A note on distribution of lipolytic activity in the digestive tract of veal calves

Six Holstein calves, 3 to 4 weeks of age were fed a milk replacer and a starter concentrate. Calves were slaughtered after 105 days, without fasting.


INTRODUCTION
Fat covers a major part of the calf energy requirement, thus digestion of lipids in preruminant calves deserves steady attention.Fat digestibility in young calves is generally high but may be decreased if inexpensive vegetable fats were added to 167 MAROUNEK M. ET AL. skimmed milk to replace the milk fat in the diet.Digestibility of fat depends on its fatty acid profi le, being lower in fats containing saturated long-chain fatty acids, and high in fats with unsaturated and/or short-chain fatty acids (Roy and Stobo, 1975).Fatty acids are released from their ester bonds by lipases, which represent a large family of hydrolases, ubiquitous throughout living organisms.A comprehensive review on lipid digestion in young ruminants was published by Noble (1980).In calves previous studies worth attention were concerned with lipase present in saliva (Grosskopf, 1965;Edwards-Webb andThompson, 1977, 1978;Toothill and Thompson, 1977), abomasum (Guilloteau et al., 1983(Guilloteau et al., , 1985)), and pancreatic juice (Edwards-Webb and Thompson, 1977;Toothill and Thompson, 1977;Guilloteau et al., 1983Guilloteau et al., , 1985)).Villeneuve et al. (1996) investigated pregastric lipases secreted by the lingual and pharyngeal tissues of young ruminants.Šimůnek et al. (1995) published a paper on ontogenesis of hydrolytic activities in the gastrointestinal tract of young goats.Activities of lipases were very variable and rather low in almost all samples examined.We are not aware of a comparative study dealing with distribution of lipolytic activity in individual segments of the digestive tract of veal calves.Thus, we measured the lipolytic activity in samples of digesta taken from the rumen, abomasum, small intestine and large intestine of 4 months-old calves fed a milk replacer and a starter concentrate.The effect of pH on the lipolytic activity was also investigated.

Animals and diets
Six Holstein bulls, 3 to 4 weeks of age at the start of the experiment, were fed a milk replacer Telasan V (Bodit Tachov, Czech Republic) and a starter concentrate Telstar (Zea Sedmihorky, Czech Republic).The composition of feeds is presented in Table 1.Calves received the milk replacer twice a day at 0.4 kg in 3 l of water.Starter was available ad libitum and its consumption was measured.Animals were slaughtered after 105 days.Feed was available up to the slaughter.

Sampling
The abdominal cavity was immediately opened, the digestive tract was applied to separate rumen, abomasum, small intestine and large intestine (including the caecum).The contents of the rumen, abomasum, the fi rst sixth of the small intestine (presumably the duodenum), the remaining part of the small intestine (presumably the jejunum and ileum), and the large intestine were removed, weighed and pH was measured.Digesta samples were taken and stored at -70°C until analysis.

Lipolytic activity assay
Three methods of lipase activity measurement were compared.A method based on determination of oleic acid released from emulsifi ed 4.5 mM triolein (Fluka, cat.no.62314) was not suitable for measurement of low lipolytic activities in the rumen and abomasal contents.Lipase activity measurement using the refl ectometer RQfl ex plus ® (Merck KGaA, local supplier Merck Ltd., Říčany, Czech Republic) was rapid, but results were given in relative units only.Thus, the lipolytic activity was measured by the modifi ed method of Bier (1955).Samples of digesta (1 g) were incubated with 0.2 M citrate/0.4M phosphate buffer (9 ml) and emulsifi ed tributyrin (10 ml) at pH corresponding to the respective parts of the digestive tract.The emulsion was prepared by sonication from tributyrin (10 ml), Tween 80 (1 ml) and water (100 ml).The reaction mixture was incubated (37°C, 1 h) and then centrifugated.Liberated butyrate was determined by titration, after steam distillation in the Markham apparatus.Specifi c activity of lipases was expressed as mmol butyrate liberated per 1 g of digesta in 1 h.Total activity was calculated as the product of specifi c activity and weight of digesta in individual segments.It was assumed that duodenal content represented 15% of the small intestinal digesta weight.To assess the effect of pH on lipolytic activity, mixed samples were prepared and pH of the reaction mixture was adjusted to a desirable value (4.0 to 6.5, 2.2 to 4.5, 5.0 to 10.5, and 5.0 to 9.0 for rumen, abomasal, small intestinal and large intestinal contents, respectively).

RESULTS AND DISCUSSION
Calves consumed milk replacer and starter concentrate at 84 and 202±34 kg, respectively, and increased the average weight from 45.4 to 167.0 kg in the course of the experiment.2.03 ± 0.87 0.25 ± 0.33 0.61 ± 0.24 0.93 ± 0.46 0.48 ± 0.33 mean values ± SD 1 expressed as mmol butyrate liberated from tributyrin/h per g digesta 2 expressed as mol butyrate liberated from tributyrin/h per segment total lipolytic activity in segments of the digestive tract.Specifi c lipolytic activity (per g digesta) was higher in the duodenum than in other digestive segments.The rumen, however, thank to its greater volume contained almost one half of the total lipolytic activity of the whole digestive tract.The data in Table 2 illustrate a high potential role of the rumen in hydrolysis of dietary lipids.However, during drinking the rumen is by-passed due to the refl ex of oesophagal groove closure (Guilhermet et al., 1976).Thus, it is obvious that the role of the rumen in fat digestion was limited to lipids present in the solid feed.The lowest specifi c lipolytic activity was that of abomasal contents, in spite of the fact that tributyrin is the ideal substrate for lipases which hydrolyse fat in the abomasums of milk-fed ruminants (Noble, 1980).
The pH optima of lipases in individual digestive segments were greatly different (Figure 1).The highest activity of lipases towards tributyrin in pooled samples of digesta from the rumen, abomasum, duodenum and jejunum/ileum was observed at pH of 5.5, 2.5, 8.5 and 9.0, respectively.The rate of hydrolysis of tributyrin in the rumen contents of grazing cows reached a maximum at pH 7.0 (Faruque et al., 1974), which is a value higher than pH 5.5 observed in the present experiment.On Figure 1.pH-activity curves for the lipolytic activity in sections of the digestive tract of veal calves the other hand, the pH optimum for hydrolysis of tributyrin in the small intestine (9.0) agrees with the pH optimum reported by Shahani et al. (1976) for bovine pancreatic lipase (8.8).There was no clear pH optimum for hydrolysis of tributyrin by the caecal/ colonic contents.The pH optima of lipases in the rumen and abomasum corresponded to digesta pH in these organs.Lipids in the small intestine, however, were hydrolysed at pH values which were about two pH units below the optimal level.In spite of it, the small intestinal lipolytic activity seems to be high enough to hydrolyse dietary fat completely.Consequently, the role of the large intestine may be limited to hydrolysis of lipids of desquamated epithelial and lysed bacterial cells.As various microbial lipases participate in this process, no distinct pH optimum was obvious.
It can be concluded that lipolytic enzymes are present in all sections of the calf digestive tract, but individual digestive segments differ substantially in specifi c and total lipolytic activity, and in nutritional signifi cance of these activities for the animal.

Table 1 .
Composition of feeds of calves, g/kg 2Telstar contained cereals, cereal by-products, oilseed cake, by-products of the sugar industry, antioxidant, vitamin and mineral supplements Table 2 presents weights of digesta, pH, specifi c and

Table 2 .
Weight of digesta, pH, specifi c and total lipolytic activity in segments of the digestive tract of veal calves fed milk replacer and starter concentrate