Effect of stocking density and feeding regimes on haematology, stress and histological recovery in stunted Lebeo rohita fingerlings reared in cage culture

doi:10.21203/rs.3.rs-1827294/v1

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Effect of stocking density and feeding regimes on haematology, stress and histological recovery in stunted Lebeo rohita fingerlings reared in cage culture

https://doi.org/10.21203/rs.3.rs-1827294/v1

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A 330-day field trial was conducted to evaluate the effect of stocking density and feeding ration on stunted Labeo rohita (Hamilton, 1822) fingerling reared in floating net cages. The stunted fingerling of L. rohita with an average body weight 38.23 ± 1.90 g were stocked in cages (3 × 3 × 3 m) at different densities viz. T₁ (10), T₂ (15), T₃ (20) and T₄ (25) fish/m³ and fed with varying levels of feeding rations at 3, 4, 5 and 6% at each stocking density. The 4×4 factorial design was used, and each density and feeding level kept in triplicate, and fed with commercial pellet feed (crude protein 28% and fat 5%) twice in a day. The significant recovery was observed in the T₁ (10*5 and 10*6) treatment groups, which showed highest Hb, RBC, PCV and lowest WBC content at the end of experiment. Similarly, significant changes were observed in glucose, cortisol and lactate dehydrogenase (LDH), which were highest in higher stocking density (T₄), and normalized in lower stocking T₁ (10*5 and 10*6) treatment groups. Histological examination showed, almost complete recovery of structural changes such as swelling/ edema primary lamellae, hyperplasia of basal epithelium between the secondary lamellae of gill and necrosis in liver of lower stocking densities (10 and 15 fish/m³). In contrast, greater structural changes in both the organs were observed in the higher stocking density groups (20 and fish nos/m³). Based on the results, it can be concluded that maximum growth and complete recovery (in term of haematology, stress and histological changes) were observed in lower stocking density (10 fish/m³) fed with 5 to 6% feeding levels. Hence, in an Indian reservoir cage-based cultures, 10 fish/m³ fed with 5 to 6% feeding rations can be recommended at commercial scale to get maximum and sustainable profit by using the stunted L. rohita fingerling.

Stocking density

Feeding ration

Cage

L. rohita

Histological changes

In the recent past, aquaculture production has increased nearly 12 times, at an average annual growth rate of 10%, which indicates the present contribution and imminent demand of aqua food in the future (FAO 2020). India is the third largest producer of fish in the world and second in aquaculture which producing 11.25 MT of farmed fish, sharing 7.96% to the world fish production (DADHF 2021). The major contribution comes from the Indian Major Carps (IMC), contributes around 89% of the total freshwater aquaculture production (DADHF 2018). The Indian major carps, namely, catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala) contributing 70–80% follwed by exotic carps 20–30% of the total fresh water fish production (FAO 2017). Among the three major carps, rohu is the most preferred species due to its better growth, meat quality, consumer demand, disease resistant and high market demand in India.

India has huge amount of inland water resources such as rivers, reservoirs, lakes, ponds, and tanks. India has a total number of 19,370 reservoirs which covers around 3.15 million ha area. But the production of large and medium reservoirs are very low due to poor or under utilization and rapid population growth coupled with over-exploitation of reservoirs stocks threatening the inland capture fisheries. The average production of large and medium reservoirs is 20–25 kg/ha while small reserviors having 50–60 kg/ha. Overall, reservoir contributes aroound 1% in total indian fisheries production due to its low fish production (Ayyappan et al., 2011). To increase the production of large, medium and small reservoirs, management activities such as reduction of over exploitation, ranching, close of fishing in breeding season were prioritized as a major step to an enhance the capture fisheries. Simultaneously, in order to utilize the potential of these water bodies, cage culture was introduced in reservoirs to increase the production per unit area.

It has been observed that the growth and production of normally reared fingerling were less as compared to the stunted fingerling. The stunted seed defined as the seed produced by rearing normal fingerling at higher stocking densities than usual with sub-optimal level of feeding and fertilization for 8–12 months (Ravi et al. 2012). This reduces the normal growth, resulting from stunting, due to less food availability and overcrowding. When these stunted yearlings are stocked under normal conditions of rearing, they show compensatory growth, which leads to faster growth than normal fingerling (Ali et al. 2003). The capacity of an animal to grow rapidly after a period of reduced growth resulting from inadequate food availability, or some other unfavorable environmental condition, is often termed as compensatory growth when shifted to a favorable condition with adequate feeding. (Jobling et al. 1994, Jobling and Kaskela 1996; Melard et al. 1998; Jobling 2010; Ali et al. 2003). This compensatory growth is obtained through the phenomenon called as hyperphagia (excess feeding). They are less vulnerable to predation and diseases and are more tolerant to environmental fluctuations; require less time to reach marketable size leading to higher production (Suresh Babu et al. 2019). The aim of stunting is to remove the unhealthy seeds, weak and sick fry and to produce healthy seed, which is larger, as a stocking material for grow-out culture (Jobling 2010).

During the stunted seed production, the feeding strategy (sub-optimal feeding or starvation) have a significant effect on the physiological and biochemical alteration in fish (Falahatkar 2012). In the stunted fish, the haematological parameters (Hb, RBC, PCV, and WBC), stress (serum glucose, cortisol and LDH) and histological changes play an important role during compensatory growth phase which also indicates the physiological status of aquatic organisms (Kumar et al. 2022).

The degree of compensation attained is classified as no compensation, partial compensation and complete compensation or over compensation, depending on the method and species selected for stunting (Ali et al. 2003; Lingam et al. 2019). Before starting the commercial level of stunted fingerling rearing in cages, a right candidate species is to be identified and its optimum stocking density and feeding ration need to be standardized in order to maximize the production and profitability. Preliminary information on the culture of stunted fish in open water cage culture is very limited; especially there is a lack of information on the stocking density and feeding ration to be followed. Thus, the present study was conducted to standardize the stocking density of stunted L. rohita fingerling reared in cage with optimum feeding ration.

Experimental site and animal

The experiment was conducted at Dimbhe Reservoir, Ambegaon, Pune, Maharashtra. The reservoir is situated at the basin of Ghod river located at 19.1146738°N, 73.7220996°E which covers total surface area 17,547 km². The 8 months stunted L. rohita fingerling were procured from the private fresh water fish seed farm located in Bharuch district of Gujarat and the fish were carefully transported in 1000 L capacity of syntax water tank. The oxygen level was maintained at saturation level during the transportation using the oxygen cylinder to minimize the stress due to temperature fluctuations and the transportation was carried out in the night time. Water quality parameters were checked regularly at experimental site following standard protocols (APHA 2005).

Experimental design

In the present study, the effect of stocking density and feeding level were evaluated. The stunted fingerling of L. rohita with an average body weight of 38.23 ± 1.90 g were stocked in cages at different densities viz. T₁ (10), T₂ (15), T₃ (20) and T₄ (25) fish/m³ and fed with varying levels of feed rations of 3, 4, 5 and 6% at each stocking density. The 4×4 factorial design was used, and each density and feeding level kept in triplicate, and fed with commercial pellet feed (crude protein 28% and fat 5%) twice in a day. The 48 numbers of square shaped floating net cage, made up of high density poly ethylene (HDPE) used during the trial. The size of each cage was 3 × 3 × 3 m with 6 mm mesh size were used as an experimental unit.

Stocking of experimental animal and rearing

Before stocking of experimental animal, the cages were properly cleaned and washed for proper exchange of water. The experimental cages and animal were disinfected with KMnO₄ (4 ppm) solution and released into cages for acclimatization for next 15 days. At the end of acclimatization, a total 7560 numbers of stunted fingerling of L. rohita were randomly stocked in 48 cages at different stocking densities such as 10, 15, 20 and 25/m³.

A commercial floating fish feed having the nutrients composition of Crude protein 28%, fat 4%, fiber 8.5% and moisture content 11.5% of was used and the fish fed twice in a day (morning (9.00 hrs) and evening (17.00 hrs) as per their respective feeding rations viz. 3, 4, 5 and 6% of body weight at each stocking density throughout the experimental period.

The experimental cages were cleaned manually once in a week to avoid the attachment of barnacles and algae for proper movement of water to maintain the optimum water quality parameters throughout the experimental trial. The experimental cage was covered with a covering net to prevent the escaping of fish from cages and to save the fish from predatory birds.

Sample collection and processing

Blood samples were collected on 0th, 30th, 165th and 330th day during the experimental trial from each experimental cage (n = 3). Blood was withdrawn from caudal vein of the fish by inserting a 25-gauge needle attached with 1 ml of syringe. The collected blood sample was immediately pooled and transferred to an eppendorf tube containing 0.2% EDTA (as an anti-coagulant) to prevent clotting of blood. The collected blood was used for determination of differential blood cells count. For serum preparation, the blood was collected in a syringe without using an anticoagulant and transferred to serological tubes. The blood samples were allowed to clot. The clotted samples were spun down at 6000 rpm for 10 min and supernatant was collected and stored at -20°C until used for further analysis. After drawing the blood, fish were treated with 5 ppm KMnO₄ and released back in to the same cage. To know the tissue level physiological recovery in stunted rohu, gills and liver were collected at 0th, 165th and 330th days. In each sampling, three fish from each cage (n = 3) were sacrificed for tissues collection. The tissues were collected by using sterilized scissor and collected tissues were preserved in 10% neutral buffered formalin for histological analysis. From 10% NBF solution, tissues were changed intermittently and small slits were made in the tissues to increase the penetration of NBF into the tissues.

Haematological parameters

Haemoglobin (Hb)

The Hb level of blood was analyzed by the cyanmethemoglobin method using Drabkins’ fluid (Qualigens). 20 µl blood was mixed with 5 ml of Drabkins working solution. The absorbance was measured using a spectrophotometer at a wave length of 540 nm. The final concentration was calculated by comparing with the standard cyanmethemoglobin. Hb concentration was calculated by using the following formula.

Hb (g/dl) = [OD (T) / OD (S)] X [251 / 1000] X 60

OD (T) = Absorbance of test sample

Red blood cell (RBC) and white blood cell (WBC) counts

For calculation of RBC and WBC, 20 µl blood was diluted with 3980 µl of RBC diluting fluid (Hayem’s) and WBC diluting fluid in a separate clean test tube, respectively. The mixture was shaken well to suspend the cells uniformly in the solution. Then 10 µl red blood cell mixture was placed in the Neubauer counting chamber. Care was taken to avoid the formation of air bubbles inside the chamber. The red blood cells were counted in the five groups of small squares. The cells lying inside the five small squares were counted under high power objective (40x). Similarly, white blood cell mixture was poured and the cells were counted in four big squares under high power (40×) magnification of light microscope. The number of RBC (× 10⁶/mm³) and WBC (× 10³/mm³) of blood sample were calculated by the standard formulae.

Number of RBC/µL = N X Dilution factor / (Area counted X Depth of fluid)

Number of WBC/µL = N X Dilution factor / (Area counted X Depth of fluid)

Where, N denotes the total number of RBC/WBC counted in the haemocytometer.

Number of WBC/mm³\(=\frac{\text{N} \text{x} \text{D}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n}}{\text{A}\text{r}\text{e}\text{a} \text{c}\text{o}\text{u}\text{n}\text{t}\text{e}\text{d} \text{x} \text{D}\text{e}\text{p}\text{t}\text{h} \text{o}\text{f} \text{f}\text{l}\text{u}\text{i}\text{d}}\)

= N X 200 / (0.02 X .01)

= N X 10000

Packed Cell Volume (PCV)

A known volume of blood is centrifuged to form three layers: Red cells, Buffy coat (WBC and platelets) and plasma. PCV is the ratio of the height of red cells column to that of the whole blood in the tube. Anticoagulated blood was transferred to capillary tubes of length of 75 mm and internal diameter of 1 mm. Blood was allowed to enter the tube stopping at 10–15 mm from one end. Wipe the outside of the tube and seal the dry end by pushing into the plasticine two or three times. Centrifuge the capillary tube for five minutes and read the hematocrit value in the micro hematocrit reader. The result was expressed as percentage.

Serum glucose and cortisol

Serum glucose was analyzed using the glucose kit (Trans Asia Bio-Medicals, India) following the manufacturer’s guidelines. The detection method was based on the Trinder’s method and absorbance value was measured at 670 nm using a spectrophotometer (Thermo Scientific, Genesys, 10S UV-VIS). The result was expressed in mg/dl of sample.

Serum cortisol levels were estimated using the Cortisol EIA Kit (Cayman Chemicals, USA) following kit standard protocols. It is a competitive assay, where competition between cortisol and cortisol tracer (Cortisol-acetylcholinesterase-AChE) to conjugate for a limited number of cortisol-specific mouse monoclonal antibody binding sites present in the well. At the end of assay a distinct yellow colour was produced and it was measured at 412 nm by using microplate reader. The amount of cortisol tracer bound in the well is inversely proportional to the concentration cortisol in the sample (mg/dl).

Lactate Dehydrogenase (LDH) activity)

The LDH activity was assayed by the method of Wroblewski and LaDue (1955). Total of 3 ml of the reaction mixture, comprised of 2.7 ml of 0.1 M phosphate buffer (pH 7.5), 0.1 ml of NADH solution (2 mg NADH dissolved in 1 ml of phosphate buffer solution), 0.1 ml of serum and 0.1 ml of sodium pyruvate, was prepared. The reaction was started after addition of substrate sodium pyruvate. The OD was recorded at 340 nm at 30 sec intervals for 3min. The enzymatic activity was expressed as micromoles of NAD released mg^− 1 protein min^− 1.

Histology of gill and liver

The 10% NBF preserved tissues were cut into small pieces and kept for overnight washing to replace the formalin crystals. Tissues were dehydrated with a series of alcohol gradients and cleared using xylene. Then the tissues were embedded in paraffin wax following the impregnation technique (Leica, EG 1140H, Germany). The embedded wax blocks were sectioned at 3–5 µm thickness using a microtome (Leica RM 2125RT, Germany) and stained with haematoxylin and eosin (Luna, 1968). Then the tissues were mounted using DPX and kept for proper drying. The slides were observed under microscope and microphotographs were taken using Olympus FSX 100 at 40x magnification.

Statistical analysis

Statistical analysis of different hematological and metabolic enzymes parameters were done by one -way analysis of variance (ANOVA) using SPSS version 21.0 for windows. Duncan’s multiple range test was used for post hoc comparison of mean (P < 0.05) between different groups. All the data presented in the text, figures and tables expressed as mean ± standard error and statistical significance for the test was set at 5% confidence limit.

Haematological parameters

At 0th day, there were no significant difference (P > 0.05) observed in the Hb, RBC, PCV and WBC content in all the different densities and feeding ration groups (Tables 1, 2, 3 and 4). At 30th, 165th and 330th days, the significantly (P < 0.05) higher Hb, RBC, and PCV value were observed in lowest stocking density group feeding with higher level of feeding rations such as 10*5 and 10*6 treatment groups, while the lowest value were observed in the highest stocking density and lowest feeding rations treatment groups (25*3 and 25*4) (Table-01, 02, 03). However, contrasting trend was observed in the WBC content in the treatment groups where significantly lower WBC was recorded in (10*6) and (10*5) treatment groups. The complete recovery was observed in feeding ration dependent manner with inversely proportional to stocking density of fish.

Table 1

Effect of stocking density and feeding ration on haemoglobin of stunted *L. rohita* fingerling reared in floating net cages
Treatments	0th days	30th days	165th days	330th days
10*3	5.80 ± 0.03	9.16 ± 0.09^d	10.06 ± 0.03^c	10.40 ± 0.05^bc
10*4	5.80 ± 0.03	9.73 ± 0.12^c	10.30 ± 0.05^b	10.60 ± 0.05^b
10*5	5.80 ± 0.03	10.13 ± 0.09^b	11.10 ± 0.05^a	11.20 ± 0.11^a
10*6	5.80 ± 0.03	10.70 ± 0.20^a	11.23 ± 0.03^a	11.23 ± 0.08^a
15*3	5.80 ± 0.03	8.60 ± 0.17^e	9.20 ± 0.05^f	10.03 ± 0.03^d
15*4	5.80 ± 0.03	8.63 ± 0.26^e	9.20 ± 0.05^f	10.23 ± 0.03^c
15*5	5.80 ± 0.03	8.76 ± 0.15^e	9.33 ± 0.03^de	10.36 ± 0.03^bc
15*6	5.80 ± 0.03	8.83 ± 0.03^e	9.40 ± 0.05^d	10.53 ± 0.03^b
20*3	5.80 ± 0.03	7.20 ± 0.05^g	8.26 ± 0.08^j	8.13 ± 0.08^ef
20*4	5.80 ± 0.03	7.63 ± 0.03^f	8.50 ± 0.05ⁱ	8.30 ± 0.05^e
20*5	5.80 ± 0.03	7.80 ± 0.05^f	8.63 ± 0.03^gh	8.36 ± 0.08^e
20*6	5.80 ± 0.03	7.53 ± 0.12^f	8.73 ± 0.05^g	8.13 ± 0.24^ef
25*3	5.80 ± 0.03	6.26 ± 0.03^j	7.03 ± 0.08^m	7.16 ± 0.08^h
25*4	5.80 ± 0.03	6.32 ± 0.03ⁱ	7.20 ± 0.03^l	7.23 ± 0.12^h
25*5	5.80 ± 0.03	6.40 ± 0.05^hi	7.33 ± 0.03^l	7.36 ± 0.08^h
25*6	5.80 ± 0.03	6.63 ± 0.15^h	7.76 ± 0.05^k	7.96 ± 0.07^g
In each column, mean values with different superscripts differ significantly at p < 0.05. Hb expressed in g/dL. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Table 2

Effect of stocking density and feeding ration on RBC of stunted *L. rohita* fingerling reared in floating net cages
Treatments	0 days	30th days	165th days	330th days
10*3	1.10 ± 0.02	1.96 ± 0.03^bde	2.32 ± 0.02^c	2.37 ± 0.01^cd
10*4	1.10 ± 0.02	1.84 ± 0.03^cde	2.39 ± 0.01^c	2.54 ± 0.06^b
10*5	1.10 ± 0.02	2.32 ± 0.05^ab	2.65 ± 0.04^b	2.73 ± 0.01^a
10*6	1.10 ± 0.02	2.60 ± 0.15^a	2.82 ± 0.01^a	2.83 ± 0.03^a
15*3	1.10 ± 0.02	2.08 ± 0.15^bcd	2.11 ± 0.03^d	2.26 ± 0.02^d
15*4	1.10 ± 0.02	2.25 ± 0.22^b	2.15 ± 0.02^d	2.30 ± 0.01^c
15*5	1.10 ± 0.02	2.08 ± 0.15^bcd	2.19 ± 0.01^d	2.45 ± 0.01^bc
15*6	1.10 ± 0.02	2.19 ± 0.02^b	2.32 ± 0.02^c	2.47 ± 0.01^bc
20*3	1.10 ± 0.02	1.68 ± 0.03^efg	1.63 ± 0.01^e	1.69 ± 0.01^f
20*4	1.10 ± 0.02	1.74 ± 0.13^efg	1.51 ± 0.03^f	1.75 ± 0.08^ef
20*5	1.10 ± 0.02	1.53 ± 0.06^fgh	1.48 ± 0.02^f	1.83 ± 0.08^e
20*6	1.10 ± 0.02	1.55 ± 0.02^fgh	1.62 ± 0.01^e	1.86 ± 0.01^e
25*3	1.10 ± 0.02	1.26 ± 0.01ⁱ	1.38 ± 0.02^g	1.54 ± 0.06^g
25*4	1.10 ± 0.02	1.30 ± 0.01ⁱ	1.28 ± 0.01^h	1.69 ± 0.07^f
25*5	1.10 ± 0.02	1.36 ± 0.04ⁱ	1.44 ± 0.06^f	1.65 ± 0.06^fg
25*6	1.10 ± 0.02	1.47 ± 0.04^gh	1.63 ± 0.03^e	1.87 ± 0.06^e
In each column, mean values with different superscripts differ significantly at p < 0.05. RBC expressed in 1× 10⁶/mm³. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Table 3

Effect of stocking density and feeding ration on PCV of stunted *L. rohita* fingerling reared in floating net cages
Treatments	0 day	30th days	165th days	330th days
10*3	25.61 ± 0.01	37.36 ± 0.17^b	42.30 ± 0.05^ab	45.60 ± 0.05^a
10*4	25.61 ± 0.01	37.26 ± 0.03^b	42.43 ± 0.08^ab	45.53 ± 0.12^a
10*5	25.61 ± 0.01	39.40 ± 0.05^a	42.80 ± 0.05^ab	45.40 ± 0.20^a
10*6	25.61 ± 0.01	39.63 ± 0.12^a	43.46 ± 0.21^a	45.76 ± 0.16^a
15*3	25.61 ± 0.01	37.20 ± 0.05^b	41.40 ± 0.10^ab	44.70 ± 0.45^a
15*4	25.61 ± 0.01	37.33 ± 0.03^b	40.86 ± 0.33^b	44.16 ± 0.14^a
15*5	25.61 ± 0.01	37.53 ± 0.06^b	41.63 ± 0.03^ab	44.53 ± 0.23^a
15*6	25.61 ± 0.01	37.10 ± 0.15^b	41.70 ± 0.15^ab	44.53 ± 0.18^a
20*3	25.61 ± 0.01	32.76 ± 1.63^c	36.00 ± 0.70^c	37.66 ± 0.22^b
20*4	25.61 ± 0.01	30.76 ± 0.50^d	30.26 ± 0.21^d	29.60 ± 0.70^cde
20*5	25.61 ± 0.01	31.30 ± 0.10^cd	31.93 ± 0.10^d	31.36 ± 0.42^cd
20*6	25.61 ± 0.01	28.53 ± 0.10^e	31.50 ± 0.71^d	31.90 ± 0.25^c
25*3	25.61 ± 0.01	27.63 ± 0.60^e	28.43 ± 0.15^e	28.63 ± 0.73^de
25*4	25.61 ± 0.01	28.30 ± 0.08^e	27.30 ± 0.12^e	27.56 ± 0.20^e
25*5	25.61 ± 0.01	28.30 ± 0.05^e	27.66 ± 0.12^e	28.16 ± 0.13^e
25*6	25.61 ± 0.01	28.87 ± 0.05^e	28.66 ± 0.13^e	28.30 ± 0.03^e
In each column, mean values with different superscripts differ significantly at p < 0.05. PCV expressed in %. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Table 4

Effect of stocking density and feeding ration on WBC of stunted *L. rohita* fingerling reared in floating net cages.
Treatments	0 day	30th day	165th day	330th day
10*3	78.09 ± 0.20	63.56 ± 0.75^d	72.56 ± 0.85^c	72.91 ± 0.38^c
10*4	78.09 ± 0.20	63.20 ± 0.80^d	72.24 ± 0.05^c	71.63 ± 0.05^c
10*5	78.09 ± 0.20	61.13 ± 0.40^e	70.16 ± 0.26^cd	70.15 ± 0.21^cd
10*6	78.09 ± 0.20	60.21 ± 0.73^e	68.62 ± 0.73^d	69.32 ± 0.12^d
15*3	78.09 ± 0.20	68.25 ± 0.88^bc	74.22 ± 0.28^bc	74.52 ± 0.15^bc
15*4	78.09 ± 0.20	68.12 ± 0.60^bc	74.31 ± 0.20^bc	74.24 ± 0.33^bc
15*5	78.09 ± 0.20	66.14 ± 0.52^c	72.69 ± 0.12^c	73.69 ± 0.08^c
15*6	78.09 ± 0.20	66.02 ± 0.63^c	72.31 ± 0.05^c	72.61 ± 0.12^c
20*3	78.09 ± 0.20	71.26 ± 0.66^ab	78.22 ± 0.85^b	78.33 ± 0.14^b
20*4	78.09 ± 0.20	71.16 ± 0.08^ab	78.13 ± 0.38^b	77.21 ± 0.05^b
20*5	78.09 ± 0.20	70.91 ± 0.17^b	77.02 ± 0.05^b	76.42 ± 0.11^b
20*6	78.09 ± 0.20	70.31 ± 0.28^b	76.21 ± 0.06^b	77.32 ± 0.31^b
25*3	78.09 ± 0.20	73.56 ± 0.05^a	84.16 ± 0.62^a	86.32 ± 0.66^a
25*4	78.09 ± 0.20	73.13 ± 0.08^a	84.34 ± 0.66^a	85.42 ± 0.20^a
25*5	78.09 ± 0.20	72.43 ± 0.20^a	83.31 ± 0.12^a	83.24 ± 0.05^a
25*6	78.09 ± 0.20	72.12 ± 0.15^a	82.34 ± 0.13^a	82.46 ± 0.32^a
In each column, mean values with different superscripts differ significantly at p < 0.05. RBC expressed in 1× 10³/mm³. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Serum stress parameters

There was no significant difference (P > 0.05) observed in glucose, cortisol and LDH values at 0th day of in all the experimental groups (Tables 5, 6 and 7). At 30th, 165th, and 330th days, the complete recovery was observed at lowest stocking density fed with highest feeding rations (10*5 and 10*6 groups). While, the serum glucose, cortisol and LDH values were highest observed in the highest stocking density fed with lowest feeding rations of 25*3 and 25*4 treatment groups. Similar to the haematological parameters the recovery was observed in feeding ration dependent manner with inverse stocking density treatment groups.

Table 5

Effect of stocking density and feeding ration on glucose of stunted *L. rohita* fingerling reared in floating net cages
Treatments	0 day	30th days	165th days	330th days
10*3	102.42 ± 0.51	90.52 ± 2.13^d	86.01 ± 2.21^d	79.01 ± 3.25^bc
10*4	102.42 ± 0.51	86.50 ± 2.15^de	83.21 ± 2.14^de	76.61 ± 3.33^c
10*5	102.42 ± 0.51	84.57 ± 2.14^de	81.23 ± 2.19^e	75.23 ± 3.15^c
10*6	102.42 ± 0.51	83.59 ± 2.11^e	80.16 ± 2.14^e	73.16 ± 4.23^c
15*3	102.42 ± 0.51	103.54 ± 3.21^b	96.24 ± 3.15^bc	88.11 ± 4.51^b
15*4	102.42 ± 0.51	98.53 ± 3.15^bc	93.25 ± 3.16^bc	89.02 ± 4.58^b
15*5	102.42 ± 0.51	96.53 ± 3.21^bc	91.26 ± 3.14^c	85.16 ± 2.25^b
15*6	102.42 ± 0.51	94.55 ± 3.25^cd	89.16 ± 3.24^cd	83.33 ± 2.64^b
20*3	102.42 ± 0.51	108.54 ± 1.58^a	102.28 ± 1.55^ab	84.32 ± 1.96^b
20*4	102.42 ± 0.51	103.53 ± 1.68^b	98.22 ± 1.64^b	93.21 ± 2.58^a
20*5	102.42 ± 0.51	101.50 ± 1.57^b	95.31 ± 1.39^bc	92.26 ± 2.64^ab
20*6	102.42 ± 0.51	100.49 ± 1.59^b	92.42 ± 1.66^c	92.31 ± 2.46^ab
25*3	102.42 ± 0.51	110.60 ± 0.93^a	105.31 ± 0.91^a	97.41 ± 2.54^a
25*4	102.42 ± 0.51	108.49 ± 0.68^a	101.33 ± 0.85^ab	95.17 ± 2.17^a
25*5	102.42 ± 0.51	107.51 ± 0.86^a	99.17 ± 0.59^b	94.13 ± 2.89^a
25*6	102.42 ± 0.51	105.54 ± 0.89^ab	98.12 ± 0.88^b	94.33 ± 2.64^a
In each column, mean values with different superscripts differ significantly at p < 0.05. Glucose expressed in mg/dL. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Table 6

Effect of stocking density and feeding ration on cortisol of stunted *L. rohita* fingerling reared in floating net cages.
Treatments	0th day	30th days	165th days	330th days
10*3	112.42 ± 0.51	85.71 ± 1.55^e	72.52 ± 3.11^f	61.66 ± 3.21^e
10*4	112.42 ± 0.51	81.13 ± 1.36^f	71.23 ± 3.25^f	60.21 ± 3.02^e
10*5	112.42 ± 0.51	78.34 ± 1.34^fg	63.31 ± 3.16^g	54.31 ± 3.51^ef
10*6	112.42 ± 0.51	73.07 ± 2.15^g	62.13 ± 2.25^g	51.33 ± 2.56^f
15*3	112.42 ± 0.51	102.18 ± 2.65^cd	84.21 ± 2.16^e	72.26 ± 2.41^c
15*4	112.42 ± 0.51	98.96 ± 2.31^d	83.30 ± 2.61^e	69.25 ± 2.55^d
15*5	112.42 ± 0.51	95.32 ± 1.89^d	82.12 ± 2.83^e	67.36 ± 2.11^d
15*6	112.42 ± 0.51	91.28 ± 1.98^d	87.02 ± 2.91^de	65.24 ± 2.32^de
20*3	112.42 ± 0.51	111.78 ± 1.78^b	102.16 ± 2.34^b	80.21 ± 2.15^b
20*4	112.42 ± 0.51	108.32 ± 2.21^bc	101.66 ± 1.89^b	74.13 ± 1.96^c
20*5	112.42 ± 0.51	105.21 ± 2.06^c	96.30 ± 2.01^c	72.30 ± 1.26^c
20*6	112.42 ± 0.51	98.13 ± 2.54^d	92.11 ± 2.34^cd	69.24 ± 1.54^d
25*3	112.42 ± 0.51	120.36 ± 3.13^a	108.13 ± 2.31^a	87.30 ± 1.02^a
25*4	112.42 ± 0.51	116.36 ± 3.21^ab	105.22 ± 1.96^ab	76.14 ± 1.11^bc
25*5	112.42 ± 0.51	112.31 ± 3.51^b	102.21 ± 1.59^b	72.19 ± 0.98^c
25*6	112.42 ± 0.51	110.68 ± 3.25^b	101.58 ± 1.54^b	70.18 ± 0.48^cd
In each column, mean values with different superscripts differ significantly at p < 0.05. Cortisol expressed in mg/dL. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Table 7

Effect of stocking density and feeding ration on lactate dehydrogenase of stunted *L. rohita* fingerling reared in floating net cages.
Treatments	0th day	30th days	165th days	330th days
10*3	20.03 ± 0.87	18.18 ± 1.62^cd	16.15 ± 1.22^de	19.30 ± 2.14^cd
10*4	20.03 ± 0.87	16.20 ± 1.26^e	15.60 ± 1.31^ef	17.20 ± 2.11^d
10*5	20.03 ± 0.87	14.04 ± 1.22^g	13.92 ± 1.24^fg	15.51 ± 2.61^de
10*6	20.03 ± 0.87	13.13 ± 1.85^g	12.20 ± 1.11^g	14.93 ± 2.19^e
15*3	20.03 ± 0.87	19.22 ± 1.86^bc	18.50 ± 1.17^bc	21.10 ± 2.43^bc
15*4	20.03 ± 0.87	18.03 ± 1.83^cd	16.69 ± 1.13^de	19.68 ± 2.17^cd
15*5	20.03 ± 0.87	16.86 ± 1.23^de	14.80 ± 1.21^ef	17.90 ± 2.54^cd
15*6	20.03 ± 0.87	15.55 ± 1.25^efg	14.02 ± 1.09^f	16.03 ± 2.43^de
20*3	20.03 ± 0.87	21.31 ± 1.24^ab	19.01 ± 0.75^b	22.36 ± 1.86^b
20*4	20.03 ± 0.87	19.24 ± 0.98^bc	18.40 ± 0.86^bc	20.40 ± 1.68^bc
20*5	20.03 ± 0.87	18.91 ± 0.96^bc	17.62 ± 0.79^cd	18.70 ± 1.66^cd
20*6	20.03 ± 0.87	17.15 ± 0.99^de	16.80 ± 0.46^de	17.91 ± 1.67^cd
25*3	20.03 ± 0.87	23.27 ± 0.86^a	21.10 ± 0.46^a	25.09 ± 0.85^a
25*4	20.03 ± 0.87	21.01 ± 0.81^b	19.30 ± 0.48^b	23.27 ± 0.76^ab
25*5	20.03 ± 0.87	19.87 ± 0.84^b	17.41 ± 0.49^cd	20.51 ± 0.73^bc
25*6	20.03 ± 0.87	18.01 ± 0.88^cd	17.61 ± 0.47^cd	18.73 ± 0.49^cd
In each column, mean values with different superscripts differ significantly at p < 0.05. Lactate dehydrogenase expressed in mg^− 1 protein min^− 1. Note: Treatments: T₁ (10/m³), T₂ (15/m³), T₃ (20/m³), T₄ (25/m³) fed with feeding ration 3, 4, 5 and 6% of body weight.

Histological observations of gill and liver

Gills

At 0th day, stunted fish showed the swelling of the primary lamellae, hyperplasia and complete fusion of secondary lamellae in gill section (Fig. 1B). At 165th and 330th day, in the lower stocking density group (10 fish/ m³) the healthy primary and secondary gill lamellae were observed with spacing and thickness (Figs. 2 and 3). Similarly, in 15 fish/m³ stocking density group the primary gill lamellae were slightly disturbed in term of swelling/edema and hyperplasia of basal epithelium between the secondary gill lamellae (Figs. 2 and 3). In higher stocking densities of20 and 25 fish/m³ treatment groups, the brachial disruption were observed which leads to hyperplasia and complete lamellar fusion, especially in apical part of primary gill lamellae (Figs. 2 and 3). Result indicates that the complete recovery in gill was observed from the 165th day onwards in the lower stocking density group while the stress condition were observed in all other treatment groups, including15 fish/m³.

Liver

At 0th day, the size of hepatocyte was very low which shows the necrosis in all the treatment groups (Fig. 1A). Similarly, on 165th day, the size of hepatocyte in 10 fish/m³ and 15 fish/m³ was bigger than the 20 fish/m³ and 25/m³, which shows the absence of necrosis in the 10 fish/m³ and 15 fish/m³ treatment groups (Fig. 4). The hepatocyte cytoplasmic content in 20 fish/m³ and 25/m³ treatment group was eosinophilic, indicates the accumulation of protein while 10 fish/m³ and 15 fish/m³ treatments group shows the lipid accumulation. However, at 330th day, the small hepatocyte was observed in lower stocking density (10 fish/m³ and 15 fish/m³) treatment groups. The slightly bigger hepatocyte was observed in the 20 fish/m³ and 25 fish/m³ treatments groups (Fig. 5). The hepatocyte cytoplasmic content in 15 fish/m³, 20 fish/m³ and 25 fish/m³ treatments groups was accumulated the higher amount of protein, while in 10 fish/m³ treatment group accumulated the lipid. The higher stocking groups (20 fish/m³ and 25 fish/m³) shows the hepatocyte necrosis and haemorrhage even at the end of the experimental trial which was absent in lower stocking groups.

The increase in stocking density reduces the growth of L. rohita fingerlings even in higher feeding ration groups was observed in our previous study (Paswan et al. 2021). The effects of stocking and feeding rations were visibly started appearing on 30th day of the experimental trial in different treatment groups. In general, higher stocking density reduced the space per individual animal, thereby adversely affecting social interactions, reduced the physiological need of animal and increasing stress in the fish. The elevated stress condition would lead to higher energy requirements to maintain the homeostasis of fish thereby causing reduction in utilization and feed and growth of cultured fish which ultimately affects the profit in aquaculture.

In the present study the recorded water quality parameters such as temperature, pH, dissolved oxygen, total alkalinity, ammonia, etc, inside as well as outside (50 m away) of the cages installed in Dimbhe reservoir, were within the range for optimal production conditions of L. rohita fingerling (Paswan et al. 2021; Mane et al. 2017).

The haematological parameters such as Hb, RBC, PCV, and WBC are most widely used to assess the health status of cultured animal (Khoei 2021; Kumar et al. 2022). (Rehulka et al. 2004) reported that diet composition and metabolism are two main factors which affect the hematological parameters of fish. In the present study, at 0th day, the lower Hb, RBC and PCV values were observed. It has been reported by Lingam et al. (2019), during the production of stunting fingerling i.e., restricted feeding period or malnutrition can affect the energy required for basal metabolic activities of fish and this might be the reason for decreased hematological values at the 0th day. When common carp and Persian sturgeon starved for seven and four weeks, respectively, resulted in lower level of Hb, RBC and PCV values and it was even lower than the reported values of the present study (Murachi 1959; Yarmohammadi et al. 2015). In the present study, the observed value of Hb, RBC and PCV at 0th day, increased gradually from 30th days onwards even up to 165th and 330th days, indicating the recovery of the stocked fish in cages. The lowest Hb, RBC, and PCV value were observed in the highest stocking density fed with lowest feeding ration 25*3 treatment group throughout the experimental period. Similarly, the higher stocking density decreased the Hb, RBC and PCV in African catfish (Clarias gariepinus) (Magouz et al. 2019), brook trout (Marchand and Boisclair 1998), gilthead seabream (Montero et al. 2001) and great sturgeon (Rafatnezhad et al. 2008) as observed in the present study. The significantly lower Hb, RBC and PCV values of 20 fish/m³ and 25 fish /m³ treatment groups might be due to crowding stress in cages and it was remained up to 330th day of the trial due to the existence of chronic stress. On the other side, fish stocked in lower stocking densities (10 fish/m³) regained the normal Hb, RBC and PCV values in the present study supported by the findings of (Kamal and Omar 2011) who reported higher values of Hb, RBC and PCV in silver carp reared in lower stocking density (3 fish/m³) than the higher stocking densities (6 & 9 fish/m³). Grant (2015) stated that, the production of RBC increased when the fish returned to normal conditions after experiencing a chronic stress and it was observed in the present study lower stocking (10 fish/m³) fed with higher feeding ration (5 to 6%) from 30th days onwards. Barcellos et al. (2004) also observed that, fish exposed to unsuitable environment may alters their hematological parameters, whereas returning of same fish to optimal culture condition exhibit normal blood biochemistry which might be the reason for recovery in Hb, WBC and PCV values of rohu reared in lower stocking density fed with higher feeding rations.

In contrast, the WBC value was high at 0th day in all the treatment groups. The value was significantly decreased at 30th day and it might be due to fact that, stunted fish change their blood composition after coming to the semi-normal or normal condition. The values of WBC were slightly increased at 165th day and started showing lower values up to 330th days in lower stocking density fed with higher feeding rations, while it was increased from the 165th to 330th day in higher density group due to chronic stress. Falahatkar et al. (2019) also reported the WBC level of a new hybrid strain (Leuciscus aspius × Rutilus frisi) was increased when the fish stocked in higher stocking density Similarly, Wendelaar-Bonga (1997) stated that, WBC content increased during the stress condition caused by the higher stocking density n fish. On the other side, hematological changes occurred due to crowding stress can be nullified or neutralized by the increase in the feeding ration (Martinez et al. 1994; Montero et al. 1999; Montero et al. 2001).

The higher serum cortisol and glucose level in fish has been considered as primary and secondary level stress indicator (Hattingh 1976; Manush et al. 2005; Metwally and Wafeek 2014). The secretion of corticosteroids increased due to all types of stress which in turn elevate the blood glucose level for energy production to fight the stressful condition (Nakano and Tomlinson 1967). In general, the level of cortisol and glucose were fluctuated in fish, subjected to fasting and feeding. On 0th day, the concentration of glucose and cortisol were higher and it might be because of stunted fingerling need higher amount of energy to maintain their physiological homeostasis. At 30th day, in higher stocking density fed with low feeding rate increased glucose and cortisol levels were observed while lower stocking density fed with higher feed rations decreased glucose and cortisol levels were observed which indicate the returning stunted fish normal physiological conditions. Further, on 165th and 330th days, the level of glucose and cortisol were normalized in lower stocking (10 fish /m³) group fed with higher feeding rations of 5 to 6%, while the level remains higher in higher stocking density (20 and 25 fish/ m³) and lower feeding groups (3 and 4%). The reason for elevated serum glucose and cortisol is due to the chronic stress caused by the crowding conditions which induces the production of gluconeogenesis and glycogenolysis for supplying the energy to cope with changed conditions (Iwama et al. 1999; Vinodhini and Narayanan 2009). The values of glucose and cortisol were recovered at lower stocking density (10 fish/m³) and indicated the revival of their physiological homeostasis to maintain the energy demand of fish. The levels of glucose and cortisol were increased due to crowding stress in European sea bass (Dicentrarchus labrax) and mrigal as reported by (Caruso et al. 2011) and (Tejpal et al. 2009) respectively. The enhanced levels of cortisol in high stocking density (25 fish/m³) of the present study are in agreement with similar observations in gilthead sea bream (Montero et al. 1999), zebrafish (Ramsay et al. 2006) Japanese flounder (Bolasina et al. 2006) and European sea bass (Lupatsch et al. 2010). In contrast, high stocking density showed no effect on cortisol level in great sturgeon juveniles (Rafatnezhad et al. 2008) and siberian sturgeon (Hasanalipour et al. 2013).

It has been reported that the LDH activity was increased when there is an oxygen debt in the tissue, which converts pyruvate into lactate by LDH and NADH into NAD⁺ to maintain the redox potential in tissues (Murray et al. 2000; Shamna et al. 2015; Kumar et al. 2018). The stress-induced upsurge activity of LDH was observed in several fishes (Shamna et al. 2015; Kumar et al. 2019). In the present study at 0th day, the activity of serum LDH was higher in all the treatment groups indicates the energy demand of stunted animal for survival. At 330th day, higher level of LDH was noticed in higher stocking density group fed with minimum feed rations (25*3) and it may be due catabolism of carbohydrate which possibly increased the energy demand caused by the chronic crowding stress. Similarly, the value of LDH was increased with increase in stocking density of wedge sole (Dicologoglossa cuneata) (Herrera et al. 2014). However, at the end of the experiment, lowest activity was observed in lower stocking density (10 fish/m³) fed with higher feeding ration which shows complete recovery in this treatment groups.

Chatterjee et al. (2010) reported a significantly higher activity LDH activity when rohu fingerlings were transported in higher stocking density (80 fish/L), Similarly, grey mullet reared under high stocking density (400 numbers/l) displayed an elevated LDH activity in the liver tissues (Kumar et al. 2012). LDH helps to synthesize ATP, under anaerobic conditions using lactate as substrate, through gluconeogenesis in fish which could be the possible reason for raised LDH activity in higher stocking density groups of the study. Additionally, the increased LDH values indicates the damage of liver tissues which can be well correlated with the histological examination in the present study at higher stocking density. Histological observation used to assess the fish health and stress, and it could provide the complete picture of short and long term effect of stress on organism (Hinton and Laurén 1990; Rašković et al. 2013; Wang et al. 2020). However, limited number of studies were conducted to evaluate the effect of stocking density on the histological structure of gill and liver. Major studies reported the environmental stress induced the histological changes in gill such as gill epithelial hypertrophy and hyperplasia, fusion in lamellae, goblet cell proliferation, increased mucus secretion, necrosis of secondary lamellae and haemorrhage and in liver it caused oedema, vacuoles, hypertrophy, steatosis, atrophied, and necrosis of hepatocytes (Scott and Rogers 1980; Roberts 1989; Ferguson 1989; Mallat 1985; Hinton and Laurén, 1990; Poleksić and Mitrović-Tutundžić, 1994; Chapman et al. 2000; Smith et al. 2000; Mohapatra et al. 2003; Wolf and Wolfe 2005; Yengkokpem et al. 2006; Benliet al. 2008; Fazioa et al. 2014; Raskovic et al. 2013; Aziza et al. 2015; Fregoso-López et al. 2017; Lingam et al. 2019).

In the present study, at 0th day the swelling of primary lamellae, hyperplasia, and complete fusion was observed in the secondary gill lamellae due to stunting of fish under crowding conditions with restricted feeding. At 165th and 330th days, the lower stocking density (10 fish/m³) group shows the healthy primary as well as secondary gill lamellae which indicated complete recovery and it was also supported by better growth performance (Paswan et al. 2021). However, the gills were slightly disturbed in 15 fish/m³ and completely disturbed in term of swelling/edema and hyperplasia of basal epithelium between the secondary gill lamellae was observed in the 20 fish/m³ and 25 fish/m³ treatment groups. The stocking density, especially higher stocking density, advesly affects the gill histology (Wang et al. 2019). It has been reported that, some fishes had developed stress reponse mechanism during the hyposia such as increase the number of secondary gill lamella in Crucian carp (Sollid et al. 2003), African cichlids (Chapman et al. 2000) and in Channa striatus (Natarajan 1985). Most fishes in sublethal hypoxic conditions showed stress signs such as gill epithelial hypertrophy and hyperplasia, goblet cell proliferation with increased mucus secretion, haemorrhage, etc (Scott and Rogers 1980). The higher stocking density (8.4 Kg/m³) induced hyperplasia in secondary lamellae, fusion in lamellae, necrosis of secondary lamellae and epithelia exudation in the mullet (Fazioa et al. 2014). In the present study, higher stocking density effect on the histological changes of gill were in agreement with earlier findings of (Poleksić and Mitrović-Tutundžić 1994; Raskovic et al. 2013; Fregoso-López et al. 2017).

Fish liver plays a vital role in the metabolism and considered as the first organ to be affected during stunting process (Power et al. 2000; Gambardella et al. 2012; Liu et al. 2019) due to generation of oxidative stress (Storch and Juario 1983; Ostaszewska et al. 2006). At 0th day, the prominent hepatocyte necrosis was observed in the treatment groups. It has been reported, hepatocyte necrosis, degeneration of hepatocyte and vacuoles formation in fish liver after exposing them to higher stoking density (Wang et al. 2019). In the present study at 165th and 330th days, the size of hepatocytes was larger in 10 fish/m³ and 15 fish/m³ treatment groups as compared with 20 fish/m³ and 25 fish/m³ treatment groups. The lower stocking group did not show any hepatocytes necrosis while in higher stocking density (20 fish/m³ and 25 fish/m³), the prominent hepatocyte necrosis and haemorrhage were observed. Some study reported the necrosis and haemorrhage in hepatocytes due to ammonia exposure in Nile tilapia (Benli et al. 2008), hypoxia in channel catfish (Harper and Wolf 2009) higher stocking density in largemouth bass (Wang et al. 2019).

The abrupt change in size of hepatocytes of higher stocking density was observed due to the stress altered the hepatic energy storage (Wolf and Wolfe 2005; Benli et al. 2008; Wang et al. 2020). Wolf and Wolfe (2005) reported decrease of vacuolation in hepatocyte due to loss of cytoplasmic glycogen and/or lipid caused by insufficient energy intake. Conversely, increased hepatocellular vacuolation is more commonly associated with overnutrition or toxicity (Wolf and Wolfe 2005) in the cultured fish. In the present study, larger size of hepatocytes were observed in the lower stocking density group beefed with increased feeding rations which influenced the deposition of glycogen and lipid in the liver. Similarly, some studies reported the carbohydrate levels in the diets influence the liver morphology by fulfilling the energy demand in fish (Mohapatra et al. 2003; Kumar et al. 2005; Yengkokpem et al. 2006).

The haematological parameters, stress and histological alteration were severe up to throughout the experiment in all the treatment group, except the 10 fish/m³, which recovered and established all the physiological changes. At the end of 330th days, the stocking density (20 fish/m3 and 25 fish/m³) treatment groups showed altered haematological parameters, stress and abrupt change in gill and liver histology. However, lower stocking density group 10 fish/m³, recovered and maintain the normal physiological homeostasis throughout the experiment. Similarly, in our previous study higher growth performance was observed in the 10 fish/m³ fed with 5 and 6% feeding rations. Consider in all this, it can be concluded that the stunted L. rohita fingerling can be stocked, at a rate of 10 fish/m³ and fed with 5% of feed for economically viable grow-out culture with better physiological conditions.

Acknowledgements

The authors are thankful to the Director and Vice-chancellor, ICAR-Central Institute of Fisheries Education, Mumbai, for providing facilities. The first author acknowledges the Indian Council of Agricultural Research, New Delhi, for the PhD research fellowship at ICAR-CIFE, Mumbai.

Funding details: No funding provided for this work

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

Availability of data and material: All the date are included in the manuscript

Code availability: Not applicable

Conflict of interest: No potential conflict of interest was reported by the authors.

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Effect of stocking density and feeding regimes on haematology, stress and histological recovery in stunted Lebeo rohita fingerlings reared in cage culture

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Abstract

Figures

Introduction

Materials And Methods

Experimental site and animal

Experimental design

Stocking of experimental animal and rearing

Sample collection and processing

Haematological parameters

Haemoglobin (Hb)

Red blood cell (RBC) and white blood cell (WBC) counts

Packed Cell Volume (PCV)

Serum glucose and cortisol

Lactate Dehydrogenase (LDH) activity)

Histology of gill and liver

Statistical analysis

Results

Haematological parameters

Serum stress parameters

Histological observations of gill and liver

Gills

Liver

Discussion

Conclusion

Declarations

References

Additional Declarations

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