Dietary Supplementation of Glutamine Improves Metabolic Functions in 1-14 Days Old Broilers Under Cold Stress

.


INTRODUCTION
I n the winter, in most parts of China, the outdoor temperature may be below 0 ℃. In general, cold stress can occur when the temperature is below the optimal living standard for chicks. When chicks suffer from cold stress, the body needs to consume a lot of energy to maintain body temperature to resist the external low-temperature environment, only relatively little energy is used for production and maintenance (Zhang et al., 2016), resulting in reduced growth performance (Hu et al., 2021) and even death. At the same time, cold stress usually causes physiological and biochemical changes in poultry blood (Hangalapura et al., 2006;Wang and Xu, 2008), so it is of great significance for broiler breeding to solve or relieve cold stress of chicks. Cold stress reduces the incubation efficiency of laying hens, and the weight of chicks hatched at normal temperature was significantly higher than under cold stress (Kamanli et al., 2015). Wang and Xu (2008) showed that the weight gain of pheasants in the cold stress group decreased significantly and feed efficiency increased significantly within one week.
Glutamine (Gln) is added to the chickens diet resulted O n l i n e

F i r s t A r t i c l e
in improved body weight and feed conversion ratio (Zulkifli et al., 2016). Other studies have shown that the addition of Gln in the diet of broilers led to a significant increase in body weight and immune organ weight (Szabó et al., 2014). Yi et al. (2005) also demonstrated a promoting effect of Gln on the growth performance of broilers at a later stage. Studies have shown that Gln can alleviate the effects of heat stress on the growth performance of broilers and improve their serum physiological and biochemical indices (Dai et al., 2012;Hu et al., 2016). Previous studies have shown that different content of Gln can significantly improve the performance and immune organ index of 1-14 days old chicks (Xiao et al., 2019). However, there are few reports on the effects of Gln on serum biochemical indices of cold stress chicks. The aim of the present study was to evaluate the effect of Gln on the serum biochemical parameters in the first two weeks of the broiler chickens under cold stress.

MATERIALS AND METHODS
Test materials L-glutamine (L-Gln) was purchased from Amresco, USA having purity > 99.0 %. Automatic Biochemical Analyzer and centrifuge were purchased from HATTIECS, Federal Republic of Germany.

Animals husbandry and experimental design
A total of 192 healthy, 1-day old Arbor Acres (AA) broilers were randomly divided into four groups (four replicates in each group and 12 chicks in each replicate), with group I as the control group fed with Gln-free basic diet, and groups II, III and IV fed with 1.0, 1.5 and 2.0 Gln, respectively. The temperature and humidity of the chicken coops were controlled by heaters and humidifiers, and the temperature was maintained at 25±2 ℃ and the humidity was maintained at 60% to 70% for 14 days.

Feeding management and diet formulation
Three days before the start of the experiment, the chicken house was cleaned, fumigated and disinfected. Broilers were fed and watered freely. Immunization was carried out according to broiler husbandry regulations. Daily observation of feed consumption, faeces composition and health condition of the chickens was carried out. The basic diet was corn-soybean meal, which was designed according to the recommendation of National Research Council (1994) ( Table I).

Sampling and measurement of indicators
At 7 and 14 days of the experiment, two healthy chicks were randomly selected from each replicate after a 12 h fast, and then slaughtered and sampled. Blood was collected from the carotid artery of the broiler and centrifuged in a 10 ml tube at 3000 r/min for 10 min, then the serum was divided into 0.5 ml tubes and stored at -20℃. Hitachi 7600 Automatic Biochemical Analyzer was used to measure serum biochemical indicators, namely potassium ion (K + ), sodium ion (Na + ), chloride ion (Cl -), calcium ion (Ca 2+ ), phosphorus ion (P 3+ ), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), triiodothyronine (T3), thyroxine (T4), albumin (ALB), globulin (GLB), glucose (GLU), triglyceride (TG), and uric acid (UA) with the help of commercial assay kits (Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China).

Statistical analysis of data
Data were analyzed under one-way analysis of variance (ANOVA) using SPSS 18.0. Means were separated by using Tukey test. The results were expressed as means±SEM, and P<0.05 was considered as point of significance.

Effect of glutamine on serum antioxidant ability of coldstressed chicks
The effect of Gln on serum ions of cold stressed O n l i n e

F i r s t A r t i c l e
broilers on day 7 and 14 are given in Table II. No significance (P>0.05) difference was found in serum K + , Na + , Cl -, and P 3 during the first 7 days of the experiment. Serum Ca 2+ was significantly (P<0.05) higher in group IV compared to the control. On day 14, serum K + and P 3+ were significantly (P<0.05) lower in group IV compared to the control. The rest of the ions did not change significantly (P>0.05) between the control and the treatments.

Effect of glutamine on serum enzymes activities
The effect of Gln on serum ALT, AST, LDH, and CK on day 7 and 14 are given in Table III. Serum LDH content decreased significantly (P<0.05) in the treatment groups compared to the control. No significant changes were recorded in other parameters on day 7.
On day 14, serum AST decreased significantly (P<0.05) in group II compared to the control. Similarly, LDH was also reduced significantly (P<0.05) in group IV compared to control.

Effect of glutamine on serum hormones contents
Compared with control, serum triiodothyronine level content in group II, III, and IV was significantly increased (P<0.05) on the 7 th day of the experiment (Table IV).

Effect of glutamine on serum protein content
Table V shows us that on the 14 th day, albumin and globulin decreased significantly (P<0.05) in group IV compared to the control.

Effect of glutamine on serum glucose (GLU), triglyceride (TG) and uric acid (UA) contents
On the 7 th day, compared with control, the GLU level was significantly decreased in group II (P<0.05). The serum UA content in group II was significantly (P<0.05) lower compared to the control. The concentration of UA decreased significantly (P<0.05) in group II and III compared to the control on day 14 th of the study (Table VI).

DISCUSSION
Serum biochemical index of animals can reflect the metabolism and health status to a certain extent. Huang et al. (2017) study showed that serum Ca 2+ and P 3+ levels were significantly reduced after 10 days of age and were raised at 5℃ lower than the control for 3 days. Hangalapura et al. (2006) also found that cold stress significantly increased serum T 3 and T 4 levels of broilers. Serum AST and ALT activities increased significantly in chickens exposed to chronic cold stress (Zhang et al., 2014). Zhen et al. (2011) showed that the levels of serum CK and LDH of 4-monthsold female quails were significantly increased after being exposed to cold stress for 4 h on daily basis. Different degrees of cold stress had different effects on the serum biochemical index of chickens.
With the increasing demand for animal protein, broiler farms are also increasing in many countries of the world. In the early stage, broilers are vulnerable to cold stress. Cold stress causes serious problems to chicks, ranging from reduced growth performance to high mortality. Poultry researchers have reported improvement in reducing cold stress through strengthening management improving environmental facilities and supplementation of feed additives (Su et al., 2018). Feed additives such as minerals (Yang et al., 2019;Medeiros-Ventura et al., 2020), probiotics (Huff et al., 2015;Su et al., 2018), and vitamins (Ozkan et al., 2007;Ferreira et al., 2015) to the diet can relieve cold stress in broilers. According to the results of previous studies, adding Gln to the diet of broilers can promote the growth performance and provides resistance to the cold stress (Dai et al., 2012;Hu et al., 2016).
Compared with the white phoenix chicks fed at normal temperature, when the normal feeding temperature is 5℃, the Ca 2+ and P 3+ in the serum decreased significantly after feeding for three days. After 15 days of experiment, the Ca 2+ and P 3+ in the serum did not change. When the normal feeding temperature is 10℃, three days after feeding, the serum Ca 2+ content decreases significantly, and the P 3+ does not change significantly. After 15 days of feeding, the serum Ca 2+ content does not change significantly, and the P 3+ content increases significantly (Huang et al., 2017). From the present study, it can be seen that cold stress can alter the broiler serum ions. The results of this study show that adding Gln to the diet of chicks tends to increase the serum Ca 2+ content and significantly reduce serum K + , P 3+ in 14-day-old chicks. The results of this study are similar to those of Zhang et al. (2018). Gln can maintain the serum ion balance of stress broilers and alleviate the harm caused by cold stress to broilers.
According to the results of previous studies, from the perspective of serum enzyme activity, cold stress has adverse effects on broilers, which may cause liver damage to broilers. The results of this experiment show that adding 1.0% ~ 2.0% Gln to the diet of chicks can reduce the serum LDH, CK, and ALT activity of chicks. This experiment is similar to the results of the Hu et al. (2016). Gln can reduce the serum LDH and CK activity, and relieve the damage caused by stress to the body.
T 3 and T 4 hormones play an important role in the growth and development of the body. They can promote the metabolism of proteins, sugars, and fats, thus promoting the growth and development of the body. Xiao et al. (2019) showed that dietary supplementation with different concentrations of Gln significantly increased ADFI and ADG, reduced FCR, and improved immune organ index in chicks of different ages. A certain increase in T 3 and T 4 levels in serum can relieve the damage caused by cold stress to the body. The results of this experiment show that supplementation of 1.0% ~ 2.0% Gln can improve T 3 and T 4 levels in the serum of chicks. The results of this study are similar to those of Dai et al. (2012) and Hu et al. (2016). Gln can improve T 3 and T 4 levels in the serum of stress broilers and mitigate the adverse effects of stress on broilers.
Supplementation with 1.5% Gln was added to the diet O n l i n e

F i r s t A r t i c l e
Dietary Supplementation of Glutamine Improves Metabolic Functions 5 of chicks, and the serum albumin and globulin content of 7 days old chicks increased by 10.49 and 9.51%, respectively. Gln can improve the serum albumin and globulin levels of stressed animals, which may be due to the addition of exogenous Gln, which enhances the synthesis efficiency of proteins in animals (Zhang et al., 2015). The results of this study also show that 2.0% Gln is added to the diet of chicks, and the serum albumin and globulin content of 14-days-old chicks are significantly reduced. This may be that the high doses of exogenous Gln are added to provide nutrients, and then the deposition of albumin and globulin in the serum are accelerated into body proteins, and the potential mechanism needs to be further studied. The increase in serum glucose content is due to the increase in glycogen decomposition in broilers at low temperatures (Zhang et al., 2014), which also indicates that animal bodies need to consume more sugar to convert into heat energy in cold environments to resist cold environments ( Van et al., 2010). Summarizing the results of previous studies, it can be concluded that cold stress can increase the serum glucose, triglyceride, and uric acid content of broilers. The results of this study show that add Gln to the diet, and the serum glucose content of 7-dayold chicks decreased to varying degrees, and the serum glucose content decreased significantly. Adding exogenous Gln to cold stress chicks can provide energy for the body as an energy substance. The serum glucose content of chicks in the Gln group decreased. Add 1.0% ~ 2.0% Gln to the chick's diet, and the serum triglyceride and uric acid content of chicks decreased to varying degrees.
In the chick stage (1 to 14-days old), too low temperature does have negative impact on serum biochemical indicators. Under this experimental condition, supplementation with Gln improves the serum biochemical indexes of cold stress chicks. Among them, adding 1.0 % Gln is the best dosage.