Effects of feeding diets supplemented with vitamin E and vitamin C on performance, egg quality and stereological and structural analysis of the liver of laying hens exposed to heat stress

Abstract

This study was conducted to determine the effects of vitamin E and vitamin C on performance, egg quality and histopathological effect on the liver tissue of laying hens exposed to heat stress. A total of 256 lohman LSL laying hens were randomly assigned to 16 treatment groups, four replicates of 4 birds each. The birds with a 2×2×4 factorial design kept at normal (20°C) or heat stress (30°C) and either received either two levels of vitamin C (0 and 100 mg/L of drinking water) or four levels of vitamin E (0, 45, 65 or 85 U/kg of diet) for 11 weeks, in which one week was allowed for an acclimation period. Heat stress caused significant (P<0.05) decrement in the average feed intake, egg yields, egg shell thickness and Haugh unit. Supplementation of vitamin E increased significantly (P<0.01) the feed efficiency, egg yield and Haugh unit. The heat stress condition caused an increases in density of necrotic cell, but the area of parenchyma significantly decreased by heat stress in liver. Addition of 45 and 65 U vitamin E/kg to feeds significantly decreased density of necrotic cells. In coclusion, although supplementation of 85 U vitamin E increased feed conversion, Haugh unit and egg production, resulting in the highest degree of liver damage.Therefore, we thought that a supplementation of 65 IU of vitamin E/kg in diets appeared to be the most efficacious dose.

Key Words:

• Heat stress
• Feed efficiency
• Haugh unit
• Egg hell thickness
• Histopathology.

Introduction

It has been known that decreased feed intake negatively affects laying hens exposed to heat stress, which has a highly detrimental effect on egg production; both the number and weight of eggs were markedly decreased when the bird was exposed to high ambient temperatures (Smith, 1974). Feed intake, egg production, egg weight and shell quality were decreased in heat-stressed birds (Putpongsiriporn et al., 2001; Lin et al., 2002). Similarly, Daghir (1995) expressed that the decrease in independent Ca amount, indicating bad shell quality, was because of the decrease of feed intake caused by the high temperature stress. Although it is impossible to get rid of the negative effect of heat stress completely on laying performance, considerable attention has been paid to the role of nutrition in minimizing the effects of heat stress (Shane, 1988).

Vitamin E is the most active natural antioxidant used in animal feeding; it exhibits an antioxidant activity at low concentration and pro-oxidant activity at high concentration (Chen et al., 1998). It was speculated that vitamin E protects the liver from lipid peroxidation and prevents cell membranes from damage (Yu, 1994; Bollengier-Lee et al., 1998; 1999). It was also reported that vitamin E supplementation at high levels can improve the egg performance in hens exposed to heat stress (Bollengier-Lee et al., 1998). Moreover, Putpongsiriporn et al. (2001) found that the addition of vitamin E (25, 45 or 65 U/kg) to hen diets decreased the detrimental effects of heat stress in laying hens, and it increased the egg quality.

Vitamin C was reported to have improved the performance of poultry and to increase the egg production, to improve hatchability and fertility, and to reduce the egg breakage and mortality in a hot environment (Thornton and Moreng, 1959).

In recent years, design-based stereology has become the state-of-the-art methodology in quantitative histological analysis, which helps our understanding of the functional and pathological morphology of organs such as the liver (Sahin et al., 2003).

The objective of the present study is to investigate the effects of vitamin E (0, 45, 65 and 85 U/kg of diet) and vitamin C (0 and 100 mg/L) levels on performance, egg quality, stereological, morphometrical and histopathological analysis of the liver in laying hens exposed to heat stress.

Materials and methods

Animals and experimental procedure

The trial was conducted to investigate the interaction of vitamins E and C on performance, egg quality and histopathological effect on the liver tissue of laying hens exposed to heat stress. A total of 256, 24-week-old, Lohman LSL hybrid laying hens was used in the study. The hens were randomly assigned to 16 treatment groups, four replicates of four birds each. Eight tretment groups were kept at normal poultry-house conditions (20°C) and the eight tretment groups were exposed to heat stress (30°C). In both poultry houses, groups either received either two levels of vitamin C (0 and 100 mg/L of drinking water) or four levels of vitamin E (0, 45, 65 or 85 U/kg of diet). Ingredients and chemical compositions of the basal diet are shown in Table 1. Feeds were offered to birds for 11 weeks, in which one week was allowed for an acclimation period.

Table 1 Composition of experimental diets.

Ingredients	Amount, %	Calculated	Analysed
Corn	35	Dry matter, %	90	91
Wheat	17.5	Crude protein, %	16	17
Wheat bran	12.5	Crude cellulose, %	6	6.5
Soybean meal	17	Crude ash, %	12	12.2
Sunflower meal	5	Dissolved ash in HCL, %	1	-
Meat-bone meal	2.5	Lysine, %	0.7	-
Marble meal	7.5	Methionine, %	0.33	-
Limestone	2.4	Calcium, %	3.4	-
Salt	0.3	Phosphorus, %	0.7	-
Vitamin premix°	0.2	Salt, %	0.3	-
Mineral premix^#	0.1	ME, kcal/kg	2650

° Supplied per kilogram of diet: Vit. A, 6400 U; Vit. D, 1600 U; Vit. E, 30 U; Vit. K3, 3000 mg; Vit. B¹, 1500 mg; Vit. B², 4000 mg; Vit. B¹², 0.4 mg; Nicotinamide, 4.8 mg; Cal-D, 4.8 mg; Vit. B⁶, 2 mg; choline, 160 mg; D-Biyotin, 0.8 mg; Ca, 0.28 mg;

# supplied per kilogram of diet: Mn, 80 mg; Zn, 90 mg; Fe, 50 mg; Cu, 12 mg; Se, 1.5 mg; sodium chloride, 2.5 g. ME, metabolizable energy.

During the experiment, hens were provided with feed and water ad libitum. Egg production and feed consumption data were recorded daily. Feed conversion rate was expressed as kilogram feed intake per kilogram egg production. Eggs used for evaluation of yolks and albumen were broken and the weights of yolks, albumen, shell, shellstrength and shell thickness recorded. Haugh unit values were determined biweekly (n=8). Haugh unit was calculated with the following formula: Haugh unit = 100 log (H+7.57-1.7×W0.37), where H = albumen height (mm) and W = egg weight (g). At the end of the study, five birds from each treatment groups were anesthetized via short inhalation of ether, then slaughtered and their livers were removed.

Histological and morphometrical process

Firstly, liver weights were determined. Then, livers were put in 10% formalin for stereological and histopathological examination. Following, tissue samples were processed routinely and embedded in paraffin. Sections were cut at 5 µm, and stained with hæmatoxylin and eosin. Slides were covered and photographs taken using a light microscope with camera attachment (Nikon Eclipse E600, Japan). The size of the tissue from different areas of each liver was measured using an ocular micrometer in a light microscope (Pellegrino et al., 1979; Horobin, 1996).

Stereological method

Stereology is a number of mathematical and statistical methods that allows the evaluation of three-dimensional structural information from two-dimensional sections (or slices). This allows researchers to derive important quantitative and structural knowledge, such as the volume, surface area or numbers of particles (e.g., cells) within defined regional lines. We describe some of these methods as applied to quantifying the total number of cells in defined regions of the liver formation. Two methods were used for this evaluation. First, the physical disector method (Gundersen and Osterby, 1977; Sterio, 1984; Gundersen and Jensen, 1987; Howard and Reed, 1998) was used to estimate the numerical density of hepatocytes within each dµm3. Second, the Cavalieri principle was used to determine the area of the parenchyma and sinusoids of the hens’ livers (Gundersen and Jensen, 1987; Savaş et al., 2002; Sönmez et al., 2002).

Statistical analysis

Differences between groups were analyzed with analysis of variance (ANOVA) using the statistical package SPSS for Windows (SPSS 1999), version 10.0. Significant means were subjected to a multiple comparison test (Duncan) at α=0.01 and 0.05 levels (Snedecor and Cochran, 1980).

Results and discussion

Exposure of hens to high temperature resulted in a significant decrease in feed intake (16.5 g, P<0.01). On the other hand, feed intake did not change by supplemental vitamin E level in the present study. It was found that supplementation of vitamin C causes an increase in the feed intake. The interaction between heat x vitamin E was important with respect to feed intake (Table 2). Mashaly et al. (2004) reported that feed intake significantly decreased in laying hens exposed to heat stress.Controversial results were reported by some other researchers, such as the findings of Puthpongsiriporn et al. (2001) and Scheideler and Froning (1996), which were in accordance with our study. However, supplementation of dietary vitamin E (500 mg/kg) caused a significant increase (P<0.01) in feed intake of hens (Bollengier-Lee et al., 1998) and in Japanese quail (Şahin et al., 2002) exposed to heat stress. Bollengier-Lee et al. (1999) investigated the effects of different dietary concentrations of vitamin E (125, 250, 375 and 500 mg alpha-tocopherol/kg) on laying hens from 26 to 30 weeks of age exposed to chronic heat stress at 32°C. They reported that egg weight and food intake were similar in all the groups, but egg production was increased by dietary vitamin E levels.

Table 2 Effect of dietary vitamin E and vitamin C on performance and liver weight in laying hens exposed to heat stress.

Heat	Vitamin C	Vitamin E, U/kg	Feed intake, g	Feed conversionratio	Egg production, %	Liver weight, g	Liver weight, %
Normal (20°C)	0 mg/L vitamin C	0 E	2114.6±11.8	2.00±0.5	81.9±3.6	40.5±1.2	2.8±0.010
		45 E	119.3± 12.9	2.27±0.8	83.8±4.6	41.0±1.3	3±0.011
		65 E	116.3± 11.4	1.94±0.5	88.1±3.1	41.9±1.2	3±0.012
		85 E	116.5±12.9	1.88± 0.4	87.7±3.8	42.5±1.4	3.1±0.09
	100 mg/L vitamin C	0 E	118.6± 12.7	2.20±0.5	84.9±3.7	41.0±1.0	2.8 ± 0.04
		45 E	118.7± 11.8	2.09±0.4	86.9±2.7	41.0±1.2	2.9 ±0.08
		65 E	118.2± 11.1	2.01±0.3	87.9±3.8	42.4±1.3	3.1±0.010
		85 E	121.3± 12.2	1.89±0.5	90.6±3.3	43.0±1.2	3.2±0.012
Heat stress (30°C)	0 mg/L vitamin C	0 E	100.3±9.5	1.99±0.6	69.2±4.0	37.0±0.9	2.5±0.03
		45 E	100.3±8.2	2.05±0.7	67.8±3.4	37.5±1.0	2.6±0.05
		65 E	99.5±8.9	2.02±0.8	68.7±3.9	38.0±1.1	2.7±0.04
		85 E	98.3±10.9	1.97±0.7	78.4±2.8	39.0±1.2	2.8±0.07
	100 mg/L vitamin C	0 E	108.5± 21.7	2.16±0.9	70.2±3.0	38.0±1.4	2.6±0.02
		45 E	103.5±15.1	2.04±0.4	79.5±3.5	38.0±1.2	2.8 ±0.03
		65 E	102.6±15.7	2.13±0.7	68.3±2.7	39.1±1.2	2.8±0.02
		85 E	97.9±12.5	1.92±0.7	82.4±3.1	37.5±1.0	2.7±0.002
Heat		Normal	117.9±12.0	2.04±0.5	86.8±3.1	41.9±1.2	2.98±0.08
		Heat stress	101.4±13.6	2.04±0.7	72.9±3.3	38.0±1.1	2.68±0.09
		P	^**	ns	^**	^*	^**
Vitamin E, U/kg		0	110.5±16.1	2.09±0.5^a	76.5±2.9^c	39.1±0.9	2.67± 0.07^c
		45	110.5±18.4	2.11±0.7^a	79.5±3.8^b	39.3±1.1	2.82±0.010^b
		65	109.2±14.4	2.03±0.4^ab	78.5±4.3^bc	40.3±1.1	2.90±0.012^a
		85	108.5±16.0	1.92±0.7^b	84.4±3.1^a	40.5±1.4	2.93±0.012^a
		P	ns	^**	^**	ns	^**
Vitamin C, mg/L		0	108.2±13.7	2.02±0.7	78.2±2.9	39.7±1.3	2.80±0.009
		100	111.2±16.7	2.06±0.6	81.3±3.8	40.0±1.2	2.86±0.05
		P	^**	ns	ns	ns	^**
Heat x vitamin E			^**	^**	^**	ns	^**
Heat x vitamin C			ns	ns	ns	ns	ns
Vitamin E x vitamin C			ns	^**	^*	ns	ns

a,b,c Column means with no common superscript differ significantly;

* P<0.05,

** P<0.01; ns, not significant.

It was determined that feed conversion ratio had not changed significantly between the normal and heated groups. Dietary vitamin E level significantly improved feed conversion efficiency (P<0.01). The interaction between vitamin E x vitamin C was also important with respect to feed conversion (Table 2). The present findings were supported with data reported by Sell et al. (1997) and Çiftçi et al. (2005) However, Bollengier-Lee et al. (1998) and Şahin et al. (2002) reported that supplementation of vitamin E did not affect feed conversion in male turkeys.

Egg production was significantly decreased for hens exposed to heat temperature compared with in the normal temperature (Table 2). These findings are in agreement with those of Whitehead et al. (1998), Kirunda et al., (2001),and Mashaly et al. (2004) who reported that egg production in laying hens decreased when they were exposed to high environmental temperature. Supplementation of vitamin C and vitamin E significantly (P<0.01) improved egg production. The highest egg production was determined in birds fed 85 U vitamin E/kg. The interaction between heat x vitamin E and vitamin E x vitamin C was important with respect to egg production. Therefore, it may be said that supplementation of laying hen diets with a relatively high concentration of vitamin E (85 U/kg) reduced the detrimental effect of heat stress upon egg production, as previously reported by Bollengier-Lee et al. (1998, 1999), Puthpongsiriporn et al. (2001), Şahin et al. (2002), Mashaly et al. (2004) and Çiftçi et al. (2005).

Supplementation of vitamin E and vitamin C did not have any affect on liver weight, which was affected negatively by heat stress. Exposure to high temperature caused a significant (P<0.05) decrease (10%) in the liver weight of hens (Table 2).

Overall results are presented in Table 3. Egg weights were increased significantly (P<0.01) with the 45 and 85 U vitamin E supplementation and were decreased significantly (P<0.01) by heat stress and vitamin C. The interactions between heat x vitamin C and vitamin E x vitamin C were important with respect to egg weights. However, Keshavarz et al. (1995) reported that supplementation of vitamin C significantly increased egg weight in laying hens exposed to heat stress. Mashlay et al. (2004) reported that egg weight, shell weight, shell thickness, and specific gravity were significantly decreased when the birds were exposed to heat stress

Table 3 Effect of dietary vitamin E and vitamin C on egg quality in laying hens exposed to heat stress.

Heat	Vitamin C	Vitamin E	Egg weight, g	Egg white ratio, %	Egg yolk ratio, %	Egg shellratio, %	Egg shell thickness, µm	Specific gravity, gr/cm^3	Haugh unit
Normal (20°C)	0 mg/L vitamin C	0 E	62.2±3.9	61.5±2.2	26.1±2.3	12.4±1.7	357±27.5	1.10±0.05	86.76±5.01
		45 E	63.6±3.7	61.9±2.0	25.6±1.4	12.2±1.9	375±11.4	1.12±0.03	92.98±4.95
		65 E	63.3±3.1	61.0±2.5	27.3±2.1	11.9±1.9	353±25.7	1.61±0.03	92.40±5.52
		85 E	66.4±2.9	63.6±1.9	25.1±1.7	11.6±1.3	367±21.1	1.17±0.06	94.24±7.03
	100 mg/L vitamin c	0 E	61.4±3.7	61.3±3.4	25.5±2.2	13.1±3.2	372±13.9	1.10±0.03	87.00±9.1
		45 E	63.2±3.7	61.4±1.8	26.7±1.9	12.4±2.1	377±25.0	1.14±0.05	93.38±5.88
		65 E	59.6±3.3	59.6±1.5	27.4±2.0	13.0±2.7	367±21.3	1.15±0.04	93.20±6.10
		85 E	61.5±3.7	62.0±1.7	26.2±1.6	11.8±1.4	380±23.5	1.17±0.06	91.56±4.96
Heat stress (30°C)	0 mg/L vitamin C	0 E	54.9±2.5	63.0±2.0	25.5±1.9	10.4±1.9	345±26.9	1.10±0.03	86.06±6.0
		45 E	57.9±4.0	62.7±1.8	25.2±0.8	10.8±1.9	343±22.7	1.12±0.02	89.02±4.98
		65 E	56.2±2.9	62.5±2.4	26.4±2.6	9.8±1.9	342±32.6	1.12±0.05	88.59±7.60
		85 E	57.3±3.4	62.9±1.8	26.8±2.0	10.1±0.9	345±22.7	1.13±0.03	90.51±5.57
	100 mg/L vitamin c	0 E	55.4±3.5	62.2±1.1	25.7±1.1	12.2±1.5	342±18.6	1.12±0.04	85.42±6.91
		45 E	56.8±3.8	61.7±1.6	26.5±1.5	10.9±1.9	344±22.6	1.11±0.05	87.73±7.32
		65 E	57.2±3.7	63.0±2.5	25.4±1.8	10.5±1.9	345±22.1	1.12±0.04	90.82±4.92
		85 E	55.4±3.3	63.0±1.6	25.8±1.5	10.1±1.5	336±18.8	1.09±0.05	90.43±6.66
Heat		Normal	62.7±3.9	61.6±2.4	26.2 ±3.0	12.3±2.3	369±23.1	1.20±0.05	91.44±9.17
		Heat stress	56.4±3.4	62.6±1.9	25.9 ±1.8	10.6±1.8	343±25.5	1.13±0.04	88.57±6.40
		P	^**	^*	ns	^**	^**	ns	^*
Vitamin E, U/kg		0	58.5±4.8^b	62.0±2.4^b	25.7±1.9^b	12.0±2.3^a	355±25.7	1.10±0.04	85.31±10.8^b
		45	60.4±4.9^a	61.9±1.8^b	26.0±1.7^b	11.6±2.1^ab	360±25.6	1.12±0.04	90.78±6.2^a
		65	59.1±4.2^b	61.5±2.6^b	26.6±2.2^a	11.3±2.4^bc	352±29.4	1.13±0.04	91.25±6.36^a
		85	60.2±5.4^a	62.9±1.8^a	25.9±1.9^b	10.9±1.5^c	358±28.1	1.14±0.06	91.68±6.2^a
		P	^**	^*	^**	^**	ns	ns	^**
Vitamin C, mg/L		0	60.2±5.1	62.4±2.2	26.0±2.0	11.1±1.9	354±27.7	1.19±0.04	90.07±6.4
		100	58.8±4.5	61.8±2.2	26.1±1.8	11.8±2.3	358±26.5	1.13±0.05	89.94±9.4
		P	^**	^*	ns	^*	ns	ns	ns
Heat x vitamin E			ns	^**	^**	ns	ns	ns	ns
Heat x vitamin C			^**	ns	ns	ns	^*	ns	ns
Vitamin E x vitamin C			^*	ns	^**	^*	ns	ns	ns

a,b,c Column means with no common superscript differ significantly;

* P<0.05,

** P<0.01; ns, not significant.

Egg white ratio and egg yolk ratio were not negatively influenced by the heat stress. Supplementation of vitamin C did not have any affect on egg yolk ratio. Egg white ratio was increased significantly (P<0.05) with the 85 U vitamin E supplementation and was decreased significantly (P<0.05) by vitamin C. The interaction between vitamin E x vitamin C was important with respect to ratio of egg shell and yolk. However, Puthpongsiriporn et al. (2001) reported that supplementation of vitamin E did not affect ratio of egg white, yolk and shell in chickens. Ajakaiye et al. (2011) reported that egg weight, weight of albumen, yolk and shell significantly increased supplementation of vitamin E and C when the birds were exposed to heat stress

The heat stress condition caused reductions in egg shell ratio (13.8%) compared to the normal condition. There was a tendency of greater decrease in egg shell ratio for chickens fed 85 IU vitamin E/kg than for chickens fed basal diet. But, egg shell ratio increased by vitamin C. Egg shell thickness was decreased significantly (7.1%, P<0.01) by heat stress. Since plasma calcium level was significantly decreased by the exposure to high temperatures in laying hens (Mahmoud et al., 1996), the decrease in shell quality in the present study may be partially due to a reduction in plasma calcium. There were no an effect of the vitamin C and vitamin E supplementation on the egg shell thickness. However, Şahin et al. (2002) reported that supplementation of vitamin E significantly increased egg shell thickness in Japanese quail. The interaction between heat x vitamin C was important with respect to egg shell thickness.

The heat stres did not change specific gravity. The specific gravity value was not influenced significantly by dietary vitamin C and vitamin E (P>0.05). Conversely, Şahin et al. (2002) showed that supplementation of vitamin E significantly increased specific gravity in Japan quail.

Haugh unit, a measurement made to determine albumen quality, was calculated from the height of the thick albumen and the weight of the intact egg (Amer, 1972). Vitamin C had no significant influence on Haugh unit values in this study. Heat stress had significant influence on Haugh unit values, which decreased significantly (P<0.05) in this study. Similarly, Kirunda et al. (2001) reported that Haugh units of eggs from heat-stressed birds were reduced after heat exposure. Haugh unit values were increased significantly by increasing dietary vitamin E levels at present study. These results were similar to data reported by Puthpongsiriporn et al. (2001). However, Şahin et al. (2002) and Çiftçi et al. (2005) reported that vitamin E did not affect Haugh Units in Japanese quail and laying hens.

The physical disector was developed as an unbiased and efficient stereological method to estimate the cell number in a region. This tool gives a reliable estimation of particle number and size in an anatomically defined area (Sterio, 1984; Gundersen et al., 1988). Therefore, the quantitative analyses of hepatocytes provided us very significant findings in evaluation of cell proliferation, dimensional changes and degeneration. In the second step, Cavalieri principle was used for area estimating (Gundersen and Jensen, 1987). According to this principle, area or volume of an object was estimated by cutting it into equally spaced sections with thickness. The position of the first section must be uniformly random in length. In our study, serial liver sections were obtained and used for this purpose.

In the histopathological section of the study, routine histological method was performed on all liver samples to discern different tissue particles. All slides were observed under a light microscope. At first, all slides were stained with haematoxyline-eosin to define routine histological structures. In normal temperature groups, the central vein (the initial branch of hepatic vein) occupied the longitudinal axis of each classical lobule (FIgure 1 A).

Figure 1 A, C, Healthy liver sections obtained from normal temperature groups. B, D, liver sections were obtained from heat stressed groups. It was shown that sinusoidal dilatation (enlarged spaces). Magnification: 200x.

Hepatocytes radiate, like spokes of a wheel, from the central vein, forming anatomizing, fenestrated plates of liver cells, separated from

each other by large vascular spaces known as hepatic sinusoids (FIgure 1 A). Resident macrophages, known as Kupffer cells, were associated with the sinusoidal lining cells in the sinusoids (FIgure 1 C). In all of the sinusoids of the heat stressed groups central veins and branches of portal vein were dilated (FIgure 1 B,D). Significant mononuclear cell infiltration was observed both between the cell plates and around the dilated vessels (FIgure 2 B). Also in heat stressed groups, some hepatocytes were more acidophilic than normal heat groups and there was cytoplasmic shrinkage and more dark and small nuclei in the acidophilic hepatocytes (FIgure 2 B,C,D). In heat stressed groups’ slides, more lipid drops were defined in a pattern, increasing from the first to the fourth treatment groups (FIgure 3 B,C,D). Also peri-cellular connective tissue arising (fibrosis) was found (FIgure 3 A).

Figure 2 Liver sections obtained from heat stressed groups. A, shows fibrillous connective tissue arising in peri-cellular areas. It was shown sinusoidal dilatation (enlarged spaces) and mononuclear inflammation in B. Also in B, C, D there was cytoplasmic shrinkage and more dark and small nuclei in the acidophilic hepatocytes Magnification: 200x.

Figure 3 A, liver section from normal temperature groups. B, C, D, liver sections obtained from heat stressed groups. A, normal, healthy cellular morphology. It was shown sinusoidal dilatation (enlarged spaces) and mononuclear inflammation in B. Also in B, C, D there was fatty changes as small lipid droplets (microvesicular steatosis). Magnification: 200x.

All values from the stereological assessment are presented in Table 4. There was a significant decrease in area of parenchyma with supplementation of vitamin E and vitamin C. The area of parenchyma significantly decreased in hens in heat condition compared to those of hens in normal condition (P<0.05). A possible cause of this result was that more fibrosis developed in livers of hens exposed to heat stress than those of hens in normal poultry houses due to connective tissue cells in heat stressed livers being more affected (FIgure 2), and the reduction of parenchyma was a cause of functional loss.

Table 4 Stereological estimations performed in this study.

Heat	Vitamin C	Vitamin E	Estimating parameters
			A Parenchyma area, cm^2	B Sinusoid area, cm^2	C Ratio parenchyma: area/sinusoid	D ND of all hepatocytes (living and dead)	E ND of only necrotic hepatocytes (dead)	F Ratio total hepatocytes: necrotic hepatocytes
Normal (20°C)	0 mg/Lvitamin C	0 E	32261.3	2738.8	11.78	0.029	0.007	4.1
		45 E	23161.0	988.5	23.43	0.025	0.007	3,6
		65 E	22837.5	1312.5	17.40	0.028	0,005	5.6
		85 E	22982.5	1167.5	19.68	0.031	0.005	6.2
	100 mg/L vitamin C	0 E	32192.5	2807.5	11.46	0.034	0.005	6.8
		45 E	22697.5	1448.5	15.66	0.033	0.006	5.5
		65 E	22635.0	1515.0	14.94	0.032	0.004	8.0
		85 E	22817.3	1278.7	17.84	0.032	0.010	3.2
Heat stress (30°C)	0 mg/L vitamin C	0E	32368.8	2406.3	13.45	0.038	0.012	3.2
		45 E	22440.0	2980.0	7.53	0.029	0.006	4.8
		65 E	21835.5	2317.5	9.42	0.032	0.006	5.3
		85 E	23053.8	1096.3	21.03	0.035	0.007	5.0
	100 mg/L vitamin C	0 E	22112.5	2037.5	10.85	0.025	0.008	3.1
		45 E	23078.0	2142.5	10.77	0.034	0.008	4.2
		65 E	22777.5	1372.5	16.59	0.024	0.006	4.0
		85 E	22317.5	1832.5	12.18	0.025	0.010	2.5
Heat	Normal		25198.1	1657.1	16.51	0.030	0.006	5.4
	Heat stress		23747.9	2023.1	12.72	0.030	0.008	4.0
	P		^*	ns	^*	ns	^*	ns
Vitamin E, U/kg	0		29733.8^a	2497.5^a	11.88^c	0.031	0.008^a	4.3^b
	45		22844.1^b	1889.9^b	14.34^b	0.030	0.006^b	4.5^b
	65		22521.4^c	1629.4^c	14.58^b	0.029	0.005^b	5.7^a
	85		22792.8^b	1343.7^d	17.68^a	0.030	0.008^a	4.2^b
	P		^*	^*	^*	ns	^*	^*
Vitamin C, mg/L	0		25117.5	1875.9	15.45	0.030	0.007	4.7
	100		23828.5	1804.3	13.78	0.029	0.007	4.6
	P		^*	ns	ns	ns	ns	ns
Heat x vitamin E			^*	ns	^*	ns	ns	ns
Heat x vitamin C			^*	ns	ns	ns	ns	ns
Vitamin E x vitamin C			^*	ns	ns	ns	ns	ns

ND, numerical density of cell in per m³;

a,b,c ccolumn means with no common superscript differ significantly;

* P<0.05; ns, not significant

The sinusoid area did not affected by vitamin C (P>0.05). Nonetheless, the sinusoid area was significantly decreased by heat stres and supplementation of vitamin E in this study. The rate of sinusoidal area to area of parenchyma decreased in heat stressed livers. But supplementation of vitamin E significantly (P<0.05) increased rate of sinusoidal area to area of parenchyma. This result indicates that sinusoids were enlarged (sinusoidal dilatation) by heat stress, histopathologically (FIgure 1 B, 2 B, 3 B), and as a result, liver tissue may have impeded uptake of nutrients from the blood, including nutritious venous contents for feeding requirements.

The addition of vitamin E and vitamin C to the diet and heat stres had no impact on density of all hepatocytes in this study (Table 4). The heat stress condition caused an increases in density of necrotic cell (25%) compared to the normal condition. Supplemental 45 and 65 U vitamin E/kg tended to decrease density of necrotic cell in liver of laying hens. Dietary supplementation of vitamin C had no significant effect on density of necrotic cell. In this study, we found that supplementation of 65 U

vitamin E/kg increased the ratio of necrotic cell density to total cell density (Table 4) but the ratio of necrotic cell density to total cell density was not affected by heat stress and vitamin C.

Conclusions

Consequently, feed intake, egg productioni liwer weight, egg weight, egg weight ratio, egg shell ratio, egg shell thickness and Haugh unit negatively influenced by heat stres. Vitamin E supplementation at different levels (45, 65 and 85 U/kg of diet) had no significant effects on feed intake, however, it improved egg production, feed efficiency and Haugh Unit in laying hens in the present study. Although supplementation of 85 U vitamin E and 100 mg vitamin C/l increased feed conversion and egg production, it caused higher destruction of the liver. We estimate that 85 U vitamin E/kg may reduce performance later in the production period because of the destruction of the liver.

According to our results, heat stress caused liver damage such as sinusoidal dilatation, fibrosis, cell death (necrosis), and fatty changes [lipid accumulation in cells=in microvesicular (small droplets) type]. Moreover, these damages led to functional tissue loss and may damage liver function in heat stressed hens result of this liver functions would not been assumed, for example many toxicants could not been removed, essential complements could not produced; thus performance of hens may be reduced.

Therefore, we thought that a supplementation of 65 U of vitamin E/kg in diets appeared to be the most efficacious dose. Also the addition of vitamin C may contribute to reducing liver injury caused by heat stress.

Acknowledgements:

the authors gratefully acknowledge the financial and technical assistance of Ataturk University, Erzurum-Turkey.