Newborn poultry in cold regions often suffer from cold stress,
causing a series of changes in their physiology and metabolism, leading to
slow growth and decreased production performance. However, a single
anti-stress substance cannot completely or maximally eliminate or alleviate
the various effects of cold stress on animals. Therefore, the effects of the
supplemented glutamine and L-carnitine on broilers under low temperature
were evaluated in this study. Broilers were randomly allocated into 16
groups which were respectively fed with different levels of glutamine and
L-carnitine according to the L16 (45) orthogonal experimental
design for 3 weeks (the first week is the adaptive feeding period; the
second and third weeks are the cold exposure period). Growth performance
was recorded, and blood samples were collected during cold exposure. The
results showed the supplementation had altered the plasma parameters, growth
performance and cold-induced oxidative stress. The increase of
corticosterone and suppression of thyroid hormone was ameliorated.
Supplemented groups had lower daily feed intake and feed-to-gain ratio, higher
daily weight gain and better relative weights of immune organs. Plasma
glucose, total protein, blood urea nitrogen and alkaline phosphatase
changed as well. Oxidative stress was mollified due to the improved
activities of superoxide dismutase and glutathione peroxidase, heightened
total antioxidant capacity and stable malondialdehyde. Dietary glutamine and
L-carnitine improve the growth performance, nutritional status and cold
stress response of broilers at low temperature, and their interaction
occurred.
Introduction
Within the background of global warming, the environment becomes more complex
and changeable, and the disturbance to animals becomes more frequent
(Bailey et al., 2019; Hansen et al., 2019). At present, environmental low
temperature has become one of the most common stressors in cold regions, and
the cold stress caused by it
poses a threat to the health of human beings and animals. Cold stress is able
to induce various negative responses like other stresses, affecting
neuroendocrine, reproduction and cardiovascular systems, to change the
biochemical catalytic processes of the organism, leading to oxidative
damage, apoptosis, and other physiological or pathological reactions
(García-Díaz et al., 2015; Solianik et al., 2015; Lian et al.,
2017; Zhang et al., 2017; Cong et al., 2018). Cold stress also significantly
increases systemic energy expenditure and causes substantial metabolic
changes, including decreased insulinemia, increased hepatic glucose
production, feed intake changes, and increased glucose and fat utilization
in peripheral tissues (Yao et al., 2018; Liu et al., 2019; Shi et al.,
2019). Meanwhile, cold stress affects the production performance and product
quality of livestock and poultry (Zhang et al., 2014; Hao et al., 2015;
Nguyen et al., 2015). For example, cold stress not only stunts the growth
and development of broilers, but also requires more feed to maintain their
body temperature, resulting in low feed conversion rate and waste of
resources, as well as poor weight uniformity and carcass quality of
broilers. More seriously, the morbidity and mortality will be exacerbated
(Tsiouris et al., 2015). Therefore, cold stress is one of the main
barriers limiting the development of animal husbandry and is an urgent
problem to be solved in cold regions.
At present, one of the most effective ways to resist cold stress is to
improve diet. However, stress response is a non-specific systemic adaptive
response and its physiological abnormalities are also various. Therefore,
single anti-stress additives or nutritional supplements are difficult to
promote the maintenance of body homeostasis.
Finally, we selected the combination of glutamine and L-carnitine to
increase the nutritional and energy levels of the diet to improve the
adverse reactions of cold stress. Glutamine is the most abundant free amino
acid of the body (Wellen et al., 2010; Cruzat et al., 2018; Li et al.,
2019). Besides its role as a constituent of proteins and its importance in
amino acid transamination, glutamine has many kinds of nonnutritive function
(Li et al., 2019) and has been claimed to be essential conditionally
during stress or clinical situations (Stehle and Kuhn, 2015; Coqueiro et
al., 2019; Oh et al., 2020). For example, glutamine is the principal
metabolic fuel for small intestine enterocytes, lymphocytes, macrophages
and fibroblasts and is considered an essential amino acid in some species
under inflammatory conditions such as infection and injury (Roth, 2008;
Carr et al., 2010). Moreover, glutamine is one of the most efficacious
substrates for gluconeogenesis and has a protective effect on the
physiology and function of gastrointestinal tract (Fan et al., 2015; Zuhl
et al., 2015). Glutamine is able to decrease the incidence of infection in
surgery and trauma patients and is investigated as one effective
supplementary nutrition (Moe-Byrne et al., 2012). Meanwhile,
L-carnitine is reported to have two major functions: facilitating the
transport of long-chain fatty acids across the mitochondrial membrane and
the removal from mitochondria of short- and medium-chain fatty acids that
accumulate as a result of normal and abnormal metabolism (Rehman et al.,
2017; Weinert et al., 2017). Thus, dietary L-carnitine supplementation
promotes the β-oxidation of these fatty acids to generate adenosine
triphosphate (ATP) and improves energy utilization (Madsen et al., 2018).
In addition, L-carnitine participates in biological processes, for example,
regulation of gluconeogenesis, stimulation of fatty acid synthesis and
ketone, branched-chain amino acid, triglyceride, and cholesterol metabolism
(Maruyama et al., 2019). L-carnitine therapy is also a reasonable
approach for reducing systemic inflammation and its complications
(Khalatbari-Soltani and Tabibi, 2015).
The dietary addition of glutamine and L-carnitine has been studied a lot.
But the research about the glutamine supplementation is mainly focused on
its oral, parenteral, or enteral application under clinical conditions
(Roth, 2008; Tian et al., 2017). A similar state also exists in the
research field of L-carnitine (Mongioi et al., 2016; Agarwal et al.,
2018; Gavrilova, 2018). However, there are few reports about the effects of
dietary glutamine and L-carnitine on anti-cold stress of broilers. Therefore,
the purpose of this study is to investigate the influence of the
supplemental glutamine and L-carnitine on the grow performance, the
physiological response and antioxidant capacity of Arbor Acres (AA) broiler
chickens under cold exposure.
Materials and methodsChickens and diets
In this study, 480 one-day-old Arbor Acres (AA) broiler chickens were
randomly divided into 16 treatment groups of 30 birds each. Each group was
further divided into one main and two replicate subgroups. Each group was
kept in a specially designed biotron (College of Animal Science and
Veterinary Medicine, Heilongjiang Bayi Agricultural University), where the
temperature, relative humidity and lighting were controlled. These 16 groups
were administrated according to the L16 (45) orthogonal
experimental design of two diet supplements: glutamine (0, 0.4, 0.6, 0.8
percentage of diet weight) and L-carnitine (0, 50, 75, 100 mg/kg diet
weight). The levels of glutamine and L-carnitine in the diet are shown in
Table 1. The basal meal met the National Research Council requirements. The
experimental diets and drinking water were offered ad libitum. After the
first week of adaptive feeding, all groups were exposed to cold temperatures at 23±0.5∘C (this ambient temperature went down by 10±0.5∘C compared to the optimal temperature of 33 ∘C, and the relative humidity was
40 %) for 2 weeks. Feed intake and body weight gain were recorded weekly
during the 2-week cold exposure period, and daily feed intake (DFI), daily
weight gain (DWG) and feed-to-gain ratio (F / G) were calculated. The
experimental scheme is shown in Fig. 1.
Blood samples were collected weekly from each group at 06:00 GMT+8 of each
blood collection day. Blood was drawn from a wing vein using a heparinized
syringe within 30 s, applied to the warm bath at 37 ∘C for 20 min and
then centrifuged at 3500 rpm for 10 min to obtain plasma, which was stored
at -70∘C until analysis. All groups were euthanized after 12 h of
feed deprivation at the end of cold exposure. The weights of spleen, thymus
and bursa of Fabricius were determined, and relative weights of each organ
were calculated.
All experiments were approved by animal protection and the Utilization Committee
of Heilongjiang Bayi Agricultural University. All operations were carried out
according to the regulations of animal science and veterinary medicine
college. All efforts were made to minimize animal suffering and to reduce
the number of animals used.
Measurement of biochemical parameters
Corticosterone (CORT), as a classical stress hormone, was selected to
evaluate the cold stress state of chickens in each group. Thyroid hormones
(triiodothyronine, T3 and thyroxine, T4) have been selected to
assess the functional status of the thyroid body, which is related to the
body's metabolic heat production and body heat regulation. Plasma CORT,
T3 and T4 were measured using a commercial kit (Nanjing
Jiancheng Bioengineering Institute, Nanjing, PR China).
Plasma glucose (GLU), total protein (TP), blood urea nitrogen (BUN) and
alkaline phosphatase (ALP) have been used as indicators to evaluate the
biochemical and metabolic changes of chicks in each group and were measured
using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing,
PR China).
Plasma superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), total
antioxidant capacity (T-AOC) and malondialdehyde (MDA) were used to assess
the antioxidant capacity of chicks in each group and were measured using a
commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, PR
China).
All operations were carried out according to the instructions of the kit.
Statistical analysis
The effects of glutamine and L-carnitine on the measured parameters and the
possible time-dependent effects were analyzed by the general linear model
(SPSS Ver. 19.0). Duncan's test was used to determine which specific pairs
differed. Means were considered significantly different when P<0.05.
ResultsEffects of glutamine and L-carnitine on growth performance and immune
organ index of broilers under cold stress
The DFI, DWG and F / G are presented in Table 2, the relative weights of the
spleen, thymus and bursa of Fabricius in Table 3, and the results of
orthogonal contrast and variance analysis in Table 4. The growth performance
of the broilers exposed to the low ambient temperature improved due to the
supplementation of glutamine and L-carnitine. In the first week, the added
glutamine and L-carnitine had obviously altered the DFI, DWG and F / G (P<0.05), whereas there was an interaction only on DWG and F / G (P<0.05).
Groups A1 or B1 had the highest DFI, but the lowest DWG and the
highest F / G. On the contrary, in the second week, except for DFI neither DWG
nor F / G was affected by the two additives and their interaction (P>0.05). Groups A1 or B1 still had the biggest DFI means and
relatively lower DWG and F / G but did not maintain the least levels. With regard to
the total values, the changes of DFI, DWG and F / G induced by the
supplementation were significant (P<0.05), and there was apparent
interaction (P<0.05). Groups A1 or B1 had the high DFI, the lowest
DWG and the highest F / G. Overall, the DFI and F / G of the broilers in group
A1B1 were the highest while the DWG was not the least.
The growth performance of Arbor Acres broilers under low temperature conditions
supplemented by glutamine and L-carnitine.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. Data are given as mean ± SEM.
The immune organ indexes of Arbor Acres broilers under low temperature conditions
supplemented by glutamine and L-carnitine.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. Data are given as mean ± SEM.
The effects of supplemented glutamine and L-carnitine on growth
performance and organ's relative weight of Arbor Acres broilers under low temperature conditions.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. a–c Mean values within a column with no common superscript
differ significantly (P<0.05). *P<0.05. **P<0.01.
NS: not significant.
L-carnitine had significant impact on the relative weights of thymus (P<0.05) while glutamine lacked this effect (P>0.05), and groups
A1 had low values, but groups B1 or A1B1 had the lowest
means. The relative weights of the spleen were strongly affected by
glutamine or L-carnitine (P<0.05), and groups A1 or B1 or
A1B1 had the lowest means. The influence of glutamine or
L-carnitine on the relative weights of bursa of Fabricius was not obvious
(P>0.05), but still groups A1 or B1 or A1B1
had the lowest values.
Effects of glutamine and L-carnitine on related hormones and biochemical indexes in broilers under cold stress
The plasma concentrations of CORT, T3 and T4 are shown in Table 5, the plasma concentrations of GLU, TP, BUN and ALP in Table 6, and the
results of orthogonal contrast and variance analysis in Table 7. In the
first week, CORT concentrations were not affected by different levels of
glutamine or L-carnitine (P>0.05), and there was no interaction
(P>0.05). However, in the second week the interaction appeared
(P<0.05) and glutamine created obvious impact (P<0.05), and
groups A1 or B1 had the highest values. The group A1B1
also had the highest plasma CORT, but in the 2 weeks the exposure time
did not affect plasma CORT levels (P>0.05).
The plasma corticosterone and thyroid hormone of Arbor Acres
broilers under low temperature conditions supplemented by glutamine and L-carnitine.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. Data are given as mean ± SEM.
The plasma parameters of Arbor Acres broilers under low temperature conditions
supplemented by glutamine and L-carnitine.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. Data are given as mean ± SEM.
The effects of supplemented glutamine and L-carnitine on
corticosterone, thyroid hormone and other parameters of Arbor Acres broilers
under low temperature conditions.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. a–c Mean values within a column with no common superscript
differ significantly (P<0.05). *P<0.05. **P<0.01. NS: not significant.
There was no influence of glutamine or L-carnitine or interaction on the
plasma T3 (P>0.05), and group A1B1 had the least
values. Groups A1 or B1 had the lowest T3 levels in the first
week while having relatively low levels in the second week. On the other hand, the
two additives had obviously influenced the plasma concentrations of T4
(P<0.05) except the supplementation of L-carnitine in the first week
(P>0.05), and there was the interaction (P<0.05). Groups
A1 or B1 have the significantly lowest T4 (P<0.05),
while the values of groups A3 or B3 are the highest. Moreover, the
change of T3 or T4 had the time-dependent effect (P<0.05),
and from the first week to second week T3 grew obviously (P<0.05), while T4 declined significantly (P<0.05).
The plasma concentrations of GLU, TP, BUN and ALP had been altered by the
dietary supplementation. The addition of glutamine or L-carnitine apparently
altered plasma GLU concentrations (P<0.05), though only in the
second week there was obvious interaction (P<0.05). In the first
week, groups A1 or B1 obviously had the smallest values (P<0.05),
while in the second week, the means of groups A1 were not the least but
not different from the others, and the levels of groups B1 still were
the lowest. Moreover, the other groups held relatively stable and higher
plasma GLU, and the chickens in A1B1 always had the lowest GLU
concentrations. The change profile of TP was largely identical to that of
GLU, but the glutamine had a more obvious effect (P<0.05), and all
TP contents of groups A1 or B1 or A1B1 were the lowest.
Contrarily, only in the second week, dietary glutamine had a great impact on
the plasma BUN levels (P<0.05), but the means of groups A1
were not significantly different from others (P>0.05).
Nonetheless, the BUN means of all groups decreased obviously (P<0.05) and the chickens in group A1B1 had the highest BUN levels.
The alteration of ALP activity caused by the added glutamine or L-carnitine
and the interaction became apparent only in the second week (P<0.05),
and groups A1 or B1 had the biggest means. With the exposure,
groups A1 or B1 had increased ALP activity, whereas those of the
left groups declined. Overall, group A1B1 always had the highest
level.
Effects of glutamine and L-carnitine on antioxidant capacity of broilers under cold stress
The data of plasma SOD, GHS-PX, T-AOC and MDA are presented in the Table 8,
and the results of orthogonal contrast and variance analysis can be found in Table 9. The
activity of SOD and GHS-XP was not affected by the added L-carnitine (P>0.05), but it changed obviously because of the supplemented glutamine (P<0.05)
and in the second week by the obvious interaction (P<0.05). In the
studied 2 weeks, SOD of groups A1 or B1 held the relatively
stable level, whereas those in the other groups grew significantly (P<0.05). The added glutamine had the positive dose-dependent impact on the
GHS-PX activity (P<0.05), and groups B1 had the lowest levels,
though the L-carnitine had no obvious effect (P>0.05). The
chickens in group A1B1 had the lowest activity of SOD and GSH-PX.
With regard to T-AOC, both glutamine and L-carnitine yielded the obvious
effect (P<0.05), though only in the second week the interaction
became apparent (P<0.05). The means of all groups increased with the
experimental period, and groups A4 or B4 had the highest values
and gained the most, whereas groups A1 or B1 had the lowest means
and grew the least. Moreover, group A1B1 had the lowest T-AOC.
L-carnitine had obvious influence on plasma MDA (P<0.05) and groups
B1 had the highest levels. In the second week, glutamine had the
significant effect (P<0.05), and groups A1 had the highest
levels but no significant difference from others (P>0.05). Both
additives did not exert the time-dependent effect on the change of plasma MDA
(P>0.05), but the interaction always existed (P<0.05)
and group A1B1 had the highest levels.
The antioxidant capacity of Arbor Acres broilers under low temperature conditions
supplemented by glutamine and L-carnitine.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. Data are given as mean ± SEM.
The effects of supplemented glutamine and L-carnitine on
antioxidant capacity of Arbor Acres broilers under low temperature conditions.
The groups A1, A2, A3 and A4 were treated by glutamine
at 0, 0.4, 0.6 and 0.8 percentage of diet weight, and groups B1, B2,
B3 and B4 were treated by L-carnitine at 0, 50, 75 and 100 mg/kg diet
weight. a–c Mean values within a column with no common superscript
differ significantly (P<0.05). *P<0.05. **P<0.01. NS: not significant.
Discussion
The inconspicuous effect of glutamine and L-carnitine on plasma CORT in the
first week and the strong impact of glutamine and obvious interaction in the
second week may indicate that only in the second week the ability of
glutamine and L-carnitine to offset the increasing of plasma CORT becomes
apparent. Since elevated levels of plasma CORT are a classic indicator of
stress (Quinteiro-Filho et al., 2012; Honda et al., 2015), the highest
levels of plasma CORT in groups A1 or B1 or A1B1 imply
that the dietary glutamine and L-carnitine might have the ability to reduce
the plasma CORT concentrations and thereby could ameliorate the stress
responses or improve the resistance of the broilers to cold stress.
Glutamine or L-carnitine has been used as effectively dietary fortification
to resist various stressful situations (Izgüt-Uysal et al., 2007;
Kelek et al., 2019).
The groups A1 or B1 or A1B1 have the highest DFI,
relatively lower DWG and the highest F / G, and the supplementary glutamine
and L-carnitine have the obvious interaction, though there is significant
difference among groups F / G only in the first week. It can be concluded that
the dietary supplementation elicited the positive responses of growth
performance and feed conversion in the broilers during the 2-week exposure
to low temperature, and the studies about many species under various
conditions have the same report (Yi et al., 2005; Bartell and Batal,
2007). Obviously, the groups A1 or B1 or A1B1 have poor
growth performance and feed efficiency, mainly because they have spent more
energy for thermogenesis to resist the cold stress rather than for growth,
and thus their energy metabolism has been damaged (Hangalapura et al.,
2003; King and Swanson, 2013).
One possible mechanism to maintain the normal energy consumption in the
additive-administrated groups is that the addition of glutamine or
L-carnitine might moderate the stress-induced rising of plasma CORT and
improve the impaired energy balance of broilers exposed to cold, which might
be caused by the higher plasma CORT (Yuan et al., 2008), and the obvious
interaction of the two additives strengthens the effect. The high levels of
stress-induced CORT may stimulate the higher energy to spend for various
stress responses and hurt the energy efficiency (Jiang et al., 2008; Yuan et
al., 2008; Babacanoğlu et al., 2013), which may result in the
redistribution of energy and consequently damage the growth of many species
(Ralph et al., 2015).
Another involved mechanism is the alteration of thyroid hormone that would
induce the better energy metabolism (Chatzitomaris et al., 2017; Nordio,
2017). Glutamine- or L-carnitine-supplemented groups have higher T3
and T4 than groups A1 or B1, and therefore the chickens in
the latter groups might hold poorer metabolic conditions and hence should
have worse growth performance. The obvious variance of thyroid hormone may
be negatively correlated to the CORT concentrations as some research has
found (Angelier et al., 2016). The plasma CORT in groups A1 or
B1 or A1B1 is the highest, and consequently T3 and
T4 levels are lowest. Furthermore, the reduction of T4 and
increasing of T3 with the exposure time in all groups means the
augmented transformation of inactive T4 to active T3 probably
meets the strong metabolic need of the 1-day-old chickens for growth
or stress resistance in the experimental 2 weeks.
Glutamine not only is an important precursor for the synthesis of amino
acids, nucleotides, nucleic acids, amino sugars, proteins and many other
biologically important molecules, but also has been demonstrated to be the
principal metabolic fuel for rapidly proliferating cells (Yi et al.,
2005; Roth, 2008; Wellen et al., 2010). Furthermore, diets supplemented with
L-carnitine would enhance the oxidation of long-chain fatty acids to
generate ATP, improve energy efficiency (Rehman et al., 2017),
consequently perhaps promote the protein-sparing action of fat (Li et
al., 2017), reduce the heat production in exercising pigeons (Celik
and Oztürkcan, 2003), and has been reported to have a protective effect on
gastric mucosal barrier in rats exposed to cold-restraint stress
(Izgüt-Uysal et al., 2007). Thus, there are still maybe other
approaches for glutamine and L-carnitine to improve the metabolism for the
young broilers at rapid development.
The relative weights of the spleen, thymus and bursa of Fabricius in the
groups A1 or B1 are low, and group A1B1 has the lowest
values, which implies that immunodepression happened. These results confirm
that the immunological organs are sensitive to CORT (Mehaisen et al.,
2017; Yaribeygi et al., 2017), for the highest plasma CORT levels were
observed in the groups A1 or B1 or A1B1. Broilers are a kind of homothermal animal that have a high energy requirement for thermoregulation under low temperature, which is probably a constraint to immune function
(Hangalapura et al., 2003), and hence energy-demanding immune responses
in the control group A1B1 or groups A1 or B1 may be
suppressed. Therefore, the supplementation of glutamine and L-carnitine has
mitigated the induced immunodepression by moderating augmentation of plasma
CORT and keeping the energy balance, which will consequently improve the
immunity of broilers raised under low temperature.
The glutamine or L-carnitine supplemented groups keep relatively stable
plasma GLU in the experiment period since the CORT concentration had no
obvious fluctuation, and then the CORT-induced glycogenolysis or
gluconeogenesis and hyperglycemia will not occur apparently (Zhao et al.,
2009). Moreover, glutamine or L-carnitine addition is able to arouse the
increasing of plasma glucose because glutamine is one of the most effective
substrates for gluconeogenesis and L-carnitine participates in biological
regulation of gluconeogenesis involving the reduced oxidation of
gluconeogenic precursors and the favored aerobic metabolism (Celik and
Oztürkcan, 2003; Roth, 2008; Ringseis et al., 2013). However, it is the
significantly heightened CORT in the groups A1 that incited the
increase of GLU in the second week.
Plasma TP levels are regarded as an index of protein nutritional status.
Thereby, the dietary supplemented groups obviously have better protein
nutritional conditions, and group A1B1 has the poorest status. The
addition of carnitine to animal feed can promote the oxidation of fat, which
provides the most cost-effective energy and has the potential to enhance
sparing of the protein (Rehman et al., 2017). In humans, L-carnitine
supplement was reported to improve the serum total protein concentrations in
patients in maintenance dialysis by sparing nitrogen because fewer amino
acids are required for synthesis of L-carnitine (Katalinic et al., 2018).
In addition, glutamine is 1 of the 20 proteinogenic amino acids
accounting for 5 %–6 % of bound amino acids, and it is also the most abundant
amino acid in blood and the free amino acid pool (Roth, 2008).
Glutamine has been reported to improve nitrogen balance in a catabolic state
(Buijs et al., 2013). However, there is no conspicuously
different BUN between all groups, which indicates that CORT-induced protein
catabolism does not happen because of the short stress period or the dietary
addition of glutamine or L-carnitine implies no impact on protein
catabolism, and the negative time-dependent effect of plasma BUN suggests
the dropping protein catabolism. Higher protein nutritional situations and
declined protein catabolism in the supplemented groups might result in
faster growth of these young chickens. It has been reported that glutamine may
also act as a signal or regulator of metabolic demands to increase protein
synthesis and decrease protein degradation in skeletal muscle of young
broilers (Xi et al., 2011; Lambertucci et al., 2012). Conversely, group
A1B1 without glutamine or L-carnitine supplementation owns the
lowest TP and highest BUN and then poorer growth performance.
The higher ALP activity of groups A1 or B1 or A1B1 in
the second week may illustrate the bone metabolism, and thus be induced by
bone abnormality, which is one of a series of responses to the higher CORT
levels of these broilers because the high glucocorticoid may seriously hurt
the physiological conditions of the skeletal system
(Beier et al., 2017). Although ALP activity appears
to be associated with bone activity, the intestinal isoenzyme makes the
largest contribution to plasma ALP activity, and the injury of intestinal
mucosa may cause the increasing of plasma ALP (Viveros et al., 2002).
Glutamine might play the important role in maintaining the integrity of the
intestinal mucosa as the fuel of the cells of the intestinal mucosa and the
gut-associated lymphoid tissue (Bartell and Batal, 2007; Izgüt-Uysal
et al., 2007). Therefore, the negative time-dependent effect of ALP activity
in the glutamine or L-carnitine supplemented groups may be the response to
not rising CORT levels or the protective function of the additives to the
intestinal mucosa.
The supplementation of glutamine or L-carnitine has ameliorated the
oxidative stress and improved the antioxidant capability, and their
significant interaction, especially in the second week, has fortified the
results. The corticosterone-induced or stress-induced oxidative stress, as
many studies have found, should be not serious in the supplemented groups,
for the CORT level was decreased by the additives (Lin et al., 2004).
Moreover, glutamine is a precursor of glutathione, and then its
supplementation can be used to maintain high levels of glutathione (by the
altered activity of GHS-PX) and to avoid oxidative stress damage
(Roth, 2008). In this experiment, the addition of glutamine has
obviously improved plasma SOD and GHS-PX activity and nonenzymatic
antioxidants (T-AOC) and decreased plasma lipid peroxidation (estimated by
MDA levels), and the variation of GHS-PX activity is both dose- and
time-dependent, especially in the supplemented groups.
The apparently unmodified activity of plasma SOD and GHS-PX by the added
L-carnitine is against what was observed in mammals and might be caused by
the short period of the exposure and the less reliability on the energy
production by lipids (Buijs et al., 2013; Katalinic et al., 2018).
Nonetheless, since L-carnitine would facilitate the oxidation of long-chain
fatty acids and aerobic metabolism, L-carnitine obviously reduced the plasma
concentrations of MDA and hence mollified the lipid peroxidation, which would
be supported by the trials of humans and rats (Banihani et al., 2012; Jia
et al., 2014; Kazemi Fard et al., 2015). Moreover, the vitamin C and
methionine sparing activity of L-carnitine can maintain the levels of
antioxidants, and therefore T-AOC grew with the addition of L-carnitine
(Roth, 2008). The inconspicuous changes of plasma MDA
concentrations with time in all groups indicate the stable lipid
peroxidation, which might be due to the simultaneous rise in T-AOC,
suggesting the increased antioxidant capacity with age.
Conclusion
The supplemented diet of glutamine and L-carnitine can improve the growth
performance and nutritional status and ameliorate the stress response and
oxidative stress of the young broilers exposed to low ambient
temperature. Their interaction, especially significant in the second week,
has amplified the results. The orthogonal contrast analysis shows that the
group A3B4 had the highest feed efficiency, and then the broilers
that were raised at the low ambient temperature in this experiment had
better be feed by the supplement of glutamine at 0.6 percentage of diet
weight and L-carnitine at 100 mg/kg diet weight.
Data availability
The data are available from the corresponding author upon request.
Author contributions
YL finished the experiment and wrote the manuscript. YY
corrected the manuscript for publication. RY, SL and PL performed the literature search. YH and HL completed the
data analysis. BX and SL supervised the work, evaluated the data,
and approved the final version. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no conflict of interest.
Financial support
This research has been supported by the National Natural Science Foundation of China (grant nos. 31972637 and 31772695), the Natural Science Foundation of Heilongjiang Province (grant no. ZD2019C004), and the Heilongjiang Bayi
Agricultural University for San Heng San Zong (grant no. ZRCQC202003).
Review statement
This paper was edited by Manfred Mielenz and reviewed by two anonymous referees.
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