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Chapter 3 Enzyme activity in the small intestine of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet
Introduction
The growth of poultry during the first week of life has been studied quite intensively (Mitchell & Smith, 1991; Dunnington & Siegel, 1995; Noy & Sklan, 1995; Iji et al., 2001a & b; Noy et al., 2001), as this particular period represents a large proportion of the life span of meat-type poultry. Lilja (1983) suggested that the ultimate growth of poultry can be directly correlated to the development of specifically the gastrointestinal and cardiovascular systems. Birds must be able to adapt to exogenous diets rapidly, in order to achieve their genetic growth potential (Dibner, 2000). Early growth rate may be influenced by various factors, including yolk sac residue, levels of pancreatic and intestinal enzymes, surface area of the gastrointestinal tract, nutrient digestibility and absorption, and quality of feed (Dibner, 2000). Uni (2003) suggested that poultry chicks undergo rapid development, both physical and functional, during the first week post-hatching and that this development of the gastrointestinal tract gives the chicks the capacity to digest feed and assimilate nutrients.
It is often noted that ostrich chick mortalities occur around 2-3 weeks post-hatching, due to chronic starvation (Verwoerd et al., 1997). The authors ascribe this situation to environmental, social or nutritional stress to which the ostrich chicks are exposed in the first week post-hatching. Survivors are also often left with low growth rate, inefficient feed utilisation and reduced resistance to diseases (Uni, 2003).
Noy & Sklan (1999) suggested that pancreatic and brush border enzymes have to be available in sufficient quantities for effective digestion and absorption to occur in chickens. They reported, however, that quantitative determination of digestive enzymes in the intestines of poultry immediately post-hatching was not carried out at that time (Noy & Sklan, 1999). Iji et al. (2001) conducted a study to determine the development and characteristics of certain intestinal enzymes in broiler chicks on a commercial starter diet. They found that there was a general reduction in the specific activities of intestinal enzymes as broiler chicks aged, but that the increased length and surface area of the gastrointestinal tract compensate for this reduction per unit of mucosal surface. The authors suggested that enzyme activities differ between different intestinal sites and that this may also have an influence on overall digestion (Iji et al., 2001).
In 2003 Iji et al. conducted a study on ostrich chicks at intervals of 3, 27, 41, 55 and 72 days of age intervals to test the development of enzyme activity. Based on these findings, the authors suggested evaluation of enzyme activity at closer intervals, particularly during the early stages, to obtain a clearer picture of enzyme development when changes are profound in other avian species. These changes are usually observed within the first two weeks post-hatching (Uni et al., 1998; Uni et al., 1999).
For this trial 49 ostrich chicks were slaughtered over a sixteen day period and the activity of certain enzymes was determined in the small intestine to establish the effect of growth on enzyme activity immediately post-hatching on a pre-starter broiler diet. The project had ethical approval from the Onderstepoort Animal Use and Care Committee (Protocol 36-5-623).
Materials and Methods
Animals
Forty nine chicks were slaughtered every second day for sixteen days, starting from two-day old chicks. Fifty nine ostrich chicks were originally obtained from the Oudtshoorn Experimental Farm of the Department of Agriculture Western Cape, South Africa, where they were hatched and weighed. The chicks were transported by air to the Faculty of Veterinary Science of the University of Pretoria, Onderstepoort, South Africa, for slaughtering. The average weight of the day-old chicks was 839.31 g.
Chicks were kept in a clean, disinfected room which was kept as cool and dark as possible. Noise and human contact were limited to the absolute minimum to restrict stress. Chicks were provided with clean drinking water and a poultry pre-starter diet. The minimum specifics for the commercial pre-starter diet were: CP = 24.5%; Moisture = 11.5%; Energy = 12 MJ/kg feed; Fat = 6.7%; Fibre = 3.6%; Ash = 6.2%. The average hatching and slaughter weights of the chicks are presented in Table 1.
Collection of Material
Chicks were euthanized with CO2 in a closed container. The digestive tracts were immediately removed from the carcasses after death. Each section of the digestive tract was identified. Samples of the wall of the small intestine were taken from the duodenum (ascending or distal limb of the duodenal loop, just before the secondary distal loop); jejenum (before the vitelline diverticulum, where the yolk sac stalk entered the jejunum) and ileum (approximately 5 cm before the ileo-ceacal junction). The samples were approximately 2 cm in length and weighed between 0.11 and 2.01 g. These samples were used for enzyme analyses.
Tissue samples were rinsed with ice-cold saline (0.9% NaCl), using a syringe to flush out any intestinal content. The samples were then cut open lengthwise and placed on ice with the luminal surface facing away from the surface of the ice. An 18 gauge (18G) needle was attached to a syringe and the tissue surface was thoroughly rinsed, taking special care not to damage the mucosal layer of the wall. The tissue samples were wrapped in pre-marked pieces of aluminium foil and stored in liquid nitrogen, in a thermos flask. When all samples had been collected, the tissue samples were transferred to plastic bags and stored in a freezer at approximately minus 85°C.
Measurements and Calculations
Brush-border membrane vesicles were isolated from the intestinal tissues according to the method described by Shirazi-Beechey et al. (1991) with the following additions: A crushed ice-bed of about 5 cm thick was prepared in a flat container (24 cm by 30 cm). All instruments and buffer were kept on cold crushed ice. All the steps of the procedure were completed on the ice bed. Samples (eight per day) were taken from the freezer and placed on the ice-bed to prevent thawing. Each sample was quickly weighed, cut into small pieces, with a pair of sharp scissors while still frozen, and immediately transferred to pre-marked (for identification) conical plastic tubes and placed in the ice-bed in an upright position. Depending on the weight of the tissue sample, the appropriate amount of pre-cooled Buffer A (Table 2) was added to each tissue sample (< 1-1 g tissue weight, use 10 ml of Buffer A; >1-1.5 g use 20-25 ml). The tubes were sealed with parafilm to prevent any spillage. Each sample was vibromixed with a Vortex tube mixer (Heidolph REAX top) at maximum speed for 60 seconds and filtered through a 70 mm diameter Buchner funnel, where after each sample was homogenized individually for 30 seconds with an ULTRA-Turrax T25 homogenizer at 13 500 rpm. To prevent contamination, the homogenizer dispersing head was spun in clean distilled water and wiped dry after each sample. Immediately after mixing, the homogenate was divided (± 600 µl each), into three 1.5 ml Eppendorf tubes, recapped firmly and kept on the ice-bed. After preparation of all the samples, they were placed in a freezer at -85°C until they could be analysed for alkaline phosphatase and protein content.
The remaining homogenate was weighed (to the nearest 0.5 g) into centrifuge tubes. The weights of the samples to be placed opposite each other in the centrifuge were balanced by adding cold Buffer A (Table 2). This dilution was carefully noted. Where 10 ml of Buffer A (Table 2) was added at the start of the procedure (corresponding with a ± 1 g cut sample), 50 µl of MgCl2.7H2O, from a 2.5 M Stock Solution (50.825 g/100 ml) was added to each sample. Accordingly, 100 µl of MgCl2.7H2O was added if the sample volume was between 20 ml and 25 ml. The exact amount added was taken into account during enzyme activity calculations. After the MgCl2.7H2O was added, each sample was vibromixed briskly and allowed to stand for 20 minutes on the ice-bed to aid subsequent sedimentation during centrifugation. Suspensions were centrifuged at high speed, using a Du Pont Refrigerated Sorvall Ultra-Centrifuge RC 6, Rotor SS-34, at 30 000 g (5 000 r.p.m.) and with the temperature set at 6°C for 15 minutes. The supernatant was then transferred to clean pre-marked tubes, discarding the pellets this time, and again centrifuged at 30 000 g (14 500 r.p.m.), at 6°C for 30 min. The supernatant was removed and the pellets re-suspended in cold Buffer B (Table 2). Homogenization was performed at this stage in the procedure, using a 18G needle. Samples were centrifuged for the third time at 30 000 g (14 500 r.p.m.) at 6°C for 45 minutes. The final pellets were re-suspended in 500 ml of cold Buffer C (Table 2) and passed through a 25G needle to obtain a homogenous suspension. The suspension was then divided and transferred to four pre-marked 1.5 ml Eppendorf tubes kept on ice. These homogenized tissue samples were also stored at -85°C for later analyses for chymotrypsin, trypsin, amylase and lipase activity.
Samples were analysed for alkaline phosphatase according to the method described by Forstner et al. (1968) and Holdsworth (1970). Protein was determined according to the method described by Bradford (1976). Chymotrypsin amidase and trypsin amidase were determined according to the method described by Servière-Zaragoza et al. (1997).
Amylase content was determined using the ACE™ Amylase Reagent (Reagent number AE2-5) intended for the quantitative determination of alpha (α) amylase activity in serum using the ACE™ clinical chemistry system. The method uses a modified p-nitrophenyl-maltoheptaoside as substrate. A multifunctional glucosidase cleaves the amylase reaction products and releases the p-nitrophenol. The terminal glucose of the substrate is chemically blocked preventing cleavage by the indicator enzyme. The rate of release of pNP is monitored at 408nm and is proportional to the α-amylase activity in the sample.
Lipase content was determined using the ACE™ Lipase Reagent (Reagents 1 and 2, Catalog number 11821792), intended for the quantitative determination of lipase activity in serum using the ACE™ clinical chemistry system. This chosen method uses a 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin) ester as substrate. The chromogenic lipase substrate 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin) ester is cleaved by the catalytic action of alkaline lipase solution to form 1,2-O-dilauryl-rac- glycerol and an unstable intermediate glutaric acid-(6-methylresorufin) ester. This decomposes spontaneously in alkaline solution to form glutaric acid and methylresorufin. The colour intensity of the red dye formed is directly proportional to the lipase activity and was determined photometrically.
Enzyme activity can either be expressed as mole substrate hydrolysed/mg protein/min (Shirazi-Beechey et al., 1991; Iji et al., 2001) or as Units/mg protein (Pletschke et al., 1995; Servière-Zaragoza et al., 1997). The final enzyme activity was expressed as mmole product/min/mg protein.
Statistics
The data were analysed according to a standard eight (age) x three (region of small intestine) factorial analysis (Snedecor & Cochran, 1980) for protein of the brush border membrane and the enzymes alkaline phosphatase, trypsin and chymotrypsin. For the enzymes amylase and lipase a standard eight x two (region of small intestine) factorial analysis was done, as activity of these two enzymes was only tested in the duodenum and ileum. Repeated records from the same experimental units assessed for different parts of the small intestines were accounted for by adding the random effect of animal in the mixed model analysis (Harvey, 1990). Although the interaction between age and region of the small intestine was not significant (P>0.05) in all analyses, these interactions are provided to depict a clear picture of the results.
Results and Discussion
Protein
The protein content of the brush-border membrane (BBM) was higher (P<0.05) in the duodenum and jejunum than in the ileum, at two and four days of age. Thereafter there was no significant differences (P<0.05) between the protein content of the BBM of the different parts of the small intestine up until 16 days of age (Figure 1). Iji et al. (2001a) reported that the protein content in broiler chicks was the same in all the regions on the first day post-hatching, but that it was higher thereafter in the jejunum than in the other two regions. In a study on ostrich chicks Iji et al. (2003) observed that the protein content of the BBM of the jejunum and ileum was higher than that observed in the duodenum on three and 27 days of age. This was attributed to the growth of microvilli in these regions (Iji et al., 2003). The protein reported of the BBM for this trial, was also much higher than the protein content reported by Iji et al. (2003). The protein content in the BBM of the duodenum on two days of age, for example, was 1.97 mg/g tissue, compared to 0.18 mg/g tissue reported by Iji et al. (2003) for three days of age. The ostrich chicks in the trial conducted by Iji et al. (2003) were fed a starter diet between 3 and 27 days of age, which was formulated with an ostrich feed database (CP = 17.94%; Energy = 16.2 MJ/kg feed; fat = 4.19%; fibre = 8.08%). The differences in the protein content could be due to nutritional differences within the diets of the two trials, different ages of chicks, different sites at which enzyme activities were measured and the way analyses were conducted.
Acknowledgements
Abstract
Chapter 1: General introduction
Hypotheses
References
Part 1: Yolk utilisation and the development of the small intestine of ostrich chicks
Chapter 2: The composition of egg yolk absorbed by starved ostrich (Struthio camelus L.) chicks from one to seven days post-hatching and for ostrich (Struthio camelus L.) chicks from one to sixteen days posthatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 3: Enzyme activity in the small intestine of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 4: A histological and morphometric study of the small intestine of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Part 2: Influence of various pre-starter diets on growth and the development of the small intestine of ostrich chicks
Chapter 5: A growth and digestibility study of ostrich (Struthio camelus L.) chicks on eight different prestarter diets
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 6: Enzyme activity in the small intestine of ostrich (Struthio camelus L.) chicks on eight different pre-starter diets
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Chapter 7: A histological and morphometric study of the intestinal tract of ostrich (Struthio camelus L.) chicks on eight different pre-starter diets
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Chapter 8: General conclusion and future perspective
Recommendations
References
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