Effects of GABA on the Thymus Cytokines of Wenchang Chickens Submitted to Heat Stress

Under routine feeding conditions, 1-day-old male chicks were randomly divided into control group (CK), heat-stress group (HS), and GABA + heat stress group (GABA+HS). The thymus contents of IL-1, IL-2, TGF-β1, IFN-γ, GH and HSP70 were determined by enzyme-linked immunosorbent assays. The results showed that: (1) IL-1 and TGF-β1 contents of HS group were significantly lower than those of the CK group (p<0.05), and those of the GABA+HS group were significantly higher relative to the HS group (p<0.05); (2) IL-2 and IFN-γ contents of the HS group were significantly higher than those of the CK group (p<0.05), and those of the GABA+HS group were significantly lower relative to the HS group (p<0.05);(3) the thymus GH content of all three groups first increased and then decreased. The expression levels GH of the HS and GABA+HS groups were significantly lower than CK group (p<0.05); and (4) HSP70 expression levels in the thymus were significantly higher in the HS and GABA+HS groups relative to the CK group (p<0.05). These results indicate that heat stress affected thymus development, immune functions, and overall growth of chickens. Furthermore, it was shown that feeding GABA may significantly improve the immune responses of heat-stressed chickens by increasing the expression levels of IL-1 and TGF-β1, and effectively alleviate the negative effects of heat stress on thymus development by changing HSP70 expression and GH secretion.


INTRODUCTION
Animals are frequently submitted to stress in the modern livestock industry.As a result of the intensification of livestock production and the development of large-scale farming, the rearing environment has become one of the main aspects that limits animal productivity.Among the environmental factors, high temperature has a huge impact on livestock and poultry production and may cause serious economic losses (Rispoli et al., 2011).
Most regions of southern China, especially Hainan Island, are characterized by hot temperatures, long days, high humidity, and heavy rainfall during about two-thirds of the year (Tang et al., 2008;Zou et al., 2015).Animals reared in these regions often suffer heat stress.Therefore, exploring methods to alleviate heat stress may significantly improve their productivity and reproductive performance.
When the environmental temperature is higher than the upper limit chickens are able to cope with, their physiological functions are disturbed, causing elevation of body temperature and respiratory rate, as well as accumulation of metabolites.These changes can cause intestinal tissue structure changes and affect feed digestion and absorption, leading appetite loss, diarrhea, and eventually significantly increasing morbidity and mortality (Quinteiro-Filho et al., 2012;Chand Effects of GABA on the Thymus Cytokines of Wenchang Chickens Submitted to Heat Stress et al., 2014;Chen et al., 2014).Heat stress not only affects the physiology of chickens, but also has negative effects on their immune function.It has been shown that heat stress can disrupt chicken's immune system, cause lesions in immune organs, and reduce the numbers of lymphocytes and macrophages, leading to immunosuppression (Smith et al., 2003;Khajavi et al., 2003).Therefore, the mitigation of heat stress is of great significance for the chicken industry.
Gamma-aminobutyric acid (GABA) is widely present in animals as an inhibitory neurotransmitter.GABA plays many roles in livestock.It mediates a stress reaction and participates in a variety of metabolic activities to promote amino acid balance in the body (Dai et al., 2012).GABA is currently used in animal production as a functional amino acid feed additive to promote feed intake, enhance the immune status, and to increase productivity.It was also reported that GABA improves the feed conversion ratio and the growth performance of broilers under heat stress (Chen et al., 2002) and maintains the antioxidant function of the intestinal mucosa of broilers submitted to heat stress (Chen et al., 2013).In addition, GABA is considered an effective and safe feed additive (Takeshima et al., 2014).
The thymus is an important lymphoid organ of the central immune system and the main site of T cell differentiation and development, and therefore, participates in cell-mediated immunity.Heat stress has been shown to negatively affect the thymus development and to impair the immune functions (Quinteiro-Filho et al., 2012).Consequently, mitigating the negative impacts of heat stress on thymus development in order to maintain the immune functions is key to ensure adequate broiler growth performance.In a previous study, we showed that feeding chicks with a GABA solution alleviated the effects of heat stress, increased body weight and feed intake, and regulated the immune function of the intestinal tract of broilers (Chen et al., 2015).However, the effects of heat stress on thymus immune factors in chickens are still unclear.Therefore, we aimed at investigating these effects and the roles of GABA under heat stress in order to provide experimental support for the use of GABA to reduce losses due to heat stress in the poultry industry.

Birds
A total of 108 healthy one-day-old male Wenchang chicks (Hainan Yong Ji Live Stock Co., Ltd).Upon arrival at the experimental facilities located in Haikou, Hainan province, China, chicks were individually weighed and numbered.Initial body weight was not significantly different among groups.
Birds were randomly designated to a control group (CK), a heat stress group (HS), and GABA + heat-stress group (GABA+HS), with six replicates of six birds each.The chicks of the GABA+HS group received 0.2mL of a GABA solution (50 mg/kg of body weight) daily by oral gavage.The GABA solution was purchased from Sigma, St Louis, MO, USA.The chicks of CK and HS groups received 0.2mL saline solution daily by oral gavage.
Birds were housed in a conventional broiler house with natural ventilation, and divided in three pens (one per experimental group).A 14:10 light to dark lighting program was applied.All chicks were provided ad libitum access to feed and distilled water (15-20 chicks/m 2 feeding density).The house was cleaned and disinfected during the five weeks of experimental period.
The diet was formulated according to the NRC standards (1994, Table1), and the birds were subjected to routine management practices.The experiment was conducted with the approval of the Animal Experimentation Ethics Committee of Hainan Normal University.

Heat stress treatment
The birds submitted to heat stress (HS and GABA+HS groups) were daily placed at 01:00 pm for 2 h in a LRH-800-GS artificial climate chamber (40±0.5 ºC, 70%-80% relative humidity; MingTian Environmental Protection Instrument Co., LTD, China), while the CK Effects of GABA on the Thymus Cytokines of Wenchang Chickens Submitted to Heat Stress group birds were placed in an incubator for 2 h at room temperature (25-28 ºC).After the heat-stress treatments, birds were returned to the conventional sheds.

Sample collection and processing
At 1, 2, 3, 4, and 5 weeks of age, six birds per group were randomly selected and sacrificed after 2 h of heat stress for rapid and sterile thymus collection.The fat surrounding the thymus was removed, and thymuses were then weighed and stored at -20 ºC until analyses.
The thymuses were rinsed in ice-cold PBS (pH 7.4) to remove the blood, and dried with filter paper.PBS was added (9:1 of thymus weight) for tissue homogenization on ice bath using a glass homogenizer until the tissue was completely broken down and presented uniform light color.The homogenates then were centrifuged at 3,000 r/min for 10 min at 4 ºC, and the supernatant was stored at -20 ºC until further use.

Statistical analyses
The data were analyzed by analysis of variance (ANOVA) using the software SPSS17.0 and are presented as mean ± SEM.Differences with p<0.05 were considered statistically significant.

Effect of GABA on thymus IL-1 contents of broilers submitted to heat stress
As shown in Table 2, thymus IL-1 content was first increased and then decreased as broilers aged, and all three groups presented a similar pattern.At all evaluated ages, the thymus IL-1 contents of the HS group were significantly lower than those of the CK group (p<0.05),while the GABA+HS group presented higher thymus IL-1 contents compared with the HS group at 1and 3 weeks of age (p<0.05).Values in the same column with no common superscripts are significantly different (p<0.05).

A-D
Values in the same row with no common superscripts are significantly different (p<0.05).CK: control, HS: heat stress, GABA+HS: GABA+heat stress.a-c Values in the same column with no common superscripts are significantly different (p<0.05).
A-D Values in the same row with no common superscripts are significantly different (p<0.05).CK: control, HS: heat stress, GABA+HS: GABA+heat stress.

Effect of GABA on thymus IL-2 content of broilers submitted to heat stress
As shown in Table 3, thymus IL-2 contents of the HS and GABA+HS groups were significantly higher compared with the CK group, except at 2 weeks of age for the HS group, and at 2 and 5 weeks of age for the GABA+HS group (p<0.05).Compared with HS group, GABA+HS group presented significantly higher IL-2 contents at 2, 3, and 4 weeks of age (p<0.05).

Effects of GABA on the Thymus Cytokines of Wenchang Chickens Submitted to Heat Stress
It was also reported that immune factors are involved in GH regulation in the neuroendocrine system, as IL-1 injection into brain ventricle caused the release of GH from anterior pituitary cells, while IFN-γ injection inhibited GH secretion (Tang and Qi, 1993).In the current study, heat stress reduced thymus IL-1 contents, as well as GH secretion; at the same time, IFN-γ contents significantly increased, which may be one of the main reasons for the reduction of GH levels.
Heat shock proteins (HSPs) are a group of nonspecific proteins synthesized under stressful conditions (Vidal et al., 2012).Heat stress has been reported to stimulate the expression of large amounts of HSPs.HSP70 is an important member of HSP family, it is activated under heat stress to protect cells, and its expression levels are closely related with heat stressinduced tissue damages, especially in chickens.Therefore, HSP70 is considered as a marker for heat stress-induced damages in the chicken thymus.Our results show that heat stress activated the expression of a large amount of the HSP70 protein in the thymus, leading to significant tissue damages.The feeding of GABA, on the other hand, was able to protect the thymus from heat stress-induced damages.A previous study showed that HSPs can also help to maintain the physiological balance by regulating cytokines, especially pro-inflammatory cytokines, to protect tissues from inflammatory damages ( Van et al., 2005).The cytokine IFN-γ investigated in the present study is an inflammatory cytokine, and its strong expression under heat stress indicates that the thymus suffered inflammatory infiltration.However, after week 4, IFN-γ levels were significantly reduced, indicating that HSPs blocked the expression of pro-inflammatory factors.The high expression of IL-2 induced by heat stress may also be related to HSP70, as IL-2 promotes the activity of cytotoxic T (Tc) cells, while the high expression of HSP70 may stimulate Tc cells to increase the expression of IL-2.As the feeding of GABA mitigated heat stress, the body's physiological status gradually returned to normal, reducing HSP70 expression and IL-2 content in the thymus.Therefore, HSP70 expression can be used as an indicator of changes in the immune function.

CONCLUSION
Under heat stress, the IL-1, TGF-β1, and GH contents of the thymus of chickens decreased, while IL-2, IFN-γ, and HSP70 content increased.Heat stress changed the thymus contents of immune factors, with potential negative effects on the immune functions and performance of the chickens.GABA can be used as an additive to mitigate the negative effects of heat stress on the immune function and growth performance of chickens, and may have extensive applications in animal production.

Table 1 -
Ingredients and chemical compositions of the basal diet (g/kg)

Table 2 -
Effect of GABA on the thymus IL-1 content of chickens submitted to heat stress (ng/L, n=6)

Table 3 -
Effect of GABA on the thymus IL-2 content of chickens submitted to heat stress (ng/L,n=6)