Effects of long-term postgastric infusion of casein or glutamic acid on small intestinal starch digestion and energy balance in cattle

Abstract

The objective of this experiment was to evaluate effects of postruminal flows of casein or glutamic acid on small intestinal starch digestion and to quantify changes in energy and nutrient balance. Twenty-four steers (body weight = 179 ± 4 kg) were duodenally infused with raw cornstarch (1.46 ± 0.04 kg/d) and either 413 ± 7.0 g casein/d, 121 ± 3.6 g glutamic acid/d or water (control). Measures of small intestinal starch digestion and nutrient excretion were collected across 4 d after 42 d of infusion and measures of respiration via indirect calorimetry were collected across 2 d after 48 d of infusion. Ileal starch flow was least among calves provided casein, but ileal starch flow was not different between glutamic acid or control. Small intestinal starch digestion tended to be greatest among calves provided casein, least for glutamic acid and intermediate for control. Casein increased ileal flow of ethanol soluble oligosaccharides compared to glutamic acid and control. Large intestinal starch digestion was not different among treatments. By design, N intake was greatest among cattle provided casein, intermediate among calves provided glutamic acid and least for control. Nitrogen retention was greater in response to casein compared to control and glutamic acid. Intake of gross energy from feed was similar across treatments, and gross energy from infusate was greatest for casein, intermediate for glutamic acid and least for control. Variation in gross energy intake from feed resulted in no difference in overall gross energy intake across treatments. Similar to measures of small intestinal starch digestion and N retention, casein increased calories of digestible energy and metabolizable energy, compared to glutamic acid and control, which did not differ. Postruminal infusions did not influence methane production, but heat production was greatest in steers infused with casein, intermediate for steers provided glutamic acid, and least for control. Overall, amounts of energy retained by casein tended to be nearly 34% greater than control, but glutamic acid had no impact on energy balance. Improvement in small intestinal starch digestion in response to casein increased energy and N retained; however, glutamic acid did not influence small intestinal starch digestion and energy or N balance in cattle, which seems to suggest that responses in small intestinal starch digestion to greater postruminal flows of glutamic acid become refractory across greater durations of time.

Understanding how changes in postgastric nutrient flows impact small intestinal starch digestion could allow for large improvements in performance of cattle fed starch-based diets.

Introduction

Small intestinal starch digestion is more energetically efficient and provides greater amounts of net energy in comparison to ruminal fermentation of starch in cattle (Owens et al., 1986, 2016; Huntington, 1997; Mcleod et al., 2001; Harmon, 2009). Small intestinal starch digestion also augments glucose assimilation in contrast to ruminal fermentation, and others (McLeod et al., 2006) have observed that increases in glucose assimilation in cattle have corresponded to increases in energy retained as tissues. Yet, small intestinal starch digestion in cattle is limited in comparison to ruminal fermentation of starch or small intestinal starch digestion in nonruminant animals.

Small intestinal digestion of starch in cattle is generally considered to occur by hydrolysis of starch to small chain oligosaccharides via pancreatic secretions of α-amylase. Small chain oligosaccharides are then hydrolyzed to monosaccharides by oligosaccharidases bound to the brush border before monosaccharides are absorbed from the intestinal lumen. Overall, cattle seem to have a tremendous capacity for absorption of glucose (Kreikemeier and Harmon, 1995), and it seems more likely that small intestinal starch digestion is limited most by hydrolysis of starch to monosaccharides. Several different factors (Owens et al., 1986) could contribute to limited starch hydrolysis in the small intestine of cattle, but a specific understanding of how and to what extent different factors influence small intestinal starch digestion in cattle remains elusive.

Interestingly, short-term (6 to 14 d) increases in postruminal flow of casein (Taniguchi et al., 1995; Richards et al., 2002; Brake et al., 2014a) or glutamic acid (Brake et al., 2014b; Blom et al., 2016) can improve small intestinal starch digestion in cattle. But increases in small intestinal starch digestion in response to postruminal flows of casein or glutamic seem to reflect different physiological mechanisms (Brake et al., 2014b; Blom et al., 2016). Indeed, we recently reported (Trotta et al., 2020) that cattle provided greater postruminal flows of casein with cornstarch for 60 d had more than three times greater total pancreatic α-amylase activity and nearly twice the activity of jejunal maltase and glucoamylase than cattle that received cornstarch alone. Alternatively, longer-term (i.e., 60 d) infusions of glutamic acid with cornstarch increased duodenal maltase activity but had little impact on any other pancreatic or small intestinal carbohydrases (Trotta et al., 2020).

Data are limited on the long-term influences of increased postruminal flows of casein or glutamic acid with cornstarch or cornstarch alone on measures of small intestinal starch digestion. McLeod et al. (2006) reported that increased assimilation of glucose from the small intestine in response to greater postruminal flows of a starch hydrolysate was associated with greater omental fat accretion in cattle. Greater omental fat accretion could lessen improvements in caloric efficiency of starch digested in the small intestine compared to starch that is ruminally fermented, because increased visceral adipose accretion has been associated with greater insulin resistance, inflammation, malignant transformations and impaired glucose utilization in many studies in nonruminants (Tchkonia et al., 2005; Patel and Abate, 2013; Freedland, 2004; Ibrahim, 2010; Ferrer-Lorente et al., 2014; Kredel and Siegmund, 2014). It seems possible that increases in visceral fat accretion in cattle provided postruminal infusions of starch hydrolysate alone were, at least in part, because of increases in glucose metabolized by small intestinal tissue. Glucose is poorly oxidized by bovine small intestinal tissues (El-Kadi et al., 2009) and the end products of small intestinal glucose metabolism in cattle can inhibit lipid mobilization. Alternatively, glutamic acid is the primary anaplerotic substance catabolized by small intestinal tissues in cattle (El-Kadi et al., 2009). Furthermore, peptides and amino acids provided from casein serve functionally important roles by eliciting a myriad of physiologically important responses through direct and indirect signaling mechanisms (Wu, 2013). Therefore, we hypothesized that increases in small intestinal starch digestion in response to increased postruminal flow of casein or glutamic acid could increase the energy balance of cattle in comparison to starch alone. Furthermore, we aimed to evaluate effects of greater postruminal flow of casein or glutamic acid with cornstarch or cornstarch alone on small intestinal starch digestion and energy balance in cattle.

Materials and Methods

All procedures that involved use of animals in this project were approved by the South Dakota State University Institutional Animal Care and Use Committee (protocol approval No. 15-077A).

Animals, husbandry, and treatments

Four blocks of six steers (British × Continental; initial body weight = 179 ± 4 kg) were surgically fitted with double L cannulas (Streeter et al., 1991) in the duodenum (10 cm posterior to the pyloric sphincter) and ileum (30 cm anterior to the ileocecal junction) 34 ± 2.5 d before experimentation using procedures similar to those described by Blom et al. (2016).

Beginning 10 d prior to surgery, steers were fed (4.1 kg DM/d) a diet twice daily (at 0700 and 1900 h) that was identical to the diet fed during the experimental period. Food (48 h) and water (24 h) intake were withheld prior to surgery. All surgical procedures were performed aseptically with steers standing under local anesthesia (lidocaine). Steers were provided a prophylactic antibiotic (6.6 mg ceftiofur/ kg body weight) 1 d prior to surgery and on the third d after surgery. Steers were also provided intravenous analgesia (1.65 mg flunixin meglumine/ kg body weight) and an intramuscular prophylactic antibiotic (19,824 IU Penicillin G Procaine/ kg body weight) at surgery. Subsequently, intravenous analgesia and intramuscular antibiotic were provided daily for 4 and 7 d following surgery, respectively. Rectal temperatures and local inflammation were monitored each 12 h and food intake was monitored each 8 h for 7 d following each surgical procedure; after 7 d, intake of feed was 101.3 ± 1.2% of presurgery measures of intake.

Diet and postgastric infusions

After recovery from surgery (33 ± 2.5 d), each block of six steers were randomly assigned one of three treatments within a randomized complete block design and housed in individual stanchions (1.2 × 1.5 m) in a temperature-controlled room (20.7 ± 1.7 °C) under 16 h of light (0530 to 2130 h) and 8 h of darkness daily. Steers were fed (3.36 ± 0.471 kg/d dry matter basis) a common soybean hull-based diet (Table 1; about 1.5 × maintenance energy requirement; NASEM, 2016) twice daily (at 0700 and 1900 h) and were allowed ad libitum access to fresh water. Treatments were continuous duodenal infusion of raw cornstarch (1.46 ± 0.04 kg/d) alone (control), raw cornstarch with casein (413 ± 7.0 g/d), or raw cornstarch with glutamic acid (121 ± 3.6 g/d). Sodium hydroxide (42.8 g; 40% weight/weight) was included in suspensions containing glutamic acid to adjust the pH near 7. A peristaltic pump (model CP-78002-10; Cole-Parmer, Vernon Hills, IL) delivered duodenal infusions through Tygon tubing (2.38 mm i.d.; Saint Gobain North America, Valley Forge, PA). Suspensions were maintained with continuous stirring by an electric mixer (Arrow 1750; Arrow Engineering Company, Hillside, NJ) and delivered at a rate of 563 mL/h. Cornstarch suspensions (7.5 liters) were prepared with additions of appropriate amounts of deionized water, 10% (weight/weight) casein solution, and glutamic acid by weight (884 g cornstarch; Clinton Starch 106; ADM Corn Processing, Clinton, IA) each 12 h, and the mass infused was determined by weighing residual infusate. Suspensions were placed about 1 m above the steers and each line was flushed with 100 mL of deionized water each 12 h to prevent any sedimentation of cornstarch within the infusion line.

Table 1.

Composition of the soybean hull-based diet.

Ingredients	Percent of dry matter
Soybean hulls	72.4
Brome hay	20.0
Corn steep liquor	6.0
Limestone	1.0
Salt	0.5
Mineral and vitamin premix1	0.1
Chemical composition2, % dry matter
Dry matter	82.0 ± 0.54
Organic matter	92.5 ± 0.17
Starch	0.6 ± 0.04
Crude protein	10.5 ± 0.54
Neutral detergent fiber	59.7 ± 0.76
Acid detergent fiber	43.8 ± 0.63
Rumen-degradable protein	6.2
NEm3, Mcal/kg dry matter	1.37

¹Provided to diet (per kg of diet dry matter): 100 mg Fe, 50 mg Mn, 50 mg Zn, 10 mg Cu, 0.5 mg I, 0.2 mg Se, 2200 IU vitamin A, 275 IU vitamin D, and 25 IU vitamin E.

²Mean ± SEM

³Net energy available for maintenance; calculated from tabular values (NASEM, 2016)

Sample collection

Cattle were provided duodenal infusions for 41 d and measures of nutrient disappearance from the small and large intestines and measures of total-tract nutrient balance were determined from samples of feed, infusate, orts, ileal digesta, urine, and feces collected on day 42 to 45.

Cattle were placed into metabolism crates (0.66 × 1.83 m) on day 41 to facilitate total collections of feces and urine which were used to determine total-tract nutrient balance. Feed (100 g/d) and orts (100 g, if present) were collected daily from day 41 to 44 to correlate to collections of ileal digesta, feces, and urine that were collected on day 42 to 45. Feed samples were composited across day within each block and ort samples were composited within each steer. Daily amounts of feces and urine produced from each steer were measured by weighing at 0700 h. Feces were thoroughly mixed by hand, sampled (5% by weight), composited within steer and frozen (−20 °C) prior to analyses of dry matter, ash, neutral detergent fiber, acid detergent fiber, N, and fecal energy. Urine was collected by placing a clean urine collection vessel under each steer that contained 900 mL of 10% (weight/weight) H₂SO₄ to acidify urine. Subsequently, urine within each container was mixed and 1% (weight/weight) of daily output was sampled, composited within steer and frozen prior to analyses for N and urinary energy.

Measures of small and large intestinal nutrient disappearance were determined from spot samples of ileal digesta and feces, which were obtained simultaneous to total collections of urine and feces. Beginning on day 35 and during measures of nutrient balance, CrEDTA (2.7 g Cr/L; Binnerts et al., 1968) was added to cornstarch suspensions, which served as an indigestible marker of nutrient flow. Total recoveries of CrEDTA averaged 92.6 ± 6.8%. Ileal digesta (190.63 ± 8.28 g) and spot samples of feces (237.58 ± 13.72 g) were collected each 4 h between a 12 h feeding interval (0700 and 1900 h) on day 42 to 45. Sampling of ileal digesta and spot feces was delayed 1 h each day so that composited samples of ileal digesta and spot feces represented each hour in a 12-h period. Ileal digesta was collected by fastening a plastic bag (140 × 229 mm; Fisherbrand; FisherScientific, Waltham, MA) to the ileal cannula for 0.73 ± 0.03 h, and samples of spot feces were obtained after mechanical stimulation of defecation. Ileal digesta and spot feces were alkalinized (pH near to 11) to inactivate amylolytic enzymes by adding 2.1 mL and 4.2 mL 40% (wt/wt) NaOH, respectively. Composited samples were then frozen (−20 °C) prior to analysis of dry matter, ash, Cr, N, C, starch, and ethanol soluble oligosaccharides.

After measures of total-tract nutrient balance and intestinal nutrient disappearance, steers were removed from metabolism crates and allowed 2 d of rest prior to measurement of heat production using indirect calorimetry on day 48 to 49. Samples of respired air and methane were collected over 12 h periods, and measures of respired gas and methane produced was determined by placing each steer’s head and neck into an open-circuit respiration calorimeter (76 × 76 × 180 cm) using procedures similar to those described by Petzel et al. (2019). Air flow from each calorimeter was set to a flow rate of 324 liters/min and measured by an individual mass flow meter (Dresser MicroSeries ptz+Log, GE Oil and Gas, Houston, TX). Calorimeters were run for 15 min (4.7 volumes of each calorimeter) prior to collecting samples of respired air to ensure that sampled air was reflective of gas production from steers. Samples of respired air were obtained by continuously collecting an aliquot of all inflowing and outflowing air in an air impermeable bag (61 × 61 cm Laminate, PMC, Oak Park, IL) by utilizing glass rotameters (SHO-RATE, Brooks Instrument, Hatfield, PA). Measures of air flow from each calorimeter were adjusted for temperature, relative humidity (TRH-100, Pace Scientific, Mooresville, NC), and barometric pressure (P350-D-0inch, Pace Scientific), which was measured every min (XR5-SE Data logger, Pace Scientific). Recoveries of oxygen (99.37 ± 4.11%) and carbon dioxide (90.48 ± 2.42%) were determined after combustion of ethanol in each calorimeter prior to sampling respired air.

Laboratory analyses

Samples of feces, feed, and ileal digesta were thawed at room temperature, and a portion of each sample was dried at 55 °C and ground to pass a 1-mm screen (Thomas Wiley Laboratory Mill model 4; Thomas Scientific USA, Swedesboro, NJ) for measurement of dry matter, organic matter, starch, neutral detergent fiber, acid detergent fiber, N, caloric content and the internal marker. Additionally, another aliquot of ileal digesta (75 g) or spot feces (50 g with 25 mL deionized H₂O) was neutralized using 6 M HCl prior to measures of starch and ethanol soluble oligosaccharides. Sample dry matter was analyzed by drying at 105 °C for 16 h (method no. 930.15, AOAC, 2016). Sample organic matter was analyzed by combustion (500 °C for 16 h, method no. 942.05, AOAC, 2016). Neutral detergent fiber was analyzed with addition of sodium sulfite and α-amylase as described by Van Soest et al. (1991). Acid detergent fiber was measured nonsequential to neutral detergent fiber (Van Soest et al., 1991) and measures of neutral detergent fiber and acid detergent fiber were corrected for ash content, which was determined by combustion (500 °C for 8 h).

Starch concentrations were measured using the techniques of Herrera-Saldana and Huber (1989); glucose was quantified using a glucose oxidase assay (Gochman and Schmitz, 1972) and unpolymerized glucose was measured from a subset of starch assay tubes that did not contain additions of enzyme.

Ethanol soluble oligosaccharide content was determined using techniques similar to those described by Brake et al. (2014a). Neutralized digesta and feces were centrifuged (20,000 × g for 15 min at 4 °C) and then 0.5 mL of supernatant was added to a 2-mL microcentrifuge tube (Basix microcentrifuge tube; Fisher Scientific, Waltham, MA) containing 1.25 mL anhydrous ethanol. Samples were then refrigerated (4 °C) overnight (16 h) and centrifuged (17,000 × g for 10 min at 4 °C). Supernatant was transferred to a 15-mL conical tube (Falcon Centrifuge Tube; Corning Inc., Corning, NY), and anhydrous ethanol (1 mL) was used to resuspend the remaining pellet and then centrifuged (17,000 × g for 10 min at 4 °C). Supernatant was again moved to the same 15-mL conical tube. This rinsing procedure was completed for a total of three times and then ethanol was evaporated from each tube utilizing a centrifugal concentrator (57 × g at 45 °C for 4.5 h; CentriVap; Labconco). Subsequently, measures of ethanol soluble oligosaccharide were determined using the same methods described for measures of starch concentration.

Concentration of Cr in wet feces and ileal digesta was measured by atomic absorption spectroscopy (SOLAAR S4, Thermo Fisher Scientific, Waltham, MA) from supernatant after centrifugation (20,000 × g for 15 min at 4 °C). Nitrogen and C concentration of partially dried feces, feed, orts and urine was analyzed by combustion (Vario Macro Cube, Elementar Americas, Inc., Ronkonkoma, NY). Gross energy was analyzed using a bomb calorimeter (Parr Instrument Company, Moline, IL). Urinary energy was measured after lyophilizing 15 mL of urine in small pouches (5.0 × 7.6 cm, 1 mil polypropylene bag, Associated Bag, Milwaukee, WI) and by correcting measures for the energy content of the small pouches (10.98 ± 0.058 kcal/g).

Concentrations of carbon dioxide and methane were measured by near infra-red reflectance spectroscopy and oxygen was determined by paramagnetic detection (Emerson X-Stream XE, Emerson Process Management, Solon, OH) after calibration to a standard gas containing 19.68% oxygen, 1.01% carbon dioxide, and 0.10% methane (PurityPlus, Indianapolis, IN).

Calculations

Solid and liquid components in digesta appear to pass the postruminal digestive tract at similar rates (Hogan and Phillipson, 1960; Grovum and Phillips, 1973; Sinnott et al., 2017) and previous reports indicate that liquid and particulate markers dosed in the duodenum of cattle pass to feces at nearly identical rates (Mambrini and Peyraud, 1997). Thus, flow of digesta at the ileum was calculated as described by Brake et al. (2014b). Specifically, fluid flow (g/d) was calculated as the quotient of the infusion rate of Cr to the duodenum (mg/d) and the concentration of Cr in ileal fluid (mg/g). Total flow of digesta (g/d) was calculated as the quotient of digesta fluid flow (g/d) and the proportional water content of digesta at the ileum, which was calculated as 1 minus the proportion of dry matter directly measured in ileal content by drying; dry matter flow was calculated as the difference of total digesta flow and digesta fluid flow at the ileum as determined from measures of Cr.

Ileal starch and ethanol soluble oligosaccharide flow were calculated as the product of ileal dry matter flow or fluid flow and nutrient concentration, respectively. Small intestinal starch digestion was calculated as the quotient of the amount of starch that disappeared between the duodenum and ileum (i.e., g starch dry matter infused – g ileal starch dry matter flow) and the amount of starch infused (dry matter-basis). Large intestinal starch and ethanol soluble oligosaccharide flow were calculated as the product of total fecal dry matter output and concentration of starch or ethanol soluble oligosaccharides in composited samples of spot feces and corrected for amounts of starch and ethanol soluble oligosaccharides removed in collection of spot samples of digesta or feces. Correspondingly, large intestinal starch or ethanol soluble oligosaccharide disappearance was calculated as the quotient of the amount that disappeared between the ileum and feces and the amount flowing from the ileum. Postruminal starch digestion was calculated as the amount of starch that disappeared between the duodenum and feces and the amount of starch infused (dry matter-basis) and corrected for amounts of starch removed in collection of spot samples of digesta or feces.

Amounts of oxygen consumed and carbon dioxide and methane produced were calculated as the difference in concentration of oxygen, carbon dioxide and methane from air collected from each respiration calorimeter and samples of ambient air and multiplied by the air flow from each calorimeter after standardizing for temperature, pressure, and humidity (Nienaber et al., 2009). Heat production was calculated as described by Brouwer (1965). Body weight during measures of indirect calorimetry were calculated by linearly extrapolating measures of body weight gain from initial body weight, and metabolic body weight was calculated as the product of 0.96 and body weight to the three fourths power.

Digestible energy intake was calculated as the difference between the gross energy intake from diet and infusate and energy excreted in feces. Metabolizable energy intake was calculated as the difference between digestible energy intake, energy excreted in urine, and energy exhaled as methane. Retained energy was calculated as the difference between metabolizable energy and heat of production.

Statistical analyses

One steer that received duodenal infusion of casein was removed from the experiment after day 5 because it sustained an injury to its duodenal cannula that led to a local inflammatory response and reduced feed intake (85.5% of dry matter offered was refused). Also, another steer that received duodenal infusion of casein was removed after day 35 because it had reduced feed intake (1.2 ± 0.4 kg dry matter; 39.5% of dry matter offered daily). Thus, there were only six observations for steers that received infusions of cornstarch and casein.

Data were analyzed as a randomized complete block design using the GLIMMIX procedure of SAS (version 9.4, SAS Institute, Inc., Cary, NC) with the following model:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{Y}}_{\mathrm{i}\mathrm{j}}=\mu +{\mathrm{T}}_{\mathrm{i}}+{\mathrm{B}}_{\mathrm{j}}+{\epsilon }_{\mathrm{i}\mathrm{j}}}\end{array}}$$

where Y_ij = observations for dependent variables, µ = overall mean, T_i = fixed effect of treatment, B_j = the random effect of block and ε_ij = random error. The Satterthwaite adjustment (Satterthwaite, 1946) was used to calculate denominator degrees of freedom, and the LSMEANS option was used to calculate treatment means. Differences between treatments were declared at P < 0.05 and tendencies at 0.05 ≤ P < 0.10. When the F-statistics was significant, treatments were separated by calculating a student’s t-test using the PDIFF option of SAS. Analyses of amounts of nutrients or CrEDTA removed in collection of spot samples of digesta or feces were analyzed similarly to measures of nutrient flows and energy balance, but block was considered a fixed effect because these analyses contained the entire population of interest (Green and Tukey, 1960; Dixon, 2016).

Results

Nutrient intake and apparent total tract digestion

By design, there were no differences in dry matter, organic matter, neutral detergent fiber, or acid detergent fiber intake from feed (P ≥ 0.71; Table 2), and neutral detergent fiber or acid detergent fiber intake from infusate because infusions did not contain any components of fiber; however, as expected, amounts of dry matter and organic matter provided from infusate (P ≤ 0.01; Table 2) were greatest for casein, intermediate for glutamic acid, and least from control. Yet, differences in dry matter and organic matter provided by infusate in response to added amounts of casein or glutamic acid were masked by variation in dry matter and organic matter intake from feed and overall measures of dry matter and organic matter intake were not different between treatments (P ≥ 0.59). Measures of fecal dry matter and organic matter output tended (P = 0.08) to be least for casein, but neutral detergent fiber and acid detergent fiber output were not affected by treatment (P ≥ 0.19). Correspondingly, measures of dry matter and organic matter digestibility were greatest (P < 0.01) among calves provided duodenal infusions of casein, intermediate for glutamic acid, and least for control. Even though measures of acid detergent fiber intake and fecal output were not affected by treatment, measures of total-tract acid detergent fiber (P = 0.04) digestibility were greatest for casein, least for control, and glutamic acid was intermediate; however, measures of total-tract neutral detergent fiber digestibility were not affected by treatment (P = 0.11).

Table 2.

Effect of duodenal infusion of casein or glutamic acid on total tract nutrient digestion in steers receiving 1.5 kg of duodenally infused raw cornstarch

Item, g/d	Treatment			SEM	P-value
	Control	Casein	Glutamic
Dry matter intake	4,849	5,079	5,018	213.1	0.66
Feed	3,389	3,224	3,438	212.6	0.71
Infusate	1,463A	1,845B	1,580C	20.14	<0.01
Organic matter intake	4,577	4,814	4,748	188.4	0.59
Feed	3,119	2,972	3,168	187.9	0.72
Infusate	1,462A	1,829B	1,580C	20.11	<0.01
Neutral detergent fiber intake	2,031	1,920	2,056	111.76	0.64
Feed	2,031	1,920	2,056	111.76	0.64
Infusate	–	–	–	–	–
Acid detergent fiber intake	1,517	1,441	1,538	84.49	0.67
Feed	1,517	1,441	1,538	84.49	0.67
Infusate	–	–	–	–	–
Fecal dry matter output, g/d	1,729	1,491	1,631	83.4	0.08
Fecal organic matter output, g/d	1,611	1,384	1,512	81.9	0.08
Fecal neutral detergent fiber output, g/d	450	370	411	37.2	0.29
Fecal acid detergent fiber output, g/d	337	266	308	28.51	0.19
Dry matter digestibility, %	63.7A	69.8B	67.0C	1.0	<0.01
Organic matter digestibility, %	64.8A	70.7B	68.1C	1.18	<0.01
Neutral detergent fiber digestibility, %	77.8	81.5	80.1	1.29	0.11
Acid detergent fiber digestibility, %	77.7A	82.4B	80.0AB	1.40	0.04

^A,B,CMeans in rows with different superscripts differ (P ≤ 0.05).

Small and large intestinal starch digestion

Previously, we measured duodenal starch flows in cattle of similar size fed the same (Brake et al., 2014b) in similar amounts as cattle in this experiment. Based on our previous observations, duodenal flows of dry matter and organic matter in this study were 2,478 ± 156.8 g dry matter and 1,781 ± 107.7 g organic matter, respectively (Brake et al., 2014b). Amounts of starch in feed were negligible (0.6% of dry matter; Table 1), and duodenal flows of starch from feed were likely near to 9.2 ± 0.59 g based on intake of calves and previous reports of duodenal nutrient flows from cattle fed the same feed (Brake et al., 2014b). By design, amounts of starch infused at the duodenum were not different (P = 0.21) among treatments (1,455 ± 7.5; Table 3). Flow of dry matter from the ileum (P = 0.09) tended to be least among calves provided casein but was not different between glutamic acid or control. Flow of organic matter from the ileum also appeared to numerically (P = 0.11) reflect tendencies in ileal dry matter flow. Yet, large intestinal dry matter and organic matter digestion were not affected by treatments (P ≥ 0.12).

Table 3.

Effect of duodenal infusion of casein or glutamic acid on ileal and fecal nutrient flow and small and large intestinal starch digestion in steers receiving 1.5 kg of duodenally infused raw cornstarch

Item, g dry matter/d	Treatment			SEM	P-value
	Control	Casein	Glutamic
Duodenal starch infused	1,462	1,433	1,459	18.60	0.21
Nutrient flow from the ileum
Dry matter	2,999ab	2,601a	3,044b	175.8	0.09
Organic matter	2,588	2,262	2,630	150.5	0.11
Starch	1,070A	933B	1,098A	61.17	0.04
Ethanol soluble oligosaccharides	88.7A	178.0B	60.7A	21.58	<0.01
Small intestinal starch digestion, %	27.1ab	34.3a	24.9b	3.59	0.09
Starch removed at ileum	7.7	8.5	8.3	0.63	0.60
Nutrient flow to feces
Starch	533.2	443.2	483.1	40.0	0.18
Ethanol soluble oligosaccharides	29.8	30.2	31.4	4.78	0.96
Large intestinal starch digestion, %	49.2	52.2	55.2	3.3	0.28
Large intestinal dry matter digestion, %	40.4	40.6	44.4	1.74	0.14
Large intestinal organic matter digestion, %	37.1	38.3	41.8	1.84	0.12
Postruminal starch digestion, %	63.4	69.1	66.7	2.54	0.18

^A,BMeans in rows with different superscripts differ (P ≤ 0.05)

^a,bMeans in rows with different superscripts differ (P ≤ 0.10)

Ileal starch flow was least (P = 0.04) among calves provided casein, but ileal starch flow was not different (P_t = 0.60) between glutamic acid or control. Correspondingly, small intestinal starch digestion tended (P = 0.09) to be nearly 1.3 times greater among calves provided casein, compared to glutamic acid or control. Additionally, measures of ileal ethanol soluble oligosaccharide flows were more than two times greater (P < 0.01) among calves provided casein compared to glutamic acid and control.

Similar to measures of ileal starch flow, fecal starch flow was numerically least (P = 0.18) among calves provided casein, greatest for control, and glutamic acid was intermediate. Fecal flow of ethanol soluble oligosaccharides was not different (P = 0.96) among treatments. Overall, large intestinal starch digestion was not affected (P = 0.28) in response to different duodenal infusions. Thus, postruminal starch digestion was numerically greater (P = 0.18) among calves provided casein compared to calves that received postruminal infusions of cornstarch alone, and measures of postruminal starch digestion were intermediate among calves provided duodenal infusions of cornstarch and glutamic acid.

Nitrogen balance

Nitrogen balance data are presented in Table 4. There were no differences (P = 0.60) in N intake from feed (55.8 ± 2.26 g N/d) across treatments, but N intake from infusate (P < 0.01) was greatest in calves provided duodenal infusion of casein (62.8 g N/d), intermediate in calves provided duodenal infusion of glutamic acid (14.4 g N/d), and least for control (2.9 g N/d). Total N intake (P < 0.01) reflected differences in infusate N intake and was greatest in calves provided casein (115.5 g N/d), intermediate in calves provided glutamic acid (71.9 g N/d), and least for control (60.0 g N/d; P < 0.01). Urine N excretion was greatest (P < 0.01) in response to duodenal infusion of casein (46.1 g N/d), least for control (12.2 g N/d), and glutamic acid was intermediate (21.2 g N/d); however, N excretion in feces was not different (P = 0.80; 30.8 ± 1.00 g N/d) among treatments. Thus, N retention was nearly two times greater (P < 0.01) in response to casein (38.0 g N/d) compared to control and glutamic acid (17.4 and 19.0 g N/d, respectively), which did not differ.

Table 4.

Effect of duodenal infusion of casein or glutamic acid on nitrogen balance in steers receiving 1.5 kg of duodenally infused raw cornstarch

N, g/d	Treatment			SEM	P-value
	Control	Casein	Glutamic
Intake	60.0A	115.5B	71.9C	5.59	<0.01
Feed	57.1	52.8	57.5	5.52	0.60
Infusate	2.9A	62.8B	14.4C	0.29	<0.01
Urine	12.2A	46.1B	21.2C	1.83	<0.01
Feces	30.0	31.1	31.4	2.34	0.80
Retained, g/d	17.4A	38.0B	19.0A	2.48	<0.01

^A,B,CMeans in rows with different superscripts differ (P ≤ 0.05).

Gas exchange and energy balance

Gas exchange data are presented in Table 5. Amounts of oxygen consumed daily (P = 0.03) were greatest among calves provided casein in comparison to calves provided glutamic acid or control, and numerical differences (P = 0.11) in amounts of oxygen consumed per kg of metabolic body weight seemed to reflect differences in total amounts of oxygen consumed. Similar to measures in oxygen consumption per kg of metabolic body weight, numerical differences in carbon dioxide produced daily (P = 0.15) and per kg of metabolic body weight (P = 0.13) seemed to follow differences in total amounts of oxygen consumed. Correspondingly, the respiratory quotient (0.98 ± 0.01) was not affected (P = 0.17) by postgastric nutrient flows, and postruminal infusions had no influence on amounts of daily methane produced or conversion of feed dry matter to methane (P ≥ 0.57).

Table 5.

Effect of duodenal infusion of casein or glutamic acid on consumption of oxygen (O₂) and production of carbon dioxide (CO₂) and methane (CH₄) in steers receiving 1.5 kg of duodenally infused raw cornstarch

Item	Treatment			SEM	P-value
	Control	Casein	Glutamic
MBW1, kg	51.0A	57.0B	51.4A	2.3	<0.01
O2 consumption
L/day	1375.3A	1657.5B	1462.8A	95.51	0.03
L/kg MBW	27.1	30.1	28.5	1.59	0.11
CO2 production
L/day	1360.7	1586.4	1436.9	106.62	0.15
L/kg MBW	26.78	29.29	27.99	2.54	0.13
RQ2	0.99	0.95	0.98	0.017	0.17
CH4 production
L/day	115.1	108.1	111.5	11.60	0.88
L/kg DMI	23.65	21.18	22.27	2.14	0.57

^A,B,C Means in rows with different superscripts differ (P ≤ 0.05)

¹Metabolic BW = (0.96 × BW)^0.75; NASEM, 2016

²Respiratory quotient

Energy balance data are presented in Table 6. Intake of gross energy from feed was not different (P = 0.73) across treatments, but gross energy from infusate was greatest (P < 0.01) for casein (8.32 Mcal/day), intermediate for glutamic acid (6.37 Mcal/day), and least for control (6.11 Mcal/day). Despite differences in gross energy intake from infusate, numerical differences and variation in measures of gross energy intake from feed obviated differences in overall gross energy intake (P = 0.45). Yet, gross energy intake from infusate as a proportion of dry matter intake (P < 0.01; Table 7) was greatest among calves provided duodenal infusions of casein, intermediate in calves provided glutamic acid, and least for calves provided cornstarch alone. Alternatively, gross energy intake from feed as a proportion of dry matter intake tended to be greatest for calves provided cornstarch alone, least for calves provided casein, and glutamic acid was intermediate (P = 0.09; Table 7). Consequently, overall gross energy intake as a proportion of dry matter intake was greatest for calves provided casein, least for calves provided glutamic, and control was intermediate (P < 0.01; Table 7).

Table 6.

Effect of duodenal infusion of casein or glutamic acid on energy balance in steers receiving 1.5 kg of duodenally infused raw cornstarch

Energy, Mcal	Treatment			SEM	P-value
	Control	Casein	Glutamic
Gross	19.24	20.74	19.67	1.17	0.45
Feed	13.13	12.42	13.30	1.16	0.73
Infusate	6.11A	8.32B	6.37C	0.08	<0.01
Fecal	6.61	5.60	6.23	0.54	0.14
Digestible	12.62A	15.18B	13.44AB	0.79	0.03
Urinary	0.65	0.67	0.71	0.10	0.69
Methane	1.09	1.02	1.05	0.11	0.88
Metabolizable	10.90A	13.47B	11.68A	0.69	0.01
Heat increment	6.88A	8.19B	7.29AB	0.49	0.05
Retained	3.99a	5.35b	4.38ab	0.47	0.07
Energy efficiency
Fecal energy, % of gross	34.24A	27.18B	31.57A	1.64	<0.01
Digestible, % of gross	65.76A	72.82B	68.43A	1.64	<0.01
Urine, % of gross	3.28	3.30	3.60	0.45	0.70
Urine, % of digestible	5.07	4.46	5.29	0.75	0.49
Methane, % of gross	5.68	4.90	5.39	0.53	0.38
Methane, % of digestible	8.66a	6.72b	7.85ab	0.69	0.06
Metabolizable, % of digestible	86.28A	88.91B	86.86A	0.66	0.02
Heat increment, % of digestible	54.45	54.68	54.29	2.44	0.99
Retained, % of metabolizable	36.75	38.48	37.52	2.91	0.86

^A,B,CMeans in rows with different superscripts differ (P ≤ 0.05).

^a,bMeans in rows with different superscripts differ (P ≤ 0.10).

Table 7.

Effect of duodenal infusion of casein and glutamic acid on energy as a proportion of DMI in steers receiving 1.5 kg of duodenally infused raw cornstarch

Energy, kcal/kg DMI1	Treatment			SEM	P-value
	Control	Casein	Glutamic
Gross	3,953A	4,086B	3,913C	94	<0.01
Feed	2,687a	2,411b	2,641ab	143	0.09
Infusate	1,263A	1,697B	1,272A	86	<0.01
Fecal	1,350A	1,111B	1,236C	72	<0.01
Digestible	2,597A	2,990B	2,677A	90	<0.01
Urine	130	136	141	18.9	0.80
Methane	224	200	210	20	0.57
Metabolizable	2,243A	2,653B	2,325A	86	<0.01
Heat increment	1,411A	1,639B	1,455A	90	0.03
Retained	821A	1,044B	870A	72	0.04

¹Dry matter intake

^A,B,CMeans in rows with different superscripts differ (P ≤ 0.05).

^a,bMeans in rows with different superscripts differ (P ≤ 0.10).

Fecal energy losses were numerically less (P = 0.14) for calves provided cornstarch and casein compared to glutamic acid or control, and digestible energy was greatest (P = 0.03) in calves infused with casein (15.18 Mcal/day), intermediate among calves provided glutamic acid (13.44 Mcal/d), and least for control (12.62 Mcal/d). Similarly, fecal energy losses as a percent of gross energy intake were least (P < 0.01; Table 6) among calves provided casein, compared to glutamic acid or control, but fecal energy losses as a proportion of dry matter intake were less (P < 0.01; Table 7) for cattle provided casein or glutamic acid compared to control. Digestible energy as a percent of gross energy intake (Table 6) or as a proportion of dry matter intake (Table 7) was greatest (P < 0.01) among calves provided casein, compared to glutamic acid or control.

There were no differences in urine (P = 0.69) or methane energy (P = 0.88) losses. Urine energy loss as a percent of gross energy intake (P = 0.70; Table 6), digestible energy intake (P = 0.49; Table 6), or proportion of dry matter intake (P = 0.80; Table 7) were not different among treatments. There were no differences (P ≥ 0.38) in losses of methane energy as a percent of gross energy intake (Table 6) or proportion of dry matter intake (Table 7) among treatments, but loss of methane energy as a percent of digestible energy tended (P = 0.06; Table 6) to be less among calves provided casein, compared to glutamic acid or control. Thus, measures of metabolizable energy were greatest (P = 0.01) among calves infused with casein in comparison to calves infused with glutamic acid or control, which were not different. Likewise, metabolizable energy as a percent of digestible energy (P = 0.02; Table 6) or a proportion of dry matter intake (P < 0.01; Table 7) was greatest among calves provided casein, compared to calves provided glutamic acid or control. However, measures of heat production (P = 0.05) were 19% greater (P_t = 0.05) among calves infused with casein and tended (P_t = 0.07) to be 6% greater in calves provided glutamic acid compared to calves infused with cornstarch alone. Heat production per kg dry matter intake was also greater (P = 0.03; Table 7) for calves provided casein compared to heat production in calves provided control or glutamic acid, but there was no difference (P = 0.99) in heat production as a percent of digestible energy (Table 6). Overall, retained energy tended (P = 0.07) to be 34% greater among calves provided casein and 10% greater among calves provided duodenal infusion of glutamic acid compared to control. Furthermore, retained energy as a proportion of dry matter intake was greater (P = 0.04; Table 7) in calves provided duodenal infusion of casein compared to glutamic acid or control, but retained energy as a percent of metabolizable energy was not different (P = 0.86; Table 6) between treatments.

Discussion

Small intestinal starch digestion

Small intestinal starch digestion is limited in cattle in comparison to other animals (e.g., swine, humans, canines) and is typically less than amounts of starch fermented in the rumen. Owens et al. (1986) reported that small intestinal digestion of starch in cattle only averaged 52.9% of starch flowing to the duodenum but that measures of small intestinal starch digestion in cattle were largely variable (coefficient of variation V = 35.2%). Generally, variation in measures of digestion or metabolism are small when digestion or metabolism of a specific nutrient is intrinsically limited because of limitations in genetic expression (Karasov and Douglas, 2013). Alternatively, large estimates of variance in measures of digestion are often an indication that digestive and metabolic responses are responsive to different stimuli (e.g., luminal nutrient flows; Karasov and Douglas, 2013).

Indeed, small intestinal starch digestion in cattle has been improved by postruminal flow of casein (Taniguchi et al., 1995; Richards et al., 2002; Brake et al., 2014b) or glutamic acid (Brake et al., 2014a; Blom et al., 2016). In this experiment, small intestinal starch digestion tended (P = 0.09) to be at least 27% greater among calves that received duodenal infusion of casein in comparison to glutamic acid or control. Richards et al. (2002) regressed amounts of starch digested in the small intestine of cattle on amounts of abomasally infused casein and reported that small intestinal starch digestion increased 1.6 g/d for each gram of casein flowing to the duodenum. Similarly, we (Brake et al., 2014b; Blom et al., 2016) observed linear increases in amounts of starch digested in the small intestine to increases in postruminal flows of casein that were twice as large as the amounts of casein infused by Richards et al. (2002). Yet, our measures of small intestinal starch digestion increased at a lesser rate than increases in small intestinal starch digestion observed by Richards et al. (2002). Small intestinal starch digestion increased 0.23 g/d for each gram of casein duodenally infused in this experiment, which was similar to our previous measures of increases in small intestinal starch digestion in response to greater postruminal flows of casein (Brake and Swanson, 2018). Differences in measures of small intestinal starch digestion from our previous reports (Brake et al., 2014b; Blom et al., 2016) and that of Richards et al. (2002) may be related to differences in methodologies used between our prior experiments and that of Richards et al. (2002; e.g., site of infusion, amounts of starch and casein infused, different basal diets).

Brake et al. (2014a) first reported that small intestinal starch digestion in cattle increased in response to greater postruminal flows of glutamic acid and that increases in small intestinal starch digestion to greater postruminal flows of glutamic acid were greater than to postruminal flows of casein (0.96 g/d increase in starch digested per gram of Glu infused duodenally). Later, Blom et al. (2016) reported that small intestinal starch digestion increased linearly in response to greater duodenal flows of glutamic acid (1.25 g/d increase in starch digested per gram of Glu infused duodenally); however, we did not observe any improvement in small intestinal starch digestion when we duodenally infused similar amounts of glutamic acid together with cornstarch in this experiment. Perhaps, the most salient difference between this study and our previous studies (Brake et al., 2014a; Blom et al., 2016) was the length of the infusion period. In this study, steers were infused with treatments for 42 d before measures of small intestinal starch digestion, but in our previous studies, small intestinal starch digestion was measured after 6 (Brake et al., 2014a) or 12 d (Blom et al., 2016). Differences in small intestinal starch digestion to postruminal infusions of glutamic acid across our experiments seem to suggest that effects of greater glutamic acid flows to the duodenum on small intestinal starch digestion may become refractory across greater periods of time (e.g., 42 vs. 12 d). A clear understanding of why responses to glutamic acid may become refractory but responses to casein do not remains elusive. Yet, differences in response to casein and glutamic acid in this study seem to support our previous observations that response in small intestinal starch digestion to postruminal flows of casein or glutamic seem to reflect different physiological mechanisms (Brake et al., 2014b; Blom et al., 2016).

The small intestinal epithelium interacts dynamically with the luminal environment by sensing nutrients to coordinate changes in digestive enzyme production and secretion together with sequestration of transport molecules that allow for rapid adaptation to diet changes. Responses to changes in luminal nutrient flows appears to be predominately modulated by enteroendocrine cells, which account for less than 1% of the intestinal epithelium, but collectively form the largest endocrine organ in the body by secreting peptide hormones that modulate digestion and absorption of nutrients (Sternini et al., 2008). Understanding how populations of enteroendocrine cells respond to changes in luminal nutrient flows and to what extent enteroendocrine cells are impacted by factors released from neighboring cells remains an important translational question to understanding the fundamental aspects of how changes in diet or nutrient flows to the small intestinal lumen influence digestion and nutrient absorption. Changes in amount or function of enteroendocrine cells in response to greater postruminal flows of casein or glutamic acid were not measured in this study. However, chemosensing by enteroendocrine cells is largely modulated via G-protein-coupled receptors expressed on the apical domain of enteroendocrine cells (Shirazi-beechey et al., 2014). If chemosensing by enteroendocrine cells modulates small intestinal digestive functions in cattle, then differences in small intestinal starch digestion to short- or long-term postgastric infusions of casein or glutamic acid could be related to classic responses to long-term stimulation in G-protein-coupled receptors, which are often internalized through endocytosis in response to persistent exposure to agonists (Calebiro et al., 2010; Jalink and Moolenaar, 2010; Faget et al., 2012; Magalhaes et al., 2012). Indeed, others (Daly et al., 2013) reported that endocrine secretions in response to amino acids by enteroendocrine cells were diminished when specific G-protein-coupled receptors were knocked down; alternatively, endocrine secretions in response to protein or peptides were not affected by inhibition of G-protein-coupled receptors. Thus, it seems plausible that long-term exposure to greater postruminal flows of glutamic acid could have decreased chemosensing by enteroendocrine cells, but greater postruminal protein flows, provided as casein, did not.

Ethanol soluble oligosaccharide flows were more than two times greater (P < 0.01) in calves provided postruminal infusions of casein compared to calves that were provide postruminal infusions of glutamic acid or cornstarch alone. Ethanol soluble oligosaccharides are small-chain α-glycosides (Brake et al., 2014a) and increases in ileal ethanol soluble oligosaccharide flows among cattle with similar duodenal starch flows should reflect greater increases in small intestinal hydrolysis of starch (i.e., amylolytic activity) relative to increases in hydrolysis of small-chain α-glycosides (i.e., brush border carbohydrases). Measures of small intestinal ethanol soluble oligosaccharide flows were in agreement with previous measures from our laboratory (Brake et al., 2014a, 2014b) and with measures of enzyme activities from small intestinal and pancreatic tissues from steers in this study (Trotta et al., 2020). Trotta et al. (2020) reported that duodenal infusion of casein increased pancreatic α-amylase activity (2,500 U/g protein) to a greater extent than increases in small intestinal maltase, glucoamylase, and isomaltase activities. Duodenal infusion of glutamic acid increased duodenal maltase activity but did not influence pancreatic α-amylase or jejunal or ileal carbohydrase activities (Trotta et al., 2020). Clearly, differences in small intestinal starch disappearance and ethanol soluble oligosaccharide flows from the ileum in this study combined with previous observations provide evidence that responses in small intestinal starch digestion to greater postruminal flows of casein or glutamic acid are modulated differently. However, future investigations of intestinal chemosensing in cattle will be needed to achieve a precise understanding of how small intestinal digestive functions are altered in response to changing luminal nutrient flows.

Large intestinal starch digestion

Previously, we observed (Brake et al., 2014a; Blom et al., 2016) that increases in ethanol soluble oligosaccharide flows from the ileum were related to large intestinal starch digestion. Indeed, fecal starch flow tended to be least among calves infused with casein; however, large intestinal starch digestion was not different across treatments, even though casein altered amounts and form of starch that flowed from the ileum compared to control or glutamic acid. Overall, measures of postruminal starch digestion appeared to be consistent with measures of small intestinal starch digestion, which seems to suggest that large intestinal starch flow may have exceeded capacity for large intestinal fermentation in cattle provided intragastric infusions of cornstarch regardless of treatment. Gressley et al. (2011) conducted a review of the literature and found that an average of 87 g (28%) of starch that flowed from the ileum in cattle was fermented in the large intestine. When Blom et al. (2016) infused increasing amounts of glutamic acid or casein to cattle they observed that 465 g of starch disappeared from the large intestine in steers, which accounted for 56% of ileal starch flow. In this study, an average of 611 g of starch disappeared from the large intestine in steers that corresponded to 58% of ileal starch flow.

Previously, we (Brake et al., 2014a, 2014b; Blom et al., 2016) concluded that postruminal starch digestion mirrored measures of small intestinal starch digestion in cattle provided duodenal infusions of cornstarch alone or cornstarch and casein or glutamic acid, because of limits in large intestinal starch digestion. It seems likely that large intestinal starch digestion was also exceeded in this experiment, at least in part, because neither duodenal glutamic acid nor casein had any effect on fecal N excretion. The majority of N in feces comes from microbial sources (Van Soest, 1994). If infusion of glutamic acid or casein influenced large intestinal starch digestion then one would anticipate differences in fecal N excretion. An absence of response in fecal N output to infusion of glutamic acid or casein provides additional evidence that the overall capacity for large intestinal starch digestion was exceeded by large intestinal starch flows in this study.

Energy and nitrogen balance

Duodenal infusion of casein increased amounts of starch disappearance in the small intestine at a rate of 0.23 g starch per g of duodenal casein infused, but postgastric infusion of glutamic acid had no effect on small intestinal starch digestion. Postgastric infusion of casein and cornstarch corresponded with a 20% increase in digestible energy intake and a 24% increase in metabolizable energy intake in comparison to infusion of cornstarch alone. Overall, greater duodenal flow of casein together with cornstarch tended to increase retained energy in comparison to infusion of cornstarch alone, and measures of retained energy in cattle provided cornstarch and glutamic acid were intermediate to casein and control.

Energy often limits growth in cattle of similar mass to those used in this study (Lofgreen and Garrett, 1968; Owens et al., 1995; NASEM, 2016). Consequently, increases in energy available from the diet contribute to increases in protein accretion when metabolizable protein does not limit growth (NASEM, 2016). Diets fed to cattle in this experiment were designed to exceed metabolizable protein requirements by 20% and fed in amounts to meet 1.5 × maintenance energy requirements based on measures of body weight at the beginning of the experimental period to prevent limitations in synthesis and secretion of digestive enzymes. Previously, we reported that body weight gain was increased by greater duodenal flow of cornstarch and casein in comparison to cornstarch and glutamic acid or control (Trotta et al., 2020). Indeed, postruminal infusion of casein together with cornstarch resulted in a 1.36 Mcal/d increase in retained energy and a 21.3 g/d increase in N retention in comparison to control.

It seems that increases in retained energy in response to postruminal infusion of casein and cornstarch reflected greater amounts of energy retained from increases in small intestinal starch digestion together with increases in energy retained from casein used to support protein synthesis and lipogenesis. End products of digestion of protein (i.e., casein) and cornstarch (i.e., glucose) do not have the same heat of combustion (5.65 and 4.18 kcal/g, respectively; Brody, 1945; Blaxter, 1962), and increases in retained energy in response to duodenal infusion of cornstarch and casein likely represent increases in retained energy from metabolism of glucose, the end product of small intestinal starch digestion together with energy from amino acids in casein incorporated in protein and metabolism of amino acids provided from casein in excess of requirements for protein synthesis. If the average concentration of N in protein synthesized by cattle in this study was 17% (Kleiber, 1975), then increases in retained energy accounted for by increased protein accretion should have been near to 685 kcal. Similarly, the 94 g increase in small intestinal starch digestion in response to casein infusion compared to control could have accounted for an increase in retained energy of about 393 kcal. Yet, increases in small intestinal starch digestion in response to casein compared to control more likely accounted for increases in retained energy nearer to 236 kcal because others (Harmon and McLeod, 2001) reported that the partial efficiency with which metabolizable energy is converted to retained energy for postruminal starch in cattle is approximately 60%. Regardless, energy retained in cattle in this experiment clearly represented accretion of both lipid and lean tissues.

Overall, the apparent efficiency with which metabolizable energy was converted to retained energy in this experiment averaged 37.7 ± 1.3% and was not affected (P = 0.86) by postruminal infusion of nutrients. Based on library values (NASEM, 2016), the basal diet fed to cattle in this study should have supplied 7.49 Mcal of metabolizable energy to cattle, which would account for 69, 56, and 64% of the metabolizable energy intake for cattle provided control, casein or glutamic acid, respectively. The basal diet fed to cattle in this experiment was predominately composed of soybean hulls, which generally contributes to production of ruminal short-chain fatty acids mostly comprised of acetic acid (Ipharraguerre and Clark, 2003). Therefore, it is reasonable that measures of apparent efficiency in this study seemed to largely reflect the apparent efficiency with which two-component mixtures of short-chain fatty acids containing acetic acid and butyric acid are used by ruminants (Armstrong and Blaxter, 1957; Armstrong et al., 1958).

Classically, measures of energetic efficiency with which metabolizable energy is retained from individual nutrients have been calculated as energy retained divided by metabolizable energy supplied by the nutrient (Kellner, 1900; Brody, 1945; Blaxter, 1962). Differences in energy retained and metabolizable energy intake relative to control for cattle provided postgastric infusions of cornstarch and casein or cornstarch and glutamic acid corresponded to a partial ­efficiency of energy retained from postgastric nutrient infusions of 52.9% and 50.0%, respectively. Harmon and McLeod (2001) calculated a partial efficiency of energy retention from abomasal infusions of a starch hydrolysate of approximately 60%. Measures of partial efficiency of energy retention from postgastric infusions were less in this study compared to the results of Harmon and McLeod (2001) even though postgastric infusion of casein and cornstarch resulted in an increase in small intestinal starch digestion. It is possible that measures of partial efficiency of energy retention in this study in response to postgastric infusion of cornstarch and casein were less in comparison to those reported by Harmon and McLeod (2001) because of relatively larger increases in heat increment associated with metabolism of protein provided in excess of requirements for protein synthesis (Blaxter, 1962); however, it seems more likely that the relatively lower measures of partial efficiency of energy retention were a result of amounts of starch fermented in the large intestine. While small intestinal starch digestion can provide greater energy in comparison to ruminal fermentation, large intestinal fermentation of starch can contribute to less amounts of energy retained by increasing fecal energy losses.

A lack of effect of postgastric nutrient infusion on apparent efficiency of energy retention in this experiment implies that, even though changes in postgastric nutrient flows influenced small intestinal starch digestion and energy retained, composition of body gains were likely not affected by treatments. It is, however, possible that greater postgastric flows of casein with cornstarch, but not glutamic acid influenced site of adipose accretion in this experiment. McLeod et al. (2006) reported that about 33% of increases in retained energy from small intestinal glucose absorption were accounted for by increases in adipose associated with alimentary tissues. Increased amounts of visceral adipose have been reported to contribute to various metabolic disturbances in nonruminants (Nam, 2017) that if conserved in ruminants could reduce the benefits of small intestinal starch digestion (e.g., reduced nutrient absorption, fatty liver, pancreatic dysfunction). Small intestinal mucosa predominately metabolize glucose to lactate (El-Kadi et al., 2009), which can suppress lipolysis in adipose tissues (Liu et al., 2009). Alternatively, glutamic acid is the primary anaplerotic substrate in bovine small intestinal mucosa (El-Kadi et al., 2009), and Harmon (2009) concluded that energy supplied from greater postruminal flows of protein to the small intestine was a controlling factor for small intestinal starch digestion in cattle. It seems that factors other than greater postruminal flows of energy from protein or changes in substrate available for oxidation to small intestinal mucosa contributed to increases in small intestinal digestion in this study, because greater postruminal flows of glutamic acid did not affect small intestinal starch digestion or energy retention.

Conclusions

In this study, postgastric infusion of casein in addition to cornstarch reduced ileal starch flows and tended to increase measures of small intestinal starch disappearance. Correspondingly, postgastric infusion of casein in addition to cornstarch increased digestible and metabolizable energy and tended to increase retained energy. Postgastric infusion of casein in addition to cornstarch also increased amounts of N retained when compared with postgastric infusions of glutamic acid and cornstarch or cornstarch alone. A lack of effect of postgastric infusion of glutamic acid on measures of small intestinal starch disappearance differ from previous reports. Differences in responses in small intestinal nutrient disappearance in this study compared to previous reports together with a lack of effect of postgastric flows of glutamic acid on measures of energy or nitrogen balance may suggest that effects of increased postgastric flows of glutamic acid in cattle are transient, but that effects of casein are not.

Glossary

Abbreviations

DMI

dry matter intake

MBW

metabolic body weight

RQ

respiratory quotient

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.