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Copyright ῍ ,*++, Japan Poultry Science Association.
Tohru Motoki, Hiroshi Kamisoyama, Kazuhisa Honda and Shin Hasegawa
Graduate School of Agricultural Science, Kobe University, Kobe 0/1ῌ2/*+, Japan
Potassium diformate (KDF), a chemical complex of formic acid and potassium formate, improves growth performance
in pigs. The aim of the present study was to examine the e#ects of dietary KDF on the growth, nitrogen retention, intestinal
pH, counts of Enterococcus faecalis, coliforms, and lactic acid bacteria in the cecum, and humoral immune response of
growing broiler chickens. Twenty four male broiler chicks were randomly assigned into three groups (eight birds in each
group). Each group was fed an antibiotics free commercial diet (as a control diet, ,-ῌ CP, -,*** kcal of ME/kg), a control
diet containing KDF at +ῌ, or a control diet containing antibiotics (/* g titer/t Salinomycin, /* g titer/t Avilamycin) until
,2 days of age. Dietary KDF signiﬁcantly increased the weights of body, breast muscle, thighs and wings, whereas the
weights of liver and abdominal fat were not a#ected. These ﬁndings suggest that the increase of body weight by dietary KDF
might be due to the increased muscle weight. Dietary KDF did not a#ect nitrogen retention. Dietary KDF did not a#ect
the intestinal pH, and the counts of Enterococcus faecalis, coliforms, and lactic acid bacteria in the cecum. Hemagglutination
titer was not a#ected by dietary KDF. Thus, although the mechanisms of the growth promotion by dietary KDF are not
clear, our ﬁndings suggest that the KDF might promote growth of broiler chickens at least in the early phase of growth.
Key words: broiler, growth, muscle, organic acid, potassium diformate
J. Poult. Sci., .2: ,.1ῌ,/-, ,*++
῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎῎
Introduction
In-feed antibiotics have been utilized for over half a
century to promote growth in domestic animals (Moore
et al., +3.0; Leitner et al., ,**+; Sun et al., ,**/). The
mechanisms underlying the growth promotion by in-feed
antibiotics are presumed the improvement of nutrients
availability by reducing microbial population and the prevention of pathogen infections by improving immune response (Dibner and Buttin, ,**,). Meanwhile, the extensive use of antibiotics in livestock production has increased the risk of development of resistance in human
and animal pathogens (Bates et al., +33.; Witte, +332).
Thus, the use of antibiotics to promote growth in domestic
animals has been banned in European Union. However,
the withdrawal of in-feed antibiotics suppresses growth in
domestic animals, which in turn results in the increase of
the production costs (Casewell et al., ,**-; Sun et al.,
,**/). Therefore, there is a pressing need to develop
alternative in-feed antibiotics that may be used to alleviate
the problems associated with the withdrawal of antibiotics
from feed.
Received: April ,1, ,*++, Accepted: July +-, ,*++
Released Online Advance Publication: August ,/, ,*++
Correspondence: Dr. S Hasegawa, Graduate School of Agricultural
Science, Kobe University, Kobe 0/1ῌ2/*+, Japan.
(E-mail: [email protected])
Organic acids promote growth in domestic animals.
Like in-feed antibiotics, organic acids reduce intestinal
microbial population and improve immune status (Dibner
and Buttin, ,**,). In addition, a reduction in intestinal
pH, an additional e#ect which is not induced by antibiotics, is regarded as one of the positive e#ects by organic
acids responsible for the growth promotion in domestic
animals. The use of organic acids creates an acidic environment in the gut that inhibits the growth of Escherichia
coli, Salmonella, Campylobacter and gram-negative bacteria (Dibner and Buttin, ,**,; Gunal et al., ,**0).
Potassium diformate (KDF, a chemical complex of formic acid and potassium formate, Fig. +) promotes growth
(Paulicks et al., +330; Roth et al., +330; Kirchgessner et
al., +331), increases lean percentage in the ham, ﬂank, loin
and neck and shoulder (ῌverland et al., ,***), and decreases the number of intestinal microﬂora including coliforms, lactic acid bacteria or yeast in digesta from various
segments of the gastrointestinal tract (Canibe et al., ,**+)
in pigs. Canibe et al. (,**+) described that the presence of
lactic acid bacteria in the gastrointestinal tract of pigs is
believed to be beneﬁcial for the animal, and that it is
di$cult to conclude ﬁrmly whether yeasts are detrimental
or beneﬁcial for the animal. However, they proposed that
the growth-promoting e#ect of KDF in pigs might be
explained by reducing microbial population in the gastrointestinal tract in pigs, because the reduction of the mi-
Journal of Poultry Science, .2 (.)
248
crobial population would be desirable for the dietary energy utilization by the host animal.
KDF is easier to handle than organic acids, because the
use of organic acids has been limited by problems of
handling, strong odor, or corrosion during feed processing
and during its use on the farm. Thus, KDF has been approved in European Union and Japan for use in pigs with
the claim for promoted growth performance. However,
there is little information about the e#ect of KDF on
growth performance in chickens, although studies with
chickens have indicated positive e#ects of dietary organic
acids including propionic acid, citric acid, ascorbic acid
and butyric acid on growth performance (Izat et al., +33*;
Afsharmanesh and Pourreza, ,**/; Leeson et al., ,**/;
Gunal et al., ,**0; Chowdhury et al., ,**3).
In this study, we examined the e#ects of dietary KDF
on the growth, nitrogen retention, intestinal pH, counts of
Enterococcus faecalis, coliforms, and lactic acid bacteria in
the cecum, and humoral immune response of growing
broiler chickens. We used ,2-day old broiler chickens,
because correlations among ,2-day body weight, .,-day
body weight and .1-day carcass weight were positive and
high in broiler chickens (Wang et al., +33+).
Fig. +.
Structure of potassium diformate.
Table +.
Animals and Feed
Day-old male broiler chicks (chunky) were purchased
from a local hatchery (Ishii Co. Ltd., Tokushima, Japan).
They were given free access to water and a commercial
chicken starter diet (Nosan Co., Kanagawa, Japan) and
acclimated to the facility for ﬁve days before feeding of
experimental diets (Table +). After ﬁve days of acclimatization, the chicks were fed experimental diets. All
experimental procedures followed the guidelines for the
care and use of experimental animals at the Rokkodai
Campus of Kobe University in Japan.
Preliminary Experiment
Initially, we carried out a screening experiment to examine the e#ect of dietary KDF (dose: +ῌ-ῌ) on growth
in growing male and female broiler chickens. We found
that the addition of +ῌ KDF in feed signiﬁcantly increased body weight in male chickens. Similar trends were
found in female chickens but the e#ect was not signiﬁcant.
In contrast, the addition of -ῌ KDF in feed signiﬁcantly
decreased body weight in male and female chickens.
Based on these results, the experimental diet containing
+ῌ KDF and growing male broiler chicks were used in
the following experiments (Experiments + and ,).
Assignment of Birds
Forty eight male broiler chicks were randomly assigned
into six groups (eight birds in each group). Three groups
were used to examine the e#ects of dietary KDF on the
growth, nitrogen retention, intestinal pH, counts of
Enterococcus faecalis, coliforms, and lactic acid bacteria in
the cecum in growing broiler chickens (Experiment +).
Composition of experimental diets
Feed ingredients (g/+** g)
Yellow corn
Soybean meal
Corngluten meal
Rapeseed meal
Fish meal
Soybean oil
Calcium carbonate
Calcium phosphate tribasic
Sodium chloride
Vitamin mixture+ῌ
Mineral mixture,ῌ
Potassium diformate
Cellulose
Calculated analysis
Crude protein (ῌ)
Metabolizable energy (kcal/kg)
+ῌ
Materials and Methods
Control
+ῌ
,ῌ
-ῌ
/.4.
,/4*
/4*
.4*
/4*
+4*
+4.
*41
*4,
*4,/
*4*/
*4*
-4*
/.4.
,/4*
/4*
.4*
/4*
+4*
+4.
*41
*4,
*4,/
*4*/
+4*
,4*
/.4.
,/4*
/4*
.4*
/4*
+4*
+4.
*41
*4,
*4,/
*4*/
,4*
+4*
/.4.
,/4*
/4*
.4*
/4*
+4*
+4.
*41
*4,
*4,/
*4*/
-4*
*4*
,-4*
-***
,-4*
-***
,-4*
-***
,-4*
-***
Vitamin A (,**,*** IU/g) +* g, Vitamin D (-*.*** IU/g) 1 g, Thiamine HCl +.0 g, Riboﬂavin 2 g, Prydoxine +.0 g, Choline HCl 30 g, Nicotinic acid +.0 g, Calcium panthothenate -., g,
Folic acid 2 g per kg.
,ῌ
Manganese 2* g, Zinc /* g, Iron / g, Iodine + g, Copper *.0 g per kg.
Motoki et al.: Potassium Diformate Improves Growth in Chickens
Another three groups were used to examine the e#ect of
dietary KDF on the humoral immune response in growing
broiler chickens (Experiment ,).
Experiment +
Birds (n῏2 in each group) were fed an antibiotics free
commercial diet (as a control diet), a control diet containing KDF at +ῌ, or a control diet containing antibiotics
(/* g titer/ton Salinomycin, /* g titer/ton Avilamycin)
until ,2 days of age. Birds were fed the experimental diets
containing an indigestible marker (*./ῌ chromium oxide) for the last 0-days of the experimental period. For the
last --days of the experimental period, each pen was
divided into three zones using stainless steel to create three
replicates for each group, and feces were collected,
weighed, and stored at ῍,*ῑ every day for nitrogen retention analysis. At the end of the experimental period,
chickens were sacriﬁced by decapitation. The right cecum
was immediately removed, and the digesta was used for
microbial analysis. The digesta for microbial analysis
were transferred into plastic bags, and brought to a separate place for microbial analysis. The liver, abdominal fat,
breast muscle, thighs, and wings were excised and
weighed. The digesta samples from the gizzard, duodenum (*./ cm caudally from gizzard to major duodenal
papilla), jejunum (major duodenal papilla to Meckel’s
diverticulum), ileum (Meckel’s diverticulum to ileocecal
junction), and left cecum were collected, and the pH was
measured after adding +3 volumes of distilled water and
stirring for +* min.
Nitrogen retention analysis was conducted as follows.
Nitrogen contents in feces and experimental diets were
measured by the standard methods (AOAC, +33/). For
the determination of chromium concentration, feeds and
feces were ashed by a wet-ash digestion with sodium
molybdate, sulfuric acid and nitric acid. Chromium concentration was determined at a wavelength of ..* nm
(Bolin, +3/,).
Microbial analysis was conducted as follows. All sam-
249
pling was completed within the ﬁrst two hours after sacriﬁce. Enterococcus faecalis were enumerated on the EF
Agar Base (Nissui Pharmaceutical Co. LTD. Tokyo,
Japan) after aerobic incubation at -1ῑ for .2h. Coliform
bacteria were enumerated on MacConkey Agar (Nissui
Pharmaceutical Co. LTD. Tokyo, Japan) after aerobic
incubation at -1ῑ for ,. h. Lactic acid bacteria were
enumerated on the Plate Count Agar with BCP (Nissui
Pharmaceutical Co. LTD. Tokyo, Japan) after anaerobic
incubation at -1ῑ for 1, h.
Experiment ,
Birds (n῏2 in each group) were fed the experimental
diets as described in Experiment +. Birds were inoculated
intravenously with a /ῌ sheep red blood cell (SRBC)
suspension (+ ml/kg body weight) at +2 days of age.
Blood samples were collected from a wing vein on .ῌ3
days after inoculation, and sera were separated and inactivated for -* min at /0ῑ for direct hemagglutination
analysis. The hemagglutination analysis was carried out
in U-shaped wells of disposable microplates. Sera were
serially diluted two-fold in a PBS solution. The +ῌ SRBC
suspension was added to the wells in *.*,/ ml volume,
and the microplates were incubated at -1ῑ for 0* min.
The observed agglutination was recorded by a negativethrough--ῌ system, and the highest dilution with ,ῌ was
taken as endpoint. Results are expressed in the reciprocal
of that dilution.
Statistical Analysis
All data were analyzed using analysis of variance
(ANOVA), and signiﬁcant di#erences among groups were
analyzed by the Tukey-Kramer test. All statistical analyses were performed using the commercial package (StatView version /, SAS Institute, Cary, NC, USA, +332).
Results
Dietary KDF and antibiotics signiﬁcantly (Pῐ*.*/)
increased body weight, whereas the weights of the liver
and abdominal fat were not a#ected (Table ,). The
E#ects of potassium diformate on weights of body, liver, abdominal fat, breast muscle, thighs and wings in broiler chickens
Table ,.
Body weight (g)
Liver weight (g)
Liver weight (g/+** g BW)
Abdominal fat weight (g)
Abdominal fat weight (g/+** g BW)
Breast muscle weight (g)
Breast muscle weight (g/+** g BW)
Thighs weight (g)
Thighs weight (g/+** g BW)
Wings weight (g)
Wings weight (g/+** g BW)
Control
Potassium diformate
Antiboitics
+*2242῎,,41
,+4-2῎ *411
+431῎ *4*3
+/4**῎ *42,
+4-2῎ *4*1
+/.43῎ 14,
+.4,῎ *40
,,.4*῎ 04,
,*40῎ *4.
0+42῎ ,42
/41*῎ *4-*
++3+4-῎-+43*
,,4.*῎ +4,,
+422῎ *4+*
+14-.῎ +4++4.0῎ *4+*
+3-4*῎ 143*
+04,῎ *4/*
,/-43῎ 04**
,+4-῎ *4-*
0342῎ .4,*
/420῎ *4-.*
+,*+4-῎-34-*
,/4*.῎ +4..
,4+*῎ *4+.
+/40,῎ +4-*
+4-*῎ *4+*
+2*4+῎ 04/*
+/4*῎ *4/*
,//4/῎ /41*
,+4.῎ *42*
1,4+῎ ,4,*
04*.῎ *4,/*
Values are means῎SEM for eight chickens per group (n῏2).
* Signiﬁcant with respect to control group (Pῐ*.*/).
250
Journal of Poultry Science, .2 (.)
E#ects of dietary potassium diformate and antibiotics on nitrogen retention in broiler chickens.
Values are meansῌSEM for three replicates per group.
* Signiﬁcant with respect to control group (P῍*.*/).
Fig. ,.
weights of breast muscle, thighs and wings were signiﬁcantly (Pῌ*.*/) increased in the KDF and the antibiotics
groups, and the weights of breast muscle, thighs, and
wings per +**g body weight were also signiﬁcantly (Pῌ
*.*/) increased in the KDF and the antibiotics groups
(Table ,). Nitrogen retention was not a#ected by dietary
KDF but signiﬁcantly (Pῌ*.*/) increased by dietary
antibiotics (Fig. ,). Dietary KDF did not a#ect pH of the
gastrointestinal contents (Table -). There were no signiﬁcant di#erences in the counts of Enterococcus faecalis,
coliform bacteria and lactic acid bacteria in the cecal
content among groups (Fig. -). There were no signiﬁcant
di#erences in the hemagglutination titer among groups
Fig. -. E#ects of dietary potassium diformate and
antibiotics on microﬂora in the cecum of broiler chickens. Values are meansῌSEM for eight chickens per
group.
(Fig. .).
Discussion
ῌverland et al. (,***) reported that the addition of
+.,ῌ KDF to a diet improved growth performance of
growing-ﬁnishing pigs. Paulicks et al. (+330) reported
that the addition of ,ῌ KDF to a diet for growing pigs
Motoki et al.: Potassium Diformate Improves Growth in Chickens
251
E#ects of dietary potassium diformate on pH at the
di#erent part of the intestine in broiler chickens
Table -.
Gizzard
Duodenum
Jejunum
Ileum
Cecum
Control
Potassium diformate
Antibiotics
-4,3ῌ*4*3
04-+ῌ*4*0
/432ῌ*4+*
14+2ῌ*4,.
04.-ῌ*4,/
-4-,ῌ*4+,
04,1ῌ*4*,
/422ῌ*4*14+*ῌ*4+/
/412ῌ*4+,
-4-1ῌ*4*1
04,-ῌ*4*/423ῌ*4*0
1401ῌ*4*3
/410ῌ*4*0
Values are meansῌSEM for eight chickens per group.
E#ects of dietary potassium diformate and antibiotics on
hemagglutination titer in broiler chickens. Values are meansῌ
SEM for eight chickens per group.
Fig. ..
increased body weight gain but ,.. and ,.2ῌ KDF did not
a#ect body weight gain. Based on these reports, we tested
a wider range of doses (from * to -ῌ) in the preliminary
experiment and found that the body weight of male broiler
chickens was signiﬁcantly increased only in the +ῌ KDF
group. Recently, Mikkelsen et al. (,**3) reported that
*../ῌ dietary KDF did not a#ect weight gain in broiler
chicks. This evidence and our results suggest that optimum KDF supplementation in broiler chicken feed might
be +ῌ. Cumulative feed intake of the KDF group was
numerically higher than that of the control group (calculated values were +,/*0.1 g/bird and +,.*1./ g/bird, respectively). However, these values can not be statistically
analyzed, because we did not measure individual feed
intake. In order to investigate whether the KDF-increased feed intake in broiler chickens is involved in its
growth-promoting e#ect, further studies will be needed to
analyze the relation ship between feed intake and body
weight gain individually.
The weights of breast muscle, thighs and wings were
increased in the KDF group, whereas dietary KDF did
not a#ect the weights of abdominal fat. The abdominal fat
is known to be a good indicator of fat deposition in chicks
(Shigeno, +31-; Havenstein et al., ,**-). These ﬁndings
suggest that the increase of muscle weight might be involved in the increase of body weight by KDF in chickens.
This observation raises the question whether the increase
of muscle weights by KDF in chickens is due to the improvement of nitrogen availability. However, nitrogen
retention was not a#ected by KDF. Also in pigs, KDF
did not promote the ileal digestion of dietary amino acids
(except for phenylalanine), or nitrogen retention (Mroz et
al., ,**,). These ﬁndings and our results suggest that
KDF might not a#ect nitrogen retention in both chickens
and pigs.
In the present study, +ῌ KDF did not a#ect the intestinal pH of broiler chickens. Mikkelsen et al. (,**3)
reported that *../ῌ dietary KDF did not a#ect intestinal
pH in broiler chickens. It is generally accepted that dietary organic acids decrease intestinal pH, which in turn,
results in the decrease of intestinal microbial populations.
However, it seems likely that dietary KDF, at least in this
experimental procedure, might not decrease intestinal pH
in broiler chickens.
There is evidence that dietary organic acids activate the
immune system and improve growth performance in broil-
Journal of Poultry Science, .2 (.)
252
er chickens. For example, dietary acetic acid, citric acid
and lactic acid increased serum globulin level in broiler
chicks (Abdel-Fattah et al., ,**2). Chowdhury et al.
(,**3) reported a higher density of lymphocytes in the
cecal tonsils and ileum in citric acid-fed broiler chickens.
These ﬁndings raise the possibility that dietary organic
acids improve both humoral and cellular immunity. In the
present study, we examined the e#ect of KDF on hemagglutination titer, which indicates the humoral immune
response to SRBC. However, dietary KDF did not a#ect
the hemagglutination titer in broiler chickens, suggesting
that humoral immune response might not be a#ected by
KDF. Further studies will be needed to examine the
e#ects of KDF on cellular immune responses in broiler
chickens.
In summary, in growing broiler chickens, the dietary
KDF signiﬁcantly increased body weight and muscle
weight. Dietary KDF did not a#ect nitrogen retention,
intestinal pH, the counts of Enterococcus faecalis, coliforms and lactic acid bacteria in the cecum, and hemagglutination titer. It seems likely that the growth-promotion by KDF might not be caused by the e#ects on
intestinal microﬂora, nitrogen availability and humoral
immunity. Our ﬁndings suggest that the KDF might
promote growth of broiler chickens at least in the early
phase of growth. Further studies will be needed to examine the long term (e.g. .3 days) e#ect of dietary KDF on
the growth of broiler chickens.
Acknowledgments
This work was supported by a Grant-in-Aid (Number
,+/2*-.2) for Scientiﬁc Research (C) from the Ministry
of Education, Culture, Sports, Science, and Technology of
Japan.
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