agriculture

Article
Effect of Chestnut Tannins and Vitamin E Supplementation to
Linseed Oil-Enriched Diets on Growth Performance, Meat
Quality, and Intestinal Morphology of Broiler Chickens
Lidija Perić 1, *, Dragan Žikić 1 , Mirjana Ðukić Stojčić 1 , Vladimir Tomović 2 , Jakob Leskovec 3 , Alenka Levart 3 ,
Janez Salobir 3 , Zdenko Kanački 4 and Vida Rezar 3

1 Department of Animal Science, Faculty of Agriculture, University of Novi Sad, 21000 Novi Sad, Serbia
2 Faculty of Technology, University of Novi Sad, 21000 Novi Sad, Serbia
3 Department of Animal Science, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia
4 Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, 21000 Novi Sad, Serbia
* Correspondence: lidija.peric@stocarstvo.edu.rs

Citation: Perić, L.; Žikić, D.; Stojčić,
M.Ð.; Tomović, V.; Leskovec, J.;
Levart, A.; Salobir, J.; Kanački, Z.;
Rezar, V. Effect of Chestnut Tannins
and Vitamin E Supplementation to
Linseed Oil-Enriched Diets on
Growth Performance, Meat Quality,
and Intestinal Morphology of Broiler
Chickens. Agriculture 2022, 12, 1772.
https://doi.org/10.3390/
agriculture12111772
Abstract: The objective of this study was to establish the effects of chestnut tannin extract or vitamin
E added to linseed oil-enriched diets on growth performance, meat quality, and intestinal morphol-
ogy of broiler chickens. A total of 240 day-old Ross 308 male broiler chicks were included in trial.
5% of cold-pressed linseed oil was included in finisher diets (21–40 days), and three feeding treat-
ments with four replicates were formed: finisher without additives; finisher + 200 IU vitamin E/kg;
finisher + 500 mg/kg of chestnut wood tannin extract. No significant influence of treatments was
established on body weight or feed conversion ratio. A negative effect on feed intake (p < 0.05) was
found in the vitamin E group. The addition of vitamin E increased the dressing percentage (p < 0.05)
and increased the breast meat yield (p < 0.01) compared to the control group. No significant effects
were found on the water holding capacity or pH of breast meat. The highest level of AST (p < 0.01)
and ALT (p < 0.05) was recorded in vitamin E group. The addition of chestnut tannin extract in feed
increased villus height, villus height: crypt depth ratio, and villus area compared to the other two
groups (p < 0.05). It can be concluded that vitamin E supplementation improves carcass percentage
and breast meat yield, while chestnut tannins improve the intestinal morphology of broiler chickens
when added to oil-enriched diets.
Keywords: tannins; vitamin E; broiler; performance; meat; gut morphology

Academic Editor: Hai Lin

Received: 16 September 2022 1. Introduction

Accepted: 21 October 2022
Published: 25 October 2022
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4.0/).
In 2019, chicken meat production achieved the highest growth, becoming the most
produced meat with a share of 35% of the global market [1]. This rapid growth was
possible due to the high genetic potential of broilers, good management practices, and
well-balanced and high-quality feed. At the same time, consumers started to pay more
attention to the importance of nutrition for their health. Today, the consumption of n-3
polyunsaturated fatty acids (PUFA) is recommended for human nutrition because they
exert numerous benefits on their health [2]. Also, the ratio of n-6/n-3 fatty acids in the diet
is critical especially if it is known that the intake of n-6 fatty acids is excessive, mainly in
Western countries [2]. Since humans are not efficient in synthesizing long-chain PUFA, the
production of n-3 PUFA-enriched poultry meat is gaining more importance. However, the
inclusion of oils rich in n-3 PUFA in broiler diets will increase the content of n-3 PUFA in
broiler meat which can raise oxidative stress [3]. Therefore, the inclusion of n-3 PUFA also
requires a higher level of antioxidants in broiler feed [4]. In this context, tannin-containing
products are gaining importance among poultry nutritionists [5,6] since the antioxidant
activity of chestnut tannins was reported by many authors [4,5].

Agriculture 2022, 12, 1772. https://doi.org/10.3390/agriculture12111772 https://www.mdpi.com/journal/agriculture

Agriculture 2022, 12, 1772 2 of 12

Tannins are polyphenolic compounds with different molecular weights and vari-
able chemical complexity [3,5]. Tannins can be classified into two groups: condensed
tannins and hydrolyzable tannins [7]. Tannins are usually considered antinutritional sub-
stances for monogastric animals and poultry [8,9], but recent research confirmed beneficial
biological effects of tannins such as antioxidant, antimicrobial, anti-parasitic, anti-viral,
anti-inflammatory, etc. [5,9,10]. Hydrolyzable tannins extracted from chestnut wood have
been used in broiler diets to improve litter quality [11,12], foot pad lesion score [13], and
the fatty acid composition of breast meat and act as a biological antioxidant [8,14]. One
of the key effects of tannins is the modulation of the intestinal microbiota, which can be
very beneficial for animals [5,15,16]. However, despite the mentioned positive effects, some
authors have not found a positive effect of tannin extracts on growth performance [11,14].
As opposed to tannins, which antioxidative properties are still being studied, vitamin
E has been effectively used in poultry nutrition because it is a very potent lipid-soluble
antioxidant [17,18]. Vitamin E has been established to mitigate the detrimental effect
caused by heat stress [19–21] or increased dietary intake of oils rich in polyunsaturated
fatty acids [3,22]. The addition of vitamin E to broiler diets enriched with n-3 PUFA in-
creased α-tocopherol content in plasma and meat, lowered plasma malondialdehyde, and
protected DNA from oxidative damage [23,24]. Besides vitamin E or tannin extracts, other
antioxidants such as selenium [25,26], vitamin C, or their combination [4,27] could also be
included in the oil-enriched feed.
On the other hand, high prices of feedstuffs and feed additives put much pressure on
poultry production, so the cost-effectiveness of the supplements is significant for poultry
producers. Some authors established that the return on investment could be as high as 10.9
when vitamins and minerals are used as feed supplements [28]. It has been reported that the
addition of 0.5 kg/t of tannin extracts lowered the feed cost per 1 kg of growth by 3.8% [29].
However, it is essential to emphasize that cost-effectiveness depends on many factors, and
it is difficult to state whether adding the increased dosage of vitamin E or tannin extract
will result in increased profit. The main benefit of adding these two antioxidants should be
reflected in preserving the animals’ health and improving the meat’s quality.
This study aimed to determine the effects of the dietary addition of chestnut tannin
extract or vitamin E to linseed oil-enriched diets on growth performance, meat quality, and
intestinal morphology of broiler chickens.

2. Materials and Methods

The trial was conducted at the Experimental poultry farm of the Faculty of Agriculture,
University of Novi Sad.

2.1. Experimental Design and Dietary Treatments

A total of 240 one-day-old Ross 308 male broiler chicks were randomly allocated to
three treatments with four replicates (20 birds per replicate). Birds were reared in floor
pens covered with chopped straw. Stocking density was16 birds/m2 . The temperature
regime was as follows: 32 ◦ C (0–2 d), 30 ◦ C (3–7 d), 28 ◦ C (8–14 d), 26 ◦ C (15–21 d), 23 ◦ C
(22–28 d), and 21 ◦ C thereafter. The lighting program was 18 h of light and 6 h of dark.
Consumption of feed and water was provided ad libitum. Broilers were fed a starter diet
(1–12 days) and a grower diet (13–20 days), and feed was composed to meet the needs
recommended by the company Aviagen for Ross 308 [30]. Feed was provided in mash
form. Finisher diets, which were given to the birds from the 21st to the 40th day of the
trial, were enriched with 5% of cold-pressed linseed oil, and three feeding treatments
were formed: feed without additives (Control); feed + 200 IU vitamin E/kg (Rovimix
Stay-C35 (DSM)) (Vitamin E); feed + 500 mg/kg of sweet chestnut wood extract Farmatan®
(Tanin Sevnica, Slovenia) (Tannin). Farmatan® is obtained from the sweet chestnut wood
(Castanea sati-va Mill.) by the water extraction process. It contains about 75% tannins,
mostly ellagitannins [31]. The dose of Farmatan® in animal feed recommended by the
Agriculture 2022, 12, 1772 3 of 12

manufacturer is from 0.4 to 0.7 kg/t, so an average dose of 0.5 kg/t was chosen for this
experiment. The composition of the basal diets is shown in Table 1.

Table 1. Composition and nutrient content of the starter, grower and finisher diets 1 .

Ingredients, % Starter (1–12 d) Grower (13–20 d) Finisher (21–40 d)

Maize 42.13 49.00 53.70
Wheat 5.00 3.00 4.00
Wheat bran 6.00 / /
Soybean meal 2 28.46 15.45 32.00
Full fat soybean
13.67 28.00 1.00
(extruded)
Linseed oil / / 5.00
Lysine 0.24 0.15 0.13
Methionine 0.37 0.20 0.17
Threonine 0.09 / /
Monocalcium
1.01 1.50 1.30
phosphate
Limestone 1.39 1.40 1.40
Salt 0.27 0.30 0.30
Sodium bicarbonate 0.13 / /
Phytase 0.02 / /
Ronozyme VP 3 0.02 / /
Captex T2 4 0.20 / /
Premix 5 1.00 1.00 1.00
Nutrient analyses
Metabolizable energy
12.45 13.00 13.40
(MJ/kg) 6
Crude protein, % 23.00 21.65 19.69
Lysine, % 1.44 0.93 1.50
Methionine, % 0.68 0.44 0.47
Calcium, % 0.96 1.30 0.88
Available
0.48 0.52 0.42
Phosphorus, %
1 Finisher experimental diets were supplemented with either 200 IU vitamin E/kg (Rovimix Stay-C35 (DSM))
(Vitamin E group) or 500 mg/kg sweet chestnut wood extract Farmatan® (Tanin Sevnica, Slovenia) (Tannin
group). 2 44% of crude protein. 3 DSM Nutritional Products Europe Ltd. 4 Medro–Doxal © . 5 Composition of
premix (/kg): vitamin D (4500 IU), vitamin A (10,000 IU), vitamin E (50 IU), vitamin K (3 mg), choline (1600 mg),
biotin (0.18 mg), niacin (60 mg), folic acid (1.9 mg), pantothenic acid (18 mg), vitamin B1 (2.5 mg), vitamin B2
(6.5 mg), vitamin B6 (3.2 mg), vitamin B12 (0.017 mg), Cu (16 mg), I (1.25 mg), Mn (120 mg), Fe (20 mg), Zn
(110 mg), Se (0.25 g). 6 Calculated values.

2.2. Performance and Carcass Quality

Body weight and feed intake were measured on a pen basis. Body weight and feed
intake were recorded at the end of the starter period (day 12), at the end of the finisher
period (day 20), and the end of the trial (day 40). Mortality was recorded daily. At 40 days
of age, birds were individually weighed, and 12 animals per group were killed by cervical
dislocation. Blood samples for the analyses were taken from the jugular vein. Upon slaugh-
ter, the processed carcasses were cooled for 24 h at 4 ◦ C. After the chilling; the carcasses
were weighed to calculate the dressing percentage. The ready-to-cook carcass weight was
recorded after removing the viscera, giblets, shanks, and head from the eviscerated carcass.
The dressed cold carcasses were then dissected into primal cuts (breast, legs, wings, and
back). The abdominal fat pad was removed from the carcass and measured.

2.3. Meat Analyses

After the slaughtering and carcass dissection, the left side of the breast muscle was
separated from the bone and stored in a refrigerator at 4 ◦ C. The pH analysis was carried
out 24 h post-mortem and 48 h post-mortem. Water holding capacity was determined 24 h
Agriculture 2022, 12, 1772 4 of 12

post-mortem according to the method described by Tomović et al. [32]. The pH value was
measured with a portable pH meter (Consort T651, Turnhout, Belgium) equipped with an
insertion electrode (Mettler Toledo, Greifensee, Switzerland) [32].

2.4. Blood Analyses

The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) were determined using a spectrophotometer (Clima MC-15) and test kits (Bioanalyt-
ica) by kinetic reaction, with absorbance measured at 340 nm. Activities of AST and ALT
were determined using the spectrophotometric method [33,34].

2.5. Gut Morphology

Gut samples were taken from the jejunum at the midpoint between the end of the
duodenal loop and the location of Meckel’s diverticulum. Samples of jejunum were washed
with saline solution and placed in 10% formaldehyde. Samples were stained with hema-
toxylin and eosine after the histological procedure. For determining parameters repre-
senting jejunal histology light microscope and software for image analysis (IM1000 Image
Manager, Leica, Wetzlar, Germany) were used. For the evaluation of villus height, villus
width, crypt depth, and thickness of tunica muscularis externa, at least 15 measurements
per bird were made. The villus was determined by measurements at three points: close to
the bottom, at the midpoint, and close to the tip of the villus. After that, the average values
were calculated and used for the statistical analyses.

2.6. Statistical Analyses

The statistical analysis was done using the General Lineal Model of STATISTICA 14
(TIBCO Software Inc., Palo Alto, CA, USA, 2019). The analysis was done as a complete ran-
dom design with three groups and four replicates. Percentage data were transformed using
arcsine transformation, and transformed values were used for analyses. A Levene’s test
was used to test the homogeneity of variances. One-way ANOVA was used to determine
the effects of treatment. The overall model used was:

Yij = µ + Ai + εij

where Yij represents the dependent variable, µ represents the overall mean, Ai is the effect
of the treatment (control, addition of 500 mg/kg sweet chestnut wood extract, addition of
200 IU vitamin E/kg), and εij is the error. Fisher LSD post hoc test was used to separate LS
means. Differences between treatments were considered significant at p < 0.05.

3. Results
Growth performances of broilers fed linseed oil-enriched diets with or without added
antioxidants are presented in Table 2.
No significant influence of treatments was established on body weight, average daily
gain, or feed conversion ratio. Negative effect on feed intake (p < 0.05) was found in the
vitamin E group, from 21 to 40 days. That was also reflected in total feed intake, which
was lower in the vitamin E group compared to the control group (p < 0.05). Feed intake of
birds fed chestnut tannin extract was not different from the other two groups. The lower
feed intake in the vitamin E group did not significantly influence the feed conversion ratio
(FCR), although it was numerically lowest in the vitamin E group. The mortality rate was
very low in all groups. Only one chick died in the control and tannin groups, while none
died in the vitamin E group.
Dietary supplements significantly affected dressing percentage (p < 0.05), as well as
percentages of breast (p < 0.01) and legs (p < 0.01) yields (Table 3).
Agriculture 2022, 12, 1772 5 of 12

Table 2. Effect of chestnut tannins and vitamin E on the growth performances of broiler chickens.

Control 1 Tannin 2 Vitamin E 3 SEM p Value

Body weight, g
Day 20 760 785 766 8.83 0.538
Day 40 2394 2354 2312 21.65 0.329
Average daily gain, g
1–20 days 34.98 35.78 34.44 0.43 0.477
21–40 days 89.73 86.62 85.89 0.99 0.259
1–40 days 60.25 59.25 58.19 0.55 0.338
Average daily feed intake, g
1–20 days 49.30 49.99 48.10 0.41 0.160
21–40 days 155.32 a 152.50 ab 147.83 b 1.40 0.048
1–40 days 98.23 a 97.30 ab 94.13 b 0.79 0.047
Feed conversion ratio
1–20 days 1.41 1.40 1.40 0.007 0.605
21–40 days 1.73 1.75 1.72 0.011 0.598
1–40 days 1.63 1.64 1.62 0.009 0.853
Mortality rate, %
1–40 days 1.17 1.17 0.00 / /
a–bMeans within the same row with no common superscript differ significantly (p < 0.05). 1 Control diet. 2 Control
diet + 200 IU vitamin E/kg, Rovimix Stay-C35 (DSM). 3 Control diet + 500 mg/kg sweet chestnut wood extract
Farmatan® (Tanin Sevnica, Slovenia).

Table 3. Effect of chestnut tannins and vitamin E on the dressing percentage (ready to cook) and
carcass yields (%) of breast, legs, wings, back, and abdominal fat.

Control 1 Tannin 2 Vitamin E 3 SEM p Value

Dressing percentage, % 69.53 b 69.64 b 71.17 a 0.30 0.042
Carcass yield, %
Breast 37.11 B 37.80 AB 38.87 A 0.25 0.009
Legs 30.49 A 30.13 A 29.11 B 0.16 <0.001
Back 20.41 20.25 20.71 0.17 0.543
Wings 11.05 10.88 10.85 0.08 0.327
Abdominal fat 0.92 0.92 0.92 0.06 0.998
a–b Means within the same row with no common superscript differ significantly (p < 0.05). A–B Means within
the same row with no common superscript differ significantly (p < 0.01). 1 Control diet. 2 Control diet + 200 IU
vitamin E/kg, Rovimix Stay-C35 (DSM). 3 Control diet + 500 mg/kg sweet chestnut wood extract Farmatan®
(Tanin Sevnica, Slovenia).

The results showed that adding vitamin E increased the dressing percentage compared to
the other two groups and increased the breast meat yield (%) compared to the control group.
The legs yield (%) was lowered in the vitamin E group compared to the control and tannin
group (p < 0.01). These results indicate a positive effect of vitamin E on carcass quality.
No significant differences were found between groups in the water holding capacity
or pH of breast meat (Table 4).
Analyses of the enzyme profiles of ALT and AST showed significant differences
between groups (Table 5). Precisely, the highest level of AST (p < 0.01) and ALT (p < 0.05)
was recorded in the vitamin E group, which implies the lower protective role of vitamin E
against a high level of PUFA in feed. However, it is essential to emphasize that all values
were within the normal physiological range.
Agriculture 2022, 12, 1772 6 of 12

Table 4. Effect of chestnut tannins and vitamin E on the water holding capacity and pH of the breast
meat of broiler chickens.

Control 1 Tannin 2 Vitamin E 3 SEM p Value

WHC 4 0.46 0.45 0.48 0.01 0.228
pH 24 h post mortem 5.96 6.04 6.07 0.05 0.192
pH 48 h post mortem 5.88 5.91 5.95 0.05 0.342
1Control diet. 2 Control diet + 200 IU vitamin E/kg, Rovimix Stay-C35 (DSM). 3 Control diet + 500 mg/kg sweet
chestnut wood extract Farmatan® (Tanin Sevnica, Slovenia). 4 WHC = water holding capacity.

Table 5. Effect of chestnut tannins and vitamin E on the activity of alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) in the blood of broiler chickens.

Control 1 Tannin 2 Vitamin E 3 SEM p Value

AST (IU/L) 279.21 B 270.53 B 372.06 A 14.55 0.004
ALT (IU/L) 28.74 b 31.91 b 43.33 a 2.39 0.026
1 Control diet. 2 Control diet + 200 IU vitamin E/kg, Rovimix Stay-C35 (DSM). 3 Control diet + 500 mg/kg sweet
chestnut wood extract Farmatan® (Tanin Sevnica, Slovenia). a–b Means within the same row with no common
superscript differ significantly (p < 0.05). A–B Means within the same row with no common superscript differ
significantly (p < 0.01).

The results of the intestinal morphology measurements are presented in Table 6. The
addition of chestnut tannin extract in feed improved the development of morphological
structures of the intestine, as indicated by an increase in villus height, increased villus
height:crypt depth ratio, and increased villus area compared to the other two groups
(p < 0.05). The differences between the control and the vitamin E group were not significant.

Table 6. Effect of chestnut tannins and vitamin E on the intestinal morphology of broiler chickens.

Control 1 Tannin 2 Vitamin E 3 SEM p Value

Villus height (µm) b 2207.66 a b 39.43 0.014
1940.82 2027.60
Crypt depth (µm) 439.90 ab 426.23 b 485.87 a 10.78 0.046
Villus height: crypt
4.53 b 5.35 a 4.37 b 0.15 0.015
dept ratio
Villus area (mm2 ) b 0.47 a b 0.01 0.002
0.35 0.40
Tunica muscularis
342.84 323.60 337.69 7.76 0.591
thickness (µm)
a–bMeans within the same row with no common superscript differ significantly (p < 0.05). 1 Control diet. 2 .Control
diet + 200 IU vitamin E/kg, Rovimix Stay-C35 (DSM). 3 Control diet + 500 mg/kg sweet chestnut wood extract
Farmatan® (Tanin Sevnica, Slovenia).

4. Discussion
Numerous studies have shown that broilers fed with diets with high content of PUFA
have a higher demand for dietary antioxidants [3,22,23,35]. Antioxidants added in broiler
feed have a positive effect on maintaining the oxidative balance, beneficial to the intestinal
mucosa, and increase growth performance [36]. The hormone levels like corticosterone and
catecholamines increase under stress conditions, and lipid peroxidation in cell membranes
is initiated, indicating that a higher level of vitamin E in feed is needed [35]. The present
study evaluated the effects of vitamin E as a proven antioxidant and chestnut tannins as
natural plant antioxidants. Adding vitamin E or chestnut tannins to broiler diets did not
affect growth performance. The differences between the vitamin E and tannin group were
insignificant (p > 0.05). It could be stated that the vitamin E content in standard broiler diets
is sufficient to meet their needs, even when the amount of PUFA in the diet increases. A
negative impact of a higher dosage of vitamin E on feed consumption compared to both the
control and tannin groups, which has been noticed in the present study, is not supported
by the findings of other researchers. Most studies showed that increasing vitamin E in the
feed had no significant effect on the final live weight, feed intake, and FCR under heat
Agriculture 2022, 12, 1772 7 of 12

stress conditions [35,37,38] or in standard rearing conditions [39]. A negative impact on

feed consumption, which has been noticed in the present study, is not supported by the
findings of other researchers.
Supplementing dietary tannins in feed has different outcomes in growing monogastric
animals [9]. The positive effect of low doses of chestnut tannins on body weight and
FCR were found in young birds [7]. However, the negative effect of tannin supplemen-
tation on body weight and FCR is mainly related to a higher dosage of tannins in broiler
feed [6,7,10,11]. Feed intake and body weight gain declined, and FCR increased with an
increase in dietary tannin content up to 15 g kg−1 diet [40]. Choi et al. [41] established that
tannin acid at levels below 972 mg/kg could be added to broiler feed without a negative
effect on productive performance. The negative effect of higher doses of tannin extracts
can be explained by the significant reduction in feed intake, resulting in lower weight
gain [6,7,11,40]. The negative effects of high concentrations of tannins on feed intake in
broilers can be caused by irritation of the esophagus and necrosis in crop, gizzard, and
duodenum [42]. Some authors [8,43] reported a reduction in growth performance and FCR
even with an addition level of 10 g/kg of tannin extract. These inconsistencies support the
standpoint that it is not only the dose but also the type of tannin in the feed that matters [9].
Slaughter traits are essential for poultry producers, and even the smallest differences
in the dressing percentage and carcass part yields can lead to changes in the economic
efficiency of production [27]. Since the feed composition and nutrient and energy level
in the present experiment was similar in all groups, the differences in carcass part yields
between groups were not expected. Most researchers found that dietary supplementation
with vitamin E did not affect the carcass, breast, and thigh yields [35,38,39]. However, in the
present study, adding vitamin E improved the dressing percentage compared to the other
two groups (p < 0.05). Also, vitamin E increased the breast meat yield (%) compared to the
control group (p < 0.01) but not compared to the tannin group. This is in line with Mazur-
Kuśnirek et al. [37], who reported that broilers fed diets supplemented with 200 mg/kg of
vitamin E had a higher yield of breast muscles in the carcass. An increased percentage of
breast meat of breast meat in broilers which were fed with dietsdiets with an increased
level of vitamin E was also reported by others [44,45]. To the best of our knowledge, no
clear explanation can be found in the available literature on the association between dietary
vitamin E supplementation and increased breast meat percentage. The positive effects of
vitamin E on the quality of breast meat have been proven [6,17], but the influence on the
percentage of breast meat yield has not yet been scientifically supported. Regarding the
addition of chestnut tannins, the dressing percentage was lower compared to the vitamin
E group (p < 0.05), but there were no significant differences in breast meat yield between
tannin and vitamin E groups. Other researchers also reported the lack of the effect, who
found that dressing percentage and carcass parts yields were not affected by the addition
of tannins [6,7,11,46].
Besides the dressing percentage and carcass parts yields, the most critical issue for
the consumers is the meat quality. n-3 PUFA-enriched broiler meat is prone to oxidative
damage [27], and dietary antioxidants could alleviate the adverse effects of oxidative stress,
and consequently improve the meat quality [47,48]. The addition of higher dosage of
vitamin E in broiler feed could ameliorate the resistance against pale, soft, and exudative
meat [49], improve the cell integrity, decrease lipid oxidation, reduce pigment oxidation,
and positively affect the water holding capacity [17,39]. The addition of chestnut tannins in
broiler feed could increase meat pH and result in lower drip loss [6]. However, no positive
effects of a higher level of vitamin E or addition of chestnut tannins on the water holding
capacity or pH of breast meat were found in the present study. The lack of the effect of
tannins on meat quality was also reported by Pascual et al. [46].
When examining oxidative stress in poultry, liver enzymes are a good indicator of an
altered metabolism. The main target organ in many negative conditions is the liver. Heat
stress, aflatoxicosis, and infections could cause impairing or disturbance in the metabolism
of lipids and other important nutrients such as proteins, vitamins, amino acids, nucleic
Agriculture 2022, 12, 1772 8 of 12

acids, and liver enzymes [50,51]. A level of ALT indicates non-specific cell damage, and
AST is a sensitive avian indicator for liver damage and muscle damage [26,52]. In the
present study, the highest level of AST and ALT activity was recorded in the vitamin E
group. The differences were significant compared to control and tannin group (p < 0.01
for AST and p < 0.05 for ALT). This finding implies that the protective role of vitamin E
in reducing the liver damage was lower than chestnut tannin extract. This contrasts with
findings that the best protective effect on the liver enzymes (ALT and AST) was found by the
addition of vitamin E and selenium in the feed of birds exposed to high aflatoxin levels in
feed [50]. However, Ebraimzadeh et al. [53] didn’t find differences in AST and ALT activity
between the control group and vitamin E enriched group (200 mg/kg of αtocopherol
acetate), which confirms the fact that vitamin E is not always effective in protecting the liver
from damage. Sodium selenite and vitamin E somewhat have reduced the damaging effect
of cyclophosphamide in rats, but the effect on the level of ALT and AST were not significant
(p > 0.05) [54]. It has been demonstrated that chestnut tannins have strong antioxidant
activity [55,56]. The tannin group’s lower AST and ALT values could be explained by
the protective effect of tannins on body lipid oxidation and DNA integrity [57]. Chestnut
tannins added in broiler feed can decrease the damage of lymphocyte DNA caused by
oxidative stress [3].
Normal digestion and absorption of nutrients can be realized only in a healthy gas-
trointestinal tract that is not harmed or damaged [58,59]. Gut morphology is susceptible to
a stressor [60], and the gastrointestinal tract has several possibilities to adapt to unfavorable
conditions [53]. By changing the height of its villi, the intestine will change its surface
to adapt to the changed circumstances [61]. Villus height is significant for absorption of
nutrients and their transport in the gastrointestinal tract. Elongated villi and a higher vil-
lus/crypt ratio point that the rate of migration of enterocyte-cell from the crypt to the villus
is lower [59]. The addition of chestnut tannin extract improved the development of mor-
phological structures of the intestine in the present study. The positive effect of tannins was
reflected in increased villus height, increased villus height:crypt depth ratio, and increased
villus area compared to the control and vitamin E group (p < 0.05). The differences between
the control and the vitamin E group were not significant. Elongated villi and a higher
villus/crypt ratio would be expected to reflect improved growth performance in the tannin
group, but this effect has not been demonstrated. However, the intestine morphology was
not compromised in the other groups either, so under the given circumstances, all groups
achieved similar growth performances. It can be assumed that the positive effect of tannins
could be very significant in some other challenging conditions, which could be detrimental
to gut morphology. In that case, the positive effect of elongated villi on growth performance
would probably be significant, as has been shown in many studies [16,58–60]. Elongated
villi and increased villus height:crypt depth ratio were found in broilers supplemented with
chestnut tannins exposed to heat stress [60]. It has been demonstrated that the addition
of chestnut tannin can reduce the severity of gut damage, and efficiently control necrotic
enteritis and the proliferation of Clostridium perfringens [16]. The effect of chestnut tannins
on gut morphology could be dose-dependent [11]. At lower levels of inclusion (250 mg
and 500 mg of chestnut tannins per kg of feed), the villi were formed correctly, but the
dose of 1.0 g/kg caused changes in the morphology of the intestinal wall and lowered
proliferation rate in the mother-cell zone. Range of tannic acid supplementation between
500 and 900 mg/kg improves gut health in broilers under antibiotic-free conditions [41].
Vitamin E was demonstrated to have positive effects on gut morphology and absorption
area [62–64]. Chickens fed diets supplemented with 100, 200, or 400 IU/kg vitamin E for
42 days had significantly increased villus height and width [62]. Feeding broilers with
10 mg vitamin E/kg, supplemented with glutamine for the first week of life, resulted in
better intestinal mucosa development [63]. Ebrahimzaeh et al. [53] found no effect on
villus height, crypt depth, or tunica muscularis thickness by the addition of 200 mg/kg of
α-tocopherol acetate in the feed of broiler chickens, which is consistent with the results of
the present study.
Agriculture 2022, 12, 1772 9 of 12

5. Conclusions
Based on the results of the trial, it was assessed that the addition of chestnut tannin
extract and vitamin E does not affect body weight, average daily gain, or feed conversion
ratio. A negative effect on feed intake was found in the vitamin E group, but without nega-
tive consequences on body weight gain or FCR. No effect was found on the water holding
capacity or pH of breast meat. Vitamin E supplementation was found to improve carcass
percentage and breast meat yield, while chestnut tannins improve intestinal morphology of
broiler chickens when added to oil-enriched diets. Although in this experiment, the positive
effects of tannins and vitamin E were not reflected in improved growth performance, these
effects could be beneficial in other challenging conditions in poultry production.

Author Contributions: Conceptualization, L.P., D.Ž., M.Ð.S., J.S. and V.R.; methodology, L.P., V.T.,
J.L., A.L., J.S. and Z.K.; investigation, D.Ž., M.Ð.S., J.L., V.T., Z.K. and A.L.; writing—original draft
preparation, L.P., J.L., V.T. and Z.K.; writing—review and editing, M.Ð.S., D.Ž. and V.R.; supervision,
A.L., J.S., Z.K. and V.R.; project administration, L.P., V.R. and J.S. All authors have read and agreed to
the published version of the manuscript.
Funding: This work was financially supported by a grant from the Slovenian Research Agency
(Ljubljana, Slovenia, grant No. P4-0097) and the Ministry of Education, Science and Technological
Development of the Republic of Serbia (No. 451-03-68/2020-14/200117).
Institutional Review Board Statement: All the procedures were conducted according to the ethical
norms of the EU Convention for the protection of vertebrate animals used for experimental and other
scientific purposes, according to Annex 4 of the Rulebook on the conditions for entry in the register
for inspections on animals, the content and manner of keeping that register, the welfare training
program experimental animals, the application form for approval for conducting experiments on
animals, the manner of care, treatment and deprivation of life of experimental animals, as well as
content and manner of keeping records on keeping, reproduction, trade, and conducting animal
experiments (Annex 4, Official Gazette of RS No. 39/10).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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