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Ruminal and host adaptations to changes in frequency of protein supplementation^1,2

C. G. Farmer^*, R. C. Cochran^3,*, T. G. Nagaraja, E. C. Titgemeyer^*, D. E. Johnson and T. A. Wickersham^*

^* Departments of Animal Sciences and Industry, and Diagnostic Medicine/Pathobiology, and and Statistics, Kansas State University, Manhattan 66505

Abstract

The effect of altering supplementation frequency on host N balance and key N transactions in the ruminal ecosystem were monitored. Four ruminally fistulated beef steers (BW = 513 kg; SEM = 6.5) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Supplementation frequencies were 2 and 7 d/wk. Steers were fed tallgrass prairie hay (73.1% NDF, 5.3% CP) ad libitum. Supplement (42% CP; DM basis) was fed at 0.36% BW/d to steers supplemented 7 d/wk. Steers supplemented 2 d/wk received the same amount of supplement per week, but it was equally split among the two supplementation events. Steers supplemented 7 d/wk had higher forage (P < 0.02) and total digestible OM intake (P < 0.06), total N intake, fecal N excretion, and N retention. Although both supplementation frequencies were characterized by positive N balance, the decrease in N retention in the steers supplemented 2 d/wk was due to higher (P < 0.01) urinary N loss. Ruminal fluid was sampled at 0, 2, 4, 6, 12, 24, 48, and 72 h after supplementation beginning on a day when both treatments were supplemented. Frequency x hour interactions (P < 0.02) were observed for ruminal N metabolism criteria. Counts of peptide- and AA-fermenting bacteria peaked at 2 h and returned to nadir by 12 h for steers supplemented 7 d/wk. Steers supplemented 2 d/wk peaked at 6 h with a greater population and returned to nadir at 48 h. Ruminal ammonia concentrations followed a similar trend. Specific activity of ammonia production was lower (P 0.05) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h was the same as for 7 d/wk steers. Ruminal peptides and free AA peaked at 2 h for steers supplemented 2 d/wk and were generally higher (P 0.05) during the first 6 h compared with steers supplemented 7 d/wk. Total VFA concentration was not different (P = 0.35) due to supplementation frequency. Frequency x hour interactions (P < 0.01) were observed for all molar proportions of VFA. The molar proportion of acetate and acetate:propionate ratio were lower (P < 0.01) and the molar proportions of propionate and butyrate were higher for steers supplemented 2 d/wk from 4 h to 24 h. In conclusion, forage use and N balance improved with supplementation 7 d/wk, but supplementation 2 d/wk was associated with some desirable shifts in select ruminal events that may contribute to moderating potential negative impacts of supplementing infrequently.

Key Words: Ammonia • Forage • Frequency • Nitrogen • Supplementation • Volatile Fatty Acids

Introduction

Although daily delivery of high-protein (30% CP) supplements to beef cattle consuming low-quality forage has maximized forage utilization compared with less-frequent supplementation (Farmer et al., 2001b; Bohnert et al., 2002), it appears that some mechanism(s) exists that buffer the impact of infrequent supplementation and thereby minimize differences in performance of cattle supplemented at different frequencies (Melton and Riggs, 1964; Beaty et al., 1994; Huston et al., 1999). One mechanism that buffers the impact of infrequent supplementation is N recycling. In addition, it is possible that some changes in select intraruminal events may also play a role in buffering the effect of infrequent supplementation. For example, Beaty et al. (1994) and Farmer et al. (2001b) documented a lag in peak ruminal ammonia concentration and a prolonged elevation (up to 24 h after supplementation) of ammonia with less frequently supplemented (2 or 3 d/wk) cattle compared with cattle supplemented daily. It is feasible that such patterns reflect both recycling as well as time-series differences in the prevalence of ruminal bacteria that can ferment peptides and free AA and produce ammonia (Yang and Russell, 1993). An attenuated peak and prolonged maintenance of elevated ruminal ammonia would facilitate the maintenance of fibrolytic activity and the conservation of nitrogen, thereby reducing potential negative effects from infrequent supplementation. However, such ruminal changes (i.e., changes in ruminal microbial populations and the fate of nitrogen in the rumen) in cattle supplemented at different frequencies have not been thoroughly described. Therefore, the objective of this study was to characterize ruminal nitrogen transactions in cattle receiving protein supplements at very different frequencies (7 d/wk vs. 2 d/wk). In addition, the impact on whole-animal N balance was monitored.

Materials and Methods

Four ruminally fistulated, Hereford x Angus steers (average initial BW = 513 kg) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Given that each steer received each treatment, there were ultimately four replications per treatment. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Kansas State University and included the use of anesthesia when surgical procedures were performed. Each steer was kept in an enclosed barn in tie stalls (1.2 m x 2 m) with ad libitum access to fresh water and plain white salt and offered tallgrass-prairie hay (Table 1) at 130% of its average voluntary intake for the preceding 5 d. Each steer was given an intramuscular vitamin injection (vitamin A, D, and E; Vital E A+D, Schering-Plough Animal Health, Omaha, NE) at the initiation of the trial. The two frequencies of supplementation evaluated were 2 and 7 d/wk. The same high-protein supplement was used for both supplementation frequency treatment groups (Table 1). Supplement composition (DM basis) was: 68.2% soybean meal, 22.3% ground sorghum grain, 4.5% cane molasses, and 4.5% mineral mix. Supplement was fed to deliver enough degradable intake protein (DIP) to maximize estimated total digestible OM intake (TDOMI) of a low-quality, tallgrass-prairie diet (Köster et al., 1996). Supplement was fed at 0.36% of BW/d (DM basis) for steers supplemented 7 d/wk and 1.26% of BW on Tuesdays and Fridays (DM basis) for steers supplemented 2 d/wk. Both treatment groups received the same quantity of supplement on a weekly basis, but the weekly allotments were divided into equal portions in accordance with the number of supplementation events each week. Supplements were fed at 0630, and prairie hay was offered at approximately 0700 (supplements were completely consumed before forage was fed). Orts were collected and weighed daily for each steer before supplements and hay were offered. The hay fed to the steers was ground to pass through a 75 x 75 mm screen.

Subsequent to intake and excreta output determinations, ruminal evacuations were performed with the steers on d 23, 24, and 25 for determination of ruminal ADIA passage rate. These days were used to represent the day of supplementation, the day after supplementation, and 2 d after supplementation for the group supplemented 2 d/wk. On each day, evacuations were performed immediately before feeding and 4 h after feeding. At each evacuation, ruminal contents were weighed, hand mixed, and sampled in triplicate. Ruminal digesta samples were dried at 55°C for 96 h in a forced-air oven. Following partial DM determination, ruminal digesta samples were ground (No. 2 Wiley mill) to pass a 1-mm screen. The triplicate samples were composited within steer and time period. Passage rate of ADIA was calculated by dividing the intake rate of ADIA (kg/h) by the ADIA amount in the rumen (kg). Intake rate of ADIA was measured by determining the average daily ADIA intake over the preceding 7-d period and dividing it by 24.

Subsequent to ruminal evacuations, d 26, 27, and 28 were used to measure ruminal liquid passage rate. Again, these days were used to represent the day of supplementation, the day after supplementation, and 2 d after supplementation for the group supplemented 2 d/wk. On each day, steers were dosed with 1.13 g of Cr (as CrEDTA; Binnerts et al., 1968) in 500 mL of deionized water as a marker. Dosing was performed just before feeding with the CrEDTA solution being deposited into various locations in the rumen of each steer via tubing attached to a funnel. Twenty milliliters of each ruminal fluid sample was collected prior to dosing (0 h) and at 3, 6, 9, 12, and 24 h after dosing with a suction strainer (Raun and Burroughs, 1962; 19 mm diameter; 15 mm mesh). These samples were then immediately frozen and retained for Cr analysis. Liquid passage rate was calculated by regressing the natural logarithm of the Cr concentrations (based on samples taken at 3, 6, 9, 12, and 24 h after supplementation) measured in ruminal fluid on time of sampling (Warner and Stacy, 1968).

Subsequent to sampling for liquid passage determination, an additional ruminal sampling period (d 37 through 40) was used to monitor select microbial changes in the rumen as well as changes in key nitrogenous constituents and VFA. Ruminal samples were taken immediately before supplementation (0 h) and at 2, 4, 6, 12, 24, 48, and 72 h after supplementation, relative to d 37 for both treatment groups. Ruminal fluid samples were collected with a suction strainer (Raun and Burroughs, 1962; 19 mm diameter; 15 mm mesh). At each sampling hour, the following procedures were accomplished. Ruminal pH was measured with a portable pH meter equipped with a combination electrode (Orion Research, Boston, MA) immediately after each sample was collected. Eight milliliters of ruminal fluid from each sample was added to 2 mL of 25% (wt/vol) metaphosphoric acid and frozen for subsequent VFA analysis. Two milliliters of ruminal fluid was added to 8 mL of 0.1 N HCl and frozen for subsequent ammonia analysis. Twenty milliliters of ruminal fluid was collected for peptides and free AA and prepared for analysis according to Chen et al. (1987). These samples were immediately centrifuged at 500 x g for 20 min to remove protozoa and feed particles. The supernatant was then centrifuged at 30,000 x g for 15 min to remove bacteria. The supernatant was mixed with HClO₄ (perchloric acid) and then frozen for subsequent analysis of peptides and AA.

At the same times that samples were collected for analyses of peptides and free AA, approximately 10 mL of ruminal fluid was transported in an anaerobic (CO₂-filled) screw-cap tube to the laboratory for enumeration of peptide- and AA-fermenting bacteria (Yang and Russell, 1993). Serial 10-fold dilutions of the sample were made in anaerobic dilution blanks (Anderson et al., 1987) and 0.5 mL of each dilution was inoculated in triplicate into medium 10 of Caldwell and Bryant (1966) containing 15 g/L Trypticase (BD Diagnostic Systems, Franklin Lakes, NJ) and 15 g/L Caseamino acids (BD Diagnostic Systems) as major sources of energy. The inoculated tubes were incubated for 2 wk at 39°C and enumeration was made by the most probable number method (Yang and Russell, 1993). Also, at each sampling hour, specific activity of ammonia production was determined according to Yang and Russell (1993). Ruminal fluid was strained through four layers of cheesecloth into a 500-mL flask, stoppered, and incubated for 1 h at 39°C to allow for separation of the bacterial fraction from protozoa and feed particles (which fall to the bottom of the flask). Twenty milliliters of ruminal fluid from the bacterial fraction was incubated at 39°C for 6 h with 62.5 mg of casein hydrolysate (Sigma Chemicals, St. Louis, MO) in triplicate. Ruminal fluid samples (2 mL) collected before (0 h) and after (6 h) incubation were acidified with 0.1 N HCl (8 mL) and frozen for subsequent ammonia analysis. Also, with 10 mL of rumen fluid from the bacterial fraction of the ruminal fluid sample, bacterial cells were collected by centrifugation at 10,300 x g for 20 min. The cells were then washed twice with 1 mL of 0.9% saline solution before freezing for the bacterial cell protein assay.

Laboratory Analyses
Ground forage, supplement, orts, and feces were dried overnight (16 h) at 105°C in a convection oven for determination of DM and were ashed at 450°C for 8 h in a muffle oven for determination of ash. The N contents of ground forage, supplement, orts, wet feces, and urine were determined by combustion (nitrogen analyzer; LECO FP-2000; St. Joseph, MI). The NDF concentrations of the ground forage, supplement, orts, and feces were determined as described by Van Soest et al. (1991). The analyses were conducted without sodium sulfite; however, amylase (Sigma Chemical) was added to the NDF solution (5 mL/100 mL) to aid in filtering. All NDF values included ash. The DIP content of forage and orts was determined by the 48-h Streptomyces griseus protease procedure described by Mathis et al. (2001). Supplemental DIP concentration was calculated using published values for the concentration of DIP in the constituent feedstuffs of the supplement (NRC, 1996).

After thawing, perchloric acid-treated supernatant from samples used to determine peptides and free amino acids were centrifuged at 20,000 x g for 20 min as described by Chen et al. (1987). Ten milliliters of this supernatant was added to 0.93 mL 6 N KOH. Samples were kept on ice for 30 min and then centrifuged at 20,000 x g for 20 min. These samples were used to measure -amino nitrogen and ammonia via a colorimetric procedure. Finally, 2.5 mL of the same supernatant was added to 2.5 mL of 12 N HCl in a screw-cap tube. The tubes were heated to 105°C for 22 h. After removal from the oven, 5 mL of 6 N NaOH was added to the tubes. This solution was centrifuged at 20,000 x g for 20 min and the supernatant was used for determining the increase in amino acid concentration resulting from peptide hydrolysis.

After thawing, the bacterial cells were mixed with 0.2 N NaOH (10 mL) and heated to 100°C for 15 min. Cell protein was then measured using the bicinchoninic acid procedure (Pierce Kit No. 23225, Rockford, IL) with BSA as the standard (Smith et al., 1985).

After thawing, ruminal fluid samples used for ruminal ammonia, VFA, and liquid passage rate were centrifuged at 30,000 x g for 20 min. Ruminal ammonia concentrations were determined in the supernatant using a colorimetric procedure (Broderick and Kang, 1980). Ruminal VFA were measured in the supernatant by gas chromatography (model 5890, Hewlett-Packard, Avondale, PA) as described by Vanzant and Cochran (1994). The Cr concentration in the supernatant was determined by atomic absorption spectrophotometry with an air/acetylene flame (model 3110, Perkin-Elmer, Norwalk, CT).

Statistical Analyses
Intake, digestion, and nitrogen retention data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). An F-test was used to assess treatment effects. Model terms were sequence (i.e., order in which treatments were applied), supplementation frequency, and period. The random term in the model was steer nested within sequence. Treatment means were determined using the LSMEANS procedure. The MIXED procedure of SAS was used to analyze ADIA passage rate and liquid passage rate. An F-test was used to assess treatment effects. Model terms were sequence, supplementation frequency, period, day, and supplementation frequency x day. Day served as a repeated measure. Supplementation frequency x steer within sequence served as the subject in the repeated measures statement for testing day and supplementation frequency x day. The covariance structure used in the repeated measures analysis was compound symmetry. Treatment means were determined using the LSMEANS procedure. Contrasts were used to examine the linear and quadratic responses across different days for passage data. The MIXED procedure was also used to analyze ruminal fermentation characteristics, which were measured at different hours after supplementation. An F-test was used to measure treatment effects. Model terms were sequence, supplementation frequency, period, hour, and supplementation frequency x hour. Hour served as a repeated measure. Supplementation frequency x steer within sequence served as the subject for the repeated-measures analysis used for testing hour and supplementation frequency x hour. The covariance structure used in the repeated measures analysis was compound symmetry. Treatment means were determined using the LSMEANS procedure. When the frequency x hour interaction was significant, pairwise comparisons of treatment means within a given hour were made using the PDIFF option to determine significant changes in ruminal fermentation characteristics over time.

Results

Forage and total OM intake were lower (P = 0.02), and TDOMI tended to be lower (P = 0.06) for steers supplemented 2 d/wk (Table 2). Total-tract OM (P = 0.77) and NDF (P = 0.72) digestion did not differ as a result of supplementation frequency.

Steers supplemented 2 d/wk had lower (P < 0.01) N intake (due to lower forage intake) and fecal N excretion (Table 2). However, there was no difference (P = 0.25) in absorbed nitrogen due to supplementation frequency. Steers supplemented 2 d/wk had higher (P < 0.01) urinary N excretion and, therefore, lower (P = 0.01) overall grams of N retained. Similarly, steers supplemented 2 d/wk had lower N retention as a proportion of N intake (P = 0.02) and N retention as a proportion of N absorbed (P < 0.01).

There was a supplementation frequency x hour interaction (P = 0.02) for the most probable number of peptide- and AA-fermenting bacteria (Figure 1). The counts peaked at 2 h after supplementation and returned to nadir by 12 h after supplementation for steers supplemented 7 d/wk, whereas steers supplemented 2 d/wk peaked at 6 h after supplementation and returned to nadir by 48 h after supplementation. There was also a supplementation frequency x hour interaction (P < 0.01) for specific activity of ammonia production (Figure 1). Specific activity of ammonia production was lower (P 0.01) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h after supplementation differences were not significant among treatments.

View larger version (21K): [in this window] [in a new window]   Figure 1. Influence of supplementation frequency on peptide- and amino acid-fermenting bacteria and specific activity of ammonia production by ruminal bacteria for beef steers fed low-quality, tallgrass prairie hay. Frequency x hour interactions (bacterial concentrations, P = 0.02); specific activity of ammonia production (P < 0.01, SEM = 0.82). *Represents difference (P 0.05) between supplementation frequencies at each time period.

View larger version (15K): [in this window] [in a new window]   Figure 2. Influence of supplementation frequency on ruminal ammonia, free AA, and peptides for beef steers fed low-quality, tallgrass prairie hay. Frequency x hour interactions (ammonia [P < 0.01], SEM = 0.67; free AA [P < 0.01], SEM = 0.16; peptides [P = 0.01], SEM = 0.24). *Represents difference (P 0.05) between supplementation frequencies at each time period.

View larger version (15K): [in this window] [in a new window]   Figure 3. Influence of supplementation frequency on the molar proportions of acetate, propionate, and the acetate:propionate ratio for beef steers fed low-quality, tallgrass-prairie hay. Frequency x hour interactions (acetate [P < 0.01], SEM = 0.49; propionate [P < 0.01], SEM = 0.27; acetate:propionate ratio [P < 0.01], SEM = 0.11). *Represents difference (P 0.05) between supplementation frequencies at each time period.

View larger version (15K): [in this window] [in a new window]   Figure 4. Influence of supplementation frequency on the molar proportions of butyrate, isobutyrate, and isovalerate for beef steers fed low-quality, tallgrass-prairie hay. Frequency x hour interactions (butyrate [P < 0.01], SEM = 0.37; isobutyrate [P < 0.01], SEM = 0.043; isovalerate [P < 0.01], SEM = 0.050). *Represents difference (P 0.05) between supplementation frequencies at each time period.

The increase in forage OM intake and TDOMI with the increase in supplementation frequency is consistent with other supplementation frequency research with low-quality forage as the basal diet (Beaty et al., 1994; Farmer et al., 2001b; Bohnert et al., 2002). Previously, Farmer et al. (2001b) and Krehbiel et al. (1998) observed that forage DM intake was most depressed on the day of supplementation for groups supplemented infrequently (i.e., 2 d/wk or every third day). This observation was suggested to account for much of the overall depression in forage intake. Although we did not observe a significant frequency x day interaction with regard to forage intake, our data tended to display (data not shown) a similar pattern to that described by Farmer et al. (2001b) and Krehbiel et al. (1998). Forage OM and NDF digestion in our study were consistent with values observed when ruminal nitrogen deficiency has been corrected in similar forage-based diets via protein supplementation (Köster et al., 1996; Heldt et al., 1999). However, OM and NDF digestion were not different due to supplementation frequency. The ability to sustain forage fiber digestion, even when supplemental protein is provided infrequently, attests to the fact that ruminants possess mechanisms that buffer the effects of infrequent nutrient supply.

Decreasing supplementation frequency resulted in a decrease in nitrogen retention in our study. This concurs with the findings of Bohnert et al. (2002), who fed sheep low-quality forage and provided supplement daily, every third day, or every sixth day. In contrast, Coleman and Wyatt (1982) observed no difference in N retention when beef steers were supplemented daily, on alternate days, and every fourth day. However, their basal forage was 8% CP and their average daily supplement allotment was only about half (0.15 vs. 0.36% BW) of that offered in our study. These factors may have moderated potential negative effects on N use efficiency in their study. For example, the lower amount of supplemental protein may have been less likely to produce the large spike in ruminal ammonia concentration seen with large doses of protein supplement at a single event. Given that the amount of N absorbed was similar for steers supplemented at the different frequencies in our study, the increased urinary nitrogen excretion in the 2 d/wk group (and, hence, lower N retention) verifies an inability to capitalize on the erratic nitrogen supply with the same efficiency as when the nitrogen supply is more consistent. It is possible that the urea threshold was exceeded during brief periods of surplus N, which resulted in increased excretion of urea in the urine (VanSoest, 1994). However, the fact that the 2-d/wk group was able to maintain positive nitrogen balance in the face of erratic N supply does highlight the ability of these animals to conserve nitrogen. Certainly, the ability to recycle N is known to be pivotal in this regard (Lapierre and Lobley, 2001). In fact, it is possible that under more extreme conditions (e.g., supplementing at levels known to be significantly less than the requirement), differences in N balance may be less evident between different supplementation frequencies due to increased efficiency in the use of recycled N.

Ruminal catabolism of short peptides and AA are carried out by two groups of bacteria (Wallace et al., 1997). The first group is comprised of species of ruminal bacteria that are primarily carbohydrate fermenters but possess low deaminase activity. The second group, referred to as hyper ammonia-producing bacteria, is comprised of a smaller population that is asaccharolytic and possesses high deaminase activity (Chen and Russell, 1989). The procedure used in this study to measure the bacterial population that can degrade short peptides and free amino acids does not discriminate between populations that have either high or low specific activities of ammonia production. Russell et al. (1988) and Chen and Russell (1989) indicated that three species of hyper ammonia-producing bacteria (Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum) had an average of 20-fold higher specific activity for ammonia production compared with other ammonia producers of the rumen. Given that specific activity was lower in the 2 d/wk group during the first few hours after supplementation in the prescence of increasing ruminal ammonia and overall numbers of peptide- and AA-fermenting bacteria, may indicate that the group with low deaminase activity was more prevalent in the 2 d/wk group immediately after supplementation. Also, because the low-activity, ammonia-producing bacteria are also carbohydrate fermenters, it may have been that fermentation activities were heavily oriented toward carbohydrate fermentation in the first few hours after supplementation in the 2-d/wk group due to the large influx of carbohydrate at that time. Given that specific activity was low in the 2-d/wk group in the first 12 h after supplementation it appears that the rapid increase in ammonia during this period was accomplished via low-activity ammonia producing bacteria. A significant accumulation of peptides and free AA, substrate for hyper ammonia-producing bacteria, was observed at 2 h after supplementation for the steers supplemented 2 d/wk. This also suggests that a lower number of the hyper ammonia producers may have been present immediately after supplementation for this group. Perhaps the population of hyper ammonia-producing bacteria began to increase in these steers in response to the presence of their preferred substrate. The potential attenuation in hyper ammonia-producing bacteria may have contributed to the observed lag in peak ruminal ammonia for the group supplemented 2 d/wk. However, the diversion of fermentative activity in the low-deaminase population toward carbohydrate fermentation immediately following supplementation also may have contributed to the delay in peak concentration. The overall pattern of ruminal ammonia concentration for steers offered a relatively large amount of high-protein supplement at infrequent intervals (i.e., delayed peak concentration, reduced peak amplitude, and prolonged elevation of concentration before returning to nadir) is similar to that reported in other supplementation frequency studies (Beaty et al., 1994; Farmer et al., 2001b). The lack of a dramatic increase in ruminal ammonia may have facilitated improved nitrogen conservation for steers supplemented less frequently. The extended, elevated concentrations of peptide- and AA-fermenting bacteria in the rumens of steers supplemented 2 d/wk also may have served as a N source (as cells died and were lysed) along with N recycling to maintain higher ruminal ammonia levels over an extended period of time in the less frequently supplemented steers. For less frequently supplemented animals, maintaining ruminal ammonia concentration between supplementation events would be beneficial to fibrolytic bacteria and help maintain their fiber digestion because of their preference for ammonia as a nitrogen source (Russell et al., 1992).

The most probable number of peptide- and AA-fermenting bacteria at nadir (immediately before supplementation) was not different between steers supplemented 2 and 7 d/wk. The rapid increase in peptide- and AA-fermenting bacteria in the 7 d/wk group accompanied by higher specific activity of ammonia production suggests that some amount of hyper ammonia-producing bacteria were present and ready to respond to moderate influx of protein associated with daily feeding. It also appears that there may have been an increase over time of hyper ammonia producers, which resulted in similar specific activity of ammonia production among treatments by 12 h after supplementation.

The positive association of higher molar proportions of propionate, lower molar proportions of acetate, and lower acetate:propionate ratios with infrequent supplementation, particularly during the 24-h period following supplementation, concurs with previous research (Farmer et al., 2001a). This association is consistent with reports that ruminal starch infusion significantly elevated the molar percentage of propionate and decreased the proportion of acetate in the rumen (Olson et al., 1999; Klevesahl et al., 2003). The extended improvement in the acetate:propionate ratio may be a contributing factor to the maintenance of acceptable performance of beef cattle even when intake is somewhat depressed with less frequent supplementation. One mechanism by which this could occur is by the direction of reducing equivalents into propionate, thereby maintaining conditions favorable to the maintenance of robust ruminal fermentation (Baldwin and Allison, 1983) and, potentially, diverting them from incorporation into methane. In addition, fermentations with increased propionate relative to acetate have been shown to be more efficiently used by the animal for productive purposes compared with fermentations characterized by less propionate relative to acetate (Blaxter, 1989).

It is well established that branched-chain VFA serve as growth factors for fibrolytic bacteria (Baldwin and Allison, 1983). It is also clear that provision of precursors of branched-chain VFA in the form of ruminally degradable true protein is associated with significant increases in branched-chain VFA proportions in the rumen (Köster et al., 1996; Olson et al., 1999). In our study, it is interesting to note that time-series changes in branched-chain VFA were affected by supplementation frequency and appeared to follow a pattern that was somewhat similar to the changes in specific activity of ammonia production. Russell et al. (1988) and Chen and Russell (1989) reported that the end products of at least two of the hyper ammonia-producing bacteria included branched-chain VFA. It is feasible that changes in the populations of these organisms may have affected the time-series patterns in branched-chain VFA proportions across treatments.

Implications

Daily provision of a high-protein supplement to cattle subsisting on low-quality forage increases total digestible organic matter consumed daily compared with less frequent supplementation schedules. Moreover, efficiency of utilization of dietary nitrogen consumed was positively associated with an increased frequency of feeding of such supplements. However, it seems that select ruminal events (notably time series responses for ruminal ammonia and select volatile fatty acids) may contribute to buffering the effects of infrequent supplementation on forage utilization (particularly fiber digestion). Nitrogen recycling plays a role in this regard; however, time series responses in the population of peptide- and amino acid-fermenting bacteria may be important as well.

Footnotes

¹ Article No. 03-232-J from the Kansas Agric. Exp. Stn.

² The authors graciously thank N. Wallace for his assistance.

³ Correspondence—e-mail: rcochran{at}oznet.ksu.edu.

Received for publication June 15, 2003. Accepted for publication November 10, 2003.

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