Research Article

Received: 27 October 2009 Revised: 22 October 2010 Accepted: 11 March 2011 Published online in Wiley Online Library: 16 September 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4541

Performance, carcass, gastrointestinal tract

and meat quality traits, and selected blood
parameters of broilers fed diets supplemented
with humic substances
Ergin Ozturk,∗ Nuh Ocak, Alev Turan, Guray Erener, Aydin Altop
and Soner Cankaya

Abstract
BACKGROUND: The effects of humic substances (HS) on quality traits (colour, water-holding capacity, and pH) of breast and
thigh meats and on blood minerals (calcium and phosphorus) and on metabolites (glucose, total protein, triglyceride and
cholesterols) as well as on the performance, carcass, and gastrointestinal tract (gut) traits of broilers were studied. In total, 480
male chicks (ROSS 308) were allocated to four treatments, each with five replicates containing 24 birds. While control birds
were fed a diet (mash form) with no additives (0 HS), other treatment birds were fed with diets containing HS at 0.5 (0.5 HS), 1.0
(1 HS) or 1.5 (1.5 HS) g kg−1 from 1 to 42 days of age.

RESULTS: The body and carcass weights and feed efficiency increased (P < 0.05) by 1.5 HS, and blood cholesterol decreased
(P < 0.05) by 1 HS and 1.5 HS compared to 0 HS. The 0.5 HS decreased (P < 0.10) low-density lipoprotein compared to 0 HS.
The 0.5 HS resulted in thigh meat and breast meat that were lighter and darker than those found in the other groups and 1.5 HS
respectively (P < 0.01).

CONCLUSION: Feeding with a diet containing HS caused a measurable variation in the meat quality and blood cholesterol as
well as the performance, carcass, and gut traits of broilers.


c 2011 Society of Chemical Industry

Keywords: poultry; chickens for fattening; feed additive; organic acid; blood metabolite; chemical composition

INTRODUCTION necessary for the organism.7,10 Mineral components, e.g. calcium

There is an increasing awareness that supplemental humic and phosphorus, enter the composition of tissues and body fluids,
substance (HS) as a growth-promoting agent has multiple fulfilling a body-building function. Furthermore, they participate
health effects and nutritional benefits for domestic animals. in the maintenance of homeostasis, pH, activating enzymes, vi-
This knowledge has resulted primarily from studies on the use tamins and blood enzyme activity.18 Therefore, in the search for
of humic acids to improve the immune system of calves,1,2 to alternatives to banned in-feed antibiotics, effects of natural feed
treat digestive disorders and diarrhoea of cats and dogs3,4 and additions – including humates or HS – to poultry diets on meat
on humates, including those in feed and water of poultry, to quality characteristics and selected blood parameters, which are
promote growth,5 – 9 carcass yield and characteristics,8 – 11 and related directly to bird health and functional poultry products for
egg production.12 – 15 These studies have, however, shown that specialty poultry markets, should be researched.
the assumption that humates or HS might improve growth It has been reported that there are measurable changes
performance of poultry has not yet been confirmed, and thus in pH and colour parameters of breast and thigh meats in
a systematic approach to the efficacy and safety of compounds response to dietary humate supplementation10,12 and this can be
used as feed additives for poultry is still missing. considered in poultry production units for benefiting consumer
Colour, pH, and water-holding capacity (WHC) are important acceptance.12 However, there is incomplete knowledge on the
characteristics of meat quality that can affect consumer prefer- influence and potential physiological mode of action of HS
ences. Blood metabolites, such as glucose, protein, triglyceride,
high-density lipoprotein (HDL), low-density lipoprotein (LDL), and
total cholesterol concentrates, are very important in evaluating ∗
Correspondence to: Ergin Ozturk, Ondokuz Mayis Universitesi, Ziraat Fakultesi,
and interpreting the results of investigations related directly to Zootekni Bolumu Kurupelit, TR55139 Samsun, Turkey.
E-mail: [email protected]
animal health16 and meat quality.17 Moreover, including natural
additions to chickens’ diet, such as humates or HS, influences Department of Animal Science, Faculty of Agriculture, Ondokuz Mayis
59

the increase in absorption of mineral components which are University, TR55139 Samsun, Turkey

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 www.soci.org E Ozturk et al.

on quality characteristics and nutrients of meat, and mineral

Table 1. Ingredients of basal diet and calculated nutrient composi-
and metabolite contents of blood in broilers. Accordingly, the tion (g kg−1 , as fed basis)
objectives of the present study were twofold: first, to investigate
the growth-promoting effects of HS on broiler performance, Starter Grower Finisher
(1–21 days) (22–35 days) (36–42 days)
carcass and gastrointestinal tract (gut) traits; but the main aim was
to investigate the changes in colour characteristics (Commission Ingredients
Internationale de l’Éclairage (CIE) lightness (L∗ ), a∗ , and b∗ ), pH, Maize bran 400.0 400.0 400.0
and WHC values, in nutrients (dry matter, ash, protein, and fat) Yellow maize 134.0 150.0 185.0
of thigh and breast meats and selected blood minerals (calcium Full fat soybean 210.0 210.0 230.0
and phosphorus), and in metabolite (glucose, protein, triglyceride, Soybean meal (48%) 123.0 103.0 53.0
HDL, LDL and total cholesterol) contents as influenced by HS Meat-bone meal 68.0 69.5 74.0
included in the broiler diet. Fish meal 15.0 12.0 –
Wheat 41.0 40.0 40.0
Vegetable oil 1.37 7.40 11.10
MATERIALS AND METHODS Salt 1.5 1.5 1.5
For the trial, 480 day-old male broiler chicks (ROSS 308) with Vitamin premixa 2.5 2.5 2.0
a mean (±SEM) body weight of 53.6 ± 0.02 g per bird were Mineral premixb 1.0 1.0 1.0
allocated to four treatments each with five replicates containing DL-Methionine 1.35 1.80 1.30
24 birds. While one group was fed a diet with no additives (0 L-Lysine HCl 0.28 0.30 0.60
HS), other groups were fed diets with liquid HS at 0.5 (0.5 HS), 1.0 Sodium bicarbonate 0.5 0.5 0.5
(1 HS) or 1.5 (1.5 HS) g kg−1 until 42 days of age. In this study, Coccidiostat 0.5 0.5 –
HS produced in the common method of treatment of Leonardite Calculated nutrient compositionc
with concentrated alkalines were used. On a dry matter basis, Metabolizable energy 13.21 13.31 13.57
polymeric polyhydroxy acids, ballast silicon dioxide (SiO2 ) and ash (ME, MJ kg−1 )
contents of this product are 297, 393 and 310 g kg−1 respectively. Crude protein 220.0 210.0 190.0
The polymeric polyhydroxy acids, with a ratio of 2.08 humic acid to Lysine 13.0 12.0 11.0
fulvic acid, here are in active form. The Na, K, Ca, Mg, Fe, P, Mn, Cu, Methionine 5.0 4.6 4.2
Al and Zn contents of this product are 22.27, 8.49, 8.65, 6.59, 0.93, Methionine + cystine 9.2 7.2 6.0
0.17, 0.03, 0.02, 0.01 and 0.01 mg kg−1 respectively (Surtonic , Available phosphorus 5.2 5.2 4.8
Samsun, Turkey). All animals were housed in floor pens with wood Calcium 9.0 9.0 8.5
shavings. All birds were fed ad libitum a commercial diet for the a
Provided per kilogram of diet: retinyl acetate, 6 000 000 IU; cholecal-
starter (from day 1 to day 21), grower (from day 22 to day 35) and ciferol, 800 000 IU; DL-α-tocopheryl acetate, 8 000 mg; menadione,
finisher (from day 36 to day 42) periods (Table 1). All diets were 2000 mg; riboflavin, 1000 mg; D-calcium pantothenate, 4000 mg;
formulated to meet the minimum NRC19 requirements for broilers niacin, 10 000 mg; pyridoxine, 2000 mg; folic acid, 300 mg; D-biotin,
20 mg; thiamine, 3000 mg; cyanocobalamin, 8 mg; ascorbic acid,
and were provided as a mash feed. The liquid HS, measured by 20 000 mg; choline chloride, 400 000 mg.
measuring cylinder for each treatment, was sprayed on to a small b Provided per kilogram of diet: Mn, 80 000 mg; Zn, 60 000 mg; Fe,

amount of basal diet and then this premix was added to a sufficient 30 000 mg; Cu, 5 mg; I, 2000 mg; Co, 500 mg; Se, 200 mg.
c
amount of feed to achieve the desired final concentration. All diets Based on feed composition of NRC.19
were prepared daily.
During the trial, continuous lighting was provided. Ambient
temperature was gradually decreased from 33 ◦ C on day 1 to 21 ◦ C throughout the study using a white and pink ceramic tile. A white
on day 21 and was then kept constant. Health status was evaluated tile (L∗ = 92.30, a∗ = 0.32 and b∗ = 0.33) was used as standard.
daily, whereas live weight and feed intake were measured at 21 and Lightness could range from 100 (white) to 0 (black). Positive a∗ and
42 days of age. At 42 days of age, birds were starved for 8 h before b∗ values are a measure of redness and yellowness, respectively,
slaughtering and four birds (closest to the mean pen weight) per whereas negative a∗ and b∗ values indicated greenness and
replicate or 20 birds per treatment were commercially processed blueness. The pH was measured with a pH meter (Cyberscan
at the slaughterhouse of the agricultural faculty. Plucked and PC 510, Singapore) equipped with an insertion electrode (S175CD
eviscerated carcasses were weighed after removal of the head, Spear Tip; Sensorex, Garden Grove, CA, USA) calibrated with buffers
neck, feet and abdominal fat to obtain ready-to-cook carcasses at pH 4.01 and 7.01 (Mettler Toledo, Tampa, FL, USA) at ambient
and refrigerated for 6 h at 4 ◦ C. Yield from chilled carcasses was temperature. Three replicate measures were performed on breast
evaluated. Right breast (pectoralis major) and thigh (ilio tibialis) and thigh meats, representing the whole surface of the muscles;
samples were collected at 12 h post mortem for proximate analysis thus mean pH values were calculated for each sample. The WHC
and evaluation of pH and colour traits. These samples were then of meats was determined using a press technique explained by
vacuum packaged and kept frozen (−20 ◦ C) until proximate and Oztan and Vural21 with a slight modification. A sample (0.5 g for
analyses. each sample) was placed on a filter paper (Whatman No. 1, stored
The CIE colour20 values of meats representing lightness (L∗ ), for 24 h in a desiccator containing anhydride CaCl2 ), which was
redness (a∗ ) and yellowness (b∗ ) were measured at 12 h post set between two Plexiglas plates (Ildam Cam AS, Ankara, Turkey)
mortem using a Minolta CR 300 Chroma Meter (Minolta Camera, and pressed for 20 min by a 1 kg weight. The outline areas of
Osaka, Japan). Four replicate measures were made on the ventral the expressible juice and the pressed meat film were traced, and
(anterior) aspects of both the breast and thigh meats, representing the two areas were measured using a Placom Digital Planimeter
the whole surface of the muscles, and mean colour values (Model KP-90, Sokkisha Planimeter Inc., Kanagawa, Japan). WHC
60

were calculated for each sample. The colorimeter was calibrated was calculated as the ratio of meat film area to total juice area.

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Humic substances in broiler nutrition www.soci.org

Therefore, it should be noted that a higher expressible juice area RESULTS

was related to reduced (higher drip loss) WHC.22 Mortality during the study was low (1.6%) and deaths were
Proximate analysis was performed on the raw breast and thigh not associated with any specific treatment. Body weight gain,
meats. The breast and thigh samples were mashed before analysis feed consumption and feed : gain ratio of broilers receiving HS-
and analysed in duplicate for each replicate. Dry matter, protein, fat supplemented diets are presented in Table 2. Although HS at
and ash contents were determined by AOAC-approved methods23 the tested doses had no effect on body weight gain and feed
and results were expressed as percentages on a dry matter basis. efficiency only in the first 3 weeks (1–21 days of age) of fattening,
For analyses of blood parameters, at the end of the experiment body weight gain increased with 1.5 HS compared to 0 HS and
10 birds were randomly selected for each treatment (i.e. two 1 HS, and feed efficiency improved with 1.5 HS compared to 0
birds per replicate) and fasted overnight. Blood samples (40 in HS and 0.5 HS in the grower (22–42 days of age) and over the
total) were collected (about 4 mL) from the bronchial vein into entire period (1–42 days of age) of broiler chicken fattening. Feed
Vacutainer sterilized tubes (BD Bioscience, Franklin Lakes, NJ, USA) consumption decreased (P < 0.10) with 1.5 HS compared to 0 HS
containing heparin during slaughter at 42 days of age. Plasma was and 0.5 HS in the first 3 weeks of fattening and (P < 0.10) with
separated at 4000 rpm for 20 min and stored at −20 ◦ C until further 1 HS compared to 0.5 HS in the grower period of fattening. Also
analysis. The collected plasma samples were analysed for glucose, feed consumption decreased (P < 0.01) with 1 HS compared to 0
triglyceride, total cholesterol, HDL, LDL, calcium and inorganic HS and 0.5 HS and with 1.5 HS compared to 0.5 HS (P < 0.05) over
phosphorus using an automatic analyser (Airone-200, RA, Italy) the entire period of fattening. From 0 to 42 days of age for body
according to the recommendation of the manufacturer (Biolab, weight gain, feed consumption and feed efficiency, the effects
were linear, cubic and linear, respectively.
Maizy, France).
Means for the carcass weight, relative weight and length of gut
For performance data, pen means served as the experimental
and the relative weight of edible inner organs (gizzard + heart
unit for statistical analysis. For data of relative weights and
+ liver), gizzard, proventriculus and abdominal fat pad at 42 days
length of gut, meat quality traits, chemical composition, and
of age are shown in Table 3. The carcass weight and dressing
blood parameters, slaughtered individual birds were considered
percentage (76.8%, 77.1%, 76.6% and 77.7% for 0 HS, 0.5 HS, 1
as the experimental unit. All percentage data were transformed
HS and 1.5 HS) of broilers in 1.5 HS was higher (P < 0.05) than
by taking arcsine square roots prior to analysis. In order to those of 0 HS and 1 HS. The relative proventriculus weight and
evaluate statistically the measured data, one-way analysis of abdominal fat weight decreased by 1.5 HS and increased by 0.5 HS
variance (ANOVA) was performed in a completely randomized in relation to 0 HS (P < 0.10) respectively. Therefore, the effects for
design: Ŷij = µ + αi + eij , where Ŷij are observation values (body the carcass and relative proventriculus weights were linear, while
weight gain, feed consumption, feed to gain, carcass traits, colour the effect for the relative abdominal fat weight was cubic. HS at
measurements, blood parameters, etc), µ is the overall mean, αi the tested doses had no effect on the relative weight and length
is the effect of the ith treatment (i: 1, . . ., 4; 0 HS, 0.5 HS, 1 HS of gut or the relative weight of edible inner organs and gizzard
and 1.5 HS) and eij = residual error. Duncan’s multiple range (P > 0.05).
test was then utilized to separate these differences. Also, results The L∗ , a∗ and b∗ values and the WHC of thigh and breast
from feeding treatment diets 1–4 were analysed as an orthogonal meats, and pH value of breast meat, were affected by HS dietary
polynomial. Linear, quadratic and cubic effects were determined supplementation (Table 4). The 0.5 HS resulted in thigh meat
by orthogonal polynomial contrasts. For all tests, the level of colour that was lighter than that found in the other treatment
significance was set at 0.05. Trends are discussed when P-values birds (P < 0.01). Thigh meat from the 0.5 HS birds had higher
were <0.10. All the computational work was performed by means a∗ and b∗ values than that from 0 HS and 1 HS birds (P < 0.10)
of MINITAB.24 respectively. The effect for L∗ and a∗ of thigh meat was quadratic,

Table 2. Body weight, feed consumption and feed : gain ratio of broilers receiving HS-supplemented diets

Diet Effects

0 HS 0.5 HS 1 HS 1.5 HS SEM P L Q C

Body weight gain (g per bird)

0–21 days 909 909 896 900 4.7 NS NS NS NS
∗ ∗
21–42 days 1756b 1801ab 1779b 1871a 14.8 NS NS
∗ ∗
0–42 days 2718b 2764ab 2729b 2824a 16.2 NS NS
Feed consumption (g per bird)
† ∗
0–21 days 1265a 1270a 1249ab 1234b 12.4 NS NS
21–42 days 3875ab 3896a 3792b 3841ab 15.1 † NS NS ∗

0–42 days 5130ab 5155a 5007c 5064bc 17.1 ∗∗ NS NS ∗∗

Feed to gain (g g−1 )

0–21 days 1.40 1.40 1.40 1.37 0.026 NS NS NS NS
21–42 days 2.21a 2.17a 2.14ab 2.06b 0.019 ∗ ∗∗ NS NS
∗ ∗∗
0–42 days 1.89a 1.87a 1.84ab 1.80b 0.012 NS NS

Values are means of five pens of 24 birds. Means in the same row not sharing a common letter are significantly different (NS, P > 0.05; † P < 0.10;
∗ P < 0.05; ∗∗ P < 0.01). SEM, standard error of mean. L, linear; Q, quadratic; C, cubic.
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Table 3. Carcass weight (g) and cut-up parts (g or cm 100 g−1 body weight) of broilers receiving HS-supplemented diets

Diet Effects

0 HS 0.5 HS 1 HS 1.5 HS SEM P L Q C

Carcass weight 2093b 2147ab 2106b 2209a 15.8 ∗ ∗ NS NS

Relative weight of
Gastrointestinal tract (gut) 5.89 5.94 6.08 5.91 0.05 NS NS NS NS
Edible inners organs 3.54 3.60 3.69 3.60 0.03 NS NS NS NS
Empty Gizzard 1.15 1.07 1.09 1.08 0.02 NS NS NS NS
Proventriculus 0.41a 0.40ab 0.40ab 0.38b 0.01 † ∗ NS NS
Abdominal fat 2.17b 2.39a 2.27ab 2.25ab 0.03 † NS ∗ NS
Gut weight (g cm−1 ) 0.80 0.80 0.83 0.80 0.01 NS NS NS NS
Relative length of gut 7.38 7.41 7.34 7.34 0.05 NS NS NS NS

Values are means of 20 birds per treatment. Means in the same row not sharing a common letter are significantly different (NS, P > 0.05; † P < 0.10;
∗ P < 0.05). SEM, standard error of mean. L, linear; Q, quadratic; C, cubic.

Table 4. Colour measurements (L∗ , a∗ and b∗ ), pH and WHC of breast and thigh meats from broilers receiving HS-supplemented diets

Diet Effects

0 HS 0.5 HS 1 HS 1.5 HS SEM P L Q C

Thigh meat
L∗ , lightness 58.7b 60.4a 59.3b 59.2b 0.19 ∗∗
NS ∗∗
NS
a∗ , redness 4.64b 5.68a 4.94ab 4.85ab 0.16 NS NS ∗
NS
b∗ , yellowness 5.18ab 5.84a 4.80b 5.13ab 0.15 †
NS NS ∗

PH 6.58 6.65 6.64 6.61 0.02 NS NS NS NS

WHC 0.32b 0.41a 0.35ab 0.38ab 0.01 † NS NS ∗

Breast meat
L∗ , lightness 62.5ab 60.9b 61.3b 63.7a 0.30 ∗∗ NS ∗∗ NS
a∗ , redness 1.71b 2.03a 1.94ab 2.05a 0.08 †
NS NS ∗

b∗ , yellowness 4.97a 3.96b 4.57ab 5.31a 0.13 ∗∗

NS ∗∗
NS
† ∗
PH 6.05ab 6.07a 6.07a 6.00b 0.01 NS NS
† ∗
WHC 0.31b 0.34ab 0.39a 0.35ab 0.012 NS NS

Values are means of 20 birds per treatment. Means in the same row not sharing a common letter are significantly different (NS, P > 0.05; † P < 0.10;
∗
P < 0.05; ∗∗ P < 0.01). SEM, standard error of mean. L, linear; Q, quadratic; C, cubic.

while the effect for b∗ values of thigh meat was cubic. The 1.5 HS (Table 6). The plasma phosphorus concentration increased by 1.5
resulted in breast meat colour that was lighter than that found HS in relation to 1 HS (P < 0.05); also, plasma total cholesterol
in 0.5 HS and 1 HS birds (P < 0.01). Breast meat from 0 HS birds level decreased by 1 HS and 1.5 HS compared to 0 HS (P < 0.01).
had lower (P < 0.10) a∗ value than that from 0.5 HS and 1.5 HS The HDL and LDL levels decreased with 1 HS and 0.5 HS compared
birds, while breast meat from 0.5 HS birds had lower (P < 0.05) to 0 HS (P < 0.10) respectively, and as a result, plasma HDL : LDL
b∗ value than that from 0 HS and 1.5 HS birds. The effects for L∗ cholesterol ratio (0.74, 0.77, 0.63 and 0.73 for 0 HS, 0.5 HS, 1 HS
and b∗ values were quadratic, while that for the a∗ value of breast and 1.5 HS) was the lowest and highest in the 1 HS and 0.5 HS
meat was cubic. The pH value of breast meat decreased with 1.5 groups, respectively. The effect of feeding with HS was quadratic
HS in relation to 0.5HS and 1 HS (P < 0.10) and this effect was for plasma phosphorus concentration and HDL level, linear for
quadratic. Thigh meat from 0.5 HS and breast meat from 1 HS birds total cholesterol level and cubic for LDL level.
had higher (P < 0.10) WHC value in relation to 0 HS, respectively.
The effect of feeding with HS was cubic for the WHC of both thigh
and breast meats. Table 5 shows that 1 HS decreased the protein DISCUSSION
content of thigh meat in relation to 0 HS and 1.5 HS, and the The results of the present study show that feeding with diets
protein content of breast meat compared to 0 HS (P < 0.10), and containing HS caused a measurable variation in the meat
that the ash content of breast meat increased by 0.5 HS in relation quality characteristics and blood cholesterols level, as well as
to 1.5 HS (P < 0.10), while the ash content of thigh meat increased the performance, carcass and gut traits of broilers, in which the
by 0.5 HS in relation to 0 HS and 1.5 HS (P < 0.10). For the ash 1.5 g kg−1 level tended to be more effective. The main objective of
contents of thigh and breast meats, the effects were quadratic. using these substances in broiler diets6,8,9,12 or drinking water10,25
No significant difference was observed in plasma calcium, is to improve their feed efficiency as well as body weight.8,10,12,25
62

protein, triglyceride and glucose levels among the treatments However, these effects of HS may be variable because of differences

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Table 5. Chemical composition (% of fresh matter) of breast and thigh meats from broilers receiving HS-supplemented diets

Diet Effects

0 HS 0.5 HS 1 HS 1.5 HS SEM P L Q C

Thigh meat
DM 29.95 29.99 30.07 30.10 0.058 NS NS NS NS
∗ ∗
Protein 17.50a 17.36ab 17.11b 17.59a 0.061 NS NS
Fat 11.19 11.26 11.43 11.36 0.091 NS NS NS NS
† ∗
Ash 1.11b 1.15a 1.14ab 1.11b 0.006 NS NS
Breast meat
DM 26.46 26.47 26.55 26.36 0.047 NS NS NS NS
Protein 22.01a 21.77ab 21.59b 21.79ab 0.058 † NS ∗∗ NS
Fat 2.81 2.75 2.67 2.76 0.028 NS NS NS NS
Ash 1.05ab 1.01b 1.03ab 1.06a 0.008 † NS ∗ NS

Values are means of 20 birds per treatment. Means in the same row not sharing a common letter are significantly different (NS, P > 0.05; † P < 0.10;
∗ P < 0.05; ∗∗ P < 0.01). SEM, standard error of mean. L, linear; Q, quadratic; C, cubic.

Table 6. Selected plasma minerals and metabolites (mg dL−1 ) of blood from broilers receiving HS-supplemented diets

Diet Effects

0 HS 0.5 HS 1 HS 1.5 HS SEM P L Q C

Selected minerals
Calcium 8.4 8.2 9.0 8.9 0.19 NS NS NS NS
∗ ∗
Phosphorus 5.2ab 5.4ab 5.1b 5.8a 0.13 NS NS
Selected metabolites
Glucose 239.9 231.6 231.5 243.3 4.15 NS NS ∗ NS
Protein 552.1 554.2 563.0 546.1 0.07 NS NS NS NS
Triglyceride 118.1 120.0 121.8 119.8 1.45 NS NS NS NS
∗∗ ∗∗
Cholesterol 148.8a 141.3ab 132.9b 132.8b 1.98 NS NS
† ∗
HDL 49.6a 46.7ab 41.5b 46.9ab 2.31 NS NS
† ∗ ∗
LDL 67.3a 60.8b 65.5ab 64.4ab 1.79 NS

Values are means of 20 birds per treatment. Means in the same row not sharing a common letter are significantly different (NS, P > 0.05; † P < 0.10;
∗ P < 0.05; ∗∗ P < 0.01). SEM, standard error of mean. L, linear; Q, quadratic; C, cubic.

in the inclusion level and form (water or feed), in characteristics (e.g. study were kept in a clean environment, possibly not leading to
chain length and composition, hydrophobicity) and in origin (e.g. diminished efficacy, if any, of the dietary additives.26 – 28
plant, soil, peat, coal) of organic acids, including humic acid and/or The improvement in weight gain achieved with HS supplement
digestibility of the diet used.10,25 The improvement in body weight at 1.5 g kg−1 to the diet was reflected in the dressed birds’ weight
gain and feed efficiency of birds fed a diet containing 1.5 g HS or dressing percentage. This improvement may be attributed to
kg−1 shows that the use of these products is a feasible alternative increased nutrient absorption and feed intake, as was evident
to antimicrobial feed additives used as growth promoters. These with improvements in feed conversion ratio compared to 0 HS
findings are in general agreement with previous studies using and 0.5 HS, respectively. However, it was not clear whether HS
humic acid in broiler chickens diets.6,8,12 improves nutrient absorption due to lack of significant difference
The result with respect to mortality is in agreement with previous in plasma protein, triglyceride and glucose levels between
studies,7,8,10,25 indicating that there was no alteration of mortality treatments. Moreover, no significant difference was observed
in humic acid-treated groups. These results show also that there in plasma protein levels between the treatments, although the
was no clear effect of HS or humic acid on health-related problems blood ammoniac and plasma uric acid concentrations were
in broiler chickens, as reported by Ozturk et al.10 Furthermore, we correlated with protein intake.29 Reduction in passage rate of
investigated the effects of drinking water with humic acids on ingesta through the gut and enhanced pancreatic enzymatic
caecal coliform bacteria count under the housing conditions used activity leads to increased digestibility of the diet, which can in
in the present study.25 In this study we found that counts of aerobic turn improve feed efficiency.10,27,28 The beneficial effect of feeding
mesophilic bacteria (4.0%), M17 lactococci (3.3%), coliforms (4.8%) a diet with HS at 1.5 g kg−1 on the relative proventriculus weight
and Escherichia coli (5.3%) tended to be decreased by humic acid compared to feeding a diet without HS may result from the
supplementation at doses of 25.5 mg kg−1 body weight provided acidifier effect of HS,30 and this can result in significant savings.
through drinking water. Therefore, the results with respect to Furthermore, the differences observed in carcass yields may be
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mortality and blood parameter indicate that broilers in the present related to amount of nutrients ingested by the birds fed with

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HS10,12 due to the fact that the feed intake was similar among physiology of the bird when the diet is limiting in phosphorus.18
treatments. Thus it may be explained why supplement of HS at 1.5 g kg−1
Meat quality is related directly to stress and energy metabolism to the diet produced higher weight gain and feed efficiency.
because a reliable indicator of stress and energy metabolism31,32 However, Rath et al.7 reported that there was a trend for reduction
in chickens is blood metabolites such as glucose, triglyceride and in the plasma concentrations of calcium and phosphorus by
total nitrogen. Therefore, one of the primary aims of the present humic acid, contrary to the augmentation in plasma phosphorus
study was to investigate the changes in colour characteristics (CIE concentration at the higher level of humic acid offered in the
L∗ , a∗ and b∗ ) of pH, WHC and chemical compositions of both present study. Such a case may be due to facilitatory action
breast and thigh meats, and in blood metabolites and minerals as in inorganic ion transport through biological membranes or a
influenced by feeding with diets containing HS at different levels. metal-chelating effect of humic acids, which is affected by a large
The conducted colour evaluation of thigh and breast muscles of number of carboxylic acid side chains and the mineral content of
the chickens demonstrated that addition of HS to the diet may HS used in the present study.7,10,36 Therefore, it may be said that
not only influence breeding effectiveness, but also the colour HS offers advantages on growing performance due to its effects
of their carcasses, as reported by Esenbuga et al.11 and Ozturk of carbon mineral source on feed efficiency, and of antimicrobial
et al.10,25 Despite augmentation in the L∗ value of thigh muscle and/or of acidifier in maintaining the gut health of broilers.26,30
from the supplement at 0.5 g kg−1 in comparison to the control, The effects observed in relation to the nature of HS used in the
according to CIE lightness values of meats in poultry,33 the L∗ present study and its presumed mode of action may be related to
values for all treatments in the current study were not in a normal the reported properties of HS,5,7,36 such as general improvement
range and would be considered excessively pale. However since of physiological processes, stimulation of immune response, and
the redness (index) increased by HS, which are inherently brown impairment of pathogens by reducing their capacity to adhere
to dark colour, it was possible to speak about colour darkening. and penetrate.1 – 4
Darkening in thigh muscles and an increase in their redness might At high energy demands adenosine triphosphate (ATP) will also
indicate an increase in haem pigments because the red colour in be formed anaerobically, resulting in formation of lactate. This
meat is due mainly to a protein pigment called myoglobin and, to influences muscle pH, which is an important parameter for meat
a lesser extent, haemoglobin.12 Other intrinsic factors (e.g. muscle quality.17,31,33 A similar pattern in the pH, glucose and triglyceride
and pH) can also influence meat colour.17 Our result with regard to levels indicated that there was no difference in the amount of
a∗ values may resulted from differences in iron content of breast stress exposed in each treatment. Indeed it has been reported
and thigh meats, because HS provided through drinking water that dietary inclusion by organic acids had no significant effect
enhanced the iron content of broiler meat.10 Such a case may on the serum levels of cholesterol, glucose and protein.27 In the
be important for coloration of breast and thigh meats in broilers present study, the blood protein levels of the treatments indicate
because it affects acceptability for human consumption, and is that there was no effect of HS on the acute phase response
an important feature for poultry viscera inspection.12 The colour and/or proteins, which is the modulation of the immune system of
of muscle tissue was lighter from chickens with a lower pH, in the broiler chicken.37 Reducing the amount of cholesterol in the
accordance with other studies on broilers in which final pH was diet helps lower blood cholesterol level.38,39 Increased HDL/LDL
determined.17 The results with respect to proximate analysis of cholesterol ratio in the HS-supplemented group indicate that HS
the thigh and breast meats confirmed the proposition of Qiao supplementation in the diet may have beneficial effects on the
et al.,33 who reported that there were no significant differences in blood lipid profile. Therefore, it may be said that meats from
moisture and lipid contents among broiler breast meats selected broilers fed with diets containing HS are suitable for human health
as lighter or darker than normal, but not in protein content. and for specialty poultry markets due to their total cholesterol
Generally, pH is a direct reflection of muscle acid content, and LDL levels, although the effects observed were too small and
which affects drip loss and meat colour.18 In the present study inconsistent between HS treatments.
we observed a measurable influence of HS on breast muscle
colour due to the fact that the L∗ value is important in white
muscles. The dark colour of HS may be possibly be given a better CONCLUSIONS
L∗ value. Indeed the addition of HS to the diets of broilers had When compared to no HS, feeding a diet containing HS at
no effect on thigh muscle pH, and thus a∗ and b∗ values of this 1.5 g kg−1 increased the weight gain, feed efficiency, carcass
meat appeared variable compared to the control. Lower WHC in weight and relative proventriculus weight without affecting feed
muscles can induce liquid outflow, and loss of soluble nutrients consumption, and decreased the total cholesterol level of blood in
and flavour. Therefore, meat becomes dry, hard and tasteless. The broilers. Feeding a diet containing HS at 1.0 g kg−1 decreased the
pH is important in determining the WHC of meat. The addition feed consumption, WHC value and lightness of breast meat, the
of HS to the diet not only influenced pH, but also the WHC value protein content of both thigh and breast meats, and also the total
in breast meat samples. However, it has been reported that the cholesterol and HDL levels. Feeding with a diet containing HS at
overall lower final pH did not result in an overall decrease in 0.5 g kg−1 increased the WHC value and lightness of thigh meat
WHC.34 This impact was not reflected in thigh meat because of a and the relative abdominal fat weight, and decreased LDL level.
difference in muscle fibre type, and also due to the fact that breast Although the growth-promoting effects of HS used in the present
muscle is more sensitive than thigh muscle.35 study were not substantial, the findings suggest that HS may be a
Calcium and phosphorus have important roles in the structure potential feed additive used to improve meat quality of poultry.
and metabolism of bone. Phosphorus also plays an important
role in energy accumulation. In the current study, because
the supplement of HS at 1.5 g kg−1 to broiler diets increased ACKNOWLEDGEMENTS
the plasma phosphorus concentration, it may have a beneficial This study was supported by the Research Fund of Ondokuz
64

influence on the level of phosphorus, being so significant for the Mayis University (Z-484), approved by the local Ethical Committee

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c 2011 Society of Chemical Industry J Sci Food Agric 2012; 92: 59–65
Humic substances in broiler nutrition www.soci.org

of Ondokuz Mayis University for Experimental Animals, which phosphorus, magnesium and iron levels in broiler chicken serum.
ascertained that the experiment was not an unnecessary repetition Elect J Polish Agric Univ 8:#22 (2005).
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