Effect of acute cold stress on the body temperature in chicks
Cold stress is a common challenge in broilers. Chickens allocate more energy to maintain body temperature when a heat exchange process between the body and the ambient environment is induced by the dropping of ambient temperature. Neonatal broiler chicks are highly sensitive to low temperature because of the underdeveloped feathers and weak thermoregulatory system [11]. Acute cold exposure is more likely to cause cold stress in broiler chicks, which will lead to the decline of core body temperature. In the present study, we measured the core body temperature of 7-day-old broiler chicks after acute cold exposure. Results shown that abdominal temperatures in chicks dropped to 35.49 ± 0.77℃, 34.38 ± 0.51℃, 34.48 ± 0.84℃ from 39.26 ± 0.36℃ after receiving an acute cold exposure for 4 h, 8 h, 12 h, respectively, which was in line with the previous study that had shown acute cold exposure significantly decreased the core body temperature in mice [12].
Acute cold stress upregulated the gene expression of CIRP-TLR4-IRE1 signaling pathway
The CIRP-TLR4 signaling pathway is a key pathway in signal transduction associated with inflammatory response and cold stress. As a cold shock protein, CIRP expression will be upregulated under the condition of stress, adapting cells or tissues to environmental changes [13]. As shown in Fig. 1A, compared with the control group with no (0 h) cold exposure, acute cold exposure for 8 h and 12 h significantly increased the mRNA expression of CIRP and TLR4 (P < 0.05), similar to those of previous studies [5, 12], which had revealed the function of CIRP-TLR4 signaling pathway in cold stress.
A mice model of acute lung injury had shown that CIRP induces ERS via TLR4 activation [7]. To explore the relationship between CIRP-TLR4 signaling and ERS in 7-day-old broiler chickens, we investigated the gene expression of ERS makers under acute cold stress. As one of the main branches of UPR, IRE1 senses the unfolded or misfolded proteins in ER, initiates the UPR, and drives the expression of genes regulating ER homeostasis [9]. GRP78 is an ER-resident Chaperone that is associated with IRE1. When high amounts of unfolded or misfolded proteins are present in ER, GRP78 binds to these proteins, dissociates from IRE1, which allows the autophosphorylation and activation of IRE1 [10]. The current study shown that the mRNA expression of IRE1 and GRP78 (Fig. 1B) in ileum increased significantly (P < 0.05) in response to acute cold exposure, which indicated the occurrence of ERS. No significant difference in the gene expression of XBP1 was observed between the control and experimental groups, suggested that the signaling cascade may not depend on XBP1. NF-κB is a key transcription factor that is related to inflammatory and immune responses, and can be activated by environmental stress [14]. We know that CIRP increases NF-κB expression and inflammatory response [7]. Indeed, similar to the changes of CIRP and TLR4 gene expression, cold exposure for 8 h and 12 h significantly improved the mRNA expression level of NF-κB (P < 0.05, Fig. 1C). It may provide further evidence for the link between CIRP-TLR4 and NF-κB. Similarly, the gene expression of inflammatory cytokines, IL-1β, IL-6, and IL-10 (Fig. 1D), increased significantly (P < 0.05) in response to acute cold exposure, which may be the result of the upregulation of NF-κB, suggested that acute cold exposure induced intestinal inflammatory response. In addition, a significant increase of TNF-α (P < 0.05) mRNA expression level was observed in the group exposed to cold exposure for 12 h.
Acute cold stress upregulated the gene expression of tight junction protein
Tight junction protein is crucial component of intesinal tight junction barrier. Upregulation of tight junction protein expression is a momentous cell protective mechanism against tight junction damage [15]. In the present study, we detected the gene expression of three tight junction proteins, ZO-1, Occludin, and Claudin-1 in ileum mucosa. As shown in Fig. 1E, the mRNA expression levels of ZO-1 and Occludin increased after an acute cold exposure (P < 0.05), which might represent tight junction remodeling. Acute cold exposure for different times did not altered the gene expression of Claudin-1 (P > 0.05), which was in accordance with a previous study [16].
Acute cold stress damaged intestinal structure
Small intestine is the organ where nutrients digestion and absorption mainly take place in animals, and the integrity of small intestine is the basic guarantee for intestinal function. The villi of small intestine are the first portal of nutrients digestion and absorption, while a higher villus means a stronger absorption function [17]. Crypt is a tubular structure between the base of villus and the submucosa, which is mainly composed of undifferentiated cells. The deeper the crypt, the more undifferentiated cells and the less mature the intestinal development. Because of the high turnover rate, intestinal mucosal cells are highly susceptible to various environmental and physiological stresses [18]. It has been shown that acute cold stress could cause intestinal tissue damage [19]. As shown in Table 2, cold exposure decreased the villus height, crypt depth, and V/C of small intestine. Compared with the control group, cold exposure for 8 h significantly decreased the villus height and crypt depth of duodenum (P < 0.05), and significantly decreased the villus height and V/C of jejunum (P < 0.05); A longer cold exposure for 12 h decreased the villus height, crypt depth, and V/C of duodenum (P < 0.05), decreased the villus height and V/C of jejunum (P < 0.05), and decreased the villus height of ileum (P < 0.05). All the results showed above suggested impaired intestinal structure caused by acute cold exposure, which were consistent with a previous study that had shown cold stress decreased the number, height and integrity of jejunum villus of broilers [20].
Table 2
The effect of acute cold stress on the structure of small intestine (n = 6)
Item | Acute cold exposure time | P value |
0 h | 4 h | 8 h | 12 h |
Duodenum | Villus height, mm | 1.30 ± 0.13a | 1.32 ± 0.03a | 0.97 ± 0.14b | 0.89 ± 0.17b | 0.010 |
Crypt depth, mm | 0.24 ± 0.02a | 0.22 ± 0.01ab | 0.18 ± 0.04b | 0.19 ± 0.04b | 0.048 |
V/C | 5.38 ± 0.66a | 5.95 ± 0.52a | 5.72 ± 1.45a | 4.30 ± 0.62b | 0.040 |
Jejunum | Villus height, mm | 1.05 ± 0.09a | 1.04 ± 0.07a | 0.72 ± 0.07b | 0.72 ± 0.05b | 0.001 |
Crypt depth, mm | 0.17 ± 0.01 | 0.17 ± 0.02 | 0.16 ± 0.01 | 0.16 ± 0.01 | 0.260 |
V/C | 6.06 ± 0.56a | 6.10 ± 0.36a | 4.56 ± 0.33b | 4.57 ± 0.50b | 0.001 |
Ileum | Villus height, mm | 0.61 ± 0.08a | 0.56 ± 0.03ab | 0.62 ± 0.08a | 0.48 ± 0.06b | 0.037 |
Crypt depth, mm | 0.14 ± 0.01 | 0.13 ± 0.01 | 0.14 ± 0.01 | 0.12 ± 0.02 | 0.168 |
V/C | 4.31 ± 0.54 | 4.17 ± 0.26 | 4.32 ± 0.42 | 3.81 ± 0.15 | 0.236 |
In the same row, values with a same or no lowercase letters mean no significant difference (P > 0.05), whereas with different lowercase letters mean significant difference (P < 0.05). Abbreviation: V/C, villus height to crypt depth ratio.