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J. Anim. Sci. 2002. 80:3336-3346
© 2002 American Society of Animal Science

Effects of oscillating dietary protein on ruminal fermentation and site and extent of nutrient digestion in sheep1

P. A. Ludden2, T. L. Wechter and B. W. Hess

Department of Animal Sciences, University of Wyoming 82071-3684

2 Correspondence:
AS/MB Rm 123B (phone: (307) 766-4213; fax: (307) 766-2355; E-mail:
ludden{at}uwyo.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Eight cannulated wethers (BW = 52.5 ± 5.7 kg) were used in a replicated 4 x 4 Latin square designed experiment to evaluate the effects of oscillating dietary protein concentrations on ruminal fermentation, site and extent of digestion, and serum metabolite concentrations. Four treatments consisted of a 13, 15, or 17% CP diet fed daily or a regimen in which dietary CP was oscillated between 13 and 17% on a 48-h basis (ACP). All diets consisted of 65% bromegrass hay (10.5% CP, 61.9% NDF, 37.2% ADF) plus 35% corn-based supplement and were formulated to contain the same amount of degradable intake protein (9.6% of DM) plus additional undegradable intake protein (SoyPLUS, West Central Cooperative, Ralston, IA) to accomplish CP levels above 13%. Each of four experimental periods were 16 d in duration with 12 d for diet adaptation followed by 4 d for sample collection. All wethers were fed at 3.0% of initial BW (DM basis) throughout the experiment, resulting in an average organic matter intake of 1.39 kg/d across treatments. When compared to the 15% CP daily treatment, feeding ACP had no effect (P >= 0.10) on ruminal or lower tract N, NDF, ADF, or OM digestion. True ruminal OM digestion responded quadratically (P = 0.07) to increasing dietary CP, reaching a maximum of 52.0% of OM intake with the 15% CP treatment. Sheep fed ACP tended to have lower (P = 0.08) ruminal NH3 N concentrations and an overall higher (P = 0.0001) molar proportion of acetate compared to those fed 15% CP daily. Total VFA concentrations were not affected (P >= 0.45) by increasing dietary CP. Microbial efficiency did not differ (P >= 0.55); thus, bacterial N flow at the duodenum responded quadratically (P = 0.04) to increasing dietary CP. Nonbacterial N (P = 0.001) and total N (P = 0.01) flows at the duodenum and total tract N digestibility (P <= 0.04) increased linearly as dietary CP increased. Wethers fed ACP maintained a lower (P = 0.002) serum glucose and lower (P = 0.0006) serum urea N compared to those fed 15% CP daily. Because the CP content of the diet was increased at the expense of corn, the response to increased CP observed in this experiment is most likely due to negative associative effects of supplemental starch on ruminal fermentation and microbial growth. Oscillating the CP content of the diet on a 48-h basis has little effect on digestion or N utilization in sheep compared with feeding the same quantity of protein on a daily basis.

Key Words: Digestibility • Protein • Sheep


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Winter supplementation of ruminant livestock with protein, energy, or both is often required to maintain desired levels of production. Protein supplementation has been shown to increase cow weight gain and body condition score (Clanton and Zimmerman, 1970; Rusche et al., 1993, Beaty et al., 1994) and forage intake and digestibility (Kartchner, 1980; Köster et al., 1996). However, the sustainability of such practices is often limited by the cost of supplemental feed inputs, as well as the labor and equipment required for supplement delivery. Decreasing the frequency of supplementation, in some cases up to weekly intervals, is one management practice that decreases labor and equipment costs, while producing little detrimental effect on animal performance (McIlvain and Shoop, 1963; Coleman and Wyatt, 1982; Hunt et al., 1989). Hunt et al. (1989) suggested that alternate day protein supplementation may stimulate recycling of endogenous N into the rumen thereby enhancing N utilization. Krehbiel et al. (1998) demonstrated that mature ewes receiving supplemental protein every 3 d had greater transfer of urea to the rumen vs those supplemented daily. Similarly, Cole (1999) found that oscillating the CP content of the diet at 48-h intervals resulted in improved N retention in lambs, which was further enhanced with provision of natural protein vs urea. Our hypothesis is that intermittent supplementation with undegradable intake protein (UIP; Collins and Pritchard, 1992) may further enhance N utilization by providing the animal with a source of recyclable N thereby enhancing fiber utilization and microbial protein synthesis in the rumen. This study was conducted to examine the effects of oscillating dietary protein with a UIP source on ruminal fermentation and site and extent of nutrient digestion in sheep.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
Animals and Diets
Eight mature wethers (52.5 ± 5.7 kg) previously fitted with ruminal and T-type cannulas in the proximal duodenum and terminal ileum were used in a replicated 4 x 4 Latin square design experiment. All animal care followed procedures that were previously approved by the University of Wyoming Animal Care and Use Committee. The wethers were maintained in individual metabolism crates (1.4 x 0.6 m) under continuous lighting and allowed free access to water. The wethers were weighed at the beginning of the experiment, and DM intake was restricted to 3.0% of initial BW throughout the trial. The diets (Table 1Go) were fed once daily at 0700 and consisted of 65% chopped (2.54 cm) bromegrass hay (10.5% CP, 61.9% NDF, 37.2% ADF; DM basis) and 35% corn-based supplement. Four treatments consisted of a 13, 15, or 17% CP diet fed daily or a regimen in which dietary CP was oscillated between 13 and 17% on a 48-h basis (i.e., 13, 13, 17, 17, 13, 13...; ACP). The diets were formulated to contain the same amount of degradable intake protein (DIP; 9.6% of DM) using a combination of soybean meal and urea. This level was chosen because other research has demonstrated that DIP at this level will support maximal forage digestion (Hollingsworth-Jenkins et al., 1996; Mathis et al., 2000). Treatment CP concentrations above 13% were accomplished with the addition of a UIP source (SoyPLUS, West Central Cooperative, Ralston, IA) to replace a portion of both the corn and soybean meal/urea. The resulting diets were approximately isoenergetic on a TDN basis (65.6 ± 0.3% TDN) based upon tabular values. Ammonium chloride was included in the supplement at 0.5% of the total diet DM to avoid problems associated with urinary calculi. The concentrate portion of the diet (35% of diet DM) was top-dressed at feeding.


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  		Table 1. Composition of diets fed to cannulated lambs
 
Sample Collection and Analyses
Experimental periods were 16 d in duration with 12 d for diet adaptation followed by 4 d for sample collection. In the ACP treatment, wethers received 13% CP on sampling d 1 and 2 and 17% CP on d 3 and 4 during each collection period. Animals were removed from metabolism crates for 1 d and allowed to exercise in a dry lot between experimental periods. Chromic oxide (5.0 g/d) was dosed intraruminally via gelatin capsules in two equal portions at 0700 and 1400 daily throughout the trial. On d 13 to 16, duodenal (approximately 100 mL), ileal (approximately 100 mL), ruminal fluid, and blood samples were collected at 4-h intervals with collection times advanced 1 h daily to give a sample for every 1-h interval in a theoretical 24-h clock. Duodenal and ileal samples were composited by animal, frozen (-10°C), and later freeze-dried (Genesis SQ Super ES Freeze Dryer, The Virtis Co., Gardiner, NY) and ground (1-mm screen).

Approximately 15 mL of ruminal fluid was collected using a suction strainer (Precision Machine Co., Inc., Lincoln, NE), and pH was measured immediately using a portable pH meter (Accumet Model AP5 Portable pH meter, Fischer Scientific, Pittsburgh, PA). This ruminal fluid sample (15 mL) was acidified with 1.0 mL of 6 N H2SO4 and frozen (-10°C); this sample was later thawed at room temperature, centrifuged at 10,000 x g for 10 min, and a portion of the supernatant was analyzed for NH3 using the phenol-hypochlorite assay (Broderick and Kang, 1980). The resulting NH3 concentrations were converted to NH3 N for statistical analysis, and NH3 N concentrations are reported in Table 5Go. Ruminal fluid samples representing 3, 6, 9, 12, 15, 18, 21, and 24 h after feeding were selected for analysis of VFA concentrations. Samples collected at 3, 6, 15, and 18 h after feeding corresponded to days when ACP-fed lambs received the 13% CP diet, whereas the remaining samples corresponded to days when ACP-fed lambs received the 17% CP diet. A 2.5 mL aliquot of this sample was mixed with 0.5 mL of metaphosphoric acid solution (25% wt/vol) containing 2 g/L of 2-ethyl butyrate as an internal standard (Goetsch and Gaylean, 1983), and the mixture was centrifuged at 10,000 x g for 10 min. The resulting supernatant (2.0 mL) was transferred into glass vials, and VFA concentrations were determined using a Hewlett Packard 5890 series II gas liquid chromatograph (Hewlett-Packard, Avondale, PA) equipped with a 15 m x 0.533 mm (i.d.) column (Nukol: Supelco, Bellfonte, PA) with temperature ramp of 110°C to 150°C at 8°C mL/min. Helium was used as carrier gas with a column flow rate of 20 mL mL/min. Injector and detector temperatures were 250°C.


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  		Table 5. Ruminal characteristics in cannulated wethers fed increasing dietary CP or oscillating CP concentrations
 
At the morning feeding on d 13, 250 g (wet basis) of Yb-labeled bromegrass hay (Teeter et al., 1984) and 100 mL of cobalt-EDTA solution (Uden et al., 1980) were dosed into the ruminal digesta of each wether for estimation of digesta passage rates. Samples of whole ruminal contents (approximately 250 mL) were collected at 0 (before dosing), 4, 8, 12, 16, 25, 37, 50, and 79 h from the time of dosing with passage rate markers. Whole ruminal contents were strained through four layers of cheesecloth, and 15 mL of ruminal fluid was immediately frozen (-10°C). These samples were later thawed at room temperature, centrifuged at 10,000 x g for 10 min, and the resulting supernatant was analyzed for Co concentration (Hart and Polan, 1984) using an atomic absorption spectrometer (Model 210VGP, Buck Scientific, Inc., East Norwalk, CT) with an air/acetylene flame. All particulate matter remaining in cheesecloth was dried in a 55°C oven, ground (1 mm), and stored for later analysis of Yb concentration (Hart and Polan, 1984) using an atomic absorption spectrometer (Model 210VGP, Buck Scientific, Inc., East Norwalk, CT) with a nitrous oxide/acetylene flame. Fluid and particulate passage rate constants were calculated by regressing the natural logarithm of Co or Yb concentrations on time after initial dosing (Uden, et al., 1980).

Additional samples of whole ruminal contents (approximately 150 mL) were collected on d 13 to 16 twice daily at times representing 1, 4, 7, 10, 13, 16, 19, and 22 h after feeding. Samples collected at 1, 10, 13, and 22 h after feeding corresponded to days when ACP-fed lambs received the 13% CP diet, whereas the remaining samples corresponded to days when ACP-fed lambs received the 17% CP diet. Samples were blended for 1 min with an equal volume of saline (0.9% NaCl) in a household blender (Hamilton Beach/Proctor-Silex, Inc., Zmodel 720R, Washington, DC) to dislodge particulate associated bacteria, strained through four layers of cheesecloth, and the resulting fluid was immediately frozen (-10°C). These samples were later thawed at room temperature, and a bacteria-rich fraction was prepared by differential centrifugation (Merchen and Satter, 1983). The bacteria-rich samples were freeze-dried (Genesis SQ Super ES Freeze Dryer, The Virtis Co., Gardiner, NY) and ground (mortar and pestle) for laboratory analysis. Fecal grab samples (approximately 100 g, wet basis) were taken on d 13 to 16 at 8-h intervals, composited by animal, dried (55°C) for 48 h, and ground (2-mm screen). Feed samples were also collected on d 13 to 16 of each period, dried (55°C) for 48 h, and ground (1-mm screen).

On d 12 of each period, each wether was fitted with a jugular catheter (Abbocath T. I. V. catheter over needle, Abbott Laboratory, Abbott Park, IL) approximately 12 h prior to the start of each collection period. On d 13 to 16, blood samples (10 mL) were collected via jugular catheter at 4-h intervals. Following each collection, the catheter was flushed with 10 mL of sterile saline. Blood samples were immediately refrigerated, allowed to clot for 4 h, and centrifuged at 2,000 x g for 20 min. The resulting serum was frozen for later analyses of urea N (Marsh et al., 1965) and glucose (Procedure no. 17-UV, Infinity Reagent, Sigma Diagnostics, St. Louis, MO) using a UV-Vis spectrophotometer (Beckman DU Series 600, Beckman Instruments, Inc., Fullerton, CA).

Feed, orts, duodenal and ileal digesta, ruminal bacteria, and feces were analyzed for DM, OM, and Kjeldahl N (AOAC, 1990). Feed, duodenal and ileal digesta, and feces were also analyzed for NDF and ADF content using an ANKOM 200 Fiber Analyzer (ANKOM Technology, Fairport, NY) and starch content as described by Matejovsky and Sanson (1995). The Cr content of duodenal and ileal digesta and feces was measured (Hill and Anderson, 1958) using an atomic absorption spectrometer (Model 210VGP, Buck Scientific, Inc., East Norwalk, CT). Nutrient flows within the digestive tract were determined by dividing Cr intake by the concentration of Cr in the sample taken from respective sites. The purine content of ruminal bacteria and duodenal digesta was measured (Zinn and Owens, 1986), and bacterial N flow at the duodenum was estimated by dividing the N:purine ratio of harvested ruminal bacteria by the corresponding N:purine ratio of duodenal digesta. True ruminal nutrient digestibility was calculated from apparent nutrient digestibility corrected for bacterial nutrient contributions.

Statistical Analyses
Data were analyzed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC) for a replicated Latin square design. The model included effects of lamb, period, block, and treatment. Single degree of freedom orthogonal contrasts were used to determine linear and quadratic effects of protein concentration in the diet and for the 15% CP diet fed daily vs ACP treatment. Samples collected at fixed times after feeding (i.e., ruminal pH, NH3 N, and VFA, serum glucose, and urea N) were analyzed using the REPEATED statement within the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The model included the effects of lamb, period, block, treatment, time (expressed as 0 through 96 h of collection), and treatment x time. Lamb x period x block x treatment was used to specify variation between animals using the RANDOM statement. Lamb x period x block x treatment was used as the SUBJECT and autoregression used as the covariance structure. The same contrasts described above were used to partition the treatment sums of squares. Number of observations were 32 for all variables except those with repeated measures, including ruminal pH, NH3 N, glucose, and SUN (n = 768), and VFA (n = 228). Missing observations in blood metabolite data (n = 609 for glucose; n = 756 for SUN) resulted from loss of catheter patency or assay interference due to severe hemolysis in samples.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
Because all wethers were fed at 3.0% of initial BW throughout the trial and the quantity of feed refusals (data not shown) was minimal and did not differ (P >= 0.86) among treatments, OM intake did not differ (P >= 0.19) and averaged 1,388 g/d across treatments (Table 2Go). Feeding ACP had no effect (P >= 0.46) on ruminal or total tract OM digestion compared with the 15% CP daily treatment. Similarly, Hunt et al. (1989) and Collins and Pritchard (1992) reported that total tract DM digestibility in ruminants fed low-quality forages was not affected by frequency of protein supplementation. However, true ruminal OM digestion responded quadratically (P = 0.07) to increasing CP, reaching a maximum with the 15% CP treatment. Disappearance of OM from the small intestine and lower tract was unaffected (P >= 0.28) by treatment. However, wethers receiving 15% CP daily had numerically greater ruminal OM digestion, while wethers receiving 13% and 17% CP daily treatments had numerically greater digestion of OM in the lower tract (small intestine and colon). Therefore, no difference (P >= 0.23) in apparent total tract OM digestibility was observed across treatments, which may have been due to the restricted intake regimen and(or) the quality of hay fed (10.5% CP). Matejovsky and Sanson (1995) also noted that improvements in forage DM digestion in response to protein supplementation were dependent upon forage quality, wherein protein supplementation improved DM digestibility only when low-quality forage was fed.


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  		Table 2. Digestion of OM in cannulated wethers fed increasing dietary CP or oscillating CP concentrations
 
Intake of starch linearly decreased (P <= 0.0001) with increasing dietary protein, while ACP-fed wethers, because of their numerically higher DMI, consumed a greater quantity (P = 0.0003) of starch than those receiving 15% CP daily (Table 3Go). Because the CP content of the diet was increased at the expense of corn, the changes in starch intake simply reflect the amount of supplemental corn that sheep consumed on their respective treatments. However, duodenal starch flow was not different (P = 0.23), resulting in a linear decrease (P = 0.05) in ruminal starch digestion (g/d and % of intake) as dietary protein increased. Similarly, Matejovsky and Sanson (1995) observed an increase in starch digestibility in lambs fed forage and supplemented with increasing levels of corn up to 0.75% BW. Although apparent digestion of starch in the stomach (g/d) tended to be greater (P = 0.10) for ACP-fed wethers compared to those receiving 15% CP daily, ruminal starch digestibility (% of intake) did not differ (P = 0.27). However, apparent starch digestibility in the small intestine and hindgut was similar (P >= 0.20) across treatments and averaged 16.8 and 7.7% of intake, respectively. While apparent lower tract starch digestion was unaffected by treatment (P >= 0.22), total tract digestibility of starch linearly decreased (P = 0.003) with increasing dietary protein. Also, ACP-fed sheep had similar (P = 0.56) total tract starch digestibility (% of intake) compared to those fed 15% CP daily.


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  		Table 3. Digestion of starch in cannulated wethers fed increasing dietary CP or oscillating CP concentrations
 
Due to the restricted intake regimen, intakes of NDF, ADF, and hemicellulose (Table 4Go) were not different across treatments (P >= 0.17). In contrast to the changes in OM digestion, apparent NDF and ADF disappearence in the stomach was not affected (P >= 0.52) by treatment. Apparent digestion of hemicellulose in the stomach responded quadratically to increasing dietary protein, similar to the response observed in true ruminal OM digestibility; but the response was not significant (P = 0.14). Sanson et al. (1990) reported a decrease in total tract forage DM and hemicellulose digestibility as dietary level of corn increased from 0.26 to 0.52% of BW in steers consuming low-quality meadow hay. In the present study, the protein content of the diet was increased at the expense of corn, which was fed at 0.8, 0.6, 0.5, and 0.6% of BW for the 13, 15, and 17% CP diets fed daily and ACP treatments, respectively. Other research has shown that increasing the amount of supplemental corn in forage-based diets can depress forage digestion. Pordomingo et al. (1991) found that OM digestibility of summer range forage was reduced by corn supplementation at or above 0.4% of BW. However, the response to supplementation may be dependent on forage quality. Matejovsky and Sanson (1995) reported an increase in diet DM digestibility in lambs receiving medium- and high- quality hay with increasing levels of corn up to 0.75% BW. When comparing animals fed 15% CP daily vs ACP, apparent digestion of NDF and ADF in the hindgut was not different (P = 0.19). Although increasing dietary CP did not affect (P >= 0.26) total tract NDF and hemicellulose digestibility, total tract digestion of ADF responded quadratically (P = 0.07) to increasing CP. Swanson et al. (2000) found no differences in digestibility of ADF and NDF in ewes fed forage and supplemented with increasing levels of supplemental UIP. In the present study, apparent total tract digestibilities of NDF, ADF, and hemicellulose were similar (P >= 0.41) in sheep receiving 15% CP daily compared to those fed ACP. Likewise, other researchers (Bunting et al., 1987; Collins and Pritchard, 1992; and Brown et al., 1995) have reported no differences in total tract digestibility of forage NDF and ADF in sheep receiving infrequent or alternate day protein supplements. Therefore, our data agrees with previous reports that infrequent protein supplementation has minimal impact on the digestion of OM or fiber components, which results in animal performance (i.e., BW change, body condition score, and so on) comparable to that of animals supplemented daily.


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  		Table 4. Digestion of NDF, ADF, and hemicellulose in cannulated wethers fed increasing dietary CP or oscillating CP concentrations
 
Increasing dietary protein in our experiment did not affect (P >= 0.20) particulate or fluid passage rates (Table 5Go). Feeding ACP had no effect (P = 0.90) on particulate passage rate, but increased fluid passage rate (P = 0.0001) when compared to the 15% CP daily treatment. Similar to that measured in our experiment, Hunt et al., (1989) demonstrated that feeding frequency did not influence particulate passage rate of forage. In contrast to our study, Beaty et al. (1994) demonstrated that liquid dilution rate was unaffected by frequency of supplementation. Coleman and Wyatt (1982) observed that passage rate of forage tended to decline as feeding interval increased. However, the lack of changes observed in particulate passage rate in the current study correspond to the lack of differences observed in ruminal NDF and ADF digestibility, which further suggests that starch level did not compromise ruminal fiber digestibility in the present study.

Ruminal fluid pH was not affected (P >= 0.34) by dietary treatment or by treatment x time interaction (P >= 0.69; data not shown) and averaged 6.14 across treatments. Ørskov (1992) indicated that ruminal pH below 6.0 would reduce the activity of cellulolytic bacteria, and depressions in ruminal pH associated with grain supplementation could reduce forage fiber digestibility. However, because we noted no difference (P >= 0.17) in ruminal fiber digestibility, ruminal pH was not low enough to influence ruminal fiber digestibility in the present study.

No treatment x time interaction (P >= 0.19) was observed for ruminal NH3 N concentrations; thus, only main effect means are presented in Table 5Go. Sheep fed ACP had lower (P = 0.08) ruminal NH3 N concentrations than animals consuming 15% CP daily. In contrast to our study, Collins and Pritchard (1992) reported an increase in ruminal NH3 N in animals supplemented protein every 48 h vs daily sup-plementation. Beaty et al. (1994) also reported that steers were able to sustain elevated ruminal levels of NH3 N even on days not supplemented. In our study, oscillating dietary protein lowered ruminal NH3 N but did not compromise fiber fermentation. Furthermore, supplementation with a UIP source, rather than additional DIP, may have aided in maintaining lower ruminal NH3 N levels in our experiment. Because Sultan et al. (1992) suggested that ruminal NH3 N concentrations and ruminal OM digestion are regulatory factors for increasing N recycling in the rumen, these factors may suggest a greater potential for N recycling in ACP-fed sheep in our experiment.

Ruminal NH3 N concentrations linearly increased (P = 0.0001) with the addition of UIP. Because diets were formulated to have the same amount of DIP (9.6% of DM), ruminal NH3 N should have been similar across treatments. Although protein level was increased using UIP, the changes in ruminal NH3 N concentrations in this experiment suggest an increase in ruminal N status. Based on the calculated DIP values of 8.7, 10.7, 10.2, and 10.5% for the 13, 15, and 17% CP daily and ACP treatments, respectively, the increase in NH3 N may reflect greater than anticipated ruminal degradation of our UIP source. Alternatively, the lower ruminal NH3 N concentrations observed in lambs fed the 13% CP diet may have been due to the greater consumption of starch, and thus, greater microbial uptake of the NH3 N produced. Likewise, it is also important to consider the potential of recycled N to contribute to the pool of degradable N available in the rumen (Mathis et al., 2000). Consequently, the recycling of UIP N to the rumen as urea may have contributed to the ruminally degradable N fraction thereby supplying an increase in DIP via an indirect route (Köster et al., 1996). In any case, the concentration of ruminal NH3 N was adequate and maintained fermentation of diets in this experiment, as demonstrated by the lack of changes in fiber digestibility and VFA concentrations, and was within the optimal range of 2.0 to 5.0 mg/dL ruminal NH3 N as suggested by Satter and Slyter (1974) to maintain microbial growth.

Total VFA concentrations were not affected (P >= 0.25) by increasing dietary CP. However, a significant treatment x time interaction (P = 0.04) was observed for the molar proportion of acetate, which was elevated at 9 and 12 h postfeeding in sheep fed ACP (data not shown), resulting in an overall higher (P = 0.0001) molar proportion of acetate in sheep fed ACP vs 15% CP daily. Thus, overall acetate concentrations (millimolar data not shown) were greater in ACP-fed wethers, leading to higher (P = 0.05) total VFA concentrations in ACP-fed sheep compared to wethers receiving 15% CP daily. The greater concentration of acetate in ACP-fed sheep may reflect a change in the pattern with which fiber was fermented, due to a potentially greater influx of recycled N at times removed from feeding thereby sustaining fiber digestibility. Likewise, a faster liquid passage rate is often associated with higher acetate production (Owens and Goetsch, 1988). In contrast to our study, Collins and Pritchard (1992) reported that total VFA concentrations were not affected by frequency of protein supplementation, but did note that the pattern of VFA concentrations after feeding was less dramatic in wethers fed corn gluten meal than soybean meal. In our study, molar proportions of butyrate (P = 0.002) responded quadratically to increasing dietary CP. Likewise, a quadratic response observed in the molar proportions of propionate (P = 0.003) contributed to the quadratic (P = 0.0009) response found in the acetate:propionate ratio. Sheep fed ACP also had greater (P = 0.0005) acetate:propionate ratios compared to those consuming 15% CP daily. Our data indicate that oscillating dietary protein may alter ruminal fermentation patterns in favor of increasing acetate production, perhaps by delaying or sustaining fiber fermentation at times removed from feeding. Alternatively, because the ACP-fed lambs were consuming the 17% CP diet when the 9 and 12 h samples were collected, the greater acetate concentrations at times removed from feeding for the ACP treatment may simply reflect a greater OM or fiber digestion when the 17% CP diet was fed, rather than a true alteration in the pattern of ruminal fermentation.

A significant treatment x time interaction was also observed in molar proportions of isovalerate (P = 0.01; data not shown). At times beyond 12 h postfeeding, the molar proportion of isovalerate was higher in wethers fed 17% CP daily vs the other treatments. Because DIP was balanced using different protein sources (i.e., soybean meal, urea, and SOYPLUS), this response may be due to changes in the pattern of ruminal protein degradation after feeding, and(or) an increase in total ruminal protein degradation (g/d) associated with that treatment. Overall, increasing dietary CP linearly increased (P = 0.0001) molar proportions of isovalerate and valerate and tended (P = 0.14) to linearly increase isobutyrate. In our experiment, increasing levels of ruminal NH3 N and branched-chain VFA with provision of UIP suggest that either proteolysis within the rumen was not inhibited, or that less was incorporated into bacterial cells. Since branched-chain VFA are used by cellulolytic bacteria for cell membrane synthesis and production of branched-chain amino acids (Purser and Buechler, 1966), the increase in branched-chain VFA may have supported cellulolytic bacteria, thus, supporting fiber fermentation. This further suggests that DIP level was adequate to maintain microbial growth.

Due to our treatment regimen, N intakes linearly (P = 0.0001) increased with the addition of UIP (Table 6Go). Flows of N fractions to the small intestine and N digestion were unaffected (P >= 0.14) by feeding ACP compared with the 15% CP diet fed daily. Nonmicrobial and total N flows at the duodenum and ileum increased linearly (P <= 0.003) as dietary CP increased from 13 to 17%. Bacterial N flow at the duodenum responded quadratically (P = 0.04) to increasing dietary CP, with greater bacterial N flow associated with both the 15% CP and ACP treatments. Therefore, bacterial N flow at the small intestine simply reflected the pattern of ruminal OM digestion observed in this experiment. The greater quantity of microbial N flow to the small intestine in the 15% treatment could be attributed to the greater OM fermentation in the rumen for that treatment (Clark et al., 1992). Although total bacterial N flow increased, efficiency of microbial protein synthesis did not differ (P >= 0.55) across treatments and averaged 27.3 g of N/kg of OM truly digested.


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  		Table 6. Digestion of nitrogen in cannulated wethers fed increasing CP or oscillating CP concentrations
 
True ruminal N digestibility tended to respond quadratically (P = 0.09) to increasing dietary protein. Loerch et al. (1983) observed that in situ rates of N disappearance were not only affected by type of protein but decreased with increasing level of dietary corn. These researchers noted that ruminal N disappearance from soybean meal and dehydrated alfalfa pellets (high DIP) tended to be more affected by level of corn than were blood meal, meat and bone meal, and corn gluten meal (UIP sources). In our study, the pattern of true ruminal N digested parallels the trend observed in true ruminal OM digestion. Because protein concentrations above 13% were accomplished using UIP, ruminal N digestibility (% of intake) in the 13% treatment should have been greater than the 15% treatment, and the decreased ruminal protein degradability associated with the 13% CP treatment contributed to the depressed OM fermentation for that treatment.

Nitrogen disappearance from the small intestine tended (P <= 0.11) to increase linearly with increasing dietary protein. When combined with the linear increase (P = 0.08) in N disappearance in the hindgut, we observed a linear (P <= 0.04) increase in total tract N digestibility with increasing CP content of the diet. This suggests that our UIP source (SoyPLUS) provided additional protein at the small intestine that was readily digested by the animal.

No treatment x time interaction (P >= 0.13) was observed in serum glucose concentrations (data not shown); thus, only main effects are discussed. Lambs fed ACP maintained an overall lower (P = 0.002) serum glucose concentration compared with animals consuming 15% CP daily (main effect means of 62.3 vs 65.8 mg/dL, respectively), which suggests that feeding protein on alternate days may influence serum glucose concentration, which may affect overall glucose utilization. Leng (1970) reviewed several studies and observed that dietary protein and feeding regimen have considerable effect on glucose entry rates. However, increasing dietary CP in the present study did not influence (P = 0.15) serum glucose concentrations, suggesting that protein level either as DIP or UIP, does not affect glucose utilization. Sletmoen-Olson et al. (2000) reported that gestating cows fed a high UIP supplement had higher plasma glucose concentrations than cows fed a medium UIP supplement. However, Swanson et al. (2000) reported no differences in serum glucose level in sheep consuming low, medium, and high levels of supplemental UIP.

As noted for serum glucose, no treatment x time interaction (P = 0.11) was observed in serum urea N (SUN) concentrations (data not shown); thus, only main effects are discussed. Increasing the protein level in the diet produced a linear increase (P <= 0.0001) in SUN (10.7, 13.4, and 12.6 mg/dL for the 13, 15, and 17% CP diets, respectively). These results agree with other researchers (Caton et al., 1994; Sletmoen-Olson et al., 2000; Swanson et al., 2000) who demonstrated the effects of supplemental UIP on SUN concentrations. However, the mean SUN concentration in ACP-fed sheep was lower (P = 0.0006) than for those consuming 15% CP daily (12.1 vs 13.4 mg/dL, respectively), which also parallels the response observed in ruminal NH3 N. In contrast to our study, Cole (1999) reported that mean plasma urea N concentrations of lambs fed 10 and 15% CP oscillated at 48-h intervals were similar to those continuously fed 12.5% CP. Sletmoen-Olson et al. (2000) suggested that plasma urea N concentration in beef cows is proportional to N intake. In our experiment, ACP-fed wethers received the same quantity of protein as those fed 15% daily, but ACP-treated sheep had lower SUN and ruminal NH3 N concentrations. Owens and Zinn (1988) suggested that the quantity of N recycled to the rumen is dependent on ruminal NH3 N and plasma urea concentration. Furthermore, transfer of blood urea into the rumen is affected by ruminal NH3 N concentration, blood urea concentration, and the quantity of OM fermented in the rumen (Kennedy and Milligan, 1980). In our study, despite these lower ruminal NH3 N and SUN concentrations, ACP-fed animals had similar quantities of true ruminal OM digested and microbial N flow compared with animals fed 15% CP daily. Because ammonia N supply did not appear to limit microbial fermentation in ACP-fed sheep, these results suggest that oscillating dietary protein may have stimulated N recycling on days when ruminal N was low. In a companion study employing similar dietary treatments as those used in this study (Ciminski et al., 2000), we observed a treatment x day interaction in SUN concentrations with SUN of the ACP-fed steers being lower on days when an 11% CP diet was fed and subsequently increasing when a 15% CP diet was fed. As we observed in the current experiment, steers fed the ACP treatment in that trial had lower SUN than those receiving 15% CP daily. Because ruminal NH3 N should have been similar across treatments, feeding UIP to wethers on the ACP treatment may have indirectly supplied a greater quantity of ruminal N to the rumen via recycling. Thus, providing protein in the form of UIP in the ACP treatment may have provided a "time-delay" source of DIP. The amount of DIP should have been adequate to maintain rumen microbial function for each day, while the UIP provided an increased amount of amino acids available for absorption in the small intestine. Therefore, on days when the 13% diet was fed, those amino acids circulating in the blood from the days in which UIP was supplemented may be deaminated and converted to urea by the liver. This urea can then be recycled back to the rumen to be used as a N source for microbial growth and protein synthesis. In effect, the UIP supplement may have served as a source of N with which to enhance N recycling in the ruminant animal. However, this hypothesis deserves further investigation before any conclusions on the benefits of supplemental UIP on N recycling can be made.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
Oscillating dietary protein does not negatively influence ruminal or postruminal digestion in sheep; thus, this feeding regimen can be used under practical feeding conditions and incorporated into supplementation programs with minimal impact on the performance of ruminant animals. Sheep fed oscillating dietary protein may maintain lower levels of ruminal ammonia and serum urea N, which may indicate a greater efficiency of N utilization. The role of supplemental undegradable intake protein for enhancement of N recycling in the infrequently supplemented ruminant animal deserves further investigation.


   Footnotes
 
1 We gratefully acknowledge the assistance of Eric Scholljegerdes and Jason Heeg for their assistance with sample collection associated with this project. Appreciation is also extended to Dan Rule for his assistance with manuscript preparation. We also thank West Central Cooperative, Ralston, IA, for donation of the SoyPLUS used in this project. Back

Received for publication February 28, 2002. Accepted for publication July 23, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


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