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Heat exposure alters the mRNA expression of growth- and stress-related genes in chicks
Author’s Accepted Manuscript Heat exposure alters the mRNA expression of growth- and stress-related genes in chicks Hirofumi Okuyama, Md. Sakirul Islam Khan, Akira Tsukada, Tetsuya Tachibana www.elsevier.com/locate/livsci
PII: DOI: Reference:
S1871-1413(17)30037-9 http://dx.doi.org/10.1016/j.livsci.2017.02.010 LIVSCI3149
To appear in: Livestock Science Cite this article as: Hirofumi Okuyama, Md. Sakirul Islam Khan, Akira Tsukada and Tetsuya Tachibana, Heat exposure alters the mRNA expression of growthand stress-related genes in chicks, Livestock Science, http://dx.doi.org/10.1016/j.livsci.2017.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heat exposure alters the mRNA expression of growth- and stress-related genes in chicks Hirofumi Okuyama1, Md. Sakirul Islam Khan2, Akira Tsukada3, Tetsuya Tachibana1* 1
Department of Agrobiological Science, Faculty of Agriculture, Ehime University,
Matsuyama 2
Department of Anatomy and Embryology, Graduate School of Medicine, Ehime University,
Toon 791-0295, Ehime, Japan 3
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Science,
Nagoya University, Chikusa 464-8601, Nagoya, Japan *Corresponding address: Tetsuya Tachibana. Laboratory of Animal Production, Department of Agrobiological Science, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan. Tel./ Fax. +81-89-946-9820. [email protected]
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Abstract High ambient temperature, a major stressor, impairs the growth of chickens. In this study, we examined the effect of heat exposure on the mRNA expression of various growth related genes, such as growth hormone (GH), insulin-like growth factor-1 (IGF1), GH-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), and somatostatin (SST) in layer-type chickens (Gallus gallus domesticus) at an early age. In addition, we examined the reactivity of the corticotrophin-releasing hormone (CRH) system, one of the stress-regulating pathways, to heat exposure and its role in altering the growth related genes. Four-day heat exposure reduced the body weight gain, feed intake, and feeding efficiency and increased the rectal temperature of chicks (P < 0.05). The mRNA expression levels of pituitary GH, liver IGF1, and diencephalic GHRH decreased with heat exposure (P < 0.05) whereas the levels of TRH or SST did not change. Heat exposure also reduced the diencephalic mRNA expression level of CRH and increased pituitary CRH receptor-2 mRNA and the plasma corticosterone (CORT) concentration (P < 0.05), suggesting that heat exposure affected the hypothamic-pituitary adrenal gland (HPA) axis. Similar to the heat-exposure study, subcutaneous injection of CORT for 4 days decreased body weight, and the mRNA expression of pituitary GH and liver IGF1 (P < 0.05). The present study demonstrated that heat exposure reduced the mRNA expressions of pituitary GH and liver IGF1, and suggested that the change in mRNA expression may have been partly caused by CORT. Keywords: Chick; Corticosterone; Growth hormone; Heat exposure; Insulin-like growth factor-I
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Introduction Stress is an important factor affecting the economic and health aspects of poultry production. Some stressors severely retard growth, decrease production, and finally cause death in poultry (Lara and Rostagno, 2013). For example, high ambient temperature, which is regarded as an important stressor, decreases feed intake, growth rate, feeding efficiency, and the carcass quality of broiler-type chickens (Geraert et al., 1996; McKee et al., 1997; Temim et al., 2000; Quinteiro-Filho et al., 2010; 2012; Lara and Rostagno, 2013). In layer-type chickens, heat stress depresses body weight (Scott and Balnave, 1988) and egg production and shell quality (Whitehead et al., 1998; Balnave and Muheereza, 1997; Muiruri and Harrison, 1991; Emery et al., 1984), which are assumed to be caused by heat-induced reduction in feed intake (Mashaly et al., 2004). Therefore, understanding the stress-response mechanisms of chickens is essential to overcoming the problems related to stress. However, these mechanisms have not been completely clarified. The hypothalamus, a brain region of the diencephalon that regulates the endocrine and autonomic nervous systems, as well as appetite, body temperature, and so on, plays a critical role in regulating growth. There is considerable evidence that various hormones secreted from the hypothalamic-pituitary regions regulate the growth of chickens. For example, growth hormone (GH) from the anterior pituitary is believed to be required for the growth of chickens, and GH exerts its somatic effect by increasing circulating insulin-like growth factor-I (IGF-I, the gene is abbreviated as IGF1) via stimulating the synthesis of IGF-I in the liver of chickens (Cogburn et al., 1999; Scanes, 1999; Scanes et al., 1999; Kühn et al., 2005). The release of GH from the anterior pituitary is stimulated and inhibited by the hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SST), respectively (Cogburn et al., 1999; Scanes, 1999; Kühn et al., 2005). In addition, thyrotropin-releasing hormone (TRH) in the 3
hypothalamus has been shown to stimulate the release of GH in chickens (Cogburn et al., 1999; Kühn et al., 2005). The hypothalamus also plays an important role in regulating stress response. In fact, the hypothalamic-pituitary-adrenal (HPA) axis is thought to be a major pathway in regulating stress by activating the corticotropin-releasing hormone (CRH) system (Carsia and Harvey, 1999). Because CRH has been shown to inhibit GH release in rats (Rivier and Vale, 1984; Thomas et al., 1997), stress-induced release of CRH might suppress GH release and then inhibit growth in chickens. In fact, it has been reported that heat stress suppresses the release of GH and IGF-I in black-boned chickens (Liu et al., 2014). However, little is known about the effect of heat stress on the gene expression of growth-related hormones in layer-type chicks. The purpose of the present study was to investigate whether heat exposure affects the expression of the growth-related genes in the pituitary, diencephalon and liver of layer-type chicks. The involvement of the HPA axis in response to heat stress was also investigated. Subsequently, the effect of subcutaneous (SC) injection of corticosterone (CORT) on the expression of the growth-related genes was investigated.
Materials and Methods Animals One-day-old male layer-type chicks (Gallus gallus, Julia, Nihon Layer, Gifu, Japan) were raised in a room kept at 30°C with continuous lighting. They were given free access to a commercial diet (crude protein, 24%; metabolizable energy, 3050 kcal/kg; Toyohashi Feed Mills Co. Ltd, Aichi, Japan) and water. All animal experiments were carried out in accordance with protocols approved by the Ehime University Animal Care and Use Committee (No. 4
08-o3-10). Experiment 1. Effect of heat exposure on mRNA expression of the growth-related genes in layer-type chicks At 5 days of age, 16 chicks were assigned to either the heat exposure group or the control group (8 chicks in each group) such that the average body weight was as uniform as possible. The two groups were then transferred to separate rooms kept at 30°C. One group was exposed to high temperature, and the other was used as the control. Chicks were transferred to the experimental cages at least 2 days before the experiment. One day before the experiment, chicks were individually reared in the experimental cages. The room temperature for the heat-stress group was set at 38°C when the chicks were 7 days of age, continuing until they were 11 days of age. Chicks in the control group continued to be raised at 30°C. The temperature for the heat-stress group was chosen based on previous studies on chicks (Halevy et al., 2001; Chowdhury et al., 2014). During this experimental period, body weight, rectal temperature, and feed intake were measured daily. Rectal temperature was measured by inserting a 19-mm stainless steel sensor connected to an electronic thermometer (BAT7001H, Physitemp Instruments Inc., New Jersey, USA) into the rectum. Feed and water were available ad libitum during the experiment. Feed intake was measured using a digital balance with an accuracy of 1 mg, and the feeding efficiency was calculated. After the final measurement of feed intake, the blood of chicks was collected from the jugular vein into tubes containing ethylenediaminetetraacetic acid dipotassium salt dehydrate. The collected blood was centrifuged (9000 × g at 4°C for 5 min), and the plasma was collected and stored at −30°C. Plasma CORT concentrations were later measured by enzyme immunoassay using a commercial kit (AssayPro LLC, MO, USA) according to the manufacturer’s instructions. The detection limit was 0.4 ng/ml, and intra- and inter-assay 5
variations were 2.0 and 8.4%, respectively. Immediately after blood collection, the chicks were sacrificed by decapitation, and then the liver, surface breast muscle, inner breast muscle, wing muscle, and thigh muscle were collected and weighed. In addition, the diencephalon was collected after removing the whole brain. In brief, the telencephalon, cerebellum, and optic tectum were excised from the whole brain. The diencephalon was collected by removing the mesencephalon, pons, and medulla oblongata at the caudal end of the median eminence. The collected diencephalon was quickly frozen with liquid nitrogen and stored at −80°C until analysis. The pituitary and liver were also collected and frozen. The diencephalon, pituitary and liver were used to investigate mRNA expression. These tissues (except for the pituitary) were homogenized with Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan), and the total RNA were then isolated as per the manufacturer’s instructions. Total RNA of the pituitary was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA treated with DNase I (Ambion, Austin, TX, USA) using the reverse transcription kit ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan) with random primers. To investigate the expression of the growth-related genes, cDNAs for pituitary GH, liver IGF1 and GH receptor (GH-R), and diencephalic GHRH, TRH, and SST were amplified and quantified by semi-quantitative PCR using specific primers (Table 1). To investigate the expression of the HPA-axis related genes, diencephalic CRH, urocortin-3 (UCN3), and CRH-binding protein (CRH-BP), and pituitary CRH-BP, CRH receptor-1 (CRH-R1), and CRH receptor-2 (CRH-R2) cDNA were amplified using specific primers (Table 1). Subsequently, semi-quantitative PCR was carried out in duplicate using a real-time PCR instrument (LightCycler®Nano, Roche Applied Science, Germany) with SYBR Green 6
Real-time PCR Master Mix (Toyobo, Osaka, Japan). As an internal control, polymerase (RNA) II (DNA directed) polypeptide B (RPII) was also amplified (Table 1). 18S rRNA from the pituitary was amplified as the internal control (Table 1). PCR parameters for the assays were as follows: degradation at 95ºC for 10 min followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. The accuracy of these primers was checked by melting curve analysis and the nucleotide sequencing of each PCR product. 2−ΔCT value was calculated for gene expression from real-time quantitative PCR experiments (Livak and Schmittgen, 2001) with the threshold cycle (CT) value calculated by the software of real-time PCR instrument (LightCycler®Nano, Roche Applied Science, Germany). For 2−ΔCT value, the data were analyzed using the equation, where ΔCT = (CT, Target – CT, internal control). 2−ΔΔCT method was also used to investigate the relative changes in gene expression (Livak and Schmittgen, 2001). In 2−ΔΔCT method, the data were analyzed using the equation, where ΔΔCT = (CT, Target – CT, internal control)Heat stress
– (CT, Target – CT, internal control)Control.
Experiment 2. Effect of repeated SC injection of CORT on mRNA expression of growth related genes in layer-type chicks At 6 days of age, 16 chicks were transferred to their individual cages. On the following day, the chicks were assigned to the CORT group or the control group (8 chicks in each group) so that the average body weight was as uniform as possible. Chicks were transferred to the experimental cages at least 2 days before the experiment. One day before the experiment, chicks were individually reared in the experimental cages. Chicks in the CORT group were SC injected with 1.2 mg CORT (Wako Pure Chemical Industries, Ltd., Osaka, Japan), which was dissolved in dimethyl sulfoxide and then diluted with polyethylene glycol, at a volume of 0.1 ml twice a day (8:00 and 20:00) for 4 days. The dose of CORT was decided by referring to a previous study (Song et al., 2011a) and by our preliminary experiment. Our preliminary 7
experiment found that single SC injection of 1.2 mg CORT increased plasma CORT concentration at 1 h after the injection (control group, 7.2 ± 2.1 ng/ml; CORT group, 1163.6 ± 159.5 ng/ml; P < 0.05, Student’s t-test) and 3 h after the injection (control group, 5.6 ± 1.2 ng/ml; CORT group, 14.9 ± 3.1 ng/ml; P < 0.05, Student’s t-test) in 7-day-old chicks, although the effect disappeared at 6 h (control group, 3.7 ± 0.4 ng/ml; CORT group, 4.1 ± 0.5 ng/ml). Therefore we performed the SC injection twice a day. Chicks in the control group were SC injected with the same volume of the vehicle. During the experimental period, body weight and feed intake were measured as noted above. At the end of the experiment, the chicks were sacrificed, and thereafter their liver, surface breast muscle, inner breast muscle, wing muscle and leg muscle were collected and weighed. To measure the mRNA expressions of GH, IGF1, GHRH, TRH and SST, the diencephalon, pituitary, and liver were removed and used for semi-quantitative real-time PCR as noted in Experiment 1. Statistical analysis Daily data on body weight, body temperature, and feed intake were statistically analyzed using two-way repeated measures analysis of variance (ANOVA) with respect to treatment (heat exposure or CORT injection) and time, and then analyzed using the Student’s t-test. Other data were statistically analyzed using the Student’s t-test. Data are presented as the mean ± standard error of the mean (SEM), and statistical significance was set at P < 0.05. The number of chicks is noted in the figure legends.
Results Experiment 1. Effect of heat exposure on mRNA expression of the growth-related genes in layer-type chicks Body weight, feed intake, feeding efficiency, and organ weights 8
Heat exposure reduced body weight gain (P < 0.01) and the effect was time dependent (P < 0.01). The body weight of the chicks exposed to heat stress tended to decrease at 1 day after the experiment, and the effect became significant from 2 days later (P < 0.05, Fig. 1 and Table 2). Rectal temperature increased with heat exposure (P < 0.01) and the effect was time dependent (P < 0.01). The increase in rectal temperature was observed from day 1 and continued to the end of the experiment (P < 0.05, Fig. 1). Daily feed intake decreased with heat exposure (P < 0.01) and the effect was time dependent (P < 0.01) (Fig. 1). Feed intake decreased with heat exposure compared with the control group throughout the experiment. Feeding efficiency during the 4-day experimental period also decreased with heat exposure (P < 0.05, Table 2). At the end of 4 days of heat exposure, the weight of the liver, breast muscle, inner breast muscle, wing muscle, and leg muscle in the heat exposure group was lower than that of control (P < 0.05, Table 2). Although the relative weight of the liver to body weight also decreased with heat treatment (P < 0.05), the relative weight of all the muscles was comparable to that of the control group (Table 2). mRNA expression of the growth-related genes Four days of heat exposure reduced the mRNA expression of pituitary GH and liver IGF1 (P < 0.05), whereas liver GH-R mRNA expression tended to increase (P = 0.09) with heat exposure (Fig. 2). In the diencephalon, heat exposure reduced GHRH mRNA levels whereas the TRH (P < 0.05) and SST mRNA levels did not change (Fig. 2). Plasma CORT concentration and mRNA expression of stress related genes Heat exposure increased the plasma CORT concentrations (P < 0.05, Fig. 3). In addition, heat exposure reduced CRH mRNA expression in the diencephalon (P < 0.05) but did not affect UCN3 mRNA expression (Fig. 4). The mRNA expression of CRH-BP in the diencephalon decreased with heat exposure (P < 0.05, Fig. 4). The decreasing tendency for 9
mRNA expression of CRH-BP was also observed in the pituitary (P = 0.08, Fig. 4). CRH-R2 mRNA expression but not CRH-R1 mRNA expression in the pituitary increased with heat exposure (P < 0.05, Fig. 4). Experiment 2. Effect of repeated SC injection of CORT on mRNA expression of the growth-related genes in layer-type chicks Body weight, feed intake, feeding efficiency, and organ weights The main effect of CORT on body weight was nearly significant (P = 0.06), whereas the interaction between CORT and time was significant (P < 0.01). The body weight of the CORT-treated chicks was comparable with control at 1 day after injection, but decreased at 2 days after injection of CORT (P < 0.05) (Fig. 5 and Table 3). There was no significant effect of CORT on feed intake (P = 0.48) although a significant interaction was observed between CORT and time (P < 0.01). Feed intake tended to decrease with CORT treatment, but it did not reach significance (Fig. 5 and Table 3). As the result, feed efficiency in the CORT group was lower than that in the control group (P < 0.05, Table 3). Four-day CORT treatment increased the actual and relative weights of the liver (P < 0.05, Table 3). On the other hand, the weight of all the muscles investigated decreased with CORT (P < 0.05, Table 3). CORT also reduced the relative weights of the wing and thigh muscles to body weight (P < 0.05). mRNA expression of the growth-related genes The mRNA expression of pituitary GH and liver IGF1 decreased with the SC injection of CORT (P < 0.05, Fig. 6). The mRNA expression level of liver GH-R and diencephalic TRH increased with CORT treatment (P < 0.05), while the mRNA level of GHRH and SST in the diencephalon did not change.
Discussion 10
Exposure to high temperature induced hyperthermia in the chicks, indicating they were not able to adapt to the experimental temperature used in the present study. It is likely that the physiological and molecular changes observed in the present study are due to the effect of chronic heat exposure. We confirmed that 4 day heat exposure reduced body weight including liver and muscle weight in the layer-type chicks. In addition, the present study provided the first evidence that mRNA expression of diencephalic GHRH, pituitary GH, and liver IGF1 decreased with exposure to high temperature. On the other hand, the diencephalic mRNA expression of TRH or SST did not change with exposure to high temperature. These results suggest that the diencephalic GHRH, but not TRH or SST, might be related to the suppression of GH and IGFI production under a heat-stress condition. In rodents, similar to high temperature-induced changes in the GH axis, feed deprivation reduces blood GH and IGF-I levels and suppresses hypothalamic GHRH and liver IGF1 mRNA expression (Park et al. 2004; Tannenbaum et al. 1979). Feed deprivation also reduces IGF-I concentration in blood and IGF1 mRNA expression in the liver of young layer chickens (Kita et al., 1998; Morishita et al., 1993). Because heat exposure reduced feed intake in the present study, we cannot exclude the possibility that reduced feed intake might alter mRNA expression of the growth-related hormones in the diencephalon and liver. Either way, these findings suggest that stress induced by high temperature or feed deprivation alters the growth-related genes in the GH axis, which might impair the growth of chicks. In addition to the growth-related genes, heat exposure also altered the plasma CORT concentration and gene expression of the CRH system in the hypothalamic-pituitary regions. Plasma CORT concentration increased with heat stress as was also shown in a previous study (Quinteiro-Filho et al., 2010). Heat exposure reduced mRNA expression of CRH and 11
CRH-BP in the diencephalon and increased CRH-R2 mRNA expression in the pituitary. These results seem to be contradictory, but they can be explained by the effect of CORT released after exposure to heat. It has been shown that intravenous injection of CORT reduces CRH mRNA expression in the diencephalon in chicks (Vandenborne et al., 2005). Also, Calefi et al. (2016) found negative feedback during heat stress in chicks. Therefore CORT released during heat stress might suppress mRNA expression of CRH as the negative feedback, and changes in mRNA expression of CRH-R2 might be induced to compensate for the action of CRH. Because CRH-BP is reported to bind to CRH with a higher affinity than its receptors (Sutton et al., 1995), CRH-BP is considered to inhibit the biological activity of CRH. Therefore, the decrease in mRNA expression of CRH-BP indicated that heat exposure suppressed the inhibitory effect of CRH-BP on the activity of CRH. Changes in the gene expression of the CRH system in the hypothalamic-pituitary regions and the increase in plasma CORT concentration suggested that exposure to high temperature affected the HPA axis in the layer-type chicks. Supplementation of CORT into the diet retards the growth of skeletal muscle by suppressing protein synthesis and stimulating protein catabolism in young broiler chickens (Dong et al. 2007). Chronic heat stress has also been shown to suppress protein synthesis and breakdown in young broiler chickens (Temim et al., 2000). Furthermore, SC injection of dexamethasone, a synthesized glucocorticoid, for 3 or 7 days reduces IGF1 mRNA expression in the leg muscle in 7-day-old broiler chicks (Song et al., 2011b). Therefore, heat exposure-induced release of excess CORT might downregulate the gene expression of GH and IGF1 and thereby suppressed the growth of the chicks. To examine whether the elevation of CORT affected the growth and mRNA expression of the growth-related genes, the effect of repeated SC injection of CORT was investigated. SC 12
injection of CORT reduced the growth rate and weight of muscles as observed in the heat-exposure study. These changes are in good agreement with previous reports on laying hens and young broiler chickens (Buyse et al., 1987; Lin et al., 2006; Shini et al., 2009). The increased liver weight would reflect the accumulation of fat because the injection of CORT is thought to induce fatty liver in chickens (Davison et al., 1985; Buyse et al., 1987; Malheiros et al., 2003; Lin et al., 2006; Shini et al., 2009). The repeated injection of CORT reducing mRNA expression of pituitary GH and liver IGF1 indicated that CORT inhibited the hypothalamic-pituitary-somatotropic axis. The increased mRNA expression of diencephalic TRH and liver GH-R suggested the changes occur to compensate for the release and action of GH and IGFI. In our preliminary experiment, single SC injection of CORT reduced mRNA expression of liver IGF1 but did not reduce expression of other growth-related hormones at 1 h after injection, although body weight did not change at this time (data not shown). This decrease in liver IGF1 mRNA expression preceded the retardation of growth. All our findings suggest that change in IGF1 mRNA expression might be the first information on retarding growth in chicks. Although either heat exposure or CORT injection reduced the mRNA expression of pituitary GH and liver IGF1 and retarded the growth of the chicks, we did not find the same results in other parameters. For example, CORT did not affect feed intake whereas heat exposure decreased it. Therefore, decreasing body weight under CORT treatment might be influenced by digestion of the diet, absorption and utilization of nutrients rather than the amount of feed consumption. In addition, heat exposure reduced diencephalic mRNA expression of GHRH but CORT did not alter it. On the other hand, CORT injection increased mRNA expression of TRH in the diencephalon, whereas heat exposure had no effect on it. These differences suggest that heat stress-induced physiological changes can not be explained 13
by CORT alone. In summary, the present study demonstrated that heat exposure reduced the mRNA expressions of pituitary GH and liver IGF1, concomitantly with decrease in the body weight and increase in plasma CORT concentration in the layer-type chicks. These changes were similar to those induced by repeated CORT treatment. All our findings suggest that the change in mRNA expression of the growth-related hormones might be explained by the increase of plasma CORT, at least partly. Conflict of interest
There is no conflict of interest.
Acknowledgement This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 25450398 and 23/01407.
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expression of genes related to the growth of skeletal muscle in chickens (Gallus gallus domesticus). J. Mol. Endocrinol. 46, 217–225. Sutton, S.W., Behan, D.P., Lahrichi, S.L., Kaiser, R., Corrigan, A., Lowry, P., Potter, E., Perrin, M.H., Rivier, J., Vale, W.W., 1995.
Ligand requirements of the human
corticotropin-releasing factor-binding protein. Endocrinology. 136, 1097–1102. Tannenbaum, G.S., Rorstad, O., Brazeau. P., 1979. Effects of prolonged food deprivation on the ultradian growth hormone rhythm and immunoreactive somatostatin tissue levels in the rat. Endocrinology. 104, 1733–1738. Temim, S., Chagneau, A.M. Peresson, R., Tesseraud, S., 2000. Chronic heat exposure alters protein turnover of three different skeletal muscles in finishing broiler chickens fed 20 or 25% protein diets. J. Nutr. 130, 813–819. Thomas,
G.B.,
Fairhall,
K.M.,
Robinson.
I.C.,
1997.
Activation
of
the
hypothalamo-pituitary-adrenal axis by the growth hormone (GH) secretagogue, GH-releasing peptide-6, in rats. Endocrinology. 138, 1585–1591. Vandenborne, K., De Groef, B., Geelissen, S.M., Kühn, E.R., Darras, V.M., Van der Geyten, S.,
2005.
Corticosterone-induced
negative
feedback
mechanisms
within the
hypothalamo-pituitary-adrenal axis of the chicken. J. Endocrinol. 185, 383–391. Whitehead, C.C., Bollengier-Lee, S., Mitchell, M.A., Williams, P.E.V., 1998. Alleviation of depression in egg production in heat stressed laying hens by vitamin E. Proceedings of 10th European Poultry Conference, Jerusalem, Israel. pp. 576–578.
19
Legends of figures Fig. 1. Body weight, feed intake and rectal temperature during 4-day heat stress. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group at each day (P<0.05).
Fig. 2. Effect of heat exposure on the mRNA expression of pituitary GH, liver IGF1 and GH-R, and diencephalic GHRH, TRH and SST. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 3 Effect of heat exposure on plasma CORT concentration. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 4 Effect of heat exposure on the mRNA expression of diencephalic CRH, UCN3 and CRH-BP, and pituitary CRH-R1, CRH-R2, and CRH-BP. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 5. Body weight and feed intake during 4-day SC injection of CORT. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the 20
control group at each day (P<0.05).
Fig. 6. Effect of SC injection of CORT on the mRNA expression of pituitary GH, liver IGF1 and GH-R, and diencephalic GHRH, TRH and SST. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Table 1. Nucleotide sequences of specifi primers for semi-quantitative PCR Target gene
GenBank accession
Primer sequences
GH
NM_204359
Forward: 5’-ggaggaccagaggtacacca-3’
PCR product (bp) 202
Reverse: 5’-ggtgccaaaaaccaagttgt-3’ IGF1
NM_001004384
Forward: 5’-cagggtatggatccagcagt-3’
158
Reverse: 5’-catatcagtgtggcgctgag-3’ GH-R
NM_001001293
Forward: 5’- cttcagtgcaagcgacacat-3’
186
Reverse: 5’- ggccatgactctctgctttc-3’ GHRH
NM_204454
Forward: 5’-tacctgagtgggagctgatc-3’
164
Reverse: 5’-ctgcatccttttctcagtgg-3’ TRH
NM_001030383
Forward: 5’-taaacatgcctctgccacaa-3’
235
Reverse: 5’-gactggtcccacagtgacct-3’ SST
X_60191.1
Forward: 5’-tcagagccaagccagacag-3’
164
Reverse: 5’-ggaggacaggtgggtttca-3’ CRH
NM_001123031
Forward: 5’- ctccctggacctgacttt-3’
117
Reverse: 5’-cctcacttcccgatgatt-3’ UCN-3
XM_001231710
Forward: 5’-gccttccgtctctacaatgc-3’
225
Reverse: 5’-ctgtgcctgggaggtgtatt-3’ CRH-BP
XM_003642958
Forward: 5’-ggcaatgggtttacgatcac-3’
210
Reverse: 5’-gcccactgacggattcttta-3’ CRH-R1
NM_204321
Forward: 5’-cagccatcgtcctcacctat-3’
246
Reverse: 5’-tggtcatgagaatccgaaca-3’ CRH-R2
NM_204454
Forward: 5’-tgtcggtgcattaccactgt-3’ Reverse: 5’-attgggcaaggaatacacca-3’ 21
164
RP II
NM_001006448
Forward: 5’-cagaatttgccgacctcttc-3’
172
Reverse: 5’-ggccagcatcacagtctctt-3’ 18S rRNA
AF_173612
Forward: 5’-cccctcgatgctcttaactg-3’
250
Reverse: 5’-ggcaaatgctttcgctttag-3’
Table 2. Effect of heat exposure on body weight, feed intake and the weights of liver and muscles in chicks Control
Heat stress
8
8
Final body weight (g)
97.2 ± 1.4
77.0 ± 3.3*
Body weight gain (g/ 4 days)
30.5 ± 1.9
11.4 ± 2.0*
Feed intake (g/4 days)
61.2 ± 1.4
37.0 ± 1.3*
Feed efficiency
0.50 ± 0.02
0.30 ± 0.05*
Liver (g)
4.48 ± 0.17
2.99 ± 0.22*
% of body weight
4.60 ± 0.07
3.87 ± 0.29*
Surface breast muscle (g)1
2.12 ± 0.10
1.57 ± 0.16*
% of body weight
2.20 ± 0.14
2.09 ± 0.28
0.57 ± 0.02
0.46 ± 0.05*
0.59 ± 0.02
0.59 ± 0.04
2.30 ± 0.05
1.90 ± 0.10*
2.36 ± 0.03
2.46 ± 0.06
6.11 ± 0.15
5.17 ± 0.30*
6.29 ± 0.12
6.70 ± 0.13
n
Inner breast muscle (g)1 % of body weight Wing muscle (g)1,2 % of body weight Thigh muscle (g)1,2 % of body weight 1
Each sample was collected only from the left side.
2
Wing and thigh include each bone.
Data are expressed as mean ± SEM. *Significantly different from the control group (P<0.05). 22
Table 3. Effect of repeated SC injection of CORT on body weight, feed intake and the weights of liver and muscles in chicks Control
CORT
8
8
Final body weight (g)
82.6 ± 2.4
73.9 ± 2.8*
Body weight gain (g/ 4 days)
26.9 ± 1.8
20.0 ± 1.8*
Feed intake (g/4 days)
52.2 ± 2.0
49.6 ± 3.0
Feed efficiency
0.51 ± 0.03
0.39 ± 0.02*
Liver (g)
3.73 ± 0.13
4.56 ± 0.28*
% of body weight
4.52 ± 0.14
6.13 ± 0.14*
Surface breast muscle (g)1
1.49 ± 0.09
1.16 ± 0.10*
% of body weight
1.79 ± 0.06
1.56 ± 0.03
0.45 ± 0.03
0.36 ± 0.02*
0.54 ± 0.02
0.49 ± 0.03
1.75 ± 0.09
1.28 ± 0.06*
2.11 ± 0.07
1.73 ± 0.05*
4.96 ± 0.15
4.26 ± 0.20*
6.00 ± 0.06
5.75 ± 0.10*
n
Inner breast muscle (g)1 % of body weight Wing muscle (g)1,2 % of body weight Thigh muscle (g)1,2 % of body weight 1
Each sample was collected only from the left side.
2
Wing and thigh include each bone.
Data are expressed as mean ± SEM. *Significantly different from the control group (P<0.05).
Highlights
23
Four-days consecutive heat stress reduced body weight and food intake in chicks.
Heat stress reduced mRNA level of pituitary GH and liver IGF1.
Heat stress increased plasma CORT level and affected mRNA level of CRH.
CORT might be related to heat stress-induced change in mRNA level of GH and IGF1.
24
Figure Click here to download Figure: Fig.1.pptx
Control
Body weight (g)
110
Heat exposure
Body weight
100 90 80 70
P=0.05
*
*
1
2
3
60 0
Feed intake (g)
25
4
Daily feed intake
20 15 10 5
*
* 1
44
Body temperature (˚C)
*
2
*
*
3
4
Body temperature
*
42
*
*
*
3
4
40
38 0
1
2
Days after the onset of experiment
Fig. 1 Okuyama et al.
Figure Click here to download Figure: Fig.2.pptx
Control
Arbitrary unit
2.0
Pituitary
Heat exposure
Liver
Diencephalon
1.5
P=0.09 1.0
0.5
*
*
*
0.0
GH
IGF1
Fig. 2 Okuyama et al.
GH-R
GHRH
TRH
SST
Figure Click here to download Figure: Fig.3.pptx
Concentration (ng/ml)
Control
Heat stress
15
10
5
0
Fig. 3 Okuyama et al.
*
Figure Click here to download Figure: Fig.4.pptx
Control
Heat exposure
Diencephalon
2.5
Pituitary
*
Arbitrary unit
2.0 1.5
P=0.08 1.0 0.5
* *
0.0
CRH
UCN3
Fig. 4 Okuyama et al.
CRH-BP CRH-R1 CRH-R2 CRH-BP
Figure Click here to download Figure: Fig.5.pptx
Control 100
CORT
Body weight
Body weight (g)
90
80 70
*
60
*
*
50 0
Feed intake (g)
20
1
2
3
4
Daily feed intake
15
P=0.10
10
P=0.07
5
1
2
3
4
Days after the onset of experiment
Fig. 5 Okuyama et al.
Figure Click here to download Figure: Fig.6.pptx
Control 2.5
Pituitary
Liver
2.0
Arbitrary unit
CORT
Diencephalon
*
1.5
* 1.0
* 0.5
* 0.0
GH
IGF1
Fig. 6 Okuyama et al.
GH-R
GHRH
TRH
SST
PII: DOI: Reference:
S1871-1413(17)30037-9 http://dx.doi.org/10.1016/j.livsci.2017.02.010 LIVSCI3149
To appear in: Livestock Science Cite this article as: Hirofumi Okuyama, Md. Sakirul Islam Khan, Akira Tsukada and Tetsuya Tachibana, Heat exposure alters the mRNA expression of growthand stress-related genes in chicks, Livestock Science, http://dx.doi.org/10.1016/j.livsci.2017.02.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heat exposure alters the mRNA expression of growth- and stress-related genes in chicks Hirofumi Okuyama1, Md. Sakirul Islam Khan2, Akira Tsukada3, Tetsuya Tachibana1* 1
Department of Agrobiological Science, Faculty of Agriculture, Ehime University,
Matsuyama 2
Department of Anatomy and Embryology, Graduate School of Medicine, Ehime University,
Toon 791-0295, Ehime, Japan 3
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Science,
Nagoya University, Chikusa 464-8601, Nagoya, Japan *Corresponding address: Tetsuya Tachibana. Laboratory of Animal Production, Department of Agrobiological Science, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan. Tel./ Fax. +81-89-946-9820. [email protected]
1
Abstract High ambient temperature, a major stressor, impairs the growth of chickens. In this study, we examined the effect of heat exposure on the mRNA expression of various growth related genes, such as growth hormone (GH), insulin-like growth factor-1 (IGF1), GH-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), and somatostatin (SST) in layer-type chickens (Gallus gallus domesticus) at an early age. In addition, we examined the reactivity of the corticotrophin-releasing hormone (CRH) system, one of the stress-regulating pathways, to heat exposure and its role in altering the growth related genes. Four-day heat exposure reduced the body weight gain, feed intake, and feeding efficiency and increased the rectal temperature of chicks (P < 0.05). The mRNA expression levels of pituitary GH, liver IGF1, and diencephalic GHRH decreased with heat exposure (P < 0.05) whereas the levels of TRH or SST did not change. Heat exposure also reduced the diencephalic mRNA expression level of CRH and increased pituitary CRH receptor-2 mRNA and the plasma corticosterone (CORT) concentration (P < 0.05), suggesting that heat exposure affected the hypothamic-pituitary adrenal gland (HPA) axis. Similar to the heat-exposure study, subcutaneous injection of CORT for 4 days decreased body weight, and the mRNA expression of pituitary GH and liver IGF1 (P < 0.05). The present study demonstrated that heat exposure reduced the mRNA expressions of pituitary GH and liver IGF1, and suggested that the change in mRNA expression may have been partly caused by CORT. Keywords: Chick; Corticosterone; Growth hormone; Heat exposure; Insulin-like growth factor-I
2
Introduction Stress is an important factor affecting the economic and health aspects of poultry production. Some stressors severely retard growth, decrease production, and finally cause death in poultry (Lara and Rostagno, 2013). For example, high ambient temperature, which is regarded as an important stressor, decreases feed intake, growth rate, feeding efficiency, and the carcass quality of broiler-type chickens (Geraert et al., 1996; McKee et al., 1997; Temim et al., 2000; Quinteiro-Filho et al., 2010; 2012; Lara and Rostagno, 2013). In layer-type chickens, heat stress depresses body weight (Scott and Balnave, 1988) and egg production and shell quality (Whitehead et al., 1998; Balnave and Muheereza, 1997; Muiruri and Harrison, 1991; Emery et al., 1984), which are assumed to be caused by heat-induced reduction in feed intake (Mashaly et al., 2004). Therefore, understanding the stress-response mechanisms of chickens is essential to overcoming the problems related to stress. However, these mechanisms have not been completely clarified. The hypothalamus, a brain region of the diencephalon that regulates the endocrine and autonomic nervous systems, as well as appetite, body temperature, and so on, plays a critical role in regulating growth. There is considerable evidence that various hormones secreted from the hypothalamic-pituitary regions regulate the growth of chickens. For example, growth hormone (GH) from the anterior pituitary is believed to be required for the growth of chickens, and GH exerts its somatic effect by increasing circulating insulin-like growth factor-I (IGF-I, the gene is abbreviated as IGF1) via stimulating the synthesis of IGF-I in the liver of chickens (Cogburn et al., 1999; Scanes, 1999; Scanes et al., 1999; Kühn et al., 2005). The release of GH from the anterior pituitary is stimulated and inhibited by the hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SST), respectively (Cogburn et al., 1999; Scanes, 1999; Kühn et al., 2005). In addition, thyrotropin-releasing hormone (TRH) in the 3
hypothalamus has been shown to stimulate the release of GH in chickens (Cogburn et al., 1999; Kühn et al., 2005). The hypothalamus also plays an important role in regulating stress response. In fact, the hypothalamic-pituitary-adrenal (HPA) axis is thought to be a major pathway in regulating stress by activating the corticotropin-releasing hormone (CRH) system (Carsia and Harvey, 1999). Because CRH has been shown to inhibit GH release in rats (Rivier and Vale, 1984; Thomas et al., 1997), stress-induced release of CRH might suppress GH release and then inhibit growth in chickens. In fact, it has been reported that heat stress suppresses the release of GH and IGF-I in black-boned chickens (Liu et al., 2014). However, little is known about the effect of heat stress on the gene expression of growth-related hormones in layer-type chicks. The purpose of the present study was to investigate whether heat exposure affects the expression of the growth-related genes in the pituitary, diencephalon and liver of layer-type chicks. The involvement of the HPA axis in response to heat stress was also investigated. Subsequently, the effect of subcutaneous (SC) injection of corticosterone (CORT) on the expression of the growth-related genes was investigated.
Materials and Methods Animals One-day-old male layer-type chicks (Gallus gallus, Julia, Nihon Layer, Gifu, Japan) were raised in a room kept at 30°C with continuous lighting. They were given free access to a commercial diet (crude protein, 24%; metabolizable energy, 3050 kcal/kg; Toyohashi Feed Mills Co. Ltd, Aichi, Japan) and water. All animal experiments were carried out in accordance with protocols approved by the Ehime University Animal Care and Use Committee (No. 4
08-o3-10). Experiment 1. Effect of heat exposure on mRNA expression of the growth-related genes in layer-type chicks At 5 days of age, 16 chicks were assigned to either the heat exposure group or the control group (8 chicks in each group) such that the average body weight was as uniform as possible. The two groups were then transferred to separate rooms kept at 30°C. One group was exposed to high temperature, and the other was used as the control. Chicks were transferred to the experimental cages at least 2 days before the experiment. One day before the experiment, chicks were individually reared in the experimental cages. The room temperature for the heat-stress group was set at 38°C when the chicks were 7 days of age, continuing until they were 11 days of age. Chicks in the control group continued to be raised at 30°C. The temperature for the heat-stress group was chosen based on previous studies on chicks (Halevy et al., 2001; Chowdhury et al., 2014). During this experimental period, body weight, rectal temperature, and feed intake were measured daily. Rectal temperature was measured by inserting a 19-mm stainless steel sensor connected to an electronic thermometer (BAT7001H, Physitemp Instruments Inc., New Jersey, USA) into the rectum. Feed and water were available ad libitum during the experiment. Feed intake was measured using a digital balance with an accuracy of 1 mg, and the feeding efficiency was calculated. After the final measurement of feed intake, the blood of chicks was collected from the jugular vein into tubes containing ethylenediaminetetraacetic acid dipotassium salt dehydrate. The collected blood was centrifuged (9000 × g at 4°C for 5 min), and the plasma was collected and stored at −30°C. Plasma CORT concentrations were later measured by enzyme immunoassay using a commercial kit (AssayPro LLC, MO, USA) according to the manufacturer’s instructions. The detection limit was 0.4 ng/ml, and intra- and inter-assay 5
variations were 2.0 and 8.4%, respectively. Immediately after blood collection, the chicks were sacrificed by decapitation, and then the liver, surface breast muscle, inner breast muscle, wing muscle, and thigh muscle were collected and weighed. In addition, the diencephalon was collected after removing the whole brain. In brief, the telencephalon, cerebellum, and optic tectum were excised from the whole brain. The diencephalon was collected by removing the mesencephalon, pons, and medulla oblongata at the caudal end of the median eminence. The collected diencephalon was quickly frozen with liquid nitrogen and stored at −80°C until analysis. The pituitary and liver were also collected and frozen. The diencephalon, pituitary and liver were used to investigate mRNA expression. These tissues (except for the pituitary) were homogenized with Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan), and the total RNA were then isolated as per the manufacturer’s instructions. Total RNA of the pituitary was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA treated with DNase I (Ambion, Austin, TX, USA) using the reverse transcription kit ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan) with random primers. To investigate the expression of the growth-related genes, cDNAs for pituitary GH, liver IGF1 and GH receptor (GH-R), and diencephalic GHRH, TRH, and SST were amplified and quantified by semi-quantitative PCR using specific primers (Table 1). To investigate the expression of the HPA-axis related genes, diencephalic CRH, urocortin-3 (UCN3), and CRH-binding protein (CRH-BP), and pituitary CRH-BP, CRH receptor-1 (CRH-R1), and CRH receptor-2 (CRH-R2) cDNA were amplified using specific primers (Table 1). Subsequently, semi-quantitative PCR was carried out in duplicate using a real-time PCR instrument (LightCycler®Nano, Roche Applied Science, Germany) with SYBR Green 6
Real-time PCR Master Mix (Toyobo, Osaka, Japan). As an internal control, polymerase (RNA) II (DNA directed) polypeptide B (RPII) was also amplified (Table 1). 18S rRNA from the pituitary was amplified as the internal control (Table 1). PCR parameters for the assays were as follows: degradation at 95ºC for 10 min followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. The accuracy of these primers was checked by melting curve analysis and the nucleotide sequencing of each PCR product. 2−ΔCT value was calculated for gene expression from real-time quantitative PCR experiments (Livak and Schmittgen, 2001) with the threshold cycle (CT) value calculated by the software of real-time PCR instrument (LightCycler®Nano, Roche Applied Science, Germany). For 2−ΔCT value, the data were analyzed using the equation, where ΔCT = (CT, Target – CT, internal control). 2−ΔΔCT method was also used to investigate the relative changes in gene expression (Livak and Schmittgen, 2001). In 2−ΔΔCT method, the data were analyzed using the equation, where ΔΔCT = (CT, Target – CT, internal control)Heat stress
– (CT, Target – CT, internal control)Control.
Experiment 2. Effect of repeated SC injection of CORT on mRNA expression of growth related genes in layer-type chicks At 6 days of age, 16 chicks were transferred to their individual cages. On the following day, the chicks were assigned to the CORT group or the control group (8 chicks in each group) so that the average body weight was as uniform as possible. Chicks were transferred to the experimental cages at least 2 days before the experiment. One day before the experiment, chicks were individually reared in the experimental cages. Chicks in the CORT group were SC injected with 1.2 mg CORT (Wako Pure Chemical Industries, Ltd., Osaka, Japan), which was dissolved in dimethyl sulfoxide and then diluted with polyethylene glycol, at a volume of 0.1 ml twice a day (8:00 and 20:00) for 4 days. The dose of CORT was decided by referring to a previous study (Song et al., 2011a) and by our preliminary experiment. Our preliminary 7
experiment found that single SC injection of 1.2 mg CORT increased plasma CORT concentration at 1 h after the injection (control group, 7.2 ± 2.1 ng/ml; CORT group, 1163.6 ± 159.5 ng/ml; P < 0.05, Student’s t-test) and 3 h after the injection (control group, 5.6 ± 1.2 ng/ml; CORT group, 14.9 ± 3.1 ng/ml; P < 0.05, Student’s t-test) in 7-day-old chicks, although the effect disappeared at 6 h (control group, 3.7 ± 0.4 ng/ml; CORT group, 4.1 ± 0.5 ng/ml). Therefore we performed the SC injection twice a day. Chicks in the control group were SC injected with the same volume of the vehicle. During the experimental period, body weight and feed intake were measured as noted above. At the end of the experiment, the chicks were sacrificed, and thereafter their liver, surface breast muscle, inner breast muscle, wing muscle and leg muscle were collected and weighed. To measure the mRNA expressions of GH, IGF1, GHRH, TRH and SST, the diencephalon, pituitary, and liver were removed and used for semi-quantitative real-time PCR as noted in Experiment 1. Statistical analysis Daily data on body weight, body temperature, and feed intake were statistically analyzed using two-way repeated measures analysis of variance (ANOVA) with respect to treatment (heat exposure or CORT injection) and time, and then analyzed using the Student’s t-test. Other data were statistically analyzed using the Student’s t-test. Data are presented as the mean ± standard error of the mean (SEM), and statistical significance was set at P < 0.05. The number of chicks is noted in the figure legends.
Results Experiment 1. Effect of heat exposure on mRNA expression of the growth-related genes in layer-type chicks Body weight, feed intake, feeding efficiency, and organ weights 8
Heat exposure reduced body weight gain (P < 0.01) and the effect was time dependent (P < 0.01). The body weight of the chicks exposed to heat stress tended to decrease at 1 day after the experiment, and the effect became significant from 2 days later (P < 0.05, Fig. 1 and Table 2). Rectal temperature increased with heat exposure (P < 0.01) and the effect was time dependent (P < 0.01). The increase in rectal temperature was observed from day 1 and continued to the end of the experiment (P < 0.05, Fig. 1). Daily feed intake decreased with heat exposure (P < 0.01) and the effect was time dependent (P < 0.01) (Fig. 1). Feed intake decreased with heat exposure compared with the control group throughout the experiment. Feeding efficiency during the 4-day experimental period also decreased with heat exposure (P < 0.05, Table 2). At the end of 4 days of heat exposure, the weight of the liver, breast muscle, inner breast muscle, wing muscle, and leg muscle in the heat exposure group was lower than that of control (P < 0.05, Table 2). Although the relative weight of the liver to body weight also decreased with heat treatment (P < 0.05), the relative weight of all the muscles was comparable to that of the control group (Table 2). mRNA expression of the growth-related genes Four days of heat exposure reduced the mRNA expression of pituitary GH and liver IGF1 (P < 0.05), whereas liver GH-R mRNA expression tended to increase (P = 0.09) with heat exposure (Fig. 2). In the diencephalon, heat exposure reduced GHRH mRNA levels whereas the TRH (P < 0.05) and SST mRNA levels did not change (Fig. 2). Plasma CORT concentration and mRNA expression of stress related genes Heat exposure increased the plasma CORT concentrations (P < 0.05, Fig. 3). In addition, heat exposure reduced CRH mRNA expression in the diencephalon (P < 0.05) but did not affect UCN3 mRNA expression (Fig. 4). The mRNA expression of CRH-BP in the diencephalon decreased with heat exposure (P < 0.05, Fig. 4). The decreasing tendency for 9
mRNA expression of CRH-BP was also observed in the pituitary (P = 0.08, Fig. 4). CRH-R2 mRNA expression but not CRH-R1 mRNA expression in the pituitary increased with heat exposure (P < 0.05, Fig. 4). Experiment 2. Effect of repeated SC injection of CORT on mRNA expression of the growth-related genes in layer-type chicks Body weight, feed intake, feeding efficiency, and organ weights The main effect of CORT on body weight was nearly significant (P = 0.06), whereas the interaction between CORT and time was significant (P < 0.01). The body weight of the CORT-treated chicks was comparable with control at 1 day after injection, but decreased at 2 days after injection of CORT (P < 0.05) (Fig. 5 and Table 3). There was no significant effect of CORT on feed intake (P = 0.48) although a significant interaction was observed between CORT and time (P < 0.01). Feed intake tended to decrease with CORT treatment, but it did not reach significance (Fig. 5 and Table 3). As the result, feed efficiency in the CORT group was lower than that in the control group (P < 0.05, Table 3). Four-day CORT treatment increased the actual and relative weights of the liver (P < 0.05, Table 3). On the other hand, the weight of all the muscles investigated decreased with CORT (P < 0.05, Table 3). CORT also reduced the relative weights of the wing and thigh muscles to body weight (P < 0.05). mRNA expression of the growth-related genes The mRNA expression of pituitary GH and liver IGF1 decreased with the SC injection of CORT (P < 0.05, Fig. 6). The mRNA expression level of liver GH-R and diencephalic TRH increased with CORT treatment (P < 0.05), while the mRNA level of GHRH and SST in the diencephalon did not change.
Discussion 10
Exposure to high temperature induced hyperthermia in the chicks, indicating they were not able to adapt to the experimental temperature used in the present study. It is likely that the physiological and molecular changes observed in the present study are due to the effect of chronic heat exposure. We confirmed that 4 day heat exposure reduced body weight including liver and muscle weight in the layer-type chicks. In addition, the present study provided the first evidence that mRNA expression of diencephalic GHRH, pituitary GH, and liver IGF1 decreased with exposure to high temperature. On the other hand, the diencephalic mRNA expression of TRH or SST did not change with exposure to high temperature. These results suggest that the diencephalic GHRH, but not TRH or SST, might be related to the suppression of GH and IGFI production under a heat-stress condition. In rodents, similar to high temperature-induced changes in the GH axis, feed deprivation reduces blood GH and IGF-I levels and suppresses hypothalamic GHRH and liver IGF1 mRNA expression (Park et al. 2004; Tannenbaum et al. 1979). Feed deprivation also reduces IGF-I concentration in blood and IGF1 mRNA expression in the liver of young layer chickens (Kita et al., 1998; Morishita et al., 1993). Because heat exposure reduced feed intake in the present study, we cannot exclude the possibility that reduced feed intake might alter mRNA expression of the growth-related hormones in the diencephalon and liver. Either way, these findings suggest that stress induced by high temperature or feed deprivation alters the growth-related genes in the GH axis, which might impair the growth of chicks. In addition to the growth-related genes, heat exposure also altered the plasma CORT concentration and gene expression of the CRH system in the hypothalamic-pituitary regions. Plasma CORT concentration increased with heat stress as was also shown in a previous study (Quinteiro-Filho et al., 2010). Heat exposure reduced mRNA expression of CRH and 11
CRH-BP in the diencephalon and increased CRH-R2 mRNA expression in the pituitary. These results seem to be contradictory, but they can be explained by the effect of CORT released after exposure to heat. It has been shown that intravenous injection of CORT reduces CRH mRNA expression in the diencephalon in chicks (Vandenborne et al., 2005). Also, Calefi et al. (2016) found negative feedback during heat stress in chicks. Therefore CORT released during heat stress might suppress mRNA expression of CRH as the negative feedback, and changes in mRNA expression of CRH-R2 might be induced to compensate for the action of CRH. Because CRH-BP is reported to bind to CRH with a higher affinity than its receptors (Sutton et al., 1995), CRH-BP is considered to inhibit the biological activity of CRH. Therefore, the decrease in mRNA expression of CRH-BP indicated that heat exposure suppressed the inhibitory effect of CRH-BP on the activity of CRH. Changes in the gene expression of the CRH system in the hypothalamic-pituitary regions and the increase in plasma CORT concentration suggested that exposure to high temperature affected the HPA axis in the layer-type chicks. Supplementation of CORT into the diet retards the growth of skeletal muscle by suppressing protein synthesis and stimulating protein catabolism in young broiler chickens (Dong et al. 2007). Chronic heat stress has also been shown to suppress protein synthesis and breakdown in young broiler chickens (Temim et al., 2000). Furthermore, SC injection of dexamethasone, a synthesized glucocorticoid, for 3 or 7 days reduces IGF1 mRNA expression in the leg muscle in 7-day-old broiler chicks (Song et al., 2011b). Therefore, heat exposure-induced release of excess CORT might downregulate the gene expression of GH and IGF1 and thereby suppressed the growth of the chicks. To examine whether the elevation of CORT affected the growth and mRNA expression of the growth-related genes, the effect of repeated SC injection of CORT was investigated. SC 12
injection of CORT reduced the growth rate and weight of muscles as observed in the heat-exposure study. These changes are in good agreement with previous reports on laying hens and young broiler chickens (Buyse et al., 1987; Lin et al., 2006; Shini et al., 2009). The increased liver weight would reflect the accumulation of fat because the injection of CORT is thought to induce fatty liver in chickens (Davison et al., 1985; Buyse et al., 1987; Malheiros et al., 2003; Lin et al., 2006; Shini et al., 2009). The repeated injection of CORT reducing mRNA expression of pituitary GH and liver IGF1 indicated that CORT inhibited the hypothalamic-pituitary-somatotropic axis. The increased mRNA expression of diencephalic TRH and liver GH-R suggested the changes occur to compensate for the release and action of GH and IGFI. In our preliminary experiment, single SC injection of CORT reduced mRNA expression of liver IGF1 but did not reduce expression of other growth-related hormones at 1 h after injection, although body weight did not change at this time (data not shown). This decrease in liver IGF1 mRNA expression preceded the retardation of growth. All our findings suggest that change in IGF1 mRNA expression might be the first information on retarding growth in chicks. Although either heat exposure or CORT injection reduced the mRNA expression of pituitary GH and liver IGF1 and retarded the growth of the chicks, we did not find the same results in other parameters. For example, CORT did not affect feed intake whereas heat exposure decreased it. Therefore, decreasing body weight under CORT treatment might be influenced by digestion of the diet, absorption and utilization of nutrients rather than the amount of feed consumption. In addition, heat exposure reduced diencephalic mRNA expression of GHRH but CORT did not alter it. On the other hand, CORT injection increased mRNA expression of TRH in the diencephalon, whereas heat exposure had no effect on it. These differences suggest that heat stress-induced physiological changes can not be explained 13
by CORT alone. In summary, the present study demonstrated that heat exposure reduced the mRNA expressions of pituitary GH and liver IGF1, concomitantly with decrease in the body weight and increase in plasma CORT concentration in the layer-type chicks. These changes were similar to those induced by repeated CORT treatment. All our findings suggest that the change in mRNA expression of the growth-related hormones might be explained by the increase of plasma CORT, at least partly. Conflict of interest
There is no conflict of interest.
Acknowledgement This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 25450398 and 23/01407.
14
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expression of genes related to the growth of skeletal muscle in chickens (Gallus gallus domesticus). J. Mol. Endocrinol. 46, 217–225. Sutton, S.W., Behan, D.P., Lahrichi, S.L., Kaiser, R., Corrigan, A., Lowry, P., Potter, E., Perrin, M.H., Rivier, J., Vale, W.W., 1995.
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19
Legends of figures Fig. 1. Body weight, feed intake and rectal temperature during 4-day heat stress. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group at each day (P<0.05).
Fig. 2. Effect of heat exposure on the mRNA expression of pituitary GH, liver IGF1 and GH-R, and diencephalic GHRH, TRH and SST. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 3 Effect of heat exposure on plasma CORT concentration. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 4 Effect of heat exposure on the mRNA expression of diencephalic CRH, UCN3 and CRH-BP, and pituitary CRH-R1, CRH-R2, and CRH-BP. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Fig. 5. Body weight and feed intake during 4-day SC injection of CORT. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the 20
control group at each day (P<0.05).
Fig. 6. Effect of SC injection of CORT on the mRNA expression of pituitary GH, liver IGF1 and GH-R, and diencephalic GHRH, TRH and SST. Data are expressed as mean ± SEM. The number of chicks in each group was 8. *Significantly different from the control group (P<0.05).
Table 1. Nucleotide sequences of specifi primers for semi-quantitative PCR Target gene
GenBank accession
Primer sequences
GH
NM_204359
Forward: 5’-ggaggaccagaggtacacca-3’
PCR product (bp) 202
Reverse: 5’-ggtgccaaaaaccaagttgt-3’ IGF1
NM_001004384
Forward: 5’-cagggtatggatccagcagt-3’
158
Reverse: 5’-catatcagtgtggcgctgag-3’ GH-R
NM_001001293
Forward: 5’- cttcagtgcaagcgacacat-3’
186
Reverse: 5’- ggccatgactctctgctttc-3’ GHRH
NM_204454
Forward: 5’-tacctgagtgggagctgatc-3’
164
Reverse: 5’-ctgcatccttttctcagtgg-3’ TRH
NM_001030383
Forward: 5’-taaacatgcctctgccacaa-3’
235
Reverse: 5’-gactggtcccacagtgacct-3’ SST
X_60191.1
Forward: 5’-tcagagccaagccagacag-3’
164
Reverse: 5’-ggaggacaggtgggtttca-3’ CRH
NM_001123031
Forward: 5’- ctccctggacctgacttt-3’
117
Reverse: 5’-cctcacttcccgatgatt-3’ UCN-3
XM_001231710
Forward: 5’-gccttccgtctctacaatgc-3’
225
Reverse: 5’-ctgtgcctgggaggtgtatt-3’ CRH-BP
XM_003642958
Forward: 5’-ggcaatgggtttacgatcac-3’
210
Reverse: 5’-gcccactgacggattcttta-3’ CRH-R1
NM_204321
Forward: 5’-cagccatcgtcctcacctat-3’
246
Reverse: 5’-tggtcatgagaatccgaaca-3’ CRH-R2
NM_204454
Forward: 5’-tgtcggtgcattaccactgt-3’ Reverse: 5’-attgggcaaggaatacacca-3’ 21
164
RP II
NM_001006448
Forward: 5’-cagaatttgccgacctcttc-3’
172
Reverse: 5’-ggccagcatcacagtctctt-3’ 18S rRNA
AF_173612
Forward: 5’-cccctcgatgctcttaactg-3’
250
Reverse: 5’-ggcaaatgctttcgctttag-3’
Table 2. Effect of heat exposure on body weight, feed intake and the weights of liver and muscles in chicks Control
Heat stress
8
8
Final body weight (g)
97.2 ± 1.4
77.0 ± 3.3*
Body weight gain (g/ 4 days)
30.5 ± 1.9
11.4 ± 2.0*
Feed intake (g/4 days)
61.2 ± 1.4
37.0 ± 1.3*
Feed efficiency
0.50 ± 0.02
0.30 ± 0.05*
Liver (g)
4.48 ± 0.17
2.99 ± 0.22*
% of body weight
4.60 ± 0.07
3.87 ± 0.29*
Surface breast muscle (g)1
2.12 ± 0.10
1.57 ± 0.16*
% of body weight
2.20 ± 0.14
2.09 ± 0.28
0.57 ± 0.02
0.46 ± 0.05*
0.59 ± 0.02
0.59 ± 0.04
2.30 ± 0.05
1.90 ± 0.10*
2.36 ± 0.03
2.46 ± 0.06
6.11 ± 0.15
5.17 ± 0.30*
6.29 ± 0.12
6.70 ± 0.13
n
Inner breast muscle (g)1 % of body weight Wing muscle (g)1,2 % of body weight Thigh muscle (g)1,2 % of body weight 1
Each sample was collected only from the left side.
2
Wing and thigh include each bone.
Data are expressed as mean ± SEM. *Significantly different from the control group (P<0.05). 22
Table 3. Effect of repeated SC injection of CORT on body weight, feed intake and the weights of liver and muscles in chicks Control
CORT
8
8
Final body weight (g)
82.6 ± 2.4
73.9 ± 2.8*
Body weight gain (g/ 4 days)
26.9 ± 1.8
20.0 ± 1.8*
Feed intake (g/4 days)
52.2 ± 2.0
49.6 ± 3.0
Feed efficiency
0.51 ± 0.03
0.39 ± 0.02*
Liver (g)
3.73 ± 0.13
4.56 ± 0.28*
% of body weight
4.52 ± 0.14
6.13 ± 0.14*
Surface breast muscle (g)1
1.49 ± 0.09
1.16 ± 0.10*
% of body weight
1.79 ± 0.06
1.56 ± 0.03
0.45 ± 0.03
0.36 ± 0.02*
0.54 ± 0.02
0.49 ± 0.03
1.75 ± 0.09
1.28 ± 0.06*
2.11 ± 0.07
1.73 ± 0.05*
4.96 ± 0.15
4.26 ± 0.20*
6.00 ± 0.06
5.75 ± 0.10*
n
Inner breast muscle (g)1 % of body weight Wing muscle (g)1,2 % of body weight Thigh muscle (g)1,2 % of body weight 1
Each sample was collected only from the left side.
2
Wing and thigh include each bone.
Data are expressed as mean ± SEM. *Significantly different from the control group (P<0.05).
Highlights
23
Four-days consecutive heat stress reduced body weight and food intake in chicks.
Heat stress reduced mRNA level of pituitary GH and liver IGF1.
Heat stress increased plasma CORT level and affected mRNA level of CRH.
CORT might be related to heat stress-induced change in mRNA level of GH and IGF1.
24
Figure Click here to download Figure: Fig.1.pptx
Control
Body weight (g)
110
Heat exposure
Body weight
100 90 80 70
P=0.05
*
*
1
2
3
60 0
Feed intake (g)
25
4
Daily feed intake
20 15 10 5
*
* 1
44
Body temperature (˚C)
*
2
*
*
3
4
Body temperature
*
42
*
*
*
3
4
40
38 0
1
2
Days after the onset of experiment
Fig. 1 Okuyama et al.
Figure Click here to download Figure: Fig.2.pptx
Control
Arbitrary unit
2.0
Pituitary
Heat exposure
Liver
Diencephalon
1.5
P=0.09 1.0
0.5
*
*
*
0.0
GH
IGF1
Fig. 2 Okuyama et al.
GH-R
GHRH
TRH
SST
Figure Click here to download Figure: Fig.3.pptx
Concentration (ng/ml)
Control
Heat stress
15
10
5
0
Fig. 3 Okuyama et al.
*
Figure Click here to download Figure: Fig.4.pptx
Control
Heat exposure
Diencephalon
2.5
Pituitary
*
Arbitrary unit
2.0 1.5
P=0.08 1.0 0.5
* *
0.0
CRH
UCN3
Fig. 4 Okuyama et al.
CRH-BP CRH-R1 CRH-R2 CRH-BP
Figure Click here to download Figure: Fig.5.pptx
Control 100
CORT
Body weight
Body weight (g)
90
80 70
*
60
*
*
50 0
Feed intake (g)
20
1
2
3
4
Daily feed intake
15
P=0.10
10
P=0.07
5
1
2
3
4
Days after the onset of experiment
Fig. 5 Okuyama et al.
Figure Click here to download Figure: Fig.6.pptx
Control 2.5
Pituitary
Liver
2.0
Arbitrary unit
CORT
Diencephalon
*
1.5
* 1.0
* 0.5
* 0.0
GH
IGF1
Fig. 6 Okuyama et al.
GH-R
GHRH
TRH
SST