole blood glucose levels were determined using a hand-held one touch ultraglucose meter (MD-300) and test strips. Plasma cortisol and whole blood glucose level determinations were replicated three times for O. niloticus reared under both low stocking densities (LSD) and HSD. One way Analysis of Variance (ANOVA) was performed on the data collected, and comparison of significant differences in means was carried out between LSD and HSD at 0.01%. Plasma cortisol levels revealed statistically (P≤ 0.01) significant values of HSD at 6.32 ± 1.06 ng/ml than in LSD at 4.62± 1.58 ng/ml for the O. niloticus groups studied. Whole blood glucose analysis revealed a statistical (P< 0.05) difference in the means in HSD and LSD O. niloticus groups (F � 7.946> F � 4.414; P � 0.01). Mean plasma glucose concentration was statistically (P≤ 0.01) (df,1; 8) crit higher for HSD than LSD O. niloticus groups at mean± SD, 96.84± 5.28 and 76.82± 5.92, respectively. (e findings of this study demonstrate that high stocking densities increase both cortisol and whole blood glucose concentration in tilapia fish, indicating a marked increase in stress levels. Elevated plasma cortisol and whole blood glucose concentration can be used as biomarkers for acute stress in O. niloticus produced under aquaculture systems. (e findings of this study can help inform policy on the management of stress caused by overstocking of O. niloticus and other related Cichlids under industrial aquaculture production. fish [8] and the most desired by Lake Victoria Community 1. Introduction [9]. It is also an important fish model for studies on social Fish and fisheries play an important role in addressing stress due to overcrowding [10]. nutritional and livelihood food security, especially of the Stocking density is considered a key factor in deter- poor in the developing countries [1]. Globally over 2 billion mining the productivity of fish aquaculture systems [11]. people get at least 20% of their animal protein intake from Crowding is judged as one of the most influential stressors fish [2, 3]. Nile tilapia (Oreochromis niloticus)are among the affecting fish physiology and, thus, the status of well-being in most important warm water fishes used for aquaculture aquaculture [12], and it is a common husbandry practice in production [4] and only the second most popular farmed aquaculture [13, 14]. High stocking density negatively affects fishes after Carps [3, 5, 6]. (ey are recognized as one of the both fish growth and feed utilization [1, 15]. High stocking most important species in tropical and subtropical aqua- densities have been shown to reduce feeding activity and culture [2, 5, 7]. Nile tilapia have a mild white flesh that growth rates in Coho Salmon (Oncorhynchus kisutch) and appeals to customers, making them economically important rainbow trout (Oncorhynchus mykiss) but have a positive 2 International Journal of Zoology effect on these parameters in Arctic charr (Salvelinus alpi- aquarium were fed on a carbohydrate-based feed of chick nus) [16]. It also increases the level of metabolites such as mash (18% protein) (Sigma Feeds Ltd., Nairobi, Kenya) urine and faeces in cultured Chrysichthys nigrodigitatus [17]. supplemented with crushed silver cyprinid (Rastrineobola (is stress response changes water quality [16], thus further argentea) to 25% protein at a feed portion of 10 g/kg of life subjecting fish to chemical stressors [18]. High stocking body weight, and the feeding was terminated 24 hours to density subjects fish to chronic stress [19, 20]. It is reported sampling [13]. by [21] that stocking density and social interactions between specific fish have a significant influence on stress. Stress is 2.3. Sample Collection and Storage. 21-day-old fish were considered to be a generated response, but it can be mod- anaesthetized with 2-phenoxyethanol (chemical formula: ulated by specific stressor conditions. One of the most C H O ; active substance: ethylene glycol monophenyl 8 10 2 frequent causes of chronic stress is social interaction among ether) obtained from BDH Laboratory Reagents, England at members of the same species. −1 a concentration of 0.30 ml l of water for 10 min before Stressors cause distinct stress responses in O. niloticus samples were extracted [31] to minimize suffering. A large [22] leading to elevated plasma cortisol [16, 23], the main plastic sieve was then used to draw fish (n � 5) samples from hormone that activates glucose [24], therefore elevating each of the six aquaria. Blood was then drawn through glucose levels [19, 23, 25]. Fish respond to stress by in- cardiac puncture using EDTA- (ethylene diamine tetra- creasing circulating cortisol [16, 23] and glucose [23]. acetic acid-) coated (obtained from BDH Laboratory Cortisol is released in response to chronic stressors. Elevated Reagents, England) hypodermic syringes [32] as an anti- plasma cortisol [19, 26] and glucose levels [16, 21, 26, 27] are coagulant. (is took not more than 1 min for each fish so as used as indicators of stress. (is study investigated the effect to avoid rise in blood cortisol levels due to handling stress of high stocking density-induced stress on the concentration [13, 22]. Some of the drawn blood samples (n � 20, i.e., three of cortisol and whole blood glucose in Oreochromis niloticus samples from each of the six aquaria and a further one of Lake Victoria under culture. sample from each of the second aquaria of the LSD and HSD groups) were then transferred into labelled Eppendorf tubes, 2. Materials and Methods each containing 1 ml EDTA solution, placed onto crushed ice (0 C) in an icebox, and transported to the laboratory for 2.1. Study Area. Nile tilapia fingerlings were obtained from 2 storage under refrigerated conditions. the Kenyan part of Lake Victoria (4, 100 km ), a fresh water lake. (ey were then transferred to the fish rearing facility at o o Maseno University (0 22′ 11.0″ S, 35 55′ 58.0″ E/Latitude; 2.4. Sample Analyses 0.369734, Longitude; 35.932779) located 400 km west of Nairobi where this study was conducted. Tropical climatic 2.4.1. Plasma Cortisol. Plasma cortisol levels (ng/ml) were conditions characterize this region. assayed for by Enzyme-Linked Immunosorbent Assay (ELISA) using a Neogen Corporation ELISA kit (Lansing, Ml, USA, 2010) [33] and the company’s Cortisol ELISA 2.2. Fish and Experimental Procedures. (e study protocol protocol. (is quantitative analysis of cortisol levels in the was reviewed and optimised by experts from the Department biological fluid (O. niloticus blood plasma) was performed at of Zoology, Maseno University, Kenya, and the Department the Kenya Medical Research Institute (KEMRI), Kisumu, of Biological Sciences, Masinde Muliro University of Science Kenya. An antibody-coated 96-well microplate was used. and Technology, Kenya. Healthy Nile tilapia of Lake Victoria (e standard solutions and the diluted samples were first were obtained from the fish rearing facility of the Depart- added to the microplate in duplicates. Diluted enzyme ment of Zoology, Maseno University. (ey were first conjugate was then added, and the mixture was shaken and acclimatised to the laboratory conditions (temperature, incubated at room temperature for 1 hour to allow com- dissolved oxygen, water quality, water pH, nutritional status, petition to take place between the enzyme conjugate and photoperiod, size, weight, colour, and shape of experimental cortisol in the samples for the limited number of binding aquaria) and progenesis (i.e., fish obtained from the same sites on the antibody-coated plate. (e plate was then place) as reported in [13, 26, 28]. (e fish were then reared washed with a wash buffer to remove all the unbound under natural environmental conditions (natural photoperiod material. (e bound enzyme conjugate was detected by the 12L :12D, tropical temperature, and standard water quality) by action of substrate which generated an optimal colour after the seining technique [29]. Fish of mixed sex [30] were ran- 30 minutes. Quantitative test results were obtained by domly distributed into 2 indoor glass aquaria (0.050 m ) in measuring and comparing the absorbance reading of wells of triplicate at low and high stocking densities of 150 g and 300 g, the samples against the standards with a microplate reader respectively. All fish were matched for body weight (standard set at 650 nm using SoftMax Pro Microplate Data Acqui- mass, mean± SD 15± 1 g) [13] and age [28]. sition & Analysis Software. (e samples were each diluted (e aquaria were fitted with aerator pumps (Lp Low ten (10) times before being assayed. Noise Air pumps) manufactured by Resun , China, and mercury thermometers. A sand-fine gravel filter system was also put into each aquarium. (e aquaria were filled with 2.4.2. Whole Blood Glucose. Blood glucose levels were de- equal volumes (40 litres) of rain water. (e fish in each termined from whole blood using a hand-held one touch International Journal of Zoology 3 ultraglucose meter (MD-300) and test strips manufactured (e data on cortisol concentration levels (Table 2) by TaiDoc. Technologies Corporation and supplied by MD were then organised in an a ascending order for both HSD instruments Inc., as was established by [30, 34, 35] at the and LSD and, then, used to establish a graph of cortisol sampling site. Whole blood was applied onto the test strips concentration in ng/ml against individual samples of O. fixed in the hand-held glucose meter. Glucose concentra- niloticus assayed and plotted so as to show trends (Fig- −1 ∗ tions were read in mmol. ure 2). Note that the data marked with an asterisk ( ) were obtained from samples extracted from the second aquaria for HSD and LSD groups to bring the data to 10 a piece for 2.5. Statistical Analyses. Means, standard deviations (SD), better graphing. (is was due to the fact that the second and Standard Error of Means (SEM) have been used to aquaria positions in between the first and third aquaria describe the data. (ey were determined using One-Way ensured homogeneity in experimental conditions for the ANOVA. Microsoft Office Professional plus Excel 2013 HSD and LSD groups. software was used to present the data graphically for easy Note that the sample number marked with an asterisk interpretation and understanding. (e accuracy with which did not have its cortisol values used in computing the LSD the distributions of the extracted blood samples for LSD and and HSD group means. HSD groups represents the expression of stress related to One-way ANOVA analysis of the triplicate O. niloticus stocking densities in O. niloticus under aquaculture systems control groups gave no statistical (P> 0.05) difference in is measured using SD at 2 standard deviation of the means at their means (F � 0.26< F . � 5.14; P � 0.78) at (df,2; 6) crit 95% level of confidence [36]. (e SEM shows the precision of 4.80± 0.53, 4.80± 1.12, and 3.87± 2.83 mg/dl for LSD 1, the sample means of LSD and HSD groups to the true LSD , and LSD , respectively. Similarly, no statistical 2 3 control and experimental groups’ means. One-Way (P> 0.05) difference (F � 0.43< F . � 5.14; P � 0.67) (df, 2; 6) crit ANOVA was used to test the hypotheses; H0: x̄ � x̄ was revealed between the means of the triplicate O. niloticus LSD1 LSD2 � x̄ , x̄ � x̄ � x̄ , H : not H at F experimental groups at 4.8± 0.53, 4.77± 1.12, and LSD3 HSD1 SHD2 HSD3 A 0 crit (df � 2, df � 6, α � 0.05) � 5.14 for individual 3.87± 2.83 mg/dl for HSD , HSD , and HSD , respectively, AMONG WITHIN 1 2 3 samples and H0: x̄ � x̄ H not H for the true using one-way ANOVA analysis. A comparison of the LSD1,2,3 HSD1,2,3, A: 0 sample means at F (df � 1, df � 4, α � 0.05) � statistical (P< 0.05) difference (F � 20.32> F . � 5.14; crit AMONG WITHIN (df,2; 6) crit 7.71 for cortisol. Similar statistical analyses were conducted P � 0.01) between the means 4.49± 0.54 and 6.33± 0.46 for on whole blood glucose concentrations at F (df � 2, LSD and HSD groups, respectively, revealed a sig- crit AMONG 1,2,3 1,2,3 df � 12, α � 0.05) � 3.18 for LSD , LSD , LSD , HSD , nificant difference between the two means. (e small values WITHIN 1 2 3 1 HSD , and HSD individual sample groups and at F of SEM (0.31 ; 0.27 ) for the true means (4.49 ; 2 3 crit LSD HSD LSD (df � 1, df � 8, α � 0.05) � 5.32 for LSD ; 6.33 ) confirm the fact that the true means vary negligibly AMONG WITHIN 1,2,3 HSD HSD true sample means. from the control and experimental groups’ means and are, 1,2,3 therefore, reliable (Table 3). 3. Results 3.2. Whole Blood Glucose. One-way ANOVA analysis of the 3.1. Plasma Cortisol. (e percent of maximal binding (%B/ triplicate O. niloticus control groups gave no statistical (P< 0.05) B value) was determined by dividing the averages of each difference in their means (F � 0.161< F . � 3.885; (df,2;12) crit standard absorbance value (B . . . B ) by the B absorbance 1 7 0 P≥ 0.01) at 77.76± 12.03, 74.52± 11.64, and 78.12± 9.23 mg/dl value and, then, multiplied by 100 to achieve the percentages for LSD LSD , and LSD , respectively. Similarly, no statistical 1, 2 3 (Table 1). (P< 0.05) difference (F � 0.674< F . � 3.885; (df,2; 12) crit (e data from Table 1 was used to graph the standard P≥ 0.01) was revealed between the means of the triplicate curve (Figure 1) by plotting the %B/B for each standard O. niloticus experimental groups at 100.8± 14.40, concentration on the ordinate (y) axis against concentration 94.68± 5.78, and 95.04± 5.28 mg/dl for HSD , HSD , and 1 2 on the abscissa (x) axis a using a curve-fitting routine. HSD , respectively, using one-way ANOVA analysis (e standard curve was used to determine the con- (Table 3). One-way ANOVA analysis revealed a statistical centration of the samples from their respective %B/B and, (P< 0.05) difference in the means of whole blood glucose then, multiplied with a dilution factor of 10 (Table 2). concentrations in HSD and LSD O. niloticus groups One-way ANOVA analysis revealed a statistical (F � 31.845> F . � 5.318; P � 0.01). Mean plasma (df,1;8) crit (P< 0.05) difference in the means of plasma cortisol con- glucose concentration was statistically (P≤ 0.01) higher centrations in HSD and LSD O. niloticus groups (F � for HSD than LSD O. niloticus groups at mean± SD, (df,1;18) 7.946> F . � 4.414; P � 0.01). Mean plasma cortisol con- 96.84± 5.28 mg/dl and 76.80± 5.92 mg/dl, respectively. crit centration was statistically (P≤ 0.01) higher for HSD than Like in cortisol, the small values of SEM (2.65 ; 2.36 ) LSD HSD LSD O. niloticus groups at mean± SD, 6.32± 1.06 ng/ml and for the true means (76.80± 5.92 ; 96.84± 5.28 ) LSD HSD 4.62± 1.58 ng/ml, respectively (Table 2). It is evident from similarly confirm the fact that the true means vary neg- the small values of standard error, SE, (0.33 ; 0.50 ) that ligibly from the control and experimental groups’ means LSD HSD the sample means (6.32 ; 4.62 ) are reliable indications and are, therefore, reliable (Table 4). LSD HSD of cortisol levels in O. niloticus reared under the two stocking Finally, whole blood glucose concentration levels were densities in aquaculture systems. organised in an ascending order for both LSD and HSD and, 4 International Journal of Zoology Table 1: Standard concentration (ng/ml), optical density (absorbance value), and %B/B . Standard Optical density Standard %B/B Concentration (ng/ml) (Absorbance value) S (B ) 0.0 0.764 100 o o S (B ) 0.04 0.655 86 1 1 S (B ) 0.10 0.627 82 2 2 S (B ) 0.2 0.439 57 3 3 S (B ) 0.4 0.427 56 4 4 S (B ) 1.0 0.290 38 5 5 S (B ) 2.0 0.252 33 6 6 S (B ) 10.0 0.226 30 7 7 Cortisol in EIA buffer –2 0 2 4 6 8 10 12 Concentration (ng/ml) Figure 1: Standard curve. Table 2: Plasma cortisol levels and means (P< 0.05) for HSD and LSD O. niloticus. HSD LSD Samples Cortisol, ×10 ng/ml Sample Cortisol, ×10 ng/ml Un 7.2 Un 0.6 1 11 Un 6.0 Un 5.0 2 12 Un 6.7 Un 5.4 3 13 Un 4.28 Un 3.5 4 14 Un 6.5 Un 5.2 5 15 Un 5.9 Un 5.6 6 16 Un 6.7 Un 5.2 7 17 Un 5.6 Un 5.6 8 18 Un 8.3 Un 4.2 9 19 ∗ ∗ Un 6.0 Un 5.9 10 20 Mean 6.32 Mean 4.62 SD 1.06 SD 1.58 SE 0.33 SE 0.50 Un –Un represent HSD, while Un –Un represent LSD. Un: extra samples drawn from the second aquaria from both HSD and LSD groups. 1 10 11 20 then, used to establish a graph of glucose concentration in 47.97± 9.37 ng/ml (n � 6) obtained on day 0 in an experi- −1 mgdl against individual samples of O. niloticus, as shown ment on conditioning of stress in Nile tilapia [28]. Other in (Figure 3). studies cited in [37] show the mean basal plasma cortisol ranges of 5–15 ng/ml, 16.43–39.22 ng/ml, and 5–50 ng/ml for O. niloticus and 20–60 ng/ml for a related Cichlid, O. 4. Discussion mossambicus. Studies involving other related fish families such as Cyprinid Cyprinus carpio, Salmonids Oncorhynchus 4.1. Plasma Cortisol. (e plasma cortisol concentration level clarkii, and Oncorhynchus mykiss showed similar ranges of was significantly high in HSD than in LSD for the O. basal plasma cortisol levels. niloticus groups. (e mean ± SD (i.e., 6.32± 1.06 ng/ml and (e slightly lower than normal basal corticosteroid 4.62± 1.58 ng/ml) plasma cortisol values obtained from stress response mean± SD value in LSD in this study is HSD and LSD groups (Table 2) compared favourably with not a universal phenomenon in this fish group [12]. the reported normal mean basal cortisol range of 5–60 ng/ml However, the relatively low intensities of cortisol in both for O. niloticus [22,37]. (is range also accommodates the experimental and control groups O. niloticus may the mean± SD basal value range of 31.08± 4.94 to % B/Bo International Journal of Zoology 5 Plasma cortisol 02468 10 12 Samples HSD LSD Linear (HSD) Figure 2: Plasma cortisol concentrations in High Stocking Density- (HSD-) and Low Stocking Density- (LSD-) reared O. niloticus after 21 days. Table 3: Cortisol concentrations and group means for LSD and HSD triplicates, at P< 0.05. −1 Cortisol concentrations, ×10 in ngml Sample number LSD LSD LSD Mean HSD HSD HSD Mean 1 2 3 1 2 3 1 5.2 3.5 5.6 4.8 5.6 6.7 4.28 5.5 2 4.2 5.6 0.6 3.5 8.3 7.2 6.7 7.4 3 5.0 5.2 5.4 5.2 6.0 5.9 6.5 6.1 4 — 5.9 — — — 6.0 — — Mean 4.80 4.80 3.87 4.50 6.60 6.60 5.80 6.30 SD 0.53 1.12 2.44 0.89 2.12 0.64 1.34 1.08 SE 0.31 0.65 1.41 — 1.22 0.37 0.77 — SEM — — — 0.51 — — — 0.62 Table 4: Whole blood glucose concentrations and group means for LSD and HSD triplicates, at P< 0.05. . −1 −1 Concentrations (mmol l ×18) in mgdl Sample number LSD LSD LSD Mean HSD HSD HSD Mean 1 2 3 1 2 3 1 84.6 79.2 66.6 76.8 99.0 90.0 99.0 96.0 2 57.6 77.4 88.2 74.4 126.0 95.4 93.6 105.0 3 84.6 79.2 72.0 78.6 91.8 102.6 90.0 94.8 4 86.4 82.8 86.4 85.2 91.8 88.2 91.8 90.6 5 75.6 54.0 77.4 69.0 95.4 97.2 100.8 97.8 Mean 77.76 74.52 78.12 76.80 100.80 94.68 95.04 96.84 SD 12.03 11.64 9.23 5.92 14.40 5.78 4.67 5.28 SE 5.38 5.21 4.13 — 6.44 2.58 2.09 — SEM — — — 2.65 — — — 2.36 have been as a result of extrinsic nature where response is secretion [26]. Different hormones such as alpha-melano- affected by external factors, i.e., season, time of the day, cyte-stimulating hormone (MSH), endorphin from the pars and from the intrinsic nature dependent on the genotype intermedia (PI), and some sympathetic nerve fibres [38] or phenotype of the fish such as rapid conversion of have been implicated in cortisol release during the chronic cortisol into less immunoreactive cortisone [26]. It phase in fishes, functioning as an emergency system. should also be noted that differences in corticosteroid However, if the suboptimal condition persists, this system stress responses exist among stocks of the same fish, may be deleted [16], leading to impaired cortisol release in hence the low cortisol levels recorded [12]. fish subjected to stressors. It should, however, be noted that Effects of extrinsic stress factor(s) or prolonged sub- the net effect of these apparent unknown stress factors had optimal conditions unknown to the researcher may also have no bearing on cortisol levels of the control and experimental led to the relatively low cortisol intensities in both control fish because of the standardized experimental conditions. and experimental O. niloticus groups because the interrenal However, if the effect was there, then it must have been uniform due to randomization and replication of the tissues may have become less sensitive to the action of ACTH or other pituitary hormones leading to low cortisol experiments. Cortisol concentration (ng/ml) 6 International Journal of Zoology Whole blood glucose level –5 0 5 10 15 20 Samples HSD LSD Linear (HSD) Figure 3: Glucose concentrations in Low Stocking Density- (LSD-) and High Stocking Density- (HSD-) reared O. niloticus. Both physiological and biological status of the fish auratus) [26]. During stress episodes, catecholamine acts used in this study were standardized prior to the ex- directly on the liver to stimulate glycogenolysis, which re- periment leading to a basal cortisol mean value of sults in the mobilization of glucose [43]. Catecholamines 4.62± 1.58 ng/ml in LSD-reared fish against which the promote the phosphorylation of the enzyme glycogen rise in plasma cortisol in HSD-reared O. niloticus is phosphorylase which results in increased glycogenolysis. compared. Enhanced glycogenolysis or a decreased clearance of glucose from the blood is the source for increased plasma glucose In the current study, the high plasma cortisol con- centration level in the HSD group than in the LSD group concentrations in stressed tilapia [44]. was stress-induced [39], with chronic stress (i.e., high Barreto and Volpato [22] report a mean basal glucose stocking density), producing a sustained elevation of concentration of 39.6 and 34.2 mg/dl for electroshock and cortisol in fish [40]. Elevated plasma cortisol, therefore, social stressors, respectively, both of which induced acute indicates that chronic stress occurred in HSD-reared O. stress in O. niloticus. (ese concentrations compare niloticus [16, 26]. Plasma cortisol is actually a good acute favourably with a whole blood glucose mean concentration stress marker [41], with adrenaline considered as the of 76.82± 5.92 mg/dl obtained from the triplicate LSD stress hormone and cortisol, the adaptive hormone [42]. control O. niloticus groups. In addition, the triplicate O. Figure 2 clearly demonstrates variation in the trend of niloticus groups’ whole blood concentration means for LSD , the cortisol concentrations with subsequent HSD samples LSD , and LSD , respectively, showed no statistical differ- 2 3 against lower but increasing cortisol concentrations in ence. Similarly, no statistical difference was established subsequent LSD samples. (e linear (HSD) trend line shows between the means of the triplicate O. niloticus experimental that all but one sample had cortisol concentration below the groups, HSD , HSD , and HSD , respectively (Table 4). (is 1 2 3 highest cortisol concentration recorded from LSD samples. could be attributed to the standardized experimental con- (is reinforces the fact that high stocking density induced ditions. In the present study, mean± SD for the blood stress in O. niloticus. glucose concentration level was statistically high in HSD at 96.84± 5.28 mg/dl than in LSD at 76.80± 5.92 mg/dl for the O. niloticus groups. (is is supported by strong evidence that 4.2. Whole Blood Glucose. Since glucose is an innate immune the blood glucose concentration means for both experi- parameter mediated by stress, an increase in glucose con- mental and control groups were statistically (P< 0.05) dif- centration is a secondary response to stress, and the level of ferent. Stress hormones, adrenaline, and noradrenaline in the increase is a measure of stress [21]. Glucose concen- conjunction with cortisol mobilized and elevated glucose to tration has been widely used in a variety of fish species as an cope with the energy demand in response to high stocking indicator of stress in genetic studies [26, 27]. Glucose is an density-induced stress and, hence, an increase in the blood indicator of sympathetic activation during stress [38]. plasma glucose level in HSD O. niloticus. Like in cortisol, Figure 3, a clear demonstration of (e high blood glucose concentration recorded for HSD in this study can be associated with the single independent variation in the trend of whole blood concentrations with subsequent HSD samples against lower but also increasing variable introduced, high stocking density, thus indicating the occurrence of chronic stress in the experimental O. whole blood glucose concentrations in subsequent LSD samples (Figure 3) is observed. (e linear (HSD) trend line niloticus group. (is is consistent with previous studies which have reported statistically increased blood glucose shows that only the highest whole blood concentration (88.2 mg/dl) in LSD matched the lowest whole blood con- levels in stressed fish. A three- (3-) week period of crowding stress elevated glucose in gilthead sea bream (Sparus centration recorded in HSD samples. (e linear trend line Glucose concentration (mg/dl) International Journal of Zoology 7 [3] B. O. Acosta and M. V. Gupta, “(e genetic improvement of further shows a steady rise in whole blood concentrations in farmed tilapias project: impact and lessons learned,” Success HSD samples compared to LSD samples. (is proves that Stories In Asian Aquaculture, Springer, Berlin, Germany, high stocking density induced stress in O. niloticus. [4] H. Charo-Karisa, M. A. Rezk, H. Bovenhuis, and H. Komen, 5. Conclusions “Heritability of cold tolerance in Nile tilapia, Oreochromis (e results of this study demonstrate the fact that high niloticus, juveniles,” Aquaculture, vol. 249, no. 1–4, pp. 115–123, stocking densities have a significant effect on plasma cortisol [5] A.-F. M. El-Sayed, “Effects of stocking density and feeding and whole blood glucose concentration in tilapia fish in levels on growth and feed efficiency of Nile tilapia aquaculture systems. (e elevated level of plasma cortisol (Oreochromis niloticus L.) fry,” Aquaculture Research, and whole blood glucose indicated that chronic stress in- vol. 33, no. 8, pp. 621–626, 2002. duced by high stocking density occurred in the experimental [6] G. T. Getinet, “Effects of maternal age on fecundity, spawning fish. It can, therefore, be argued that the fish suffered in- interval, and egg quality of nile tilapia, Oreochromis niloticus creased glycogenolysis or a decreased clearance of glucose (L.),” Journal of World Aquaculture Society, vol. 39, no. 5, from the blood, thus raising plasma glucose concentration in pp. 671-672, 2008. the tilapia. [7] S. F. Li, J. L. Zhao, M. Dey, and R. Dunham, “Isozyme var- It was evident that cortisol concentration in plasma iation of nile tilapia (Oreochromis niloticus) in China,” Journal elevated in response to stress and aided in carbohydrate Asian Fisheries Science, vol. 14, pp. 411–416, 2001. metabolism and promoted glucogenesis. Stress can even- [8] J. Masterson, Mosambique Tilapia, Oreochromis mossambicus, tually affect physiological activities such as feeding and the Smithsonian Marine Station at Fort Pierce, Fort Pierce, FL, immunity of the fish leading to exposure to pathogenic USA, 2007. microbes that may lead to impaired growth thus a reduction [9] M. 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International Journal of ZoologyHindawi Publishing Corporation

Published: Jul 17, 2020

References