A total of four hundred hatching eggs of broiler chicken were subjected to elevated incubation temperature for 12 hours per day during 7th to 16th day of incubation and after hatch chicks were reared up to five weeks of age. On 36th day, 192 birds were divided into 6 groups (32 birds in each group) - 2 groups were treated with normal saline as control, 2 groups with glutamine @ 0.75 mg/Kg body weight as enhancer and 2 groups with quercetin @ 5 mg/kg body weight as inhibitor. After 24 hours of treatment , one set of all three treatment groups being exposed to acute heat stress (40±1°C; 55% RH) for different duration of 0, 2, 5 and 10 hours in psychometric chamber and another set of all three treatment groups being unexposed to heat stress. The result indicated that concentrations of lipase, amylase, pepsin and trypsin were significantly (P<0.05) higher in enhancer group. Exposure of heat stress both pre and post hatch, significantly (P<0.05) increased the activity of trypsin and pepsin in jejunum and simultaneously decreased the amylase activity; however the lipase activity did not show any change in response to acute heat stress exposure. A significant difference observed (P<0.05) on enzymatic activity of pepsin and trypsin and the activity of amylase and lipase was not significant under different duration of heat stress exposure.
Heat stress causes major production and economic loss in the poultry sector worldwide (Mahmoud et al., 2003, Sejian et al., 2012). Broiler chicken is more receptive to heat stress due to higher growth rate, higher body temperature and lack of sweat gland (Geraert et al., 1996). Abrupt changes in environmental temperature affect digestion and absorption of GIT of poultry which in turn lead to loss of appetite and reduce feed intake (Itoh et al., 2001).In poultry feed efficiency diminishes from 0.485 to 0.340 as environmental temperature rises from 22ºC to 32ºC (Bonnett et al., 1997), indicating that the functional digestive tract adaptation could be associated to compensatory adjustment on production rate and enzymatic contents of digestive secretions (Osman and Tanios, 1983). Glutamine, a protein synthesis modulator is non-essential amino acid that plays an imperative role in alleviating the heat stress. Incorporation of glutamine in diet improves digestive function of small intestine in rodents (Larson et al., 2007) and pigs (Wu et al., 1996). Heat Shock Proteins play a major role in the protection of cells against heat stress stimuli (Hao et al., 2012). Glutamine promotes over-expression of heat shock protein to overcome the effect of heat stress and enhances digestive enzymes activity in chicken (Hao et al., 2012 and Devi-Priya et al., 2010). Quercetin, another protein synthesis modulator (bioflavonoid) inhibits the expression of heat shock protein. Diminution in digestive enzyme activity may be one reason for the reduction in digestive function under heat stress. Effect of protein modulator on poultry digestive enzyme activity under heat stress needs to be explored out in order to improve digestion and feed intake of broiler chicken to cope up the adverse environmental conditions such as heat stress. Manipulation of incubation temperature is currently being studied as a tool to induce epigenetic temperature adaptation to increase thermo tolerance (Tzschentke and Plagemann, 2006; Tzschentke, 2007). However, research focusing on prenatal thermal conditioning to improve resistance to heat stress in broilers is lacking. Generally chronic heat stress conditions even though cause mortality and suppress production, they improve the heat tolerance level at later stages. While acute heat stress levels may cause high mortality.
In the present study we hypothesized that both pre hatch and post hatch heat stress along with protein synthesis modulator, like glutamine would have synergistic effect on digestive enzymatic activity especially in acute heat stress conditions.
Materials and Methods
Ethical approval for the study was obtained from Institutional Animal ethics Committee of Indian Veterinary Research Institute, Izatnagar Bareilly-243122, Uttarpradesh, India.
Experimental Eggs and Birds
Four hundred hatching eggs of CARI-BRO Vishal broiler chicken were obtained from experimental broiler farm, Central Avian Research Institute, Izatnagar, India and incubated at experimental hatchery section of the institute and the chicks hatched out were housed in multi tier brooder cages up to five weeks of age and reared under uniform husbandry conditions.
A total of two hundred hatching eggs of broiler chicken were subjected to elevated incubation temperature of 39.5 °C with 55-60 % relative humidity for 12 hours per day during seventh to sixteenth day of incubation. During rest days of incubation, the eggs were incubated at normal incubation temperature. Hatch was pulled out and straight run chicks were reared up to five weeks of age in cages under standard managemental conditions. On 36th day, 192 birds were divided into 6 groups (32 birds in each group) – 2 groups were treated with normal saline via intra-peritoneal route as control, 2 groups with glutamine @ 0.75 mg/Kg body weight as HSP-70 enhancer and 2 groups with quercetin @ 5 mg/kg body weight as HSP-70 inhibitor. After 24 hours of treatment , one set of all three treatment groups being exposed to acute heat stress (40±1°C; 55% RH) for different duration of 0, 2, 5 and 10 hours in psychometric chamber and another set of all three treatment groups being unexposed to heat stress. Four birds with nearly similar body weight form each treatment group on each duration interval were selected and immediately killed by cervical dislocation. Longitudinal sections of jejunum tissue were obtained and flushed with 5 ml of saline. A homogeneous mucosal homogenate sample was farmed method given by Majumdar et al., 1992, after that stored immediately at −70°C until analysis.
Estimation of Total Protein in Intestinal Mucosal Homogenate
The total protein in intestinal mucosal homogenate was estimated by modified Lowry’s method as described by Rosenberg (1996).
Estimation of Digestive Enzymes
The digestive enzymes namely amylase, lipase, pepsin and trypsin in saline based intestinal mucosal homogenate were estimated by method of Gomori (1957), Schmid (1974), Rick and Fritsch (1974) and Rick (1974) respectively.
The data obtained from experiment were analysed by 2x3x4 factorial design using SPSS V.20 for both interaction and main effect. The means were compared using Tukey test.
Results and Discussion
The interaction and main effect of protein synthesis modulator (Mean values ± SE) on jejunal enzymatic activity are represented in Table 1 and Table 2 respectively. Results indicated significant (P<0.05) effect of protein synthesis modulator on activity of various enzymes i.e. amylase, lipase, pepsin and trypsin was observed in broilers exposed to varied periods of heat stress. The main effect of protein synthesis modulator significantly modulates activity of various enzymes in jejunum. Concentrations of lipase (92.91 ± 2.25), amylase (112.86 ± 3.16), pepsin (71.45 ± 1.34) and trypsin (83.89 ± 2.91) were significantly (P<0.05) higher in enhancer group when compared to that of control and inhibitor group.
Table 1: Effect of Protein Synthesis Modulator at both Pre and Post Hatch Heat Stress Exposure on Jejunal Enzymatic Activity (Units/mg of Protein) in Broilers
|Heat Stress||Protein Modulator||Hours||Pepsin||Trypsin||Amylase||Lipase|
|2||36.96a±2.78||19.88a±2.81||32.13a± 4.88||40.53a ±4.80|
abcde Mean values bearing different superscripts within columns differ significantly (P<0.05)
Combined effect of pre-hatch and post hatch heat stress on birds and effect of protein synthesis modulators on enzymatic activity of heat stress exposed birds has not been studied till date. The results of present study depict glutamine, protein synthesis modulator, increases the enzyme activity in birds those are subjected with both pre and post hatch acute heat stress. However, our present study are in harmony with the finding of Hao et al. (2012) and Devi-Priya et al. (2010) who reported that glutamine significantly increases the digestive enzymes activities under post hatch heat stress. Therefore, there is a possibility that the glutamine over-express heat shock protein that enhanced intestinal digestion and absorption function under acute heat stress by deploying positive effects on jejunum enzymatic activity of amylase, lipase, trypsin and pepsin with increased concentrations.
Table 2: Effect of Protein Synthesis Modulator on Jejunal Enzymatic Activity (Units/mg of Protein) in Broilers
|Protein Synthesis Modulator||Pepsin||Trypsin||Amylase||Lipase|
|Control||57.23b ± 1.46||60.43b ± 2.89||52.95b ± 1.82||48.54a ± 1.53|
|Enhancer||71.45c ± 1.34||83.89c ± 2.91||112.86c ± 3.16||92.91b ± 2.25|
|Inhibitor||38.45a ± 0.75||38.68a ± 2.88||34.18a ± 1.37||44.79a ± 1.30|
abc Mean values bearing different superscripts within columns differ significantly (P<0.05)
Results pertaining to the effect of acute heat stress exposure and duration on enzymatic activity are presented in Table 3 and Table 4 respectively.
Table 3: Effect of Duration of Heat Stress Exposure on Jejunal Enzymatic Activity (Units/mg of Protein) in Broilers
|Duration Heat Stress Exposure||Pepsin||Trypsin||Amylase||Lipase|
|0 hour||50.57a ± 1.80||50.54a ± 3.40||72.05 ± 4.16||63.23 ± 3.70|
|2 hours||58.97b ± 2.50||68.07b± 4.59||61.41 ± 3.12||59.55 ± 3.61|
|5 hours||57.63b ± 2.59||66.76b± 4.57||69.33 ± 5.04||63.63 ± 4.10|
|10 hours||55.66ab ± 2.56||58.67ab ± 4.07||65.45 ± 5.10||63.23 ± 3.43|
ab Mean values bearing different superscripts within columns differ significantly (P<0.05)
Heat stress has significantly (P<0.05) increased the activity of trypsin (72.76 ± 2.18) and pepsin (62.36 ± 1.24) in jejunum and simultaneously decreased the amylase activity (57.39 ± 3.18). However, lipase activity did not show any change in response to acute heat stress exposure.
Table 4: Effect of Heat Stress Exposure on Jejunal Enzymatic Activity (Units/mg of Protein) in Broilers
|Unexposed||50.43a ± 1.32||48.23a ± 2.12||70.54b ± 3.24||63.12 ± 2.13|
|Exposed||62.36b ± 1.24||72.76b ± 2.18||57.39a ± 3.18||62.19 ± 2.17|
ab Mean values bearing different superscripts within columns differ significantly (P<0.05)
A significant difference observed (P<0.05) on enzymatic activity of pepsin and trypsin and the activity of amylase and lipase was not significant under different duration of heat stress exposure in respect to control. Reduction in feed intake and compromised digestive functions in broiler chicken during heat stress is generally noticed. One possible reason of change in digestive function may be changes in digestive enzyme activity that why changes in activities of digestive enzymes such as pepsin, trypsin, amylase, lipase etc. of poultry needs to be explored with respect to exposure of heat stress. However; studies focused on the effects of heat stress on the poultry digestive enzyme activity are few at present. In present experiment exposure of heat stress reduce amylase activity and unaffected lipase activity in broiler chicken which reaffirm the previous findings of Routman et al., 2003, however, Osman and Tanios, (1983) found significant increase level of intestinal and pancreatic amylase in broiler chicken during exposure of heat stress. In our study, we have also evaluated the activity of pepsin and trypsin during exposure of heat stress and found significant increment of both enzyme activity in broiler chicken with respect to unexposed group while Routman and co-worker’s found trypsin activity of heat stressed exposed broiler chicken remain unaffected which might be due to different exposed experimental conditions such as heat stress mode, the duration of heat stress, and the feed and water system. We have observed a significant change in the enzyme concentrations of pepsin, and trypsin with the different duration of heat stress exposure. However, further more studies are required for establishing the exact relationship the digestive enzymes activities with respect to both pre and post hatch acute heat stress.
Exposure of acute heat stress during pre and post hatch of boiler chicken leads to elevation in enzymatic activity of pepsin and trypsin, diminution in amylase enzymatic activity and unaltered enzymatic activity of lipase. When protein synthesis modulator glutamine, combine with exposure of both pre and post hatch acute heat stress increases all the four digestive enzymes activity in broiler chicken but not synergistically.