DOI: 10.1111/jpn.

12189

ORIGINAL ARTICLE

Changes in blood profile in sheep receiving raw garlic, garlic oil

or monensin
E. Anassori1, B. Dalir-Naghadeh1, R. Pirmohammadi2 and M. Hadian1
1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran, and
2 Department of Animal Science, Faculty of Agriculture, Urmia University, Urmia, Iran

Summary
This study aimed to evaluate the effects of supplementing a basal diet (CTR) with raw garlic (GAR) or garlic oil
(GAO) on blood profile in sheep. Monensin (MON, 33 mg/kg DM) was used as positive control. Four ruminally
fistulated rams were used in three experiments each arranged in a 4 9 4 Latin square design with 28-day peri-
ods. Experiments 1 and 2 differed in the dose of GAR (75 vs. 100 g/kg DM) and GAO (500 vs. 750 mg/kg DM),
while experiment 3 was designed to compare the two doses of each additive (GAR and GAO). The animals were
fed a basal diet as TMR consisting of 77.83% forage (alfalfa hay and corn silage) and 22.17% concentrate, provid-
ing 10.50 MJ/kg DM (metabolizable energy) and 16.5% crude protein to cover maintenance energy and protein
requirements. Supplementation of monensin decreased (P < 0.05) b-hydroxybutyrate (BHB) and non-esterified
fatty acid (NEFA) concentrations in the blood compared with other treatments. There was no significant effect of
additives on serum concentration of glucose, total triglycerides, cholesterol, total protein, albumin and blood
urea nitrogen (BUN). Although the serum insulin concentration was elevated in sheep receiving MON and GAO
(P < 0.01), no change was observed in blood glucose concentration. No significant effect of GAO and GAR was
observed in key energy and protein-related blood metabolites. However, administration of monensin had a posi-
tive influence on energy indices. In conclusion, whereas parameters characterizing the energy balance did not
show a significant effect of GAR supplementation, a higher insulin concentration in GAO-treated animals was
observed.
Keywords blood, garlic, garlic oil, monensin, sheep

Correspondence B. Dalir-Naghadeh, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, P.O. Box: 57153-1177, Urmia,
Iran. Tel: +98 914 341 2489; Fax: +98 441 277 1926; E-mail: b.dalir@urmia.ac.ir

Received: 3 July 2013; accepted: 6 March 2014

resistance in humans has increased public concern on

Introduction
The factors affecting ruminal fermentation using feed
additives, such as ionophore antibiotics, have received
much attention as a useful strategy to improve pro-
duction efficiency in ruminants (Ipharraguerre and
Clark, 2003; Taghipoor et al., 2011).
Many benefits of the ionophores are due to the
improved energy status through increased propionic
acid production and a reduced methane production
(McGuffey et al., 2001; Ipharraguerre and Clark,
2003). This generally results in less mobilization of
body fat, lower levels of blood non-esterified fatty
acids (NEFA) and ketone bodies and increased glucose
production (McGuffey et al., 2001). Monensin, a
polyether ionophore antibiotic, is widely used in rumi-
nant diets, and its beneficial effects on nitrogen and
energy utilization are well known (Tedeschi et al.,
2003). However, the emergence of antimicrobial
the use of antibiotics in animal feeding, leading to
their ban in the European Union (Regulation 1831/
2003/EC). For this reason, there is an increasing
interest in evaluating ‘natural’ alternatives such as
plant extracts to modify rumen microbial fermen-
tation. The essential oils are plant secondary metabo-
lites are responsible for the antimicrobial activity of
plants and spices. They are active against a wide range
of gram-positive and gram-negative bacteria, fungi,
parasites and viruses (Reuter et al., 1996). Busquet
et al. (2005a) showed that these compounds could
influence rumen fermentation, leading to a decrease
in proportion of acetate, an increase in proportion of
propionate and butyrate and inhibition of methano-
genesis.
Few studies evaluated the effect of doses of raw gar-
lic and its essential oil contents on rumen fluid
(Busquet et al., 2005b; Yang et al., 2007; Hart et al.,

Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH 1
Blood profile of sheep in response to garlic E. Anassori et al.

2008). A recent study by Anassori et al. (2011) dem- were allowed to adapt to the additives. Within each
onstrated that raw garlic and garlic oil resulted in a experiment, there was a 1 week washout interval
range of beneficial effects on rumen fermentation that between periods, during which the sheep were fed
mimic the favourable effects of monensin on rumen basal diet with no additives.
ecosystem in sheep. Although the effects of monensin The basal diet was designed to cover 10.50 MJ of
on blood profile have been studied previously (Duf- metabolizable energy and 16.50% of crude protein
field et al., 1998; Taghipoor et al., 2011), to our per kg of DM to meet the maintenance energy and
knowledge, the effects of raw garlic and/or its ingredi- protein requirements (NRC, 1985 and AFRC, 1992)
ents on blood constituents in ruminants have not yet (Table 1). The basal diet was provided daily as a total
been addressed experimentally. The objective of this mixed ration in two equal portions at 08:00 and
work was to study the effects of dietary supplementa- 16:00. Water was freely available through individual
tion with raw garlic and garlic oil compared with drinking bowl. Additives were administered into the
monensin on blood profile in an in vivo sheep model. rumen through the rumen fistula before their morn-
As garlic has mimicked some beneficial effects of ing feeding. Raw garlic bulb was sliced, and garlic oil
monensin on rumen fermentation, we hypothesize and monensin were mixed with 20 ml of distilled
that similar effects may be induced by garlic supple- water immediately before administration.
mentation.
Sample collection and analytical procedure
Materials and methods
The diet samples (basal diet and raw garlic) were anal-
Animals, diets, additives and experimental design ysed for dry matter (DM), ash, crude protein (CP),
Details of the experimental animals, feed additives, acid detergent fibre (ADF), calcium and phosphorous
experimental design, treatments and analytical proce- using the standard methods (Association of Official
dures for basal diets, raw garlic and garlic oil have Analytical Chemists (AOAC), 1990). Neutral deter-
been described elsewhere (Anassori et al., 2011). In gent fibre (NDF) content was determined as described
brief, four rumen fistulated mature Makoui male by Van Soest et al. (1991) with the addition of alpha
sheep (55 kg 3 live weight) were used in this study.
The animals were housed in individual metabolic
Table 1 Ingredients and chemical composition of the basal diet
cages during experiments.
Raw garlic bulb (Allium sativum) was harvested from Item Amount
Hamadan County, Iran. Garlic oil was prepared Ingredient, (g/kg DM)
according to Clevenger (1928). Monensin was pur- Alfalfa hay 381.4
chased from Behroodatrak Company (Iran), which Corn silage 396.9
produces this product under a licence by Elanco Soybean meal 85.0
Division, Eli Lilly Canada. Barley grain 109.9
The study was conducted in three experiments with Wheat bran 26.8
Chemical Composition
a resting interval of 4 weeks between them. Each
DM, (g/kg of feed) 719.5
experiment was conducted as a four treatment cross- OM, (g/kg DM) 908.2
over trial with four 21 days periods. The complete set ME, (MJ/kg DM) 10.50
of studies took approximately 1 year to complete. In FME, (MJ/kg DM) 9.60
the first experiment, each of four sheep was randomly MP, (g/kg DM) 86.00
assigned to one of four diets: control diet (basal total ERDP, (g/kg DM) 112
mixed ration with no additive = CTR), control diet UDP, (g/kg DM) 20
CP, (g/kg DM) 165
with raw garlic (75 g/kg DM = GAR75), control diet
NDF, (g/kg DM) 397.3
with garlic oil (500 mg/kg DM = GAO500) and con- ADF, (g/kg DM) 209.3
trol diet with monensin (33 mg/kg DM = MON). In EE, (g/kg DM) 17.6
the second experiment, the diets were similar to Calcium, (g/kg DM) 5.80
experiment 1; however, the dose of raw garlic and Phosphorus, (g/kg DM) 2.80
garlic oil was increased to 100 g/kg DM (GAR100)
DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral
and 750 mg/kg DM (GAO750) respectively. In the detergent fibre; ADF, acid detergent fibre; EE, ether extract; ME, metabo-
third experiment, each of four sheep was fed the basal lizable energy; FME, fermentable metabolizable energy; MP, metaboliz-
diet with GAR 75, GAR 100, GAO500 and GAO750. able proteins; ERDP, effective rumen degradable protein; UDP,
During the first 7 days of each period, the animals undegradable protein.

2 Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
E. Anassori et al.
amylase and sodium sulphite, and the values were
expressed including residual ash. The metabolizable
energy (ME), effective rumen degradable protein
(ERDP), undegradable protein (UDP), metabolizable
protein (MP) and fermentable metabolizable energy
(FME) were estimated according to AFRC (1992)
(Table 1).
The identification of volatile constituents of garlic
oil was performed using a Finnigan gas chromato-
graph (Thermo Finnigan, Somerset, NJ, USA) as
described elsewhere (Anassori et al., 2011).
Blood samples were collected using evacuated tubes
from the jugular vein on day 7, 14 and 21 of each
experiment 3 h after morning feedings. Blood samples
were left at room temperature until clotting was com-
pleted (within 30 min). Tubes were then centrifuged
for 20 min at 2500 9 g. Serum samples were stored at
20 °C until analyses for determination of glucose,
non-esterified fatty acids (NEFA), b-hydroxybutyrate
(BHB), insulin, cholesterol, BUN and albumin.
Β-hydroxybutyrate and NEFA were determined by a
D-3-hydroxybutyrate kit and a NEFA Kit (Randox Lab-
oratories, Ardmore, UK) respectively. The concentra-
tion of serum glucose, cholesterol, triglyceride, BUN
and total protein was determined using an auto analy-
ser spectrophotometer (Model RA-1000; Technicon
Instruments Corporation, Tarrytown, NY, USA) using
commercial kits (Parsazmoon, Tehran, Iran). Serum
insulin concentration was determined using ELISA Kit
(kit no. 10-1202-01 Mercodia AB, Uppsala, Sweden).
Statistical analysis
A four-period, four treatment cross-over design in a
randomized sequence, based on a Latin Square design
(Williams Square), was used for each experiment. The
design was pre-arranged to balance for carry-over
affects so that every treatment is represented only
once in each column and in each row of the square
yielding to a uniform cross-over design. Data for each
parameter were analysed independently for each
experiment using a linear mixed model for a cross-
over design with the REPEATED command and
Kenward-Rogers adjustment for calculation of denom-
inator degrees of freedom. A preliminary analysis
using histograms, box plots and scatter plots was used
to examine the data for errors and outliers. No outlier
was detected. The initial model for each parameter
included period, treatment, time and all interactions
as fixed effects. Sheep were considered as random
effect. The repeated statement included time, and the
subject was sheep 9 period within treatment. The
interaction terms that were not significant were
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Blood profile of sheep in response to garlic
dropped from the reduced model. For each variable, a
number of covariance structures to model the repeated
measurements were tested with the repeated state-
ment for PROC MIXED, and the model that provided
better fit according to minimum Akaike’s information
criterion (AIC) was used. Residuals plotted against the
predicted values showed no departure from assump-
tions. Whenever the F-test was significant, differences
between least squares means were determined using
the PDIFF option and pairwise Tukey–Kramer multi-
ple comparisons with separate the means. All reported
values are least squares means and SEM. Results were
considered significant at the P < 0.05. Data were anal-
ysed using a statistical software package (SAS, Version
9.1.3, SAS Institute, Cary, NC, USA).
Results
The results of the current study indicated that crude
fat (EE) content of garlic bulb was 7.1 g/kg on a DM
basis. Also garlic bulb yielded 0.12% garlic oil on a
wet weight basis. The GC–MS analyses of the garlic oil
revealed that trisulphide di-2-propenyl (32.76%),
diallyl disulphide (28.41%) and trisulphide methyl 2-
propenyl (14.26%) are the main components of the
oil.
Glucose
Serum glucose concentrations after 21 days of treat-
ment are shown in Tables 2–4. No main effects
(P > 0.05) of treatment and time or interactions involv-
ing treatment and time were observed in concentration
of glucose throughout the study (Tables 2–4).
Β-Hydroxybutyrate
In experiment 1, supplementation of MON decreased
(P < 0.05) concentrations of BHB on day 14 compared
with GAO500. Also the serum concentration of BHB
was influenced by MON and GAO500 within experi-
mental periods and reduced (P < 0.01) (Table 2).
Non-esterified fatty acids
Overall serum level of NEFA was not altered
(P > 0.05) by raw garlic and garlic oil compared with
CTR; nevertheless, MON decreased (P < 0.01) NEFA
concentration after 14 days supplementation, without
further effects at the rest of the study (Tables 2 and 3).
There were no differences (P > 0.05) in serum NEFA
concentration between treatments in experiment 3
(Table 4).
3
Blood profile of sheep in response to garlic E. Anassori et al.

Table 2 Blood constituents of sheep-fed basal diet and diet supplemented with raw garlic, garlic oil and monensin (experiment 1)

Treatments P-value†

Variables Days CTR GAR75 GAO500 MON SE TRT Day TRT 9 Day

Serum glucose (mg/dl) 7 69.58 69.58 65.83 64.58 2.62 NS NS NS

14 67.08 66.33 62.08 63.33 2.62
21 66.33 68.33 64.08 62.83 2.62
BHB (mM) 7 0.64 0.47 0.66 0.51 0.07 * ** ***
14 0.64ab 0.36ab 0.70a 0.26b 0.07
21 0.65 0.47 0.42 0.35 0.06
NEFA (mM) 7 0.26 0.28 0.25 0.26 0.02 ** NS **
14 0.26a 0.30a 0.24ab 0.16b 0.02
21 0.24ab 0.27ab 0.31a 0.18b 0.02
Insulin (ng/ml) 7 0.41b 0.38b 0.62ab 0.87a 0.05 *** *** **
14 0.41b 0.43b 0.64b 1.18a 0.05
21 0.42c 0.45c 0.79b 1.23a 0.05
Total triglycerides (mg/dl) 7 33.44 26.19 28.19 31.94 4.45 NS NS NS
14 31.69 25.94 24.94 26.19 4.45
21 31.44 28.94 28.19 27.44 4.45
Cholesterol (mg/dl) 7 58.10 53.35 61.10 53.85 3.45 NS NS NS
14 57.35 53.35 50.60 52.10 3.45
21 55.35 46.35 54.85 55.60 3.45
BUN, (mg/dl) 7 17.06 16.84 16.76 15.76 0.47 NS NS NS
14 16.44 15.46 14.94 16.41 0.83
21 15.34 16.34 16.49 16.39 0.42
T. Protein (g/dl) 7 7.47 7.18 7.20 7.42 0.43 NS NS NS
14 7.40 7.67 7.79 7.00 0.23
21 7.64 7.10 7.52 7.45 0.28
Albumin (g/dl) 7 3.83 3.53 3.66 3.73 0.20 NS NS NS
14 3.73 3.81 4.08 3.34 0.20
21 3.98 3.49 3.81 3.61 0.20

CTR = basal diet. GAR75 = basal diet + raw garlic (75 g/kg DM). GAO500 = basal diet + garlic oil (500 mg/kg DM). MON = basal diet + monensin
(33 mg/kg DM).
TRT, treatment; BHB, b-hydroxy butyrate; NEFA, non-esterified fatty acids; BUN, blood urea nitrogen.
†*P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant.
a,b,c
Means within the same row having different letters are significantly different (P < 0.05).

Insulin interactions involving treatment and time were

The addition of GAO500 and GAO750 in experiments
1 and 2 promoted higher (P < 0.01) concentrations of
insulin than CTR after 21 days of supplementation,
while MON-treated group had the greatest serum
insulin concentration among treatments (P < 0.01)
(Tables 2 and 3). A similar response was also observed
in experiment 3 with GAO when added at 750 mg/kg
DM; moreover in a differing manner compared with
experiment 2, GAR100 supplementation increased
(P < 0.001) insulin concentration over the time
(Table 4).
Triglycerides, cholesterol, bun and total protein
Except for the cholesterol, which increased (P = 0.02)
with GAO750 compared with GAR75 in experiment
3, no effects (P > 0.05) of treatment and time or
observed in concentration of total triglycerides, cho-
lesterol, BUN, total protein and albumin throughout
the study (Tables 2–4).
Discussion
Our previous results on rumen fluid parameters
(Anassori et al., 2011) like other in vivo studies (Rich-
ardson et al., 1976) showed monensin feeding
increased propionate molar percentage while decreas-
ing butyrate molar percentage. However, in the cur-
rent study, in agreement with the others (Raun et al.,
1976; Johnson et al., 1986), MON did not significantly
alter blood glucose concentration. This contradicts
with studies of Bergman (1973) and Judson and Leng
(1973) who showed an increase in glucose level pro-
portionate to increased ruminal propionate production

4 Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
E. Anassori et al. Blood profile of sheep in response to garlic

Table 3 Blood constituents of sheep-fed basal diet and diet supplemented with raw garlic, garlic oil and monensin (experiment 2).

Treatments P-value†

Variables Days CTR GAR100 GAO750 MON SE TRT Day TRT 9 Day

Serum glucose (mg/dl) 7 71.74 71.74 67.93 66.67 2.80 NS NS NS

14 69.20 67.93 65.15 65.40 2.80
21 68.44 63.62 65.91 64.89 2.80
BHB (mM) 7 0.67 0.57 0.56 0.53 0.06 NS NS NS
14 0.67 0.70 0.63 0.28 0.08
21 0.67 0.69 0.72 0.37 0.09
NEFA (mM) 7 0.28 0.27 0.28 0.28 0.01 *** NS **
14 0.29a 0.29a 0.29a 0.17b 0.02
21 0.26 0.28 0.30 0.19 0.02
Insulin (ng/ml) 7 0.42b 0.40b 0.59ab 0.85a 0.06 *** *** **
14 0.40b 0.55b 0.63b 1.10a 0.06
21 0.41b 0.61b 1.06a 1.21a 0.06
Total triglycerides (mg/dl) 7 32.39 21.76 25.46 30.90 3.20 NS NS NS
14 30.66 22.99 27.20 25.22 4.80
21 30.41 24.97 25.71 26.45 2.90
Cholesterol (mg/dl) 7 57.99 48.41 52.10 53.82 3.20 NS NS NS
14 57.26 50.87 54.55 52.10 3.20
21 55.29 55.54 60.45 55.54 3.20
BUN, (mg/dl) 7 16.06 15.91 16.06 14.79 0.50 NS NS NS
14 15.44 14.34 15.30 15.42 0.68
21 14.37 13.78 14.61 15.40 0.32
T. Protein (g/dl) 7 7.67 7.27 7.62 7.62 0.36 NS NS NS
14 7.59 7.41 7.14 7.19 0.36
21 7.85 8.28 7.37 7.65 0.36
Albumin (g/dl) 7 3.92 3.62 3.75 3.82 0.21 NS NS NS
14 3.82 3.54 3.49 3.41 0.21
21 4.08 4.07 3.52 3.70 0.21

CTR = basal diet. GAR100 = basal diet + raw garlic (100 g/kg DM). GAO750 = basal diet + garlic oil (750 mg/kg DM). MON = basal diet + monensin
(33 mg/kg DM).
TRT, treatment; BHB, b-hydroxy butyrate; NEFA, non-esterified fatty acids; BUN, blood urea nitrogen.
†*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.
a,b,c
Means within the same row having different letters are significantly different (P < 0.05).

or absorption. The results also are in contrast to Duf-
field et al. (1998) who reported higher glucose con-
centrations in monensin-treated cows during the
post-partum period.
An increased concentration of blood insulin in
sheep supplemented with MON was consistent with
findings in other studies (Raun et al., 1976; Randel
et al., 1977). The overall, on day 7 of MON supple-
mentation, concentration of insulin elevated and
then increased progressively over the time, while
glucose levels did not change significantly through-
out the study. This unpredictable alteration in insu-
lin levels may indicate the influence of other
metabolites than glucose for stimulating insulin
release, including propionate as suggested by John-
son et al. (1986). These results support the hypoth-
esis that propionate is an insulin secretagogue in
ruminants (Hertelendy et al., 1968; De Jong, 1982).
No significant differences were observed in serum
glucose concentration. This would imply that, in
spite of an expected increased gluconeogenesis (due
to an increased propionate supply), there was also
an increase in the rate of removal of glucose from
the blood mediated probably by insulin (Brockman
and Laarveld, 1986). Another explanation as sug-
gested by Bishop et al. (1965) could be due to
insulin’s ability to decrease hepatic output of glu-
cose via promoting conversion of glucose to glyco-
gen and so keeping glucose values within normal
range.
Garlic has long been claimed to possess a hypogly-
caemic effect. However, effects of garlic preparations
on blood glucose have been controversial (Anwar and
Meki, 2003; Baluchnejadmojarad and Roghani,
2003). In the present study, our results confirmed the
findings of Chaves et al. (2008), who reported no

Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH 5
Blood profile of sheep in response to garlic E. Anassori et al.

Table 4 Blood constituents of sheep-fed basal diet supplemented with raw garlic and garlic oil (experiment 3)

Treatments P-value†

Variables Days GAR75 GAR100 GAO500 GAO750 SE TRT Day TRT 9 Day

Serum glucose (mg/dl) 7 70.75 70.75 67.00 67.00 2.60 NS NS NS

14 67.50 67.00 63.25 64.25 2.60
21 69.50 62.75 65.25 65.00 2.60
BHB (mM) 7 0.48 0.56 0.68 0.55 0.08 NS * NS
14 0.37 0.69 0.73 0.63 0.09
21 0.48 0.68 0.43 0.71 0.10
NEFA (mM) 7 0.29 0.26 0.26 0.27 0.01 NS NS NS
14 0.31 0.28 0.25 0.28 0.01
21 0.27 0.27 0.32 0.28 0.01
Insulin (ng/ml) 7 0.39 0.41 0.64 0.61 0.06 ** *** ***
14 0.45 0.50 0.65 0.65 0.06
21 0.47c 0.63bc 0.80ab 1.07a 0.06
Total triglycerides (mg/dl) 7 25.50 22.00 27.50 25.75 2.90 NS NS NS
14 25.25 23.25 24.25 27.50 2.90
21 28.25 25.25 27.50 26.00 2.90
Cholesterol (mg/dl) 7 54.25 49.25 62.00 53.00 2.80 NS NS *
14 54.25 51.75 51.50 55.50 2.80
21 47.25b 56.5ab 55.75ab 61.50a 2.80
BUN, (mg/dl) 7 16.15 16.22 16.07 16.37 0.50 NS NS NS
14 14.77 14.62 14.75 15.60 0.50
21 15.65 14.05 15.80 14.90 0.50
T. Protein (g/dl) 7 7.25 7.15 7.27 7.50 0.34 NS NS NS
14 7.75 7.30 7.87 7.02 0.24
21 7.17 8.15 7.60 7.25 0.36
Albumin (g/dl) 7 3.57 3.57 3.70 3.70 0.18 NS NS NS
14 3.85 3.50 4.12 3.45 0.18
21 3.52 4.02 3.85 3.47 0.18

GAR75 = basal diet + raw garlic (75 g/kg DM). GAR100 = basal diet + raw garlic (100 g/kg DM). GAO500 = basal diet + garlic oil (500 mg/kg DM).
GAO750 = basal diet + garlic oil (750 mg/kg DM).
TRT, treatment; BHB, b-hydroxy butyrate; NEFA, non-esterified fatty acids; BUN, blood urea nitrogen.
†*P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant.
a,b,c
Means within the same row having different letters are significant different (P < 0.05).

difference in serum glucose concentration of growing
lambs fed with diets supplemented with garlic
compared with the control. The hypoglycaemic effect
of garlic has been attributed to an increase in the
serum insulin level (Sodimu et al., 1984). In the pres-
ent study, no significant change was observed in glu-
cose level throughout the study; however, the serum
concentration of insulin elevated after 21 days of
GAO supplementation. The discrepancy between the
findings might be due to the dose, chemical composi-
tion of essential oil and experimental conditions. In
addition, in ruminants, it has been proved that propio-
nate rather than glucose is the major promoter of
insulin secretion (De Jong, 1982).
Whitaker (1997) suggested that glucose was not a
good indicator of energy balance compared with
NEFA or BHB. In the present study, blood glucose
concentration did not change, but blood NEFA and
BHB concentrations in MON-treated sheep decreased
compared with basal diet over the time, suggesting
that MON favoured a positive energy balance com-
pared with the control group. This is in agreement
with our previous published work, which showed
MON (to a greater extent) and the raw garlic and gar-
lic oil (to a lesser extent) increased propionate in the
rumen fluid in sheep while at the same time they
decreased acetate and butyrate molar concentrations
(Anassori et al., 2011).
In ruminants with positive energy balance, circulat-
ing hydroxybutyrate is largely derived from the
metabolism of butyrate during absorption across the
rumen epithelium (Pethick and Lindsay, 1982). In
contrast to Quigley et al. (1992), we previously
showed that the increased ruminal butyrate concen-
tration in GAO500-treated sheep (Anassori et al.,
2011) was associated with decreased blood BHB con-

6 Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
E. Anassori et al.
centrations. In addition, in spite of the effects of
GAR100 and GAO750 on increasing the butyrate
proportion in rumen, the blood levels of BHB
remained unchanged. This could result from the fact
that the forestomach epithelium utilizes BHB as an
energy source and hence modulating the effects of
increased ruminal production of BHB on its blood
concentrations. Relationships between BHB, NEFA
and insulin and insulin responsiveness and sensitivity
warrant further investigation.
In the present study, GAR and GAO did not affect
blood NEFA concentration. However, monensin sup-
plementation was associated with low levels of NEFA,
which may have been caused by a lesser degree of fat
mobilization in monensin supplemented sheep as sug-
gested by others (Grummer, 1993). We did not find
any effect of MON on serum concentration of triglyce-
rides and cholesterol. Taghipoor et al. (2011) also
reported no changes in serum cholesterol concentra-
tion in pre- and post-parturition period in sheep sup-
plemented with monensin. Some studies
demonstrated the hypocholesterolaemic effect of gar-
lic in human and animal (Silagy and Neil, 1994;
Chowdhury et al., 2002). Based on these studies, gar-
lic has a potential to reduce lipid accumulation in the
liver and triglycerides level in the plasma by inhibiting
hepatic fatty acid synthesis (Gofman et al., 1966). In
the current study, there were no differences in serum
cholesterol concentrations among additives compared
with CTR. However, there was an unexpected eleva-
tion of cholesterol for GAO750 vs. GAR 75. Horton
et al. (1991) reported that garlic had no effects on
plasma cholesterol and triglyceride concentrations.
Lau et al. (1987) suggested that the suppression of
lipid biosynthesis by garlic might be accompanied by
an initial mobilization of tissue lipids into circulation
increasing blood levels of cholesterol and triglycerides
during the early phase of garlic feeding. This might
explain the higher cholesterol values for GAO750 vs.
GAR 75. Alternatively, GAO750 may facilitate choles-
terol absorption from gastrointestinal tract leading to
its elevated blood level.
No differences in BUN concentrations were
detected between treatments. The reason for
unchanged BUN with MON is uncertain. In theory,
the pattern of serum urea-N concentration over time
should reflect the ruminal NH3-N concentration
because the liver essentially removes all net portal
absorption of NH3-N, which can account for 70 to
80% of urea-N released by the liver when the hepa-
tic function is not impaired (Huntington, 1990).
Monensin usually reduces protein degradation and
lowers the accumulation of NH3-N in the rumen
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Blood profile of sheep in response to garlic
(Tedeschi et al., 2003). Similarly results from previ-
ous studies showed decreased ruminal NH3-N con-
centration due to garlic supplementation (Kongmun
et al., 2010; Anassori et al., 2011). It is probable that
serum concentrations of BUN is not sensitive enough
in detecting differences in protein metabolism
between the treatments. Furthermore, a discrepancy
between serum and rumen ammonia-N has been
reported (Martineau et al., 2007). Duffield et al.
(1998) suggested that the improved energy status
with MON might reduce fatty infiltration of liver,
improving its function and enhancing its ability to
synthesize urea. The same mechanism of action
could be supposed for garlic.
Overall, the current results on the effect of garlic
and monensin on blood metabolites should be inter-
preted with caution because the values remained
within or close to normal ranges in sheep fed within
their maintenance requirements. It has been illus-
trated that the beneficial effect of monensin on
energy metabolism indicated by lowering of NEFA
levels and reduced ketones production (Stephenson
et al., 1997; Duffield et al., 1998) is more pro-
nounced when there is a negative energy balance
(early lactation or late pregnancy). The present study
was conducted without inducing negative energy bal-
ance; this could partially explain unremarkable
changes on blood constituents.
Conclusion
Garlic oil contains a variety of active ingredients,
capable of improving the nutrient utilization and
productivities in ruminants (Busquet et al., 2005a;
Anassori et al., 2011). Our previous work showed
garlic’s beneficial impact on rumen fermentation.
However, in the present study, GAO did not cause
any drastic changes in the concentrations of blood
metabolites in the sheep fed within their mainte-
nance energy and protein requirements. On the
other hand, monensin supplemented sheep had bet-
ter energy status compared with garlic oil and raw
garlic supplemented sheep as indicated by signifi-
cantly lower BHB and NEFA concentrations in
blood. This study corroborates the earlier findings of
monensin efficacy in animals with a negative energy
balance can also be observed in animals fed at their
maintenance levels.
Acknowledgements
Financial support from the Faculty of Veterinary Med-
icine (Urmia University–Iran) is fully appreciated.
7
Blood profile of sheep in response to garlic
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