Intestinal and Pancreas Enzyme Activity of Broilers Exposed to Thermal Stress

Thirty-two Hubbard broilers were raised under commercial husbandry to evaluate the effect of temperature exposure (26 and 35oC) on the activity of digestive enzymes at different ages (23 and 46 days) and different diet energy levels (2,900 and 3,200 kcal ME/kg). Data were analyzed in a 2x2x2 factorial arrangement (energy/temperature/age) in a completely randomized design. Animals were slaughtered after four hours of heat exposure for sampling of an intestinal segment, to extract alkaline phosphatase, and pancreas, to extract amylase, lipase and trypsin. The activity of intestinal alkaline phosphatase was influenced by energy level and animal age. Energy level of diet and environmental temperature interfered on the activity of pancreatic amylase. Lipase activity was affected only by broiler age. There was no effect of treatments on pancreatic trypsin activity. It was concluded that the activity of the pancreatic enzymes can be regulated by different mechanisms under stress conditions induced by heat.


INTRODUCTION
At high temperatures, birds tend to lose appetite; and feed intake, growth rate, muscle and egg production are reduced (Dale & Fuller, 1980;Cowans & Michie, 1980;Temim et al. 1999Temim et al. , 2000;;Geraert et al., 1996) and visceral fat increases in relation to carcass weight (Temim et al., 2000;Geraert et al., 1996).Feed intake and utilization are closely related to digestion and absorption, which are in turn affected by environmental temperature changes (Owen & Wiggs, 1971;Kitchin & Morris, 1971;Temim et al., 2000;Bonnet et al., 1997;Itoh et al., 2001).According to Bonnett et al. (1997), when environmental temperature increased from 22ºC to 32ºC there was a decrease in feed efficiency from 0.485 to 0.340 in poultry, indicating that the functional digestive tract adaptation could be related to compensatory adjustment on production rate and enzymatic contents of digestive secretions (Osman & Tanios, 1983).However, both the synthesis and the levels of pancreatic amylase, trypsin and lipase were shown to change due to diet modifications, induced by the presence of their respective substrates in gastrointestinal lumen (Brannon, 1990).Nevertheless, very few studies were related to putative thermal stress effects on the activity of digestive enzymes of poultry.The aim of this work was to evaluate alterations in the activity of intestinal alkaline phosphatase, pancreatic trypsin, amylase and lipase in birds fed diets containing two energy levels and submitted to thermal stress (35ºC/4h).

MATERIAL AND METHODS
The trial was conducted using 32 male Hubbard broilers, raised according to standard commercial husbandry techniques.Enzyme activity means were analyzed in a completely randomized design and a factorial scheme 2x2x2 (energy level x thermal stress x age).
Two corn and soybean-based diets were fed ad libitum, containing energy levels of 2,900 and 3,200 kcal ME/kg.Starter (0-28 days) and finisher (29-42 days) diets were formulated according to the appropriate nutritional requirements for birds in each phase and composition of diets is shown in Table 1.
At 23 and 46 days, four animals per treatment were submitted to thermal stress in a thermostatically controlled climate chamber (adjusted to keep environmental temperature at 35°C and a relative air humidity of 60%), during four hours, without food and water.Control animals were kept in cages at room temperature of 26ºC, without food and water during the same period of time.
Immediately after heat stress, control and stressed animals were slaughtered and pancreas and intestine (duodenum, jejunum and ileum) were collected.Tissues were immediately frozen in liquid nitrogen and stored at -70ºC freezer for further analysis.
Pancreas was homogenized with an Ultra-Turrax in 500 mM Tris-HCl buffer (1:20 w/v) containing 50 mM CaCl 2 , pH 8.0, at 4 °C.The homogenate was centrifuged at 14,000 x g for 30 min, at 4ºC.An aliquot of the supernatant was used for immediate lipase determination and the remaining supernatant was frozen in liquid nitrogen and stored at -70 °C freezer until the activity of the other enzymes was determined.
Pancreas α-amylase activity was measured by hydrolysis of starch followed by determination of the amount of maltose produced, in accordance to Bernfeld (1955).One unit of enzyme activity was defined and expressed as the amount of enzyme that produced one µmol of maltose/min, at 37°C.Pancreas lipase activity was assessed by titration (Tietz & Fiereck, 1966), using as substrate the olive oil emulsion (SIGMA ® ) and the colipase excess extracted from poultry pancreas.One unit of enzymatic activity was defined and expressed as the quantity of enzyme that released one µmol of fatty acid per minute.
Activation of pancreas trypsinogen was accomplished by a pre-incubation period with 0.08 units of Enterokinase (SIGMA ® ) for 30 minutes.After the activation, trypsin activity was determined at 37°C in accordance to Kakade et al. (1974) using N-α-benzoyl-L-arginine-p-nitroanilide (L-BApNA, SIGMA ® ) as substrate.One unit of enzyme activity was defined and expressed as the amount of enzyme that released one mmol of p-nitroanilide/min, at 37°C.

RESULTS AND DISCUSSION
There was no significant interaction among the factors (energy, thermal stress and age) on the activity of the pancreas enzymes amylase, lipase and trypsin and the intestinal enzyme phosphatase alkaline.As shown in Table 2, pancreas amylase activity was affected (p<0.05) by energy level of the diet and by thermal stress.Stressed birds had a higher enzymatic activity than control birds.
Details about chemical and physiological mechanisms involved in the regulation of pancreatic amylase level under normal or thermal stress conditions are still unknown.Thus, Slaughter et al. (2001) studied amylase activity in order to determine the kinetics and catalytic efficiency in relation to starch from different feedstuffs, and to establish how the enzymatic levels in gastrointestinal tract are related with glucose and insulin levels in blood.According to these authors, high levels of amylase in the gastrointestinal tract indicate that the enzymatic hydrolysis of starch could be the limiting factor in carbohydrate digestion, mainly due to factors related to its physical and chemical properties.On the other hand, Osman & Tanios (1983) reported that thermal stress causes a consistent increase in amylase activity in broilers, which could result in a significant increase in starch hydrolysis and, consequently, in the supply of glucose for internal oxidation and posterior metabolic heat production.
Thus, high pancreatic amylase activity indicates that pancreas plays an important role in the regulation of the levels of intestinal amylase and glucose supplied to achieve energetic requirements under heat stress conditions.Furthermore, higher pancreatic amylase activity in response to an increase in environmental temperature suggests that the levels of pancreatic amylase could be regulated by a complex interaction among paracrine and endocrine pancreatic secretions and neuroendocrine stimuli that could explain the abdominal fat deposition in animals exposed to chronic heat stress.Pinchasov et al. (1990) studied digestive enzymes in birds aged 14 to 83 days and fed ad libitum or intermittently.Pancreas amylase levels were reported to increase with age.These authors did not find an age effect on lipase and trypsin activities when these enzymes were expressed in units/g pancreas.Nevertheless, when the results were expressed in units/g body weight, a difference was seen between 20 and 33 days of age in the birds fed intermittently.
According to Table 2, different treatments (diet energy level, heat stress and age) did not result in different trypsin activities.Similar results were observed by Pinchasov et al. (1990) for trypsin activity in poultry from 14 to 83 days of age.
In relation to lipase activity, the results suggested that energy levels of 2,900 and 3,200kcal ME/kg were not enough to detect effects of added fat in diet.It has been suggested that the amount and type of triglycerides in the diet regulate pancreatic lipase activity but the mechanism is still unclear (Ricketts & Brannon, 1994).Other studies have shown that different fat levels added to the diet rapidly resulted in adaptive changes that reached a plateau in five days (Deschodt-Lanckman et al., 1971).
Alkaline phosphatase activity was affected by diet Considering that alkaline phosphatase activity could be modified by several factors (Komada et al., 1989), the results obtained in the current work suggest that alkaline phosphatase activity extracted from broiler gut is influenced by dietary energy level.In rats, contrasting results were found; alkaline phosphatase activity increased immediately after the rats were fed a meal rich in fat (Alpers et al., 1995).Thus, it seems reasonable to accept that these differences were originated by species characteristics, tissues and/or different methods used to prepare the enzymatic substrate, besides other unknown factors.

CONCLUSIONS
Pancreas amylase activity was affected by dietary energy level and thermal stress conditioning.There was no difference among treatments on pancreatic trypsin and lipase activity.Dietary energy level and animal age affected intestinal phosphatase alkaline activity and there was no effect of thermal stress on this enzyme.
The findings of this study suggest that the activity of pancreas amylase, lipase and trypsin, in animals under thermal stress, could be regulated by different mechanisms.However, it is still unclear if these changes are essential for poultry to better use carbohydrates, lipids and proteins under thermal stress conditions.

Table 2 -
Effect of thermal stress and dietary energy level on intestinal alkaline phosphatase (AP), pancreatic lipase (L), trypsin (T) and amylase (A) extracted from poultry.