In order to assess the effect of dietary pyridoxine supplementation on the growth performance of Nile tilapia and the haematological response under heat stress, 192 fingerlings (8.41 ± 0.22 g) were randomly distributed into eight tanks and fed practical diets supplemented with increasing levels of pyridoxine (0.0; 5.0; 10.0 and 20.0 mg of pyridoxal HCl kg⁻¹ diet) for 91 days. The fish were then weighed and the diet was quantified to determine the growth performance [weight gain (WG), feed intake, feed conversion ratio, protein efficiency rate, protein retention (PR) and survival percentage]. Haematological analyses (red blood cell count, haematocrit, haemoglobin, total leucocyte and differentiation, mean corpuscular volume, mean corpuscular haemoglobin concentration, albumin, globulin and albumin/globulin ratio) were carried out and then 32 fish were transferred and subjected to heat stress (32 °C) for 3 days, after which the haematological parameters were analysed. The fish fed the unsupplemented diet showed the lowest WG and PR. For the normal growth and health of the Nile tilapia, the Pyridoxine requirement in a practical diet is 10.0 mg of pyridoxine HCl kg⁻¹.

Introduction

Pyridoxine has been demonstrated to be an essential dietary nutrient for fish and it is related to growth and fish health. It participates in the protein and amino acid metabolism in the form of the prosthetic group of enzymes (Shiau & Wu 2003) and as a cofactor on erythropoiesis (Kaneko, Harvey & Bruss 1997). However, the relationship between pyridoxine and heat stress remains unknown.

Among other factors that can affect growth and fish health, water temperature is considered to be the most important. Fish generally experience stress, with the consequent suppression of the immune system when the temperature is inappropriate. This may lead to disease breakout, mainly when the temperature is chronically close to their maximum tolerance or fluctuates suddenly. To cope with stress, fish need to be nutritionally prepared to attend the demand and return to the normal physiological condition.

Among the nutrients required, vitamins are important especially those related to oxidative stress and erythropoiesis. Among complex B vitamins essential for erythropoiesis are pyridoxine, folate, riboflavine, niacin, tiamin and cyanocobalamin (Feldman, Zinkl & Jain 2000).

During erythropoiesis, the first step to synthesize haemoglobin is the aminolevulinic production from succinate (Krebs cycle) and glycine. At this stage, vitamin B₆ is required as a cofactor of ALA-synthetase (Kaneko et al. 1997). Pyridoxine requirements can explain the responsive anaemia in this vitamin deficiency. Thus, the absence or an inappropriate pyridoxine concentration may impair its synthesis, thus affecting fish health. Anaemia is described, according to Feldman et al. (2000), as the diminished oxygen-carrying capacity of blood, which may impair the metabolic capacity of tissues. Vitamin B₆ or pyridoxine is a hydrosoluble vitamin and its natural forms are pyridoxine, pyridoxal and pyridoximine. They are efficiently converted into pyridoxal phosphate, which is the active form for the synthesis, catabolism and interconversion of amino acids (Devlin 1998).

The quantitative requirement of pyridoxine has been determined in common carp (Cyprinus carpio) (5.4 mg kg⁻¹) (Ogino 1965), red seabream (Pagrus major) (5–6 mg kg⁻¹) (Takeda & Yone 1971), turbot (Psetta maxima) (1–2.5 mg kg⁻¹) (Adron, Knox & Cowey 1978), channel catfish (Ictalurus punctatus) (3 mg kg⁻¹) (Andrews & Murai 1979), gilthead seabream (Sparus aurata) (1.97 mg kg⁻¹) (Kissil, Cowey, Adron & Richards 1981), red hybrid tilapia Oreochromis mosambicus × Oreochromis niloticus (3 mg kg⁻¹) (Lim, Leamaster & Brock 1995), hybrid tilapia O. niloticus × Oreochromis aureus (10–15 mg kg⁻¹) (Shiau & Hsieh 1997) and Indian catfish (3.21 mg kg⁻¹) (Mohamed 2001).

The nutritional, physiological, physical and chemical changes caused by stress can be identified by the following blood parameters: red blood cells, white cells, total plasmatic protein (TPP), globulins, immunoglobulins, electrolytes and hormones. Abnormal erythropoiesis and leucopoiesis have been detected in both the absence of nutrients in diets and their inappropriate concentration (Barros, Pezzato, Falcon & Guimarães 2006). Because pyridoxine plays a role in protein synthesis, the first aim of this study was to evaluate the growth performance of Nile tilapia under dietary levels of pyridoxine. Moreover, considering its importance in red blood cell synthesis and, consequently, sufficient supply of oxygen to all tissues, especially under stress, the second purpose of this experiment was to analyse erythropoiesis, leucocyte synthesis and differentiation of fish exposed to heat stress.

Materials and methods

The experiment was carried out at the Faculdade de Medicina Veterinária e Zootecnia, Univ. Estadual Paulista, UNESP, Câmpus de Botucatu, DMNA, AquaNutri, Brasil.

Fish, feeding and experimental diets

A basal practical diet was formulated to contain approximately 32% crude protein and 18 000 kJ kg⁻¹ digestible energy based on the feedstuff values reported by Furuya, Pezzato, Barros and Miranda (2001), Guimarães, Pezzato and Barros (2008) and Pezzato, Miranda, Barros, Pinto, Furuya and Pezzato (2002) (Table 1). The vitamin premix did not contain pyridoxine hydrochloride. The nutrient levels tested were 0.0, 5.0, 10.0 and 20.0 mg pyridoxine HCl kg⁻¹ using 96% concentrate as a pyridoxine source.

Table 1. Percentage composition of the reference diet

Ingredient	g kg⁻¹
Soybean meal	622
Corn starch	40
Rice	240
Fish oil	10
Soybean oil	10
Cellulose	7
Methionine	4.5
Threonine	2.5
Dicalcium phosphate	59.9
Vitamin C (35% of ascorbic acid)	0.4
Salt	1
Vitamin B⁶-free vitamin mix^*	1
Trace mineral mix†	1
BHT‡	0.2
Total	1000

^* Vitamin premix supplied the following (IU or mg kg⁻¹ diet): vitamin B6-free, vitamin A, 16060; vitamin D3, 4510; vitamin E, 250; vitamin K (menadione sodium bisulphate), 30; thiamine, 32; riboflavin, 32; Ca-D-panthotenate, 80; niacin, 170; biotin, 10; folic acid, 10; cyanocobalamin, 0.032.
† Mineral premix supplied the following (mg kg⁻¹): Na2SeO3, 0.7; MnO, 50; ZnO, 150; FeSO4, 150; CuSO4, 20; CoSO4, 0.5; I2Ca, 1.0.
‡ Antioxidant: butylated hydroxytoluene.

Diets were mechanically mixed with water (25% of dry weight) in a KitchenAid multi-function mixer (Ação Científica, Piracicaba, Brazil) and extruded at 99–120 °C in a single-screw laboratory extrusion (Extrutec, Ribeirão Preto, Brazil) with the capacity to process 20 kg h⁻¹ of feed and excited in a 1 mm die aiming to obtain a 5.0 mm pellet diameter. Diets were air dried and stored at 18 °C until further use. The chemical composition of the experimental diets was estimated according to AOAC protocols (1995); the gross energy was measured using an adiabatic calorimeter bomb (Parr Instrument Company, Moline, IL, USA). The analysis of vitamin B₆ for the four experimental diets (Table 2) was performed in liquid chromatography (HPLC – reverse phase), according to Leenheer, Lambert and De Ruyter (1985). Samples from 1.0 to 2.0 g were finely ground, dissolved in 2% acetic acid and extracted with the aid of an ultrasonic bath for 20 min and, after cooling, were filtered in filter millex (0.45 mm). The chromatographic conditions were as follows: column ODS (C18−250 × 4.6 mm), 25 °C and detector UV 270 nm. The detection level was approximately 10 ppm and the recovery efficiency was 90.0%. At each feeding, the diet was offered two or three times until apparent satiation was reached.

Table 2. Proximate composition and pyridoxine concentration (B₆) of the experimental diets (dry matter basis)

	0.0	5.0	10.0	20.0
	Treatment (mg kg⁻¹ B₆ supplementation)
Dry matter (%)	7	7.3	8	7.9
Crude protein (%)	37.3	37.3	38.2	37.9
Crude energy (kjkg⁻¹)	18 480	18 778	18 946	19 251
B₆ mg kg⁻¹ (analysed)	ND^*	5	11	22

^* ND, not detected.

Growth performance

One hundred and ninety-two male Nile tilapia fingerlings, with an initial weight of 8.41 ± 0.22 g, were randomly assigned 24 per tank in duplicate. The tanks were supplied by a water filter system consisting of a biofilter and a thermostatically controlled heating system that maintained the temperature at 26.0 ± 1.0 °C. Each tank was considered a replicate for the growth performance trial. The physical and chemical characteristics of the water, such as pH (7.0 ± 0.5), dissolved oxygen (7.05 ± 0.15 mg L⁻¹), total ammonia (0.11 ± 0.07 mg L⁻¹) and alkalinity (100.00 ± 10.00 mg CaCO₃ L⁻¹), remained within the species' tolerance limits throughout the experimental period (Boyd 1996). The tanks were cleaned every week. After 91 days, growth performance was evaluated by determining weight gain (WG), feed intake (FI), feed conversion ratio (FCR), protein efficiency rate (PER), protein retention (PR) and survival percentage (SP).

Haematological assay

The haematological parameters were evaluated by randomly removing four fish per tank, eight per treatment, after 91 days i.e., the period before heat-induced stress. The fish were first anaesthetized with 1.5 g benzocaine in 15 L water, and after complete desensitization, blood was collected from the caudal vein using a tuberculin syringe, rinsed with an anticoagulant (3% EDTA) and then fish were sacrificed. Red blood cell and leucocyte counts were determined by dilution and enumeration using a haemocytometer. Leucocyte differentiation was performed in blood extension stained with the May–Grünwald–Giemsa–Wright stain according to Jain (1986). Differential counting was performed under a microscope at × 100 in immersion oil. Two hundred cells were counted to establish percentages for each cellular component of interest. Haemoglobin (Hb) was determined by the cyanometahemoglobin colorimetric method using a commercial kit (Gold Analisa Diagnóstica, Belo Horizonte, MG, Brazil) according to Collier (1944). The haematocrit (Ht) percentage was determined using the microhaematocrit method described by Goldenfarb, Bowyer, Hall and Brosious (1971). The total plasma protein (TPP) was measured using a manual Goldberg refractometer by breaking the microhaematocrit capillary just above the leucocyte layer after the Ht reading (Feldman et al. 2000). The mean corpuscular volume [MCV=(Ht × 10)/erythrocytes] and the mean corpuscular haemoglobin concentration [MCHC=(Hb × Ht) × 100] were calculated according to Wintrobe (1934).

The albumin concentration (ALB) was determined by the bromocresol method using the commercial kit Labtest Diagnóstica S.A.(Lagoa Santa, Minas Gerais, Brazil) for colorimetric determination. The albumin:globulin ratio (A/G) was determined using ALB and TPP values (Globulin=TPP−ALB; A/G=ALB/Globulin).

Heat stress

After haematological evaluation, fish remained in the same experimental system (eight tanks) and were then transferred to the challenge room. The experimental challenge room contained 16 40 L-plastic tanks with individual filters and aeration, as well as a heating system to increase the water temperature. Thirty-two fish were randomly distributed at two fish per tank (eight replicates per treatment). Fish were transferred at 25 °C and the temperature was increased every 2 h by 2 °C until it reached 32 °C. At each feeding, the diet was offered two or three times until apparent satiation was reached. At the end of 3 days, the same haematological parameters described previously were evaluated.

Statistical analysis

Data of growth performance were analysed by one-way analysis of variance (anova) using the general linear model (GLM), subsequent regression analysis. Data of haematological parameters were analysed by two-way analysis of variance (anova) using the GLM to test the effects of the dietary levels of vitamin B₆ and time points of heat stress and their interactions. If there was a significant F-test and normality, subsequent comparisons of treatment means were performed using Bonferroni's Multiple Range test. However, if there was a significant F-test and no normality, subsequent comparisons of the treatment means were performed using Dunn's Multiple Range test. Differences were considered to be significant at the 0.05 probability level. All analyses were performed using the sas (2002) statistical software program.

Results

Table 3 shows the data of WG, FI, FCR, PER, PR and SP. Supplementation of pyridoxine HCl did not influence SP. WG, FI, PER and PR varied quadratically (P<0.001), increasing up to the level of 9.696, 9.234, 9.709 and 14.322 mg pyridoxine HCl kg⁻¹ diet respectively. The FCR varied quadratically (P<0.001), decreasing pyridoxine to a level of 9.589 mg pyridoxine HCl kg⁻¹ diet.

Table 3. Means and standard deviations of weight gain (WG), feed intake (FI), feed conversion ratio (FCR), protein efficiency ratio (PER), protein retention (PR) and survival percentage (SP), of Nile tilapia fingerlings fed diets supplemented with levels of pyridoxine over a 91-day feeding trial

Variable	Vitamin B₆ (mg kg⁻¹)				P-value
Variable	0.0	5.0	10.0	20.0	P-value
WG (g)^*	102.43 (± 7.52)	132.95 (± 18.01)	145.09 (± 16.63)	119.85 (± 27.48)	0.0014
FI (g)^*	150.04 (± 1.88)	162.99 (± 7,15)	160.77 (± 7.08)	150.19 (± 4.82)	0.0001
FCR (g g⁻¹)^*	1.47 (± 0.13)	1.19 (± 0.13)	1.25 (± 0.17)	1.32 (± 0.31)	0.0242
PER (g g⁻¹)^*	1.96 (± 0.16)	2.44 (± 0.29)	2.57 (± 0.34)	2.28 (± 0.50)	0.0205
PR (%)^*	38.75 (± 1.32)	45.88 (± 1.65)	51.02 (± 1.68)	49.79 (± 1.19)	0.0001
SUR (%)	95.23 (± 8.15)	100.0	97.61 (± 6.31)	97.91 (± 5.90)	0.5281

Data are presented as mean ± SD.
^* Quadratic effect: WG=102.55+11.36x−0.586x², R²=0.84; FCR=1.4915−0.09685x+0.00505x², R²=0.72; FI=152.06+2.29x−0.124x², R²=0.72; PER=1.949+0.17146x−0.00883x², R²=0.78; PR=38.599+1.86225x−0.06501x², R²=0.93.

Table 4 shows the haematological data, such as RBC, Ht, Hb, MCV and MCHC, before and after the heat stress. The pyridoxine supplementation did not affect the erythrocyte count before stress was applied. After the heat stress, the Ht was higher, although 20 mg of pyridoxine HCl kg⁻¹ was the lowest value. Dietary pyridoxine supplementation affected MCHC, but did not affect Hb and MCV.

Table 4. Means and standard deviations of erythrocytes (RBC), haematocrit (Htc), haemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) of Nile tilapia fingerlings fed diets supplemented with levels of pyridoxine supplementation for 91 days and exposed to temperature stress^*

Variable	Moment	Vitamin B₆ (mg kg⁻¹)
Variable	Moment	0.0	5.0	10.0	20.0
RBC (10⁶ μL⁻¹)	Before	2.23 aA	2.30 aA	2.16 aA	2.23 aA
	Before	(± 0.26)	(± 0.38)	(± 0.22)	(± 0.33)
	After	2.32 aA	2.18 aA	2.23 aA	2.02 aA
	After	(± 0.27)	(± 0.40)	(± 0.33)	(± 0.36)
Ht (%)	Before	30.00 aA	31.31 aA	30.86 aA	30.00 aA
	Before	(± 4.67)	(± 3.54)	(± 3.08)	(± 3.15)
	After	31.93 abA	32.38 abA	34.36 bA	29.75 aA
	After	(± 2.47)	(± 3.55)	(± 6.35)	(± 6.16)
Hb (g dL⁻¹)	Before	6.76 aA	7.04 aA	7.52 aA	7.15 aA
	Before	(± 1.25)	(± 0.67)	(± 0.87)	(± 0.64)
	After	7.49 aA	7.48 aA	7.74 aA	6.94 aA
	After	(± 0.66)	(± 0.74)	(± 1.11)	(± 1.79)
MVC (fL)	Before	134.45 aA	138.15 aA	143.54 aA	139.03 aA
	Before	(± 18.20)	(± 15.27)	(± 11.11)	(± 9.85)
	After	138.56 aA	152.02 aA	156.02 aA	149.51 aA
	After	(± 11.00)	(± 29.20)	(± 32.58)	(± 29.83)
MCHC (%)	Before	22.28 aA	22.57 abA	24.37 bA	23.9 abA
	Before	(± 1.98)	(± 1.83)	(± 1.41)	(1 ± 1.40)
	After	23.47 aA	23.19 aA	22.70 aA	23.12 aA
	After	(± 1.06)	(± 1.50)	(± 2.14)	(± 1.85)

^* Small letter: comparison among treatments with moments fixed; capital letter: comparison between moments with treatments fixed.

The dietary vitamin B₆ supplementation did not affect the TPP, albumin (ALB), globulin (GLOB) and albumin/globulin ratio (A/G) before the heat stress application (Table 5). The dietary supplementation of 20.0 mg of pyridoxine HCl kg⁻¹ resulted in the lowest values of TPP and ALB after the heat stress. TPP was higher, after the heat stress, in fish fed the unsupplemented vitamin B₆ diet. Neither GLOB nor A/G was affected by the pyridoxine supplementation or by the heat stress.

Table 5. Means and standard deviations of total plasmatic protein (TPP), albumin (ALB), globulin (GLOB) and albumin/globulin ratio (A/G) of Nile tilapia fingerlings fed diets supplemented with levels of pyridoxine supplementation for 91 days and exposed to temperature stress^*

	Moment	0.0	5.0	10.0	20.0
	Moment	Vitamin B₆ (mg kg⁻¹)
TPP (mg dL⁻¹)	Before	2.93aA	2.89aA	2.96aA	2.71aA
	Before	(± 0.49)	(± 0.35)	(± 0.68)	(± 0.45)
	After	3.5bB	3.34abA	3.43abA	2.96aA
	After	(0 ± 0.46)	(± 0.40)	(± 0.45)	(± 0.59)
ALB (mg dL⁻¹)	Before	0.95aA	0.95aA	0.99aA	0.99aA
	Before	(± 0.27)	(± 0.13)	(± 0.13)	(± 0.20)
	After	1.06abA	1.04abA	1.25bA	0.85aA
	After	(± 0.17)	(± 0.26)	(± 0.47)	(± 0.21)
GLOB (mg dL⁻¹)	Before	1.98aA	1.94aA	1.97aA	1.72aA
	Before	(± 0.46)	(± 0.37)	(± 0.60)	(± 0.41)
	After	2.44aA	2.31aA	2.18aA	2.11aA
	After	(± 0.44)	(± 0.51)	(± 0.55)	(± 0.66)
A/G	Before	0.51aA	0.51aA	0.55aA	0.61aA
	Before	(± 0.21)	(± 0.11)	(± 0.21)	(± 0.22)
	After	0.45aA	0.49aA	0.67aA	0.46aA
	After	(± 0.11)	(± 0.25)	(± 0.52)	(± 0.26)

^* Small letter: comparison among treatments with moments fixed; capital letter: comparison between moments with treatments fixed.

Total leucocytes (Leuc), lymphocytes (Lymp), neutrophils (Neut) and monocytes (Mon) medians are presented in Table 6. The dietary pyridoxine supplementation did not affect Leuc, Lymp, Neut and Mon. After the heat stress, the fish fed a diet supplemented with 5.0 mg of pyridoxine HCl kg⁻¹ showed the lowest Neut and Mon values and those fed a diet supplemented with 20.0 mg of pyridoxine HCl kg⁻¹ showed the lowest Neut count.

Table 6. Medians on minimum and maximum values of leucocyte number (Leuc), lymphocyte number (Lymp), neutrophil number (Neut) and monocyte number (Mon) of Nile tilapia fingerlings fed diets supplemented with levels of pyridoxine supplementation for 91 days and exposed to temperature stress^*

Variable	Moment	Vitamin B₆ (mg kg⁻¹)
Variable	Moment	0.0	5.0	10.0	20.0
Leuc (104 cells μL⁻¹)	Before	24.15 aA	22.60 aA	19.98 aA	18.40 aA
	Before	(15.00–27.50)	(15.30–28.40)	(13.10–22.80)	(12.90–29.10)
	After	21.85 aA	23.45 aA	21.50 aA	22.55 aA
	After	(16.50–30.60)	(13.80–29.50)	(15.60–25.90)	(16.60–27.40)
Lymp (10⁴ cells μL⁻¹)	Before	21.47 aA	17.87 aA	18.54 aA	15.14 aA
	Before	(13.36–24.75)	(12.39–24.75)	(12.12–21.09)	(10.97–24.34)
	After	18.15aA	21.53 aA	20.25 aA	19.38 aA
	After	(13.69–28.92)	(12.28–28.18)	(11.54–20.98)	(15.60–24.66)
Neut (10⁴ cells μL⁻¹)	Before	1.75 aA	2.14 aB	1.18 aA	2.72 aB
	Before	(0.90–3.66)	(1.21–4.19)	(0.39–2.65)	(1.04–5.53)
	After	1.67 aA	1.23 aA	0.84 aA	1.23 aA
	After	(0.60–3.92)	(0.44–3.83)	(0.65–3.88)	(0.58–2.70)
Mon (10⁴ cells μL⁻¹)	Before	0.82 aA	1.14 aB	0.59 aA	0.57 aA
	Before	(0.24–2.22)	(0.12–1.86)	(0.26–1.57)	(0.09–2.91)
	After	0.66 aA	0.68 aA	0.65 aA	0.79 aA
	After	(0.46–1.57)	(0.22–0.88)	(0.11–1.43)	(0.19–1.76)

^* Small letter: comparison among treatments with moments fixed; capital letter: comparison between moments with treatments fixed.

Discussion

Fish exhibit limited glycogen metabolism and reserve; therefore, most of the glucose required for energy comes from protein (De Silva & Anderson 1995). The active form of B₆ vitamin, phosphate pyridoxal (PLP), is essential for energy production from amino acids, as it activates several transaminases and is considered to be the energy release vitamin as a co-enzyme of glycogen phosphorylase. This enzyme catalyses the first step of glycogen degradation, when inorganic phosphate (PLP) is necessary for α-1,4 glycoside ligation, producing glucose 1-phosphate (Devlin 1998).

Growth performance was affected by the lack of pyridoxine. The inappropriate growth may be due to compromised amino acid metabolism and glycogen catalysis (Combs 1998). However, the vitamin B₆ dietary supplementation resulted in better carcass PR. A similar result has also been reported for hybrid tilapia by Shiau and Hsieh (1997).

A low survival rate has been reported in fish fed a diet lacking vitamin B₆ (Andrews & Murai 1979; Mohamed 2001; Huang, Tian, Yang & Liu 2006). Pyridoxine deficiency in animals leads to behavioural changes and other nervous system disorders (Schaeffer 1987; Baxter 2003). Although pyridoxine HCl was not identified by chemical analysis in the unsupplemented diets, there was no significant mortality and the fish did not show any of these abnormalities. This may lead to a conclusion that 91 days may not have been sufficient to cause these disorders.

Haematological analysis is a useful tool to better understand the influence of nutritional and environmental conditions on fish health (Ranzani-Paiva & Silva-Souza 2004). The haematological characteristics of healthy fish vary according to internal and external factors. In this study, the haematological parameters were normal for healthy fish, as described by Feldman et al. (2000) for Nile tilapia and also by Barros, Ranzani-Paiva, Pezzato, Falcon and Guimarães (2009), for the same species in the same experimental facilities.

A significant RBC reduction was reported for Chinook salmon (Halver 1957), Coho salmon (Smith 1967), rainbow trout (Smith, Brin & Halver 1974) and Heteropneustes fossilis (Mohamed 2001) fed diets that were not supplemented with pyridoxine. Above normal Ht values for healthy fish were observed in this study, as well as for hybrid red tilapia fed unsupplemented vitamin B₆ diets (Lim et al. 1995). These results corroborate the idea that haematopoietic functions depend on the appropriate pyridoxine level (Feldman et al. 2000).

HPLC analyses carried out on the unsupplemented pyridoxine diet did not detect this vitamin. However, the haematological profile of fish fed such a diet was within the normal parameters for healthy Nile tilapia. This may be explained by the length of the experiment (91 days), which might have been insufficient for an accurate clinical and laboratorial diagnosis of anaemia caused by the absence of vitamin B₆.

The high temperature did not determine the release of immature cells (MCV), demonstrating that even under stress conditions, fish continue producing normal-size cells, according to Weiss and Wardrop (2010). MCHC depends on the Hb and Ht values, showing the same tendency. The antibody molecules (immunoglobulin) are glycoproteins that consist of four polypeptides chains, two different types, with two identical copies each. In the most common type, IgG, the H chains have approximately 440 amino acids and the shortest one has around 220 (Devlin 1998). Some amino acids, such as cystine, serine, threonine and methionine (transmethilation), exhibit PLP as an enzymatic cofactor and consequently supply the specific immune system of pyridoxine-deficient animals (Combs 1998). The water temperature variation is considered to be one of the most stressful factors to fish and may increase susceptibility to opportunistic pathogens. In this study, although leucocytes were not influenced by pyridoxine HCl levels, there was a slight decrease in the fish fed the unsupplemented diet after inducing the heat stress. The innate (non-specific) immunity in fish is the first line of defence against pathogen exposure, including neutrophils and macrophages (Tizard 2002).

Fish resistance compromised under stress conditions has been evaluated using plasmatic protein. TPP represents the albumin and globulin fraction present in plasma or serum that is produced in the liver. Albumin, the most abundant, is responsible for nutrient transportation and osmotic balance in the blood, and globulin is involved in the defence system. In monogastric animals, the protein restriction can cause hyperproteinaemia and hypoalbuminaemia, but the globulin concentration does not normally show alterations (Thomas 2000). Significant changes in TPP and ALB, after heat stress, may explain possible stress conditions. If the heat stress lasted for more than three days, this could probably be confirmed in the leucogram, which showed a tendency to increase in the white blood cells after stress.

In sum, dietary supplementation of pyridoxine at 10.0 mg pyridoxine HCl kg⁻¹ diet optimizes growth performance and reduces heat stress.

Acknowledgments

The authors are grateful to the São Paulo State Research Support Foundation – FAPESP, for financial support.

References

AdronJ.W., KnoxD. & CoweyC.B. (1978) Studies on the nutrition of marine flatfish: the pyridoxine requirement of turbot (Scophthalmus maximus). British Journal of Nutrition 40, 261–268.
10.1079/BJN19780121
CAS PubMed Web of Science® Google Scholar
AndrewsJ.W. & MuraiT. (1979) Pyridoxine requirements channel catfish. Journal of Nutrition 109, 533–537.
10.1093/jn/109.4.533
CAS PubMed Web of Science® Google Scholar
Association of Official Agricultural Chemists (AOAC). (1995) Official Methods of Analysis. Association of Official Agricultural Chemists (AOAC), Washington, DC, USA.
Google Scholar
BarrosM.M., PezzatoL.E., FalconD.R. & GuimarãesI.G. (2006) Nutrição e saúde de peixes. In: II Colégio Latino-Americano de Nutrição Animal. Palestra técnica em Manejo e Nutrição de Organismos Aquáticos (ed. by M.M. Barros & L.E. Pezzato), pp. 1–15. CBNA, Campinas-SP, Brazil.
Web of Science® Google Scholar
BarrosM.M., Ranzani-PaivaM.J.T., PezzatoL.E., FalconD.R. & GuimarãesI.G. (2009) Hematological response and growth performance of Nile tilapia fed diets containing folic acid. Aquaculture Research 40, 895–903.
10.1111/j.1365-2109.2009.02175.x
CAS Web of Science® Google Scholar
BaxterP. (2003) Pyridoxine-dependent seizures: a clinical and biochemical conundrum. Biochimical et Biophysica Acta 1647, 36–41.
10.1016/S1570-9639(03)00045-1
CAS PubMed Web of Science® Google Scholar
BoydC.E. (1996) Water Quality in Ponds for Aquaculture. Shrimp Mart, Songkhla, Thailand.
Google Scholar
CollierH.B. (1944) The standardization of blood haemoglobin determinations. Canadian Medical Association Journal 50, 550–552.
CAS PubMed Google Scholar
CombsG.F. (1998) The Vitamins: Fundamental Aspects in Nutrition and Health. Academic Press, New York, NY, USA.
Google Scholar
De SilvaS.S. & AndersonT.A. (1995) Fish Nutrition in Aquaculture. Aquaculture Series 1. Chapman & Hall, London, UK.
Google Scholar
DevlinT.M. (1998) Manual de Bioquímica com Correlações Clínicas. Edgard Blücher, S ã o Paulo, Brazil.
Google Scholar
FeldmanB.F., ZinklJ.G. & JainN.C. (2000) Schalm's Veterinaty Hematology, 5th edn. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
Google Scholar
FuruyaW.M., PezzatoL.E., PezzatoA.C., BarrosM.M. & MirandaE.C. (2001) Coeficientes de digestibilidade e valores de aminoácidos digestíveis de alguns ingredientes pela tilápia do Nilo (Oreochromis niloticus). Revista da Sociedade Brasileira de Zootecnia 30, 1125–1131.
10.1590/S1516-35982001000500002
Google Scholar
GoldenfarbP.B., BowyerF.P., HallE. & BrosiousE. (1971) Reproducibility in the hematology laboratory: the microhematocrit determination. American Journal of Clinical Pathology 56, 35–39.
10.1093/ajcp/56.1.35
CAS PubMed Web of Science® Google Scholar
GuimarãesI.G., PezzatoL.E. & BarrosM.M. (2008) Amino acid availability and protein digestibility of several protein sources for Nile tilapia, Oreochromis niloticus. Aquaculture Nutrition 14, 396–404.
10.1111/j.1365-2095.2007.00540.x
CAS Web of Science® Google Scholar
HalverJ.E. (1957) Nutrition of salmonoids fishes III: water-soluble vitamin requirements of Chinook salmon. Journal of Nutrition 62, 225–243.
CAS PubMed Web of Science® Google Scholar
HuangJ.W., TianZ.Y., YangH.J. & LiuY.J. (2006) Pyridoxine deficiency of grouper, Epinephelus coioides: physiological and biochemical alteration. Fish Physiology and Biochemistry 31, 331–337.
10.1007/s10695-005-2832-2
CAS Google Scholar
JainN.C. (1986) Schalm's Veterinary Haematology, 4th edn. Lea and Febiger, Philadelphia, PA, USA.
Google Scholar
KanekoJ.J., HarveyJ.W. & BrussM.L. (1997) Clinical Biochemistry of Domestic Animals, 5th edn. Academic Press, San Diego, CA, USA.
Google Scholar
KissilG.W., CoweyC.B., AdronJ.W. & RichardsR.H. (1981) Pyridoxine requirements of the gilthead bream, Spatrus aurata. Aquaculture 23, 243–255.
10.1016/0044-8486(81)90018-1
Web of Science® Google Scholar
LeenheerA.P., LambertW.E. & UyterM.G. (1985) Modern Chromatographic Analysis of the Vitamins, Chromatographic Science Series, 2nd edn, Vol. 30. Marcel Dekker, New York, NY, USA.
Google Scholar
LimC., LeamasterB.R. & BrockJ.A. (1995) Pyridoxine requirement of fingerling red hybrid tilapia grown in seawater. Journal of Applied Aquaculture 5, 49–60.
10.1300/J028v05n02_05
Google Scholar
MohamedJ.S. (2001) Dietary pyridoxine requirement of the Indian catfish, Heteropneustes fossilis. Aquaculture 194, 327–335.
10.1016/S0044-8486(00)00510-X
Web of Science® Google Scholar
OginoC.B. (1965) Vitamin requirements of carp, Cyprinus carpio: deficiency symptoms and requirements of vitamin B₆. Bulletin of the Japanese Society for the Science of Fish 31, 46–51.
10.2331/suisan.31.546
Google Scholar
PezzatoL.E., MirandaE.C., BarrosM.M., PintoL.G.Q., FuruyaW.M. & PezzatoA.C. (2002) Digestibilidade aparente de ingredientes pela tilápia do Nilo (Oreochromis niloticus). Revista Brasileira de Zootecnia 31, 1595–1604.
10.1590/S1516-35982002000700001
Web of Science® Google Scholar
Ranzani-PaivaM.J. & Silva-SouzaA.T. (2004) Hematologia de Peixes Brasileiros. In: Sanidade de Organismos Aquáticos (ed. by M.J. Ranzani-Paiva, R.M. Takemoto & M.A.P. Lizama), pp. 89–120. Varela Publishing, S ã o Paulo, Brazil.
Google Scholar
SchaefferM.C. (1987) Attenuation acoustic and tactile startles responses of vitamin B₆ deficient rats. Physiology Behavior 40, 473–478.
10.1016/0031-9384(87)90032-1
CAS PubMed Web of Science® Google Scholar
ShiauS.Y. & HsiehH.L. (1997) Vitamin B₆ requirements of tilapia Oreochromis niloticus×O. aureus fed two dietary protein concentrations. Fisheries Science 63, 1002–1007.
10.2331/fishsci.63.1002
CAS Web of Science® Google Scholar
ShiauS.Y. & WuM.H. (2003) Dietary vitamin B₆ requirement of grass shrimp, Penaeus monodon. Aquaculture 225, 397–404.
10.1016/S0044-8486(03)00304-1
CAS Web of Science® Google Scholar
SmithC.E. (1967) Progress in sport fishery research. Washington, DC, U.S. Fish Wildlife Service. Bureau Sport Fish, Wildlife Report, 54pp
Google Scholar
SmithC.E., BrinM. & HalverJ.E. (1974) Biochemical, physiological and pathological changes in pyridoxine-deficient rainbow trout (Salmo gairdneri). Journal Fisheries Research Board Canada 31, 1893–1898.
10.1139/f74-247
CAS Web of Science® Google Scholar
TakedaT. & YoneY. (1971) Studies of nutrition of red seabream. 11. Comparison of vitamin B₆ requirement level between fish fed a synthetic diet and fish fed beef liver during prefeeding period. Report of the Fisheries Research Laboratory 1, 37–47.
Google Scholar
ThomasJ.S. (2000) Overview of plasma proteins. In: Schalm's Veterinary Hematology (ed. by B.F. Feldman, J.G. Zinkl & N.C. Jain), pp. 891–898. Lippincott Williams &Wilkins, Philadelphia, PA, USA.
Google Scholar
TizardI.R. (2002) Imunologia Veterinária: Uma Introdução, 6th edn. Roca, S ã o Paulo, Brazil.
Google Scholar
WeissD.J. & WardropK.J. (2010) Schalm's Veterinary Haematology, 6th edn. Blackwell Publishing, Iowa, USA.
Google Scholar
WintrobeM.M. (1934) Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Folia Hematologica 51, 32–49.
Google Scholar

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Growth performance of Nile tilapia, Oreochromis niloticus, fed diets containing levels of pyridoxine and haematological response under heat stress

Abstract

Introduction

Materials and methods

Fish, feeding and experimental diets

Growth performance

Haematological assay

Heat stress

Statistical analysis

Results

Discussion

Acknowledgments

References

Citing Literature

References

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Growth performance of Nile tilapia, Oreochromis niloticus, fed diets containing levels of pyridoxine and haematological response under heat stress

Abstract

Introduction

Materials and methods

Fish, feeding and experimental diets

Growth performance

Haematological assay

Heat stress

Statistical analysis

Results

Discussion

Acknowledgments

References

Citing Literature

References

Related

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