Tropical Animal Health and Production (2021) 53:317

https://doi.org/10.1007/s11250-021-02758-4

REGULAR ARTICLES

Interaction effects of sunflower oil and aflatoxin at graded levels

in diet on performance, serum and tissue biochemical profile, organ
weights and immuneresponse in broiler chicken
M. V. L. N. Raju 1 & S. V. Rama Rao 1 & A. K. Panda 1

Received: 24 September 2020 / Accepted: 2 May 2021

# The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
The dietary supplementation of fat has great potential in countering the toxic effects of aflatoxin (AF) in chickens, but the issue
was less researched upon. An experiment was conducted to evaluate the response of broiler chickens to graded levels of AF B1
(0, 150 and 300 ppb) and sunflower oil (SFO) (0, 1.5 and 3.0%) in the diet in a 3 × 3 factorial manner to understand their
interaction effects. A total of 360 broiler chickens divided into 9 equal groups were fed the diets during 0 to 35 days of age, and
their response was evaluated in terms of performance, serum biochemical profile, organ weights, liver fat content and bone
mineralization. Sunflower oil at 1.5% in diet countered (P ≤ 0.01) the adverse effects of 150 ppb AF on body weight, whereas at
300 ppb AF, such a response was seen at the higher level (3%) of SFO. Aflatoxin decreased (P ≤ 0.01) feed intake by 4 and 11%
at 150 and 300 ppb concentration, respectively at 35 days of age, which was increased (P ≤ 0.01) with each incremental level of
SFO supplementation (by 3.0 and 8.8%, respectively at 1.5 and 3%, respectively). Serum protein concentration increased (P ≤
0.01) by SFO supplementation only at the higher concentration (300 ppb) of AF (by 42.4%), whereas total cholesterol and
triglyceride concentration, and immune response to SRBC inoculation increased (P ≤ 0.01) with SFO at either level of AF (by
16.8, 18.7 and 75.6% at 1.5% SFO and 33.1, 36.9 and 94.2% at 3.0% SFO, respectively at 35 days of age). Weights of the liver,
giblets, kidneys and pancreas increased (P ≤ 0.01) by 23.2, 14.7, 34.2 and 16.9%, respectively, and thymus weight decreased (P ≤
0.04) by 25.4% with 300 ppb AF, and SFO at 3% in diet countered the effect on weight of the liver and giblets. Fat deposition in
the liver increased (P ≤ 0.01) as the concentration of AF increased in diet (by 9.4 and 17.3%, respectively at 150 and 300 ppb AF),
which was significantly (P ≤ 0.05) countered by SFO at 3% in diet. Tibia bone Ca content increased by 2.4% (P ≤ 0.01) with SFO
supplementation in AF-fed chickens. It is concluded that dietary SFO supplementation countered the adverse effects of AF in
broiler chicks in a dose-dependent manner, and higher level of oil (3% in diet) was required at the higher concentration of AF
(300 ppb) in diet.

Keywords Aflatoxin . Sunflower oil . Broiler chicken diet . Interactions

Introduction (Pandey and Chauhan 2007; Khan et al. 2014) chickens.

Dietary manipulations have great potential in countering the
Aflatoxin (AF) is widely prevalent in grains and finished negative effects of AF on chickens. The high dietary concen-
feeds, particularly from the tropical and sub-tropical regions tration of protein (Chen et al. 2016) and supplementation of
of the world (Gruber-Dorninger et al. 2019). Aflatoxin is vitamin E, Se (Ulaiwi 2018), probiotics (Salem et al. 2018;
known to adversely affect the health and performance of broil- Liu et al. 2018), herbal extracts or derived agents (Khan et al.
er (Raju et al. 2005; Kurniasih and Prakoso 2019) and laying 2012; Tufarelli et al. 2016; Rajput et al. 2017; Prakoso et al.
2018) and antimicrobials like carvacrol, and trans-
cinnamaldehyde (Yin 2017) were found beneficial in reducing
* M. V. L. N. Raju the toxic effects of AF in broiler chickens. Besides, supple-
[email protected] mentation of agents that could adsorb mycotoxins (Liu et al.
2018; Saminathan et al. 2018; Jahanian et al. 2019) or bio-
1
ICAR-Directorate of Poultry Research, Rajendranagar, transform them into non-toxic metabolites (Raju et al. 2007;
Hyderabad, Telangana 500030, India Tejada-Castaneda et al. 2008) has received considerable
 317 Page 2 of 12 Trop Anim Health Prod (2021) 53:317

attention worldwide, and it is the most successful strategy that provide nutrients as per NRC (1994), except energy, and for
is being followed commercially by the poultry producers. meeting the nutrient requirements during the starter (0–21
Some of these strategies have yielded satisfactory results and days) and finisher (22–35 days) phases (Tables 1 and 2).
are being practically followed for countering the adverse ef- The individual feed ingredients were analysed for crude pro-
fects of AF either fully or partially. tein (AOAC 2016, 954.01), calcium (AOAC 2016, 927.02)
Aflatoxin causes imbalanced lipid metabolism and pro- and phosphorus (P) (AOAC 2016, 965.17) content. The
motes lipid deposition in the enlarged liver (Siloto et al. phytin P content in the feed ingredients was analysed
2013). In addition, AF decreases the secretion of pancreatic (Haugh and Lantzsch 1983), and the non-phytate P was cal-
lipase and bile (Osborne and Hamilton 1981; Matur et al. culated as the difference between the total P and phytate P
2010; Ditta et al. 2018) and thus impairs lipid digestion in content. The corresponding content of these nutrients in the
chicken causing steatorrhea and reduced availability of dietary compounded feed was arrived at by summing up the analysed
fat to the bird. Supplementation of fat, either from animal or values for the respective feed ingredients that constituted the
vegetable origin, in the diet could therefore offer the possibil- diet. The contents of ME and amino acids in the diets were
ity of overcoming this negative effect of AF in chickens, calculated using the values of the respective feed ingredients
which was however not given the desired attention. The few from the reference tables (Mandal et al. 2013).
studies conducted in the past indicated beneficial effects of
supplementation of olive oil or safflower oil at 160 g/kg in Chicks and rearing
chickens (Smith et al. 1971) and cottonseed oil at 180 g/kg in
turkeys (Hamilton et al. 1972) in countering the adverse ef- A total of 360 day-old commercial broiler chicks (Cobb 400)
fects of AF on some of the performance and serum biochem- were divided into nine equal groups, having 8 replicates of 5
ical variables. The toxic dose of AF was also reported to chicks each and housed in 3-tiered SS battery brooders in an
increase with supplemental fat in the diet (Richardson et al. open-sided house. The chicks in each group were fed ad
1987; Schaeffer and Hamilton 1990). In a previous study con- libitum one of the test diets from 0 to 35 days of age. The
ducted at this lab (Raju et al. 2005), dietary supplementation chicks in all the groups were reared under uniform manage-
of sunflower or soybean oils (at a fixed concentration of 30 mental conditions. Brooding was done up to 28 days of age
g/kg diet) totally countered the adverse effects of 300 ppb AF using incandescent bulbs. The chicks were vaccinated through
B1 on broiler chicken, whereas groundnut oil showed margin- ocular route against Newcastle disease (on 5th and 30th days
al beneficial effects. The results of the cited study (Raju et al. with LaSota strain) and infectious bursal disease (on 14th and
2005) and few other unreported studies of this lab indicated 24th days with intermediate [Georgia] strain).
that the source of oil was critical in obtaining the desired
beneficial effects, and sunflower oil (SFO) was an effective Aflatoxin
fat supplement in diet for countering the adverse effects of AF
in broiler chickens, probably due to its high concentration of The AF used in the trial was produced in the laboratory by
unsaturated fatty acids. Accordingly, SFO was chosen for the solid substrate fermentation on broken rice using Aspergillus
current study, which was conducted to evaluate graded dietary parasiticus (NRRL 2999) as per Shotwell et al. (1966). The
levels of AF and SFO in a factorial manner so as to understand aflatoxin B1 content of the dried culture was quantified
their interaction effects in broiler chicken. (AOAC 2016, 971.22) employing HPTLC (Linomat 5 and
Scanner 3, Camag, Switzerland). The required quantity of
steamed and dried rice culture was added to the diet to arrive
Materials and methods at the desired concentration of AF B1 in the test diets.

Experimental diets Data collection

A total of nine experimental diets were formulated to contain The influence of test diets on chicks was assessed in terms of
AF B1 (0, 150 and 300 ppb) and SFO (0, 1.5 and 3%) at 3 weekly body weights, feed intake and feed conversion effi-
levels each in a 3 × 3 factorial manner. These test doses of AF ciency (feed/gain). At 21 and 35 days of age, blood was col-
and SFO were selected based on the trends observed in the lected from six chicks, chosen at random from different repli-
previous study (Raju et al. 2005) and other unreported studies cates in each treatment group, and serum was separated and
of this lab. The sunflower oil used in the study was of food analysed for the concentration of total protein, total cholester-
grade (Vijaya Sunflower oil, Telangana State Co-Operative ol and the fractions of cholesterol using reagent kits
Oilseeds Growers Federation Ltd., Hyderabad, India). The (Qualigens, Mumbai, India). The reagent kits used for deter-
diets were maintained isocaloric and isonitrogenous by alter- mining the serum protein concentration were designed based
ing the levels of maize, DORB and soybean meal so as to on the principle that protein reacts with copper II ions in
Trop Anim Health Prod (2021) 53:317 Page 3 of 12 317

Table 1 Percent composition of starter diets (0–21 days of age)

Diet no. 1 2 3 4 5 6 7 8 9
SFO, % 0 1.5 3.0 0 1.5 3.0 0 1.5 3.0
Aflatoxin, ppb 0 0 0 150 150 150 300 300 300
Maize 59 53.2 47.58 59.15 53.2 47.58 59 53.49 47.58
DORB 0.3 4.83 9 0.08 4.76 8.93 0.16 4.3 8.86
Sunflower oil 0 1.5 3 0 1.5 3 0 1.5 3
Soybean meal 37 36.8 36.8 37 36.8 36.8 37 36.9 36.8
Shell grit 0.98 1.05 1.1 0.98 1.05 1.1 0.98 1.05 1.1
DCP 1.85 1.75 1.65 1.85 1.75 1.65 1.85 1.75 1.65
Constants* 0.8665 0.8665 0.8665 0.8665 0.8665 0.8665 0.8665 0.8665 0.8665
Aflatoxin** 0 0 0 0.07 0.07 0.07 0.14 0.14 0.14
Nutrients
ME, kcal/kg# 2885 2883 2885 2886 2881 2884 2882 2885 2882
Crude protein## 23.00 22.99 23.05 22.98 22.98 23.04 22.98 23.00 23.04
Calcium## 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91
Non-phytin P## 0.451 0.449 0.447 0.450 0.449 0.446 0.450 0.447 0.446
Lysine# 1.23 1.23 1.24 1.23 1.23 1.24 1.23 1.23 1.24
Methionine# 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54
Total SAA# 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90

*Contained per kg diet: NaCl 4 g, MnSO4 0.3 g, ZnSO4 0.2 g, FeSO4 0.4 g, CuSO4 35 mg, KI 0.5 mg, retinol acetate 9.08 mg, cholecalciferol 0.06 mg,
riboflavin 10 mg, vitamin K 2 mg, thiamine 1 mg, pyridoxine 1.6 mg, cyanocobalamine 8 μg, calcium pantothenate 8 mg, niacin 12 mg, choline chloride
1.0 g and coccidiostat (DOT) 0.5 g
**Culture having 217 ppm AF B1
#
Calculated
##
Analysed
SFO, sunflower oil

alkaline medium to form blue-violet complex. The absorbance petroleum ether for 24 h and assayed for breaking strength
of this blue-violet complex was read at 546 nm using a double (EZ test, Shimadzu, Japan), and calcium (AOAC 2016,
beam UV-visible spectrophotometer (MUV-61PCS, Metsar 927.02) and phosphorus (AOAC 2016, 965.17) content.
Technologies, Hyderabad, India). The concentration of total
cholesterol in serum was analysed employing the Trinder’s
method using cholesterol esterase, and the absorbance was Statistical analysis
read at 505 nm. The humoral immune response of the
chickens to sheep red blood cells (SRBC) was assessed at 21 A priori power analysis was done using G*Power software
and 35 days of age. For this purpose, 0.1 ml of 0.5% suspen- (version 3.1.9.7). The desired sample size was accordingly
sion of SRBC in normal saline was injected intravenously into calculated to be 6 birds/treatment for the variables involving
six birds in each experimental group, and the antibody titres bleeding and slaughter. The alpha value and the accepted
(log2) in serum were estimated at 5- and 10-day post-inocu- power considered were 0.05 and 0.8, respectively. Data ob-
lation (DPI) employing micro-titre haemagglutination proce- tained were subjected to statistical analyses (Snedecor and
dure (Wegmann and Smithies 1966). At the end of the trial, Cochran 1968) in a 3 × 3 factorial design (aflatoxin and sun-
six birds, selected at random from each treatment group, were flower oil) using general linear model of SPSS software
slaughtered by cervical dislocation, and the data on dressing (Version 23.0, SPSS Inc., Chicago, USA). The concentration
yields and weights of visceral and lymphoid organs were col- of AF and level of SFO in diet were the main (fixed) factors.
lected. The abdominal fat was collected from each bird The statistical model of variance analysis used was as follows.
slaughtered as per Fancher and Jensen (1989). A piece of liver Yij ¼ μ þ Fi þ T j þ ðFTÞij þ eij
and breast muscle from each of the slaughtered birds was
analysed separately for fat (AOAC 2016, 920.39) and protein where μ is the overall mean, Fi is the fixed effect of AF
(AOAC 2016, 954.01) content. The right tibia was collected concentration in diet, Tj is the fixed effect of dietary level of
from each slaughtered bird, weighed, defatted by soaking in SFO and the (FT)ij is the effect of interaction between AF
 317 Page 4 of 12 Trop Anim Health Prod (2021) 53:317

Table 2 Percent composition of

finisher diets (22–35 days of age) Diet no. 1 2 3 4 5 6 7 8 9
SFO, % 0 1.5 3 0 1.5 3 0 1.5 3
Aflatoxin, ppb 0 0 0 150 150 150 300 300 300
Maize 66.68 60.94 55.24 66.6 60.94 55.34 66.53 60.94 55.44
DORB 0 4.47 8.91 0 4.4 8.64 0 4.32 8.46
Sunflower oil 0 1.5 3 0 1.5 3 0 1.5 3
Soybean meal 29.6 29.4 29.2 29.6 29.4 29.3 29.6 29.4 29.3
Shell grit 0.98 1.04 1.1 0.98 1.04 1.1 0.98 1.04 1.1
DCP 1.9 1.81 1.71 1.9 1.81 1.71 1.9 1.81 1.71
DL-methionine 0.166 0.163 0.163 0.166 0.163 0.163 0.166 0.163 0.163
Constants* 0.6735 0.6735 0.6735 0.6735 0.6735 0.6735 0.6735 0.6735 0.6735
Aflatoxin** 0 0 0 0.077 0.077 0.077 0.154 0.154 0.154
Total 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
Nutrients
ME, kcal/kg# 2956 2955 2954 2953 2954 2955 2951 2952 2955
Crude 20.02 20.02 20.01 20.02 20.01 20.03 20.01 20.00 20.01
protein##
Calcium## 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90
Non-phytin 0.449 0.449 0.447 0.449 0.449 0.446 0.449 0.448 0.445
P##
Lysine# 1.03 1.04 1.04 1.03 1.04 1.04 1.03 1.04 1.04
Methionine# 0.48 0.47 0.47 0.48 0.47 0.47 0.48 0.47 0.47
Total SAA# 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80

*Contained per kg diet : NaCl 4 g, MnSO4 0.3 g, ZnSO4 0.2 g, FeSO4 0.4 g, CuSO4 35 mg, KI 0.5 mg, retinol
acetate 9.08 mg, cholecalciferol 0.06 mg, riboflavin 10 mg, vitamin K 2 mg, thiamine 1 mg, pyridoxine 1.6 mg,
cyanocobalamine 8 μg, calcium pantothenate 8 mg, niacin 12 mg, choline chloride 1.0 g and coccidiostat (DOT)
0.5 g
**Culture having 195 ppm AF B1
#
Calculated
##
Analysed
SFO, sunflower oil

concentration and SFO level in diet, while eij is the random concentration of AF (150 ppb), SFO supplementation at both
error. The replicate group in each pen was considered as the the levels significantly (P ≤ 0.01) increased body weight, but
experimental unit for analysing the data on body weight and at higher concentration (300 ppb) of AF, improvement in
feed efficiency, whereas for the other variables, the data of body weight was observed only at the higher level (3%) of
individual bird were considered for analysis. The means were SFO. Body weight in the group fed 300 ppb AF and 3% SFO
compared by Duncan’s multiple range test (Duncan 1955), was similar to that of the control group, while no effect was
and the differences among treatment means were considered observed by supplementing SFO at 1.5% level to the diet
significant only when the P was ≤ 0.05. containing 300 ppb AF. Aflatoxin significantly (P ≤ 0.01)
depressed feed intake only at 300 ppb AF at 21 days, while
at 35 days, feed intake decreased significantly (P ≤ 0.01) with
Results each concentration of AF. Supplemental SFO increased feed
intake similarly at both the levels at 21 days of age, but at 35
Performance of chickens days, the feed intake increased with each incremental level of
SFO. No significant interactions were found between AF and
The main effects of AF and SFO on performance at 21 days of SFO for their effects on feed intake. Feed conversion ratio at
age indicated significantly (P ≤ 0.01) lower body weight with both the ages was unaffected by AF and SFO and their inter-
AF at the higher concentration (300 ppb) and higher body actions, except for decreased FCR recorded with AF at 21
weight at both the levels of SFO (Table 3). The interaction days of age (main effect). Sunflower oil supplementation to
effects between AF and SFO levels on body weight were the control diet did not show any effect on body weight and
significant (P ≤ 0.01) at 35 days of age. At lower feed efficiency.
Trop Anim Health Prod (2021) 53:317 Page 5 of 12 317

Table 3 Body weight, feed intake

and feed conversion ratio (FCR, AF, ppb SFO, % Body wt., g Feed intake, g FCR (feed consumed, g/ body weight gain,
unit feed consumed/unit body g)
weight gain) of broiler chicken as
affected by dietary concentration 21 d 35 d 0–21 d 0–35 d 0–21 d 0–35 d
of aflatoxin (AF) and sunflower
oil (SFO) 0 0 747 1749ab 930.6 2828 1.34 1.67
0 1.5 779 1743ab 969.4 2862 1.33 1.69
0 3.0 782 1812a 975.8 2998 1.34 1.70
150 0 732 1586cd 905.7 2674 1.33 1.74
150 1.5 787 1714ab 951.5 2808 1.29 1.69
150 3.0 784 1722ab 951.2 2859 1.30 1.71
300 0 657 1468d 797.9 2440 1.31 1.72
300 1.5 706 1515d 864.1 2507 1.32 1.72
300 3.0 752 1679bc 917.6 2780 1.31 1.71
SEM 6.74 16.13 5.971 13.99 0.003 0.007
Main effects
AF, ppb 0 769a 1768a 958.6a 2896a 1.34a 1.69
150 767a 1674b 936.1a 2780b 1.31b 1.71
300 705b 1554c 859.8b 2576c 1.32b 1.72
SFO, % 0 712b 1601b 878.1b 2647c 1.33 1.70
1.5 757a 1657b 928.3a 2726b 1.32 1.71
3.0 772a 1738a 948.2a 2879a 1.32 1.71
Probability
AF 0.01 0.01 0.01 0.01 0.01 0.18
SFO 0.01 0.01 0.01 0.01 0.17 0.86
AF × SFO 0.27 0.03 0.21 0.07 0.16 0.33

Means having similar superscripts in a column do not differ significantly (Main effects : P ≤ 0.01, interaction : P ≤
0.05)
d, days

Serum biochemical profile cholesterol concentration in birds fed 150 ppb AF with SFO
supplementation at both the levels was higher than that in
Significant (P ≤ 0.01) interaction was recorded between AF control group, but at 300 ppb AF, such an effect was seen
and SFO levels for their effects on serum protein concentra- only at 3% SFO. In the control group, SFO supplementation
tion at 21 days, which increased significantly with SFO sup- increased the serum cholesterol concentration in a dose-
plementation only at the higher concentration of AF (Table 4). dependent manner. The trends observed with interaction ef-
At 35 days of age, no such interaction effects were observed, fects between AF and SFO on serum triglyceride concentra-
and the main effects of AF indicated that the serum protein tion at 21 and 35 days indicate significant effects of SFO only
concentration decreased significantly (P ≤ 0.01) at the higher at 300 ppb AF, and at the lower concentration of AF, the
concentration of AF, while the effect was marginal at 150 ppb serum triglyceride concentration was similar with 1.5 and
AF. 3% levels of SFO. Supplementation of SFO increased the
The interaction between AF and SFO levels for their effects serum concentration of total cholesterol and triglycerides,
on serum concentration of cholesterol and triglycerides was the effect being dose-dependent. The HDL, LDL and VLDL
significant (P ≤ 0.01). In comparison to the control, the serum fractions of cholesterol also showed similar response (data not
concentration of cholesterol and triglycerides was significant- shown).
ly (P ≤ 0.1) decreased by AF both at 21 and 35 days of age, the
effect being dose-dependent. Sunflower oil supplementation Immune response
raised the serum cholesterol concentration in the groups fed
AF, which was on par with that of control group at the higher The SRBC titres in both the groups fed AF at either con-
level (3%), but not at the lower (1.5%) level of supplementa- centration were significantly (P ≤ 0.01) low in compari-
tion at 21 days of age. At 35 days, on the contrary, the serum son to control at 5 days post-inoculation at 21 and 35 days
 317 Page 6 of 12 Trop Anim Health Prod (2021) 53:317

Table 4 Serum biochemical

profile of broiler chicken as AF, ppb SFO, % Proteins, g% Total cholesterol, mg/dl Triglycerides, mg/dl
affected by dietary concentration
of aflatoxin (AF) and sunflower 21 d 35 d 21 d 35 d 21 d 35 d
oil (SFO)
0 0 2.42b 3.15 126.9c 146.5d 113.4c 77.2cd
b b bc c
0 1.5 2.46 3.38 137.2 161.7 116.2 80.2bcd
0 3.0 2.81a 3.13 154.0a 181.0a 130.3b 105.8a
150 0 2.28b 2.69 103.2e 128.1e 103.5d 68.8e
150 1.5 2.55ab 3.17 114.4d 166.7b 114.0c 85.5b
150 3.0 2.49ab 3.22 123.2c 164.8b 115.8c 83.2bc
300 0 1.58c 2.80 84.21f 103.1g 92.94e 56.1f
300 1.5 2.25b 2.70 104.8e 112.4f 117.0c 74.3de
300 3.0 2.21b 2.96 124.8c 157.0c 143.0a 87.9b
SEM 0.05 0.05 2.32 3.04 1.80 1.66
Main effects
AF, ppb 0 2.56a 3.22a 139.4a 163.1a 119.9a 87.7a
150 2.44a 3.03ab 113.6b 153.2b 111.1b 79.2b
300 2.01b 2.82b 104.6c 124.2c 117.7a 72.8c
SFO, % 0 2.09b 2.88 104.8c 125.9c 103.3c 67.4c
1.5 2.42a 3.08 118.8b 147.0b 115.7b 80.0b
3.0 2.50a 3.10 134.0a 167.6a 129.7a 92.3a
Probability
AF 0.01 0.01 0.01 0.01 0.01 0.01
SFO 0.01 0.09 0.01 0.01 0.16 0.01
AF × SFO 0.01 0.09 0.01 0.01 0.01 0.01

Means having similar superscripts in a column do not differ significantly (P ≤ 0.01)

d, days

of age (Table 5), and no differences were observed be- diet, the effect however being statistically significant at
tween the 150 and 300 ppb concentrations of AF. On the the higher concentration of AF (300 ppb) (Fig. 2).
other hand, at 10 days post-inoculation, the SRBC titres Supplementation of SFO at 3.0% level alleviated the neg-
were not affected at 21 days of age, whereas at 35 days, ative effect of AF on giblets weight (Fig. 3). Weight of
the SRBC titres were significantly depressed only at the bursa, though not affected by AF, tended to increase with
higher (300 ppb) concentration of AF. Dietary supple- SFO supplementation (Fig. 4). Other parameters like
mentation of SFO either at 1.5 or 3.0% levels increased ready to cook yield and weights of gizzard, abdominal
the SRBC titres at both the concentrations of AF, i.e. 150 fat, spleen, adrenals and gall bladder were not affected
and 300 ppb at 21 and 35 days of age, and no differences by the test diets (data not shown).
were observed between the levels of SFO. Significant in-
teractions were observed between the level of AF and Tibia bone
SFO for their effects on SRBC titres. The adverse effects
of AF on SRBC titres were observed only in the absence Trends of main effects indicated that tibia bone weight de-
of SFO. creased (P ≤ 0.01) with AF feeding, whereas the bone-
breaking strength and concentration of Ca and P in tibia ash
Organ weights were not affected (Table 6). A significant interaction was ob-
served between AF and SFO levels for effects on bone Ca
Weights of the liver, giblets, kidney and pancreas in- content, which indicated that SFO supplementation at both
creased at the higher concentration (300 ppb) of AF (main the levels increased the bone Ca similarly in the groups fed
effects), while no such effect was observed at 150 ppb 150 ppb AF, but at 300 ppb AF, such a beneficial effect of
concentration of AF (Figs. 1 and 2). The weight of thy- SFO was recorded only at the higher level (3%) of
mus decreased as the concentration of AF increased in the supplementation.
Trop Anim Health Prod (2021) 53:317 Page 7 of 12 317

Table 5 SRBC tires (log2) as affected by dietary concentration of afla- Tissue fat and protein content
toxin (AF) and sunflower oil (SFO) in broiler chicken

AF, ppb SFO, % 21 days 35 days Significant (P ≤ 0.01) interaction was recorded between
AF and SFO for their effects on fat content in the skin,
5 DPI 10 DPI 5 DPI 10 DPI liver and thigh muscle (Table 7). Skin fat increased by AF
0 0 7.88a 5.50a 9.00bc 5.75ab
without SFO supplementation, but at 1.5 and 3% levels of
a bc ab SFO, the adverse effects of AF were nullified in a dose-
0 1.5 7.75 2.88 10.50 5.50ab
dependent manner. Liver fat decreased by SFO at 3% and
0 3.0 8.25a 4.63ab 9.75abc 5.63ab
300 ppb AF, whereas no specific trend could be observed
150 0 2.75b 1.75c 3.75d 2.00c
at 150 ppb AF. The muscle fat reduced with each incre-
150 1.5 8.00a 5.25a 8.00c 6.38a
mental level of SFO supplementation in the control
150 3.0 7.63a 5.50a 7.75c 6.00ab
groups (0 ppb AF), but at 150 and 300 ppb of AF, no
300 0 2.88b 1.25c 2.13d 1.13c
specific trend could be observed. The fat content in the
300 1.5 8.38a 6.13a 7.63c 2.50c
skin was higher at both the concentrations of AF, whereas
300 3.0 7.13a 5.88a 11.38a 4.38b
that of the liver increased with each incremental level of
SEM 0.29 0.26 0.39 0.27
AF, and SFO decreased the skin fat content with each
Main effects
incremental level but showed such an effect on liver fat
AF, ppb 0 7.96a 4.33 9.75a 4.29a
content only at the higher level (3%) of supplementation
150 6.13b 4.17 6.50b 4.79a
(main effects). Muscle fat content was not affected by AF
300 6.13b 4.42 7.04b 2.67b
but decreased significantly with each incremental level of
SFO, % 0 4.50b 2.83b 4.96b 1.63b
SFO.
1.5 8.04a 4.75a 8.71a 5.42a Liver protein decreased significantly with AF in a dose-
3.0 7.67a 5.33a 9.63a 4.71a dependent manner, whereas protein in the muscle was de-
Probability creased only at 300 ppb AF. SFO supplementation increased
AF 0.01 0.82 0.01 0.01 the liver protein content at the higher level of 3%, but the
SFO 0.01 0.01 0.01 0.01 muscle protein increased even at the lower level of SFO.
AF × SFO 0.01 0.01 0.01 0.01 Significant interaction was observed between AF and SFO
for their effects on protein content in the liver and muscle.
Means having similar superscripts in a column do not differ significantly
(P ≤ 0.01) The reduction in liver protein content was more conspicuous
DPI, days post-inoculation at 0 and 1.5% levels of SFO, whereas at 3% SFO, the effect
was either positive or neutral. Similarly, the reduction in mus-
cle protein was apparent at 0 level of SFO, whereas at 1.5 and
3% levels of SFO, the effects were marginal.

Fig. 1 Weights of the liver,

giblets and kidneys as affected by
aflatoxin (AF) concentration in
diet
 317 Page 8 of 12 Trop Anim Health Prod (2021) 53:317

Fig. 2 Weights of the pancreas,

bursa and thymus as affected by
aflatoxin (AF) concentration in
diet

Discussion indicating the requirement of higher levels of SFO supplemen-

tation at the higher concentration of AF.
Aflatoxin caused reduced body weight and feed intake in The beneficial effects observed with SFO supplementation
broiler chickens in a dose-dependent manner, more obviously on body weight and feed intake in broilers fed AF could be
at 35 days of age. The negative effects of aflatoxin on perfor- attributed to the prevention of AF absorption by dietary fat
mance of broiler chickens were well established (Raju and (Smith et al. 1971). Aflatoxin also is known to decrease lipase
Devegowda 2000; Solis-Cruz et al. 2019), and the significant secretion and bile concentration and increase gall bladder
depression in body weight and feed intake even at the lower weight and lipid excretion in chickens (Osborne and
concentration (150 ppb) of AF at 35 days, but not at 21 days of Hamilton 1981) and thereby hamper lipid digestion and ab-
age, indicate the cumulative/aggravated toxicity of AF with sorption. The dietary supplementation of SFO thus could
prolonged length of exposure (Yunus et al. 2011). Dietary counter the adverse effects of AF on growth and feed intake
supplementation of SFO increased body weight even at in chickens by probably decreasing the AF absorption from
1.5% level at 150 ppb AF, but such an effect at 300 ppb AF the gut and making available fat for meeting the body needs of
was observed only with the higher (3%) level of SFO chickens. Similar counteraction of AF (300 ppb B1) with

Fig. 3 Weights of the liver,

giblets and kidneys as affected by
sunflower oil (SFO) level in diet
Trop Anim Health Prod (2021) 53:317 Page 9 of 12 317

Fig. 4 Weights of the pancreas,

bursa and thymus as affected by
sunflower oil (SFO) level in diet

dietary supplementation of SFO or SBO (3%) was observed in oil used also appears to play a role, since those rich in unsat-
our previous study (Raju et al. 2005). The fatty acid profile of urated fatty acids only improved performance of chickens fed
AF (Raju et al. 2005), but not those rich in saturated fatty acid
content (Hamilton et al. 1972).
Table 6 Influence of graded dietary levels of aflatoxin (AF) and sun- Richardson et al. (1987) in their dose response study in-
flower oil (SFO) on tibia parameters in broiler chicken
volving graded concentrations of dietary AF (0.354 to 3.797
AF, ppb SFO, % Tibia μg/total AF/g feed) and fat (2 and 4% cotton seed oil) ob-
served that the apparent minimum effective dose of AF on
Wt., g Strength, N Ca, % P, %
liver lipid content and bursa weight was higher at higher level
0 0 5.37 169.4 37.4bc 18.31 of fat in diet. Furthermore, the adverse effects of AF on skin
0 1.5 5.15 172.9 38.4a 18.36 pigmentation in broiler chickens were reported to be more
0 3.0 5.15 175.9 37.8ab 17.95 pronounced at the low dietary fat levels (Schaeffer and
150 0 4.34 168.2 36.9c 18.04
Hamilton 1990). Feed conversion ratio was unaffected, except
150 1.5 5.01 172.1 38.3a 18.29
for decreased FCR recorded with AF at 21 days of age, which
150 3.0 4.80 150.9 38.2a 17.70
could be due to the reduced feed intake recorded with AF at
either concentration. Sunflower oil supplementation showed
300 0 4.35 157.9 37.3bc 18.33
no effect on body weight and feed efficiency in the control
300 1.5 4.68 161.1 37.7bc 17.30
groups (0 ppb AF), which might be due to the fact that the
300 3.0 4.58 174.9 38.3a 17.65
diets were maintained isocaloric in all the groups.
SEM 0.06 5.35 0.08 0.19
The serum protein concentration increased with SFO sup-
Main effects
plementation at 21 days only at the higher concentration of
AF, ppb 0 5.22a 172.7 37.9 18.21
AF, probably indicating the beneficial effects of fat supple-
150 4.72b 163.8 37.8 18.01
mentation only in the presence of AF, more particularly at the
300 4.54b 164.6 37.8 17.76
higher concentration. At 35 days of age, the interactions were
SFO, % 0 4.69 165.2 37.2b 18.22
absent and AF at the higher concentration of 300 ppb de-
1.5 4.95 168.7 38.1a 17.98
creased the serum protein concentration. Reduction in serum
3.0 4.84 167.3 38.1a 17.77
protein with dietary AF was reported earlier (Shukla and
Probability
Pachauri 1995; Raju and Devegowda 2000; Raju et al.
AF 0.01 0.77 0.74 0.64
2005). The serum concentration of cholesterol and triglycer-
SFO 0.17 0.97 0.01 0.63 ides was decreased by AF at both the ages in a dose-dependent
AF × SFO 0.10 0.83 0.01 0.82 manner. Responses to SFO supplementation in the serum cho-
Means having similar superscripts in a column do not differ significantly lesterol concentration were observed to be AF dose-depen-
(P ≤ 0.01) dent. Sunflower supplementation was required at the higher
 317 Page 10 of 12 Trop Anim Health Prod (2021) 53:317

Table 7 Influence of graded

dietary levels of aflatoxin (AF) AF, ppb SFO, % Fat, % Protein, %
and sunflower oil (SFO) on tissue
protein and fat deposition in Skin Liver Thigh muscle Liver Thigh muscle
broiler chicken
0 0 61.2cd 14.4d 12.2b 60.9d 79.5d
e d d c
0 1.5 60.0 14.1 7.8 63.1 82.7ab
0 3.0 57.1f 13.4e 6.9e 65.8b 81.6bc
150 0 63.2a 15.1c 8.9c 58.1e 81.5c
150 1.5 60.5de 15.7b 11.9b 57.6e 79.5d
150 3.0 56.7f 14.7cd 6.6e 72.2a 82.5abc
300 0 62.3b 16.7a 13.4a 53.7f 69.6e
300 1.5 61.6c 17.0a 6.3e 53.3f 83.1a
300 3.0 57.4f 15.2bc 8.1cd 66.2b 81.4c
SEM 0.27 0.148 0.31 0.706 0.47
Main effects
AF, ppb 0 59.4b 13.9c 9.0 63.3a 81.3a
150 60.1a 15.2b 9.1 62.6b 81.2a
300 60.4a 16.3a 9.3 57.7c 78.0b
SFO, % 0 62.2a 15.4a 11.5a 57.6b 76.9b
1.5 60.7b 15.6a 8.7b 58.0b 81.8a
3.0 57.0c 14.4b 7.2c 68.1a 81.8a
Probability
AF 0.01 0.01 0.23 0.01 0.01
SFO 0.01 0.01 0.01 0.01 0.01
AF × SFO 0.01 0.02 0.01 0.01 0.01

Means having similar superscripts in a column do not differ significantly (P ≤ 0.01)

level (3% in diet) for countering the adverse effects of 300 ppb (4.1% and 5.4% in 1.5 and 3.0% SFO diets during finisher
AF, while SFO at 1.5% in diet was sufficient at the lower level phase, for instance), which might have contributed to the ele-
of AF (300 ppb). The effects of SFO supplementation at each vated serum concentration of cholesterol in chickens.
level in diet on serum triglyceride concentration were more The SRBC titres at 5 days post-inoculation decreased with
pronounced at the higher concentration (300 ppb) of AF than AF at either concentration implying that AF depresses im-
at the lower (150 ppb) concentration. Reduced serum concen- mune response even at relatively low concentrations in broiler
tration of cholesterol and triglycerides with AF might be a diet. On the other hand, at 10 days post-inoculation, the SRBC
result of decreased fat absorption with aflatoxin feeding in titres at 35 days were depressed only at the higher (300 ppb)
chickens (Osborne and Hamilton 1981). Supplementation of concentration of AF. Aflatoxin is known to adversely affect
SFO increased the serum concentration of total cholesterol immune competence in chickens (Raju and Devegowda 2002;
and triglycerides, the effect being dose-dependent. These ef- Raju et al. 2005; Solis-Cruz et al. 2019), and the response to
fects of SFO on serum cholesterol and triglyceride concentra- SRBC inoculation indicates the humoral immuneresponse in
tion in chickens fed AF could be related to the countering chickens. Dietary supplementation of SFO increased the
ability of SFO on AF toxicity (Raju et al. 2005). The HDL, SRBC titres testifying the countering ability of SFO on the
LDL and VLDL fractions of cholesterol also showed similar toxic effects of AF on humoral immuneresponse in chickens.
response (data not shown). Weights of organs like the liver, giblets, kidney and pancreas
Sunflower oil supplementation increased the serum choles- increased, whereas that of thymus decreased with AF (300
terol concentration in the control groups in a dose-dependent ppb). These toxic effects of AF on organ weights match with
manner, which is contradicting the previous report the negative effects established in previous studies (Raju and
(Duraisamy et al. 2013), where the serum cholesterol concen- Devegowda 2000; Solis-Cruz et al. 2019). Supplementation of
tration was found to be low with SFO supplementation in SFO (3% in diet) could alleviate the negative effect of AF on
broiler chickens. In the current study, the control diet was giblet weight.
devoid of supplemental fat and the ether extract content of Aflatoxin impairs Ca metabolism through negative effects
diet (2.8% in control) increased with SFO supplementation on kidney and plasma concentration of vitamin D3 metabolites
Trop Anim Health Prod (2021) 53:317 Page 11 of 12 317

(Glahn et al. 1990, 1991), and the reduced weight of tibia due Code availability SPSS software (Version 23.0, SPSS Inc., Chicago,
U.S.A.) and G*Power software (version 3.1.9.7) were used for analysing
to AF feeding could be attributed to this effect of AF. The
the data.
interaction observed between AF and SFO levels for effects
on bone Ca content indicated that higher level of SFO supple-
Declarations
mentation was necessary for increasing the bone Ca as the
concentration of AF was increased from 150 to 300 ppb. Ethics approval The study was conducted in accordance with the insti-
The earlier report from this lab (Raju et al. 2005) indicated a tutional guidelines for the care and use of animals/chickens.
reduction in bone Ca content with AF, while only bone weight
was negatively affected by AF in the current study. Consent to participate Not applicable.
The fat content in the liver and skin increased with AF,
Consent for publication Not applicable.
which were decreased with SFO supplementation, whereas
muscle fat content, though was not affected by AF, tended
Conflict of interest The authors declare no competing interests.
to decrease with SFO supplementation. Aflatoxin causes im-
paired fat absorption (Smith et al. 1971) and serum lipid trans-
port (Tung et al. 1972) leading to fatty liver (Hamilton et al.
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