Gastrointestinal development in dairy calves

Gastric enzymes in the dairy calf

The role of gastric enzymes is important for the digestion of the diet of the young ruminant animal due to its undeveloped rumen and reliance on the abomasum and small intestine for nutrient digestion and assimilation. The ability of neonates to digest and utilize high concentrations of milk fat, especially with low concentrations of intestinal lipases, is due to a combination of enzymes called pregastric esterase (Huber et al., 1961). This complex of lipolytic enzymes provides the majority of lipid breakdown within the abomasum, similar to salivary α−amylase in monogastrics. Pregastric esterase is composed of at least six different enzymes secreted from four areas of the glosso-epiglottic area of the mouth, including the vallate papillae region of the tongue, the glossoepiglottic area, the pharyngeal end of the esophagus and the submaxillary salivary gland (Moreau et al., 1988; Ramsey et al., 1956). It is of interest to note that adult ruminants in general are not capable of digesting diets high in fat, yet it is one of the main components in the diet of a neonatal ruminant.

Although the term pregastric esterase is commonly used to describe esterases (defined as enzymes capable of hydrolyzing esters in solution) it also contains lipases, which have a more specific role in digestion (defined as specialized enzymes that hydrolyze fatty acids from water insoluble glycerol esters). Pregastric esterase has become the common term for enzymes of lipolytic or esterolytic nature secreted by mammalian oral tissues (Nelson et al., 1960).

Pregastric esterase is stimulated by nursing in the young calf (Huber et al., 1961; Moreau et al., 1988; Ramsey and Young, 1961a). During clotting of milk that occurs after ingested milk reaches the abomasum in young ruminants, pregastric esterase begins the breakdown of lipids within the casein clot (Bondi, 1987; Hill et al., 1970). Pregastric esterase hydrolyzes about 20% of all milk fat glyceride linkages, mainly short chain fatty acids, indicating its role in lipolysis (Bondi, 1987; Hill et al., 1970; Pitas and Jensen, 1970).

The preference of pregastric esterase for glyceride linkages of butyric acid has been reported to result in 59% of butyrate linkages hydrolyzed in vitro as compared with 7% of higher fatty acids (Ramsey and Young, 1961b). Butyric acid is highly soluble in water and is hydrolyzed faster than longer chained fatty acids, passing quickly from the abomasum to the small intestine. Breakdown of butyric acid and other fatty acids is higher in milk fed orally than milk that is infused into the abomasum, indicating pregastric esterase is required (Otterby et al., 1964a; Otterby et al., 1964b; Russell et al., 1980). Other less soluble fatty acids are trapped by curd particles and slowly migrate to the small intestine (Otterby et al., 1964a; Ramsey and Young, 1961a). Although pregastric esterase plays a primary role in lipolysis in the abomasum, calves fed only through infusion of milk into the abomasum, which decreases stimulation of pregastric esterase secretion, are still able to digest milk fat most likely by pancreatic lipase action (Russell et al., 1980).

Pregastric esterase may also be found in the digesta of the small intestine, which was thought to indicate a role in intestinal digestion of lipids (Otterby et al., 1964b). Later work reported that pregastric esterase has a diminished effect once it enters the duodenum and no effect at all if pancreatic enzymes are not present (Gooden, 1973).

A small part of lipolysis in the abomasum was reported to be caused by a gastric lipase secreted directly from abomasal tissues. Toothill et al. (1976) used abomasal pouch secretions to prevent oral or pancreatic contamination and found no lipolytic digestion, concluding that lipolysis in the abomasum is solely due to pregastric esterase. Other researchers also concluded that gastric lipase is similar to pregastric esterase and must have been mistakenly identified as a separate enzyme (Nelson et al., 1960; Otterby et al., 1964b).

Although most of the enzymes in the alimentary tract of the young calf seem underdeveloped, enzymes that primarily coagulate milk protein are produced in high concentrations. Chymosin, pepsin and hydrochloric acid coagulate milk, retaining casein and fat and allowing nutrients to slowly pass into the small intestine (Cruywagen et al., 1990; Guilloteau et al., 1983; Guilloteau et al., 1984). Chymosin, formerly called rennin, is found in high concentrations in the newborn calf and lamb and decreases with age and at weaning (Cybulski and Andren, 1990; Guilloteau et al., 1983; Guilloteau et al., 1984; Guilloteau et al., 1985). However, calves kept on milk for an extended period of time retain higher chymosin concentrations than weaned calves indicating abomasal enzymes are regulated by development of the rumen as well as high concentrations of lipids and casein in the diet (Cybulski and Andren, 1990; Guilloteau et al., 1985).

The bovine abomasum secretes at least three proteases, including pepsin A, pepsin B (also known as gastricsin or pepsin II), and chymosin, all secreted as proenzymes from the mucous cells of the pyloric and fundic glands and requiring a pH of less than 4 to become active (Cybulski and Andren, 1990). Pepsin A is found in high concentrations at birth and remains constant with increasing age of both calves and lambs (Guilloteau et al., 1983; Guilloteau et al., 1984; Guilloteau et al., 1985), until about 44 days (Huber et al., 1961). After weaning, the ratio of pepsin to chymosin increases the need to digest protein in solid feed rather than casein (Cybulski and Andren, 1990; Guilloteau et al., 1983; Guilloteau et al., 1985).

Pepsin A and chymosin adequately coagulate milk in the abomasum of the young calf, and therefore pepsin B is not found until weaning, when concentrations of chymosin decrease and the pH of the abomasum acquires a broader range due to varied feed (Cybulski and Andren, 1990). The potential for pepsin, trypsin, chymotrypsin and amylase to be secreted from the pancreas increases as the amounts of starch and protein increase in the diet (Garnot et al., 1977; Guilloteau et al., 1985). Therefore these generally increase with age and increasing dry matter intake of grains in the diet.


Pancreatic enzymes

During the first two days of life, high concentrations of abomasal enzymes clot colostrum and allow immunoglobulins to pass into the small intestine. Pancreatic enzymes are found in low concentrations at birth until two days of age in young ruminants, allowing immunoglobulins to remain intact. It is not until after two days of age that concentrations begin to increase until around 42 days of age (Guilloteau et al., 1983; Guilloteau et al., 1984).

Pancreatic amylase is a glycosidic enzyme found in the small intestine, making up 5-6% of total protein in human pancreatic secretions (Lowe, 1994). However, in ruminants, only 2% of total protein in pancreatic secretions is α−amylase indicating a decreased intestinal ability to hydrolyze starch (Keller et al., 1958). Starch is not part of the diet of milkfed ruminants and mature ruminants are able to hydrolyze starch in the rumen. Pancreatic amylase is found at low levels in the newborn and increases with age (Guilloteau et al., 1984; Huber et al., 1961; Le Huërou et al., 1992; Morrill et al., 1970).

Pancreatic fluid also contains two nucleases, deoxyribonuclease I (DNase) and ribonuclease (RNase). Although there is little known about either, RNase is required in weaned calves to recover phosphorus from bacterial RNA (Lowe, 1994).

Unlike other pancreatic enzymes, all peptidases are secreted as zymogens or proenzymes. The main activator of pancreatic peptidases, trypsinogen, is also secreted as a proenzyme and is cleaved by enteropeptidase to the active form of trypsin. Trypsin then cleaves other proenzymes, including chymotrypsinogen, procarboxypeptidase A, procarboxypeptidase B and procolipase, to their active forms of chymotrypsin, carboxypeptidase A and B and colipase (Lowe, 1994). Trypsin amounts secreted in pancreatic juice are low in the newborn ruminant and increase with age during the first two to four weeks in both the lamb and the calf. Chymotrypsin is higher in the young animal than trypsin but the ratio decreases with age (Guilloteau et al., 1983; Guilloteau et al., 1984; Huber et al., 1961).

Pancreatic lipases, such as colipase and phospholipase A2, are low at birth and then increase and remain constant (Gooden, 1973; Guilloteau et al., 1984; Huber et al., 1961; Le Huërou et al., 1992). These lipases are highly active at a pH of 8.5 and specifically hydrolyze triglycerides and phospholipids. Guilloteau et al. (1984) reported that from birth until three weeks of age, the colipase/ lipase ratio is higher than one indicating pancreatic activity is entirely expressed in pancreatic juice in the intestinal lumen. Although calves one to two weeks of age have a diminished capacity to absorb lipids if pancreatic enzymes are removed, there still remains lipolytic activity in the intestinal lumen (Gooden, 1973; Gooden and Lascelles, 1973). This is most likely due to enzymes present in the brush border of intestinal villi.


Brush border enzymes

There are many peptidases found in the microvilli of enterocytes. The main hydrolases found in the brush border are aminopeptidase N, aminopeptidase A, and dipeptidyl peptidase IV (Le Huërou-Luron, 2002). All of these peptidases are designed to cleave specific terminal amino acids from proteins (Palmer, 1995).

Aminopeptidase N hydrolyzes peptides in a stepwise manner up to a certain point at which dipeptidyl peptidase IV finishes the hydrolysis (Le Huërou- Luron, 2002). Aminopeptidase A, aminopeptidase N, and alkaline phosphatase are highest in the calf until two days of age and then decrease until one week of age. They remain constant until weaning, at which time they increase (Le Huërou et al., 1992).

There are four major disaccharidases found in the brush border of the small intestine including maltaseglucoamylase, sucrase-isomaltase, lactase, and trehalase (Le Huërou-Luron, 2002). Sucraseisomaltase is not present in cattle and addition of sucrose to the diet of young ruminants causes an increase in scours (Huber et al., 1961; Le Huërou et al., 1992; Le Huërou-Luron, 2002).

Maltase-glucoamylase hydrolyzes α(1-4) glucosidic bonds whereas isomaltase hydrolyzes α(1- 6) glucosidic bonds (Le Huërou-Luron, 2002). Maltase concentrations are low at birth and increase between 7 and 119 days after birth. Isomaltase is at low concentrations in the newborn and increases after weaning (Le Huërou et al., 1992). Trehalase, one of the four disaccharidases, is able to hydrolyze trehalose to produce free monomeric glucose for absorption (Le Huërou-Luron, 2002).

Lactase is the only enzyme capable of ß(1-4) glucosidase and ß(1-4) galactosidase activity. It is able to break down lactose to glucose and galactose as well as cellobiose to two monomers of glucose (Le Huërou-Luron, 2002). Lactase activity is highest at birth and decreases until about three weeks of age (Huber et al., 1961) or one week of age (Le Huërou et al., 1992) and then remains constant until levels drastically decline at weaning. However, feeding a diet high in lactose does not alter lactase concentrations (Huber et al., 1961).

Although digestive enzyme concentrations are dependent on age of the calf, early researchers reported that digestive enzyme secretions are not affected by diet of the calf (Guilloteau et al., 1983; Guilloteau et al., 1984; Huber et al., 1961; Young et al., 1960). More recent work has found alterations in secretions of digestive enzymes when different sources of protein are used in milk replacers.

Unprocessed soy protein in milk replacers often alters intestinal morphology by decreasing absorptive surface area of intestinal villi as well as concentration of disaccharidases in the brush border (Pederson and Sissons, 1984). Trypsin and chymotrypsin secretions are also inhibited by allergens and anti-trypsin factors in soy protein when ingested by the young calf (Gorrill et al., 1967; Guilloteau et al., 1986; Mir et al., 1991). Soy protein may be included in milk replacer fed to calves older than 20 days of age in low concentrations but must be processed to decrease concentrations of potential allergens as well as increase protein digestibility (Mir et al., 1991).

Digestibility of milk replacer is affected by other additions, such as using high concentrations of starch as alternative energy sources. Incorporating starch into milk replacer for preruminants is not recommended due to low concentrations of pancreatic amylase present in the intestine. Starch contains 17- 30% amylose and 70-83% amylopectin. Amylose consists of glucose units arranged in a linear polymer bound with α(1,4) glycosidic linkages and amylopectin is bound by α(1,6) branch points along the α(1,4) linked glucose polymer. These bonds must be broken before absorption can occur and the low concentrations of pancreatic amylase present do not hydrolyze enough bonds to provide adequate energy for the calf (Morrill et al., 1970).

However, once the calf is older and the rumen is more developed, starch becomes a primary part of the diet. Development of the rumen including formation and elongation of rumen papillae is critical in transforming the monogastric calf to the ruminant dairy heifer (Church, 1988). More economical diets and reduced labor required for feeding are the driving forces to demand this change as early as possible.

The primary limiting factor for weaning dairy calves from milk or milk replacer is having adequate development of the rumen in order to maintain postweaning growth at desired rates.

Rumen development is stimulated by the presence of microbial produced volatile fatty acids, primarily butyric acid (Beharka et al., 1998; McLeod and Baldwin, 2000). Volatile fatty acids introduced into the rumen as purified sodium salts, especially sodium butyrate, increase epithelial development (Tamate et al., 1962). Butyric acid provides energy for the rumen wall for maintenance and development. This development includes the formation of papillae and thickening of the rumen wall, including increased capillary development (Weigand et al., 1975). Any additional butyric acid that is not needed as energy for the rumen is transported into the blood stream as β-hydroxybutyrate. Around 90% of butyrate produced in the rumen is directly absorbed (Bergman, 1990; Weigand et al., 1975). Rumen wall growth and capillary development are needed to increase the ability of this organ to absorb rumen produced energy substrates. Since the microbial end products of starch are propionic and butyric acids, rumen development is most influenced by ingestion of grain and the digestion of the starch components in the grain. When starch is broken down in the rumen, volatile fatty acids (VFAs) are produced. (Greenwood et al., 1997; Krehbiel et al., 1992; Nocek et al., 1984; Weigand et al., 1975).

In the young pre-ruminant calf, the ability to begin lowering the pH helps provide an environment for the continuation of a normal rumen bacterial and protozoan population. Any way to augment the process of the digestion of starch may allow for faster breakdown of starch by bacteria into propionic acid for absorption as energy and butyric acid to stimulate development of the rumen. Addition of exogenous α-amylase may be one way to increase starch digestion in the ruminant animal. α-Amylase hydrolyzes α(1,4) glycosidic linkages but is unable to hydrolyze α(1,6) bonds in the branch points.

Because of this, α-amylase only begins starch breakdown and the rest is continued through microbial digestion. Thus, it was of interest to determine whether the addition of two levels of amylase (Amaize^TM, Alltech Inc.) supplied in calf starter could have an effect on rumen development by increasing the initial rate of starch digestion in the developing rumen.


A study of the effects of added amylase in Amaize^TM

Recently Gehman et al. (2003) utilized fifteen Holstein bull calves to study effects of calf starter supplemented with 0, 6, and 12 g/h/d of amylase as supplied by Amaize^TM. Calves were assigned to a treatment group on arrival with a total of five calves per treatment group. Calves were fed milk replacer (Land O’Lakes 20% all-milk protein, 20% fat product), at a rate of 10% of arrival body weight twice per day for the duration of the trial. Calves were fed calf starter (18% CP, Purina Mills, Inc.) and water once a day on an ad libitum basis. Grain was fed in the morning after milk feeding and was done in such a way that the first 200 g/d of grain contained the treatment dosage (6 or 12 g/d) of Amaize^TM. After calves ate this amount each day they were offered ad libitum grain for the remainder of the day. This was done so that calves were receiving the treatment at the desired rate of daily intake at the earliest possible age. Fecal and health scores were monitored daily. Calves were euthanized at the end of 5 weeks (35 days) of age. The reticulorumens were sampled in nine areas for papillae analysis as shown in Figure 1.

Samples were taken from the rumen from the following regions: the caudal portion of the caudal ventral blind sac (A), the right and left caudal dorsal blind sac (RB and LB), the right and left cranial dorsal blind sac (RC and LC), the right and left cranial ventral blind sac (RD and LD), and the right and left caudal ventral blind sac (RE and LE). The rumen papillae were measured according to length, width, papillae per cm2, and rumen wall thickness (Lesmeister et al., 2002). All 15 calves arrived healthy, there was no mortality, and no calves were treated for clinical disease during the study period.

The primary measure of interest of this study was rumen development. All areas of the rumen were sampled and measured as shown in Figure 1. Tables 1, 2, and 3 along with their accompanying Figures 2, 3, and 4, show the rumen papillae length, width, and density. These measurements are primary aspects of the rumen that are related to the tissue surface area available for volatile fatty acid absorption. Each area grows at a different rate and is quite different in size, even in adult ruminants. Therefore the sampling pattern as shown was used to quantify the entire reticulorumen.

Papillae length had five measured areas where the 6 g/hd/d treatment was significantly greater (P<0.10) than either the control (0 g/hd/d) or the 12 g/hd/d treatments. In 6 of 9 areas papillae width was significantly greater (P<0.05) in the 6 g/hd/d group than the control and in 4 areas was greater than the 12 g/hd/d group. In no cases was the 12 g/hd/d group greater than controls. In papillae per cm2, there were three areas where the 6 gm/hd/d group were larger (P<0.05) than the controls and two areas where the 12 g/hd/d were greater than controls along with two areas where the 12 g/hd/d group were greater than the 6 g/hd/d group. Rumen wall thickness was not affected by treatment. These data show that the amylase affected rumen growth and increased parameters related to nutrient absorption. The lack of difference in rumen wall thickness is not surprising as this is quite consistent across all areas of the rumen.

Grain intake was similar across all treatment groups and was quite typical of calves in summer temperatures and appeared adequate to support weaning of the calves by the end of the experiment.

All animal growth measurements taken including heart girth, body weight, hip width, and withers height were similar for all treatments. These appeared normal for calves of this age and feeding regime with moderate levels of milk replacer and grain intake.




Figure 1.Rumen sampling method: A = caudal portion of caudal ventral blind sac, RB and LB = right and left caudal dorsal blind sac, RC and LC = right and left cranial dorsal blind sac, RD and LD = right and left cranial ventral sac, RE and LE = right and left caudal ventral blind sac.



Table 1. Least squares means of papillae length (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.



¹Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas.



Table 2.Least squares means of papillae width (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.



¹Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas.



Table 3. Least squares means of papillae per cm2 for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.



¹Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas.





Figure 2.Least squares means of papillae length (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of Amaize^TM in grain.





Figure 3.Least squares means of papillae width (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of Amaize^TM in grain.





Figure 4.Least squares means of number of papillae per cm2 for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of Amaize^TM in grain.



Figures 2-4.Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas. Within each rumen area, bars with different letters differ at the P < 0.10 level.



Table 4.Least squares means of rumen wall thickness (cm) for 9 areas of the rumen in calves fed 0, 6, or 12 g/d of amylase in grain.



¹Rumen areas sampled: RB = right caudal dorsal blind sac, LB = left caudal dorsal blind sac, RC = right cranial dorsal blind sac, LC = left cranial dorsal blind sac, RD = right cranial ventral blind sac, LD = left cranial ventral blind sac, RE = right caudal ventral blind sac, LE = left caudal ventral blind sac, A = caudal portion of caudal ventral blind sac. Figure 1 shows location of these areas.



In summary, the enzyme systems of the neonatal dairy calf are quite complex, yet well suited for an animal that begins life as a monogastric and is transformed to a ruminant. The development of the rumen is paramount in importance from a modernday management standpoint where economics and labor management are limiting factors on progressive dairy farms.


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