Download Immunobiochemical status of sheep exposed to periods of

Published December 5, 2014
Immunobiochemical status of sheep exposed to periods
of experimental protein deficit and realimentation
A. Sahoo,*1,2 A. K. Pattanaik,* and T. K. Goswami†
*Animal Nutrition Division, and †Immunology Section, Indian Veterinary Research Institute,
Izatnagar – 243 122, India
ABSTRACT: Immunobiochemical status of sheep exposed to periods of experimental protein deficit and
realimentation was studied in 12 sheep (15 mo) randomly distributed into 2 equal groups and fed individually 2 different concentrate supplements along with
wheat straw to provide 100% (normal protein, NP) or
50% (low protein, LP) of CP requirements. The study
was comprised of 3 periods; during period 1 (0 to 13
wk) and 2 (14 to 26 wk) animals in the 2 groups were
fed NP and LP diets, respectively; during period 3 (27
to 44 wk), animals in the LP group were switched over
to NP diet to allow realimentation, whereas animals in
the NP group remained on the same NP diet. Blood
was collected from all groups at end of each period, and
serum glucose, total protein, albumin (sAlb), globulin
(sGlb), urea (sU), creatinine (sCr), cholesterol, triiodothyronine, and thyroxine concentrations were determined. During the same periods, the cell-mediated
immune (CMI) response was measured by a delayed
type hypersensitivity (DTH) assay and in vitro nitrite
production by lymphocytes. At the end of periods 2
and 3, humoral immune response (HIR) was measured
by sensitizing the sheep with Brucella abortus S99 antigen and measuring antibody titers on 0, 7, 14, 21, and
28 d postinoculation by ELISA. Feed intake decreased
with prolonged protein deficit and showed recovery
during period 3. Blood chemistry revealed reduced
sAlb concentration in the LP group resulting in narrow sAlb:sGlb ratio, increased sCr concentrations (P =
0.008) accompanying a decreased (P = 0.004) sU:sCr
ratio, and decreased glucose concentrations (P = 0.05).
Other variables did not change significantly between
the NP and LP groups. The DTH response at the end
of period 1 and 2 showed marked (P = 0.008) effect of
protein restriction on CMI. Nitrite production, basal
and after lipopolysaccharide stimulation, was greater
(P = 0.04) in the NP group. The HIR was less (P =
0.04) in the LP group during period 3. Realimentation of protein in the LP group during period 3 showed
recovery in CMI and HIR. In conclusion, protein deprivation induced a decline in CMI and HIR in sheep
accompanying alterations in related metabolic profile.
However, a marked recovery was observed after realimentation.
Key words: immunity, metabolic profile, nutrition, protein deficit, sheep
©2009 American Society of Animal Science. All rights reserved.
INTRODUCTION
The effects of nutrition on immunological functions
have been the focus of clinical and experimental studies.
Field studies in developing countries have consistently
linked severe malnutrition with immunologic crippling
and increased susceptibility to infection (Good, 1981).
Immune functions have traditionally been regarded as
part of maintenance requirements, but an increasing
body of evidence suggests sensitivity of immunity to
nutrient supply (Galyean et al., 1999).
1
Present address: Central Sheep & Wool Research Institute, Avikanagar (Rajasthan)-304 501, India.
2
Corresponding author: [email protected]
Received January 25, 2008.
Accepted April 15, 2009.
J. Anim. Sci. 2009. 87:2664–2673
doi:10.2527/jas.2008-0906
Domestic livestock in tropics are undernourished due
to chronic shortage of protein and energy-rich feeds.
Sheep and goats are often raised on grazing with little
or no supplementation. Unavailability of forage is the
main reason of underfeeding, and protein is the most
critical macronutrient. We have established that deficits in protein and reduced immune status adversely affect production of animals (Sahoo et al., 2002). Protein
deficiency decreases cytokine release and consequently
affects regulation of immune response (Klasing, 1988)
and immunoglobulin synthesis (Reddy and Frey, 1993).
Protein malnutrition is often accompanied by infections, and metabolic response to chronic protein and
energy malnutrition is complicated by the concurrent
response elicited by the stress due to infection (Jahoor
et al., 1999). The diversion of nutrients from growth to
immune-related processes is a major cause behind fail-
2664
Protein deficit and immunobiochemistry
ure to achieve optimal growth in the face of an immune
challenge (Spurlock, 1997). Epidemiological observations have established the aggravation of infections
and many instances of vaccine failure in malnourished
patients (Chandra, 1999). Sheffy and Williams (1982)
reported the impact of seasonal starvation and refeeding of cattle on frequency of foot and mouth disease
outbreaks in India. They suggested that dysfunction
in any protective mechanism results in inadequate inflammatory response and specific immunity. Periodic
starvation or malnutrition with a protein-deficit occurs
in many developing countries (Devendra and Sevilla,
2002). Our study was carried out to assess immunological and biochemical responses in sheep fed a low protein (LP) diet and after realimentation with a normal
protein (NP) diet.
MATERIALS AND METHODS
The protocol for the current study was approved by
the Animal Ethics Committee of the Indian Veterinary
Research Institute.
The experiment was conducted in 3 periods. Periods
1 (0 to 13 wk) and 2 (14 to 26 wk) were to assess the
effects of protein deficit on a short-term and long-term
basis, respectively, whereas period 3 (27 to 44 wk) was
aimed at ascertaining the effects of realimentation of
the deficit group, when switched over to normal level
of protein feeding. The study was carried out using 12
apparently healthy 15-mo-old Muzzafarnagari rams as
experimental animals. The animals were procured from
the sheep farm of Central Institute for Research on
Goats (Makhdoom, India). Before the beginning of the
experiment, all the animals were fed on a standard diet
consisting of concentrate and wheat straw for a period
of 30 d to ensure similar nutritional and metabolic status. The animals were housed in a well-ventilated shed
with facilities for exercise in the adjacent paddock and
had ad libitum access to fresh water.
Animal and Experimental Design
The details of experimental design and distribution
of sheep in different groups are presented in Figure 1.
The animals were distributed randomly in 2 groups,
NP-fed group (22.8 ± 2.0 kg) and LP-fed group (23.0 ±
1.6 kg), of 6 animals each. The NP group was fed a diet
with 13% CP (on as-fed basis) consisting of concentrate
mixture (Table 1) and wheat straw. The CP content of
the diet in LP group was reduced to 50% of that in NP
(i.e., CP 6.5% on as-fed basis) by reducing the level
of protein meal in the concentrate mixture, but the
energy content was similar. The ratio of concentrate
and roughage was regulated once every 2 wk during the
course of experimental feeding to meet the minimum
growth potential (20 to 40 g of BW gain/d) as per
NRC (1985) with minor modifications recommended
by ICAR (1998) to account for the characteristically
2665
Figure 1. Experimental design and distribution of sheep in different groups. NP = normal protein diet for maintenance; LP = low
protein diet (50% of NP). The LP group was realimented with NP diet
during 27 to 44 wk (period 3).
lesser BW and growth rate of crossbred sheep in India.
The sheep were fed individually with required concentrate mixture and wheat straw (ad libitum with 10 to
15% refusal) offered separately in 1 feeding, and daily
feed weigh backs were performed to assess the level of
feed intake. Samples of the offered feeds were collected every 2 wk to determine DM content and nutrient
composition, whereas samples of refusal were collected
and weighed daily from each pen to determine DMI.
The pooled samples of feeds and refusals were dried in
a forced-air oven (50°C), ground in a laboratory mill
(SM100, Retsch GmbH, Haan, Germany) through a
1-mm screen, and stored for laboratory analysis. The
restriction of CP to 50% level was imposed gradually
starting with 75%, then 60% of NP, with each period
lasting 3 d. The study was comprised of 3 periods; during periods 1 (0 to 13 wk) and 2 (14 to 26 wk), animals under NP and LP groups were fed the respective
NP and LP diets. However, immediately after period 2,
starting from 27 wk, LP group animals were switched
over to the NP diet, as a measure of dietary protein
realimentation, whereas the NP group was allowed to
be fed on the normal protein diet (period 3), extending up to 44 wk. Thus, the NP animals were fed the
normal protein diet for the whole duration of the study
(i.e., 44 wk), whereas LP animals were fed a proteindeficient diet for first 26 wk, which was followed by
feeding a normal protein diet for the last 18 wk of the
experiment. Period 3 was designed to assess recuperative changes after alimentation to a normal protein diet
from a state of deficit protein nutrition.
BW Recording
The animals were weighed at regular intervals (once
every 2 wk) in the morning before offering feed and
water to record pattern of BW change during the 3
periods.
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Sahoo et al.
Table 1. Physical composition (%, as-fed basis) of concentrate supplement fed to sheep
during the experiment
Ingredient
Ground maize
Wheat bran
Soybean meal, solvent extracted
Mineral mixture1
Salt
Concentrate with
normal protein
Concentrate with
low protein
12.5
18.0
68.0
1.0
0.5
26.0
69.0
3.5
1.0
0.5
1
Contained per kilogram: 280 g of Ca; 120 g of P; 5 g of Fe; 0.26 g of I; 0.77 g of Cu; 0.13 g of Co.
Blood Collection and Sampling
Blood samples were collected by jugular venipuncture at the end of each period to monitor the periodic alterations in hematobiochemical attributes viz.
hemoglobin (Hb), total serum protein (sTP), albumin
(sAlb), globulins (sGlb), urea (sU), creatinine (sCr),
glucose (sG), cholesterol (sCh), triiodothyronine (T3),
and thyroxine (T4). Blood samples were collected in
10-mL tubes without anticoagulant, left for 30 min at
room temperature, and centrifuged for 20 min at 1,000
× g at room temperature (25°C), and the serum was
stored at –20°C for analysis.
read at 525 nm (Rahmatullah and Boyde, 1980). The
sCr was measured spectrophotometrically by Jaffe’s reaction (Heinegard and Tiderstrom, 1973). The sG was
determined by O-toluidine reduction method (Hultmann, 1959), and sCh was determined through oxidation with ferric chloride under acidic condition and
measuring the purple colored complex at 570 nm (Zlatkis et al., 1953). All the measurements were done using a UV-visual spectrophotometer (UV2800, Labomed
Inc., Culver City, CA). A diagnostic kit supplied by
Bhabha Atomic Research Centre, Mumbai was used for
the RIA of T3 and T4 with counting in a Multi Crystal
Gamma Counter (LB 2103, Berthold-Wallac, Wildbad,
Germany).
Feed Analysis
Samples of concentrate mixture, wheat straw, and refusals were analyzed for DM, ash, N, and Soxhlet ether
extract (methods 930.15, 942.05, 990.02, and 920.39,
respectively; AOAC, 1990), and NDF and ADF (Van
Soest et al., 1991). Neutral detergent fiber was determined with sodium sulfite and α-amylase in the NDF
reagent. The values for NDF and ADF were expressed
with residual ash.
Blood Biochemical Assay
The Hb concentration of blood was determined immediately after collection by cyanomethemoglobin method
(Coles, 1980). Briefly, 20 µL of blood was treated with
5.0 mL of Drabkin’s solution [0.05 g of KCN, 0.20 g of
K3Fe(CN)6, 1.00 g of NaHCO3 in 1 L of H2O], and the
absorbance was read at 548 nm. The sTP concentration
was measured by treating with biuret reagent, where
the formation of blue peptide-Cu complex in alkaline
solution was measured spectrophotometrically at 540
nm (Gornall et al., 1949). The sAlb was measured by
dye-binding method, where 25 µL of blood serum was
treated with 3.0 mL of buffered (pH 4.2) dye solution
(succinate buffer, pH 4.0; bromocresol green 0.6 mM;
Brij-35 30% solution 0.06%) and the absorbance was
read at 630 nm (Doumas et al., 1971), and the sGlb
was calculated by difference (total protein minus albumin). The serum samples were analyzed for sU by
treating the protein-free serum sample (0.20 mL) with
acid-ferric solution containing diacetylmonoxime and
thiosemicarbazide (3.0 mL), and the absorbance was
Immunological Variables
The cell-mediated immune (CMI) response of the
experimental animals was assessed at the end of each
period. Delayed type hypersensitivity (DTH) was assessed from increase in skin thickness after intradermal
inoculation of phytohaemaglutinin-P (PHA-P) antigen at 24, 48, 72, and 96 h. In vitro nitrite production, basal and after simulation with lipopolysaccharides (LPS) by lymphocytes in the culture medium as
a measure of CMI, was determined by Griess’s reaction
method (Green et al., 1982). Mononuclear cells from
the peripheral blood were separated by density gradient
centrifugation (Boyum, 1968). Briefly, whole blood (5
mL) was collected into heparin-coated tubes (Hindustan Syringes and Medical Devices Limited, New Delhi,
India) by jugular venipuncture from each animal. The
blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and 7 mL of the blood/HBSS mixture
was layered over 4 mL of histopaque (Sigma, St. Louis,
MO). Samples were centrifuged at 400 × g for 30 min
at 25°C. After centrifugation, the interface cells were
removed and washed twice with sterile HBSS. Finally,
cells were dispensed in RPMI-1640 medium (phenol
free) having 10% fetal calf serum, 100 IU of penicillin, 100 µg of streptomycin, and supplemented with 5
mM of l-arginine to achieve a cell concentration of 5 ×
106 cell/mL. Ninety-six well flat-bottomed tissue culture plates were seeded with 5 × 106 cells in 100 µL
of above medium containing Brucella antigen (10 µg/
mL) followed by incubation at 37°C under 5% CO2 for
48 h. For the estimation of nitrite, culture superna-
2667
Protein deficit and immunobiochemistry
Table 2. Chemical composition (% of DM) of the concentrate supplement and wheat straw fed to sheep during the experiment
Concentrate supplement1
Item
NP
LP
Wheat straw
OM
CP
Ether extract
Total carbohydrate2
NDF
ADF
90.7
33.9
3.0
53.7
38.8
9.7
93.0
14.1
2.8
76.1
43.7
15.5
92.9
3.9
0.8
88.2
80.9
51.9
1989). The statistical difference between periods 1, 2,
and 3 was assessed separately. Analysis of covariance
was employed to ascertain the postchallenge periodic
difference in immunological parameters between the
treatments. The statistical software SPSS (SPSS Inc.,
Chicago, IL) was used for the analysis of data.
RESULTS
Nutrient Composition and Intake
1
NP = normal protein; LP = low protein.
2
Total carbohydrate = OM − CP − ether extract.
tant (50 µL) was mixed with an equal volume of Griess
reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4], and
absorbance was recorded at 570 nm using a microplate
reader (Microscan-MS5605A, Electronic Corporation of
India Limited, Hyderabad, India). Nitrite level was determined by comparison with sodium nitrite standard
curve. Similar observations were also recorded toward
the end of realimentation period (44 wk). Before that,
humoral immune response (HIR) of the animals was
assessed in both the phases by sensitizing the sheep
with Brucella abortus S99 antigen and measuring the
antibody titer at 0, 7, 14, 21, and 28 d postinoculation
by ELISA. A constant dilution of serum on different
days (1:1,000 in PBS with Tween-20) for each test serum was used for antigen-antibody reaction, and the
color development was measured at 492 nm. Greater
absorbance indicated greater antibody titer in the sample, thus indicating qualitative difference between the
treatments.
Statistical Analysis
A mixed model with repeated measures was used
for the analysis of data. The data were subjected to
ANOVA and Student’s t-test (Snedecor and Cochran,
The basal roughage of wheat straw contained 39 g of
CP/kg of DM, whereas it was 339 and 141 g/kg of DM
in concentrate mixtures for NP and LP, respectively
(Table 2). Animals in LP group did not show any sign
of gastrointestinal disorder during the course of protein restriction. The feed DMI in both the groups was
statistically similar (P = 0.91) during period 1, but
the CP intake was greater in NP (101 g/d) than LP
(53 g/d) as it was experimentally reduced in the later
group (Table 3). Prolonged protein deficit (period 2)
reduced (P = 0.007) both DMI (62.7 ± 2.59 vs. 52.0
± 1.85 g/kg of BW0.75) and CP intake of animals in
the LP group compared with animals in the NP group.
Animals in the LP group showed increased (P = 0.03)
intake of feed DM and CP during the realimentation
period (period 3) and became similar (P = 0.22) to the
animals in NP group.
Pattern of BW Change
The BW at 13, 26, and 44 wk of experimental feeding (Figure 2) was not different (P > 0.05) between the
groups. However, there was a decrease in net BW gain
of animals in the LP group compared with animals in
the NP group during periods 1 (1.4 ± 0.5 vs. 3.6 ±
0.5 kg; P = 0.02) and 2 (0.8 ± 0.3 vs. 2.8 ± 0.5 kg; P
= 0.009). During period 3, animals in the LP group
showed a comparative increase (P < 0.001) in BW gain
(3.6 ± 0.5 kg) relative to the animals in the NP group
(2.4 ± 0.4 kg).
Table 3. Pattern of DM and CP intake in sheep exposed to protein restriction and realimentation1
Period3
Item2
DMI, g/(kg of BW0.75·d)
NP
LP
CP intake, g/(kg of BW0.75·d)
NP
LP
a,b
1 (0 to 13 wk)
2 (14 to 26 wk)
3 (27 to 44 wk)
48.6 ± 4.24b
53.6 ± 3.03b
62.7 ± 2.59a,z
52.0 ± 1.85b
68.1 ± 2.32a
64.4 ± 2.58a
8.0 ± 0.108z
4.2 ± 0.071b
8.1 ± 0.077z
4.4 ± 0.052b
8.1 ± 0.098
8.0 ± 0.087a
Overall (0 to 44 wk)
60.8 ± 3.09
57.6 ± 2.52
8.1 ± 0.091z
5.9 ± 0.068
P-value4
0.03
0.03
0.65
<0.001
Within a row, means without a common superscript letter differ (P < 0.05).
Means in a column (between treatment groups NP and LP) differ (P < 0.05).
1
The data are expressed as mean ± SEM from n = 6 observations.
2
NP = normal protein diet, provided CP for maintenance as per NRC (1985) and ICAR (1998); LP = low protein diet, provided 50% CP of NP
(LP diet was replaced with NP during period 3).
3
Period 1 and 2 refer to short- and long-term protein restriction, respectively; period 3 refers to period of realimentation.
4
P-values for period effects.
z
2668
Sahoo et al.
Figure 2. Pattern of BW change in sheep subjected to protein
restriction and realimentation. NP = normal protein diet for maintenance; LP = low protein diet (50% of NP). The LP group was realimented with NP diet during 27 to 44 wk (period 3).
Blood Variables
The Hb concentration (g/dL) showed no difference
(P = 0.09) between the NP and LP groups in period
1, but declined (P = 0.04) later in period 2, thereby
revealing variation (P = 0.007) between the 2 groups
(9.0 vs. 12.0 g/dL; Table 4). In period 3, there was recuperation and the values became comparable between
LP and NP. Concentration of sTP in the LP group
declined (P = 0.04) in period 2 (5.4 g/dL) and was less
(P = 0.05) compared with the NP group (6.6 g/dL).
There was also decrease in sAlb concentration in the
LP group (2.3 g/dL) showing apparent difference (P =
0.05) from the NP group (3.2 g/dL) during period 2,
which recovered partially during period 3 (2.5 g/dL),
resulting in an overall decreased (P = 0.06) value (2.5
g/dL) compared with the NP group (3.0 g/dL). No alteration (P = 0.24) in sGlb concentration was observed
in LP during period 1 to 3, and there was no difference
between the groups (P = 0.95). The sAlb:sGlb ratio revealed similar treatment effect and treatment × period
interaction as that observed for sGlb. Prolonged feeding of LP also reflected in decreased (P = 0.05) sU concentration (18.6 ± 2.4 mg/dL during period 2), which
showed recovery during the realimentation period (21.9
± 1.85 mg/dL). There were similar (P = 0.57) sU values in the NP and LP groups during period 1, but it
declined (P = 0.05) in the LP group during period 2
and showed recovery during period 3. However, there
was no difference between the treatment groups (P =
0.81). Concentration of sCr (mg/dL) was greater (P =
0.008) in the LP group than the NP group, especially
toward the end of protein deficit state [i.e., period 2
(1.4 ± 0.137 vs. 0.8 ± 0.069 mg/dL)]. There was partial
recovery after realimentation, but the overall difference
remained (1.2 ± 0.088 vs. 0.9 ± 0.064 mg/dL; P =
0.03). The sU:sCr ratio in the LP group declined (P =
0.02) in period 2 (13.6 vs. 21.4 mg/dL), which was also
less (P = 0.004) compared with the NP group (30.8
mg/dL). Although, the sU:sCr ratio showed recuperation during period 3, the overall difference between the
treatment groups persisted (P = 0.02). There was no
difference (P = 0.92) in concentration of sG (mg/dL)
between the NP (50.9 mg/dL) and LP groups (48.6
mg/dL) during period 1, but it became apparent (P =
0.05) during period 2 (52.9 vs. 47.0 mg/dL). During realimentation, the sG became similar (P = 0.77), which
was also reflected in the overall average values (53.2
mg/dL in NP and 48.9 mg/dL in LP). Concentration
of sCh (mg/dL) did not differ between the treatments
or the periods showing an average value of 116.8 mg/dL
in NP and 111.1 mg/dL in LP groups. The concentration of T3 was comparatively less in LP than NP during
period 1 (P = 0.10) but not during period 2 (P = 0.47)
or 3. However, serum concentration of T4 did not differ
(P = 0.56) between the NP and LP groups during any
period of observation.
Immunological Response
The CMI response of sheep as assessed through DTH
reaction at the end of periods 1, 2, and 3 revealed increased (P < 0.001) skin thickness after 24 and 48 h of
intradermal inoculation of PHA-P antigen, which reduced (P < 0.001) subsequently at 72 and 96 h (Figure
3). There was a difference (P = 0.007) in the rate of
DTH reaction between the NP and LP groups in periods 1 and 2. After realimentation (period 3), the gap
was narrowed down with no difference (P > 0.05) between normal and deficient groups. Similarly, the CMI
response assessed through in vitro nitrite production
revealed greater (P = 0.04) values for basal and postLPS stimulation in the animals of NP group during
period 2 (Figure 4). The protein deficient animals in
LP group, however, showed recovery with similar (P
> 0.05) in vitro nitrite production when shifted to NP
diet during period 3.
The HIR by ELISA showed a reduced (P = 0.04) antibody titer (OD492) against Brucella abortus S99 antigen in the animals on the LP diet (Figure 5). The titer
showed an increasing trend until 21 d postinoculation
of antigen and continued to remain at peak concentration until 28 d and declined thereafter. There was no
difference (P = 0.52) in antibody titer between the NP
and LP groups during realimentation period, indicating
a recovery in HIR.
DISCUSSION
Crude protein intake by animals in the NP group
was within the range recommended by NRC (1985)
and ICAR (1998) for the maintenance requirement of
sheep. However, the CP intake in the LP group was
reduced by nearly 50% of that in the NP group. Paul
et al. (2003) in a comprehensive study recommended a
reduced protein requirement (6.98 g of CP and 4.49 g
of digestible CP per kg of BW0.75) for the maintenance
of growing Indian sheep (BW range > 15 kg) under
the tropical condition, compared with the recommendations of NRC and ICAR. Thus, when compared with
the recommendations suggested by Paul et al. (2003),
CP intake by the LP animals in the present study fell
short by nearly 35%.
2669
Protein deficit and immunobiochemistry
1
Table 4. Effects of protein restriction and recuperation on blood variables of sheep
Period3
Attribute2
Hemoglobin, g/dL
NP
LP
Total protein, g/dL
NP
LP
Albumin, g/dL
NP
LP
Globulin, g/dL
NP
LP
Albumin:globulin ratio
NP
LP
Urea, mg/dL
NP
LP
Creatinine, mg/dL
NP
LP
Urea:creatinine ratio
NP
LP
Glucose, mg/dL
NP
LP
Cholesterol, mg/dL
NP
LP
Triiodothyronine, ng/dL
NP
LP
Thyroxine, ng/dL
NP
LP
1 (0 to 13 wk)
2 (14 to 26 wk)
3 (27 to 44 wk)
12.4 ± 0.39
11.3 ± 0.57a
12.0 ± 0.33z
9.0 ± 0.41b
12.7 ± 0.43
11.2 ± 0.29a
6.8 ± 0.43
6.6 ± 0.27a
6.6 ± 0.35z
5.4 ± 0.33b
3.2 ± 0.17
2.9 ± 0.18a
Overall (0 to 44 wk)
P-value4
12.4 ± 0.38
10.5 ± 0.40
0.86
0.04
6.5 ± 0.28
6.0 ± 0.25ab
6.6 ± 0.21
6.0 ± 0.18
0.92
0.04
3.2 ± 0.21z
2.3 ± 0.16b
3.0 ± 0.21z
2.5 ± 0.11b
3.0 ± 0.18z
2.5 ± 0.12
0.98
0.04
3.8 ± 0.26
3.7 ± 0.25
3.6 ± 0.21
3.2 ± 0.19
3.5 ± 0.35
3.5 ± 0.39
3.6 ± 0.21
3.5 ± 0.21
0.93
0.24
0.81 ± 0.047
0.77 ± 0.039
0.85 ± 0.051z
0.70 ± 0.037
0.85 ± 0.057
0.71 ± 0.044
0.84 ± 0.039z
0.73 ± 0.031
0.93
0.44
22.8 ± 2.65
25.7 ± 1.58a
24.4 ± 3.16
18.6 ± 2.36b
24.2 ± 2.80
21.9 ± 1.85ab
23.8 ± 2.59
22.1 ± 2.17
0.86
0.05
0.9 ± 0.081
1.2 ± 0.108
0.8 ± 0.069
1.4 ± 0.137z
0.9 ± 0.092
1.1 ± 0.101
0.9 ± 0.064
1.2 ± 0.088z
0.42
0.33
23.9 ± 2.59
21.4 ± 2.01a
30.8 ± 2.43z
13.6 ± 1.25b
26.6 ± 2.38
18.9 ± 1.67ab
27.6 ± 1.97z
18.0 ± 1.51
0.08
0.02
50.9 ± 2.58
48.6 ± 2.13
52.9 ± 2.33z
47.0 ± 1.70
55.8 ± 1.03
51.2 ± 2.47
53.2 ± 1.45
48.9 ± 1.35
0.24
0.39
109.7 ± 5.90
107.7 ± 7.62
117.6 ± 9.44
112.7 ± 6.57
116.8 ± 5.06
111.1 ± 4.20
0.56
0.76
2.2 ± 0.19
1.9 ± 0.15
0.08
0.03
29.7 ± 2.48
27.2 ± 2.14
0.96
0.93
123.0 ± 11.31
112.8 ± 9.41
2.0 ± 0.22
1.5 ± 0.18b
30.2 ± 2.71
28.0 ± 3.97
2.0 ± 0.27
1.7 ± 0.25b
30.5 ± 5.11
28.1 ± 3.69
2.6 ± 0.28
2.6 ± 0.21a
28.6 ± 5.79
25.5 ± 4.44
a,b
Within a row, means without a common superscript letter differ (P < 0.05).
Means in a column (between treatment groups NP and LP) differ (P < 0.05).
1
The data are expressed as mean ± SEM from n = 6 observations.
2
NP, normal protein diet, provided CP for maintenance as per NRC (1985) and ICAR (1998); LP, low protein diet, provided 50% CP of NP
(LP diet was replaced with NP during period 3).
3
Period 1 and 2 refer to short- and long-term protein restriction, respectively; period 3 refers to period of realimentation.
4
P-values for period effects.
z
The decrease in feed and nutrient intake with the
long-term protein deficit might be attributed to a
complex set of metabolic alterations related to ruminal digestion and fermentative activities (MichaletDoreau and Doreau, 2001; Doreau et al., 2003). During the realimentation phase (period 3), animals in
group LP did not show hyperphagy, which may be
attributed to the roughage component of the diet providing an intrinsic limitation to voluntary feed intake
(Weston, 1996; Baumont et al., 2000; Fisher, 2002).
A decreased BW gain in LP during periods 1 and 2
was attributed to less total feed and nutrient intake.
However, a greater BW gain during period 3 by LP
group animals compared with NP animals, without
any apparent variation in DMI, may be attributable
to metabolic readjustment to prolonged LP and re-
duced feed intake, thereby economizing the available
nutrient resources for a greater rate of gain in compensation.
The Hb level decreased during period 2 as an effect of prolonged protein deficit. A severe protein deficiency has been suggested to interfere with Hb production (Whitehair, 1958). In domestic ruminants, sTP
and sAlb concentrations are used for the assessment of
nutritional status (Kaneko, 1989), and reduced concentrations are a measure of protein deficiency and malnutrition. The sAlb has a long half-life, but inadequate
dietary protein intake leads to a slow and gradual decrease in the albumin concentration when enough AA
are not supplied to the liver cells for its synthesis, and
thus, its concentration provides a long-term indicator
of protein intake in ruminants.
2670
Sahoo et al.
Figure 3. Delayed type hypersensitivity response of sheep subjected to protein restriction and realimentation. NP = normal protein
diet for maintenance; LP = low protein diet (50% of NP). The LP
group was realimented with NP diet during period 3. A treatment ×
time interaction revealed during period 1 and 2 at 48 h postinoculation; *P < 0.05.
The concentration of sGlb may increase in chronic
infections or during decreased immunity, and a reduced
sAlb:sGlb ratio may indicate an increase in sGlb concentration caused by chronic parasitism or compensation for the sAlb loss evident in protein malnutrition
(Payne, 1987). Nonetheless, the ratio should always be
interpreted with care by taking into account the concurrent changes in sTP concentration. The concentrations
of sGlb were less affected by a low protein intake than
sTP and sAlb, which could be partially attributable to
a tendency to compensate for the decrease in sAlb. It is
also likely that immunological functions are prioritized
over productive function (Coop and Kyriazakis, 1999),
and thus sGlb are better conserved during nutritional
constraints, particularly that of protein leading to narrow sAlb:sGlb ratio. Interestingly, sAlb concentration
remained depressed during realimentation in period 3
with sGlb remaining unaltered. Experimental and clinical hypoproteinaemia has been reported to result in liver atrophy (Oser, 1971) that affects albumin synthesis
in protein-deficient diets (Charland et al., 1994). Usually, the sGlb concentration is the last to be affected in
prolonged hypoproteinaemia, resulting in lowered immunity in animals (Tizzard, 1992). Infection, inflammation, and injury do not suppress the rate of synthesis
of albumin; rather, such conditions may bring about an
increase in catabolism or intravascular loss of albumin
and total protein (Fleck et al., 1985). In support of
this argument, the concentration of sCr (mg/dL) was
elevated in the LP group, especially toward the end of
protein deficit period 2.
Urea production and its concentration in blood respond to immediate changes in dietary protein intake,
thereby reflecting short-term changes in protein metabolism and complementing the information provided
by the analysis of sTP and sAlb concentrations (Payne,
1987). There was a fall in sU concentrations despite the
fact that the body has an excellent regulatory mecha-
Figure 4. Cell-mediated immunity (CMI) in sheep subjected to protein restriction and realimentation as assessed through in vitro nitrite
production both at basal (unstimulated) level and after stimulation with lipopolysaccharide (LPS). NP = normal protein diet for maintenance;
LP = low protein diet (50% of NP). The LP group was realimented with NP diet during period 3. The CMI response was greater in NP than LP
for basal and after LPS stimulation during period 2; *P < 0.05; **P < 0.01.
Protein deficit and immunobiochemistry
nism of a decreasing urinary N excretion with a major proportion of urea being recycled into the rumen
through saliva (Satter and Roffler, 1977). There exists a negative relationship between N intake and the
percentage of hepatic urea recycled from gut to lumen
(Harmeyer and Martens, 1980) consequential to a reduction in overall N flux or in its proportion. This may
result in decreased urinary N excretion due to relative
preservation of N over carbon during AA catabolism on
reduced protein intakes (Millward et al., 1991). The net
transfer of urea across portal drained viscera increases,
as a percentage of N intake, in underfed ewes (Nozière
et al., 2000), which contributes to sparing of N, together with decreased N uptake by the kidney (Cirio
and Boivin, 1990) and urinary loss (Harmeyer and
Martens, 1980; Nozière et al., 2000). The decreased sU
and increased sCr concentrations, with the consequent
reduction in the sU:sCr ratio recorded during period 2,
can be related to physiological adaptation of renal function to save protein through recycling of urea with low
dietary protein intake. The effect of protein intake on
the blood urea concentration was confounded when a
low protein intake was combined with a restricted feed
intake or by increased production of urea due to muscle
catabolism.
Nutrition-related changes in the sCr concentrations
include elevated concentrations during fasting, malnutrition, or food restriction, which have been related to
a reduction in filtration in the kidneys and increased
production due to muscle catabolism (Kaneko, 1989).
The possibilities of assessing the nutritional status of
deer based on plasma or serum urea and creatinine
concentrations have attracted considerable interest.
DelGiudice et al. (1992) observed an increase in sCr,
a decrease in sU and a reduction in the sU:sCr ratio
through autumn from above 50 to 10 mg/dL in freeranging white-tailed deer exposed to seasonal undernutrition. The increase in sCr concentration on a low protein diet might have resulted from the decrease in the
glomerular filtration rate and the decrease in the excretion of creatinine into urine (Kaneko, 1989). The sCr
concentration has also been suggested to increase when
protein derived from muscle is used as an energy source
at times of nutritional deprivation. However, the feed/
energy intake in the present experiment was similar in
NP and LP animals, and thus, the possibility of muscle
catabolism can be overruled. The observed sU:sCr ratio
is principally affected by the plasma urea concentration, and its reduction reflects a shift in metabolism
toward conserving endogenous protein (Kaneko, 1989).
The absence of differences in concentration of sG and
sCh between animals in NP and LP groups was indicative of similar energy status. The concentration of
T3 revealed some periodic alteration and was also in
response to a realimentation diet during period 3. The
treatment and period did not have significant effect on
T4, but T3 was numerically less (17 to 24%) in LP than
NP animals during protein deficit periods (1 and 2).
Less T3 with normal concentrations of T4 is observed
2671
Figure 5. Humoral immunity of sheep subjected to protein restriction and realimentation as assessed through antibody titer (OD492)
against Brucella abortus S99 antigen. NP = normal protein diet for
maintenance; LP = low protein diet (50% of NP). The LP group was
realimented with NP diet during period 3. A treatment × day interaction revealed during period 2 at d 7, 14, and 21 postchallenge with
Brucella abortus antigen; **P < 0.01; ***P < 0.001.
in nutritional (protein) limitation (Stockigt, 2004). Todini (2007) stated that circulating thyroid hormones
can be considered as indicators of the metabolic and
nutritional status of the animals, and the quantity and
quality of feed eaten is a major factor determining plasma concentrations of T3. It is also thought that thyroid
hormones play an important role in activation and proliferation of lymphoid (thymomimetic) organ and tissue
of the body (Bidey et al., 1999), which could be due to
a possible role in eliciting the production of factors such
as thymulin and IL-2, which affect the immune system
(Marsh, 1995). Singh et al. (2006) also observed a significant depression in both cell-mediated and humoral
immune response in goitrous goats and attributed this
to a reduced thyroid hormone concentration that could
have impaired the functional status of lymphoid organ.
The sTP, sAlb, sU, and sCr concentrations and the
sU:sCr ratio especially showed significant variation that
could be related to physiological adaptation to deficit
protein nutrition and therefore appear as useful indicators of the protein metabolism in sheep.
Cell-mediated and humoral immune responses are
critical to the host defense against intracellular bacterial pathogens. A variety of in vitro and in vivo immune
function tests have been developed for their monitoring
(Cheville et al., 1993; Chiodini, 1996). The immunological variables evaluated in the present investigation
were aimed at providing an indication of the potential
effects of nutritional treatments on immune function independent of any diseased state. There was a decline in
2672
Sahoo et al.
CMI with a prolonged period of protein deficit. The attainment of peak DTH response and its continuity also
declined during periods 1 and 2, which was indicative of
decreased immunological response to an unknown antigen. Similarly, the nitrite production response, both
basal and post-LPS stimulation, was less. However,
when analyzed critically, the increment in nitrite production from basal to LPS stimulation was comparable.
This shift in immunological response was a clear indication of prioritization of metabolic activation toward
antigenic stimuli. Peripheral blood mononuclear cells
(lymphocytes in the present study) produce nitric oxide (NO) in response to stimulation with antigens, and
its determination in cell cultures may thus provide an
indication of immunological response of the animal. A
key component of this response is the clonal expansion
of lymphocytes (Pugliese, 1990) and the elaboration
of cytokines that activate macrophages for killing of
pathogenic antigen located within the phagosomal compartment. The potent mediators for this are reactive N
intermediates (e.g., NO) produced via the induction of
inducible NO synthase (iNOS), often as a sequel to
IFN-γ, TNF-α, or LPS stimulation (MacMicking et al.,
1997; Kaufmann, 1999). When exposed to infectious
and inflammatory agents, the host responds by initiating a wide range of metabolic adjustments mediated
mainly by cytokines, in addition to the specific and
nonspecific immune responses (Klasing, 1988).
Several in vitro or in vivo studies in the past have
confirmed cytokine (alone or in combination)-mediated changes of nutritional importance (anorexia, fever,
increased energy expenditure, and a marked increase
in N excretion), demonstrating net protein catabolism
(Klasing, 1987). Thus, the general metabolic response
to stress of infection, inflammation, or trauma is characterized by an increased rate of whole body protein
turnover and a redistribution of protein synthetic activity away from the synthesis of muscle and tissue proteins in support of immune-related processes that are
critical for survival (Jahoor et al., 1988; Fleck, 1989).
Therefore, among the macronutrients, protein seems to
be critical to meeting the increased catabolic activity
and initiating an immunological response against the invading pathogens or any antigenic stimuli. The reduced
antibody titer (OD492) against Brucella abortus S99 antigen, reflecting a decline in HIR in LP compared with
NP animals was indicative of prolonged protein insufficiency and a disjunctive effect on immunological status
of animals. In an earlier study (Sahoo et al., 2002),
calves fed a reduced protein level (75% of normal) had
significant depression in indirect hemagglutination titer
against Pasteurella multocida (P52 strain). Further, the
observation of a decrease in feed/nutrient intake with
prolonged protein deficit, coupled with reduced energy
consumption, might have overstated the effect.
The pleiotropic activities of the cytokines released
as part of the immune response strongly indicate an
altered nutrient requirement, the quantitative nature of
which has not been well investigated. Realimentation
of LP group with normal protein diet during period 3
showed partial recovery in the blood metabolic profile
and in the cell-mediated and humoral immune response.
However, the realimentation period of 18 wk seems to
be inadequate, and it appears a longer intervention period, or an increased level of CP (greater than the normal requirement), or both would have brought about
appreciable recuperative metabolic activation, which
needs critical investigation. Because poor nutrition (low
protein) was associated with a state of compromised
immunological response (both humoral and cell-mediated) and had adverse effects on production (e.g., BW
gain) and health of the animals, a detailed investigation
involving variable degrees of protein deficit in relation
to stage of production, diseased state, and normal functioning of animals along with concurrent field observations on various production variables and response to
prophylactic regimes (disease prevention, vaccination,
etc.) is essential for making definite recommendations.
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