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Effect of rumen-degradable protein balance deficit on voluntary intake, microbial protein synthesis, and nitrogen metabolism in growing double-muscled Belgian Blue bulls fed corn silage-based diet^1,2,3

Faculté Universitaire des Sciences Agronomiques Passage des Déportés, 2 B-5030 Gembloux, Belgium

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Two trials were conducted to evaluate the effect of rumen-degradable protein balance (OEB) deficit on voluntary intake (trial 1), microbial protein synthesis, and N metabolism (trial 2) in growing double-muscled Belgian Blue bulls. In trial 1, six bulls (339 ± 26 kg of initial BW) were used in a replicated 3 x 3 Latin square and received a diet of 60% corn silage and 40% concentrate with ad libitum intake (DM basis). Three concentrates were formulated by adding urea at the expense of barley to give similar dietary contents of intestinal digestible proteins, NE for fattening, and fermentable OM, but with different levels of OEB. Thus, 2 levels of OEB deficit (–23.7 and –9.2 g of OEB/kg of DM) were compared with a diet providing a slight OEB surplus (5.3 g of OEB/kg of DM). Voluntary DMI decreased linearly (P = 0.02) with decreasing rumen-degradable protein balance. This decrease in intake could explain the linear decrease in ADG observed when negative OEB diets were fed. In trial 2, six bulls (304 ± 12 kg of initial BW) with cannulas in the rumen and proximal duodenum were used in a replicated 3 x 3 Latin square and fed diets similar to those used in trial 1 at an intake level of 85 g of DM/kg of BW^0.75. Diurnal variations of ruminal NH₃-N and plasma urea-N concentrations were greatly influenced by the level of OEB in the diet. No differences in NDF and starch degradation in the rumen, microbial N flow at the duodenum, or efficiency of microbial protein synthesis in the rumen were noted among the levels of OEB in diets. The reductions of the OEB value from 5.3 g/kg of DM to –9.2 g/ kg of DM and –23.7 g/kg of DM were associated with reductions of 26.5 and 48.8% in urinary N output. Absolute amounts of N retained by the bulls increased significantly with the level of OEB in diets. Indeed, 51.4% of the incremental supply of N was excreted between –23.7 and –9.2 g of OEB/kg of DM diets, and 74.6% of the incremental supply of N was excreted between –9.2 and 5.3 g of OEB/kg of DM diets. Feeding diets characterized by an adequate intestinal digestible protein supply and a OEB close to –10 g of OEB/kg of DM could be a feeding strategy to reduce N losses from the farm with little effect on the animal performance and voluntary intake. Reduced OEB may reduce N excretion in the environment but may also result in decreased N retention.

Key Words: bull • nitrogen utilization • rumen-degradable protein balance • rumen fermentation • voluntary intake

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Farm operations need to be carried out with minimum environmental effects. Nitrogen losses from animal production are inevitable, but N excretion can to some extent be prevented or at least controlled (Tamminga, 2003). Reducing N losses from the farm begins with proper animal feeding to reduce N excretion (Satter et al., 2002).

In ruminant N nutrition, 2 general strategies can be used to reduce N excretion. They consist of limiting the excess of protein digested in the small intestine and balancing the level of rumen-degradable protein (RDP) with the available energy supply for microbial protein synthesis (Colin-Schoellen et al., 2000; Børsting et al., 2003). The RDP balance according to the Dutch system (OEB; Tamminga et al., 1994) makes it possible to put this second strategy in practice. Indeed, the OEB value estimates the difference between the potential microbial protein synthesis based on RDP and that based on rumen-fermented OM (FOM; Yu et al., 2003). The optimal OEB value of a diet is close to zero and corresponds to a well-balanced availability of energy and N to rumen microbes. When the OEB value is positive for a diet, N losses from the rumen occur, whereas a negative value indicates a shortage of RDP, and, consequently, the microbial activity may be impaired. However, ruminants are able to recycle large quantities of urea-N into the rumen instead of excreting them in the urine, thus partially supplying bacteria with their need for N (Marini and Van Amburgh, 2003). Therefore, urea-N salvage mechanisms could be a very important key to increase N efficiency in ruminants (Stewart and Smith, 2005).

We hypothesized that feeding a diet characterized by a negative OEB value and an adequate intestinal digestible protein (DVE) supply will enhance the N efficiency of growing bulls, due to greater N salvage. The objective of this study was to investigate the effect of an OEB deficit on double-muscled Belgian Blue bull digestion, N metabolism, and voluntary intake.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All animal care, handling techniques, and surgical procedures were approved by the Gembloux Agricultural University Animal Care and Use Committee before the initiation of research and followed the guidelines issued by the committee on care, handling, and sampling of the animals.

Trial 1

Animals and Diets. Six double-muscled Belgian Blue bulls (339 ± 26 kg of initial BW; 494 ± 30 kg of final BW) were individually penned (3 x 4 m) and fed 2 equal meals at 0800 and 1600. Fresh water was available at all time. The 6 bulls were used in a replicated 3 x 3 Latin square. Three different diets were formulated to yield 3 different levels of OEB (–5, 0, and 15 g/kg of DM), but similar dietary contents of intestinal digestible proteins, NE for fattening, and fermentable OM according to the Dutch system (Van Es and Van der Honing, 1977; Tamminga et al., 1994). The OEB value is estimated as microbial protein synthesized in the rumen, based on rumen-degraded feed CP minus microbial protein synthesized in the rumen, based on rumen- FOM, with microbial protein synthesis calculated as 0.15 x FOM (Tamminga et al., 1994).

Ingredient composition, actual chemical composition, and actual nutritive value of the 3 diets are presented in Table 1. Among diets, only the urea and the dry-rolled barley contributions varied, because the different levels of OEB were obtained from dry-rolled barley substitution with urea. The rumen-degradable N:FOM ratio of the 3 diets was 17.1, 21.4, and 25.5 g of N/kg of FOM (CVB, 2000), which corresponded to OEB values of –3.7, –9.2, and 5.3 g of OEB/kg of DM. These actual OEB values were lower than those expected (i.e., –15, 0, and 15), because the actual chemical composition of the corn silage differed from our initial prediction based on a corn sample taken before beginning the experiment. The diets provided 95% of the NE and DVE requirements for a double-muscled Belgian Blue bull of 400 kg with an ADG of 1.4 kg of BW/d (De Campeneere et al., 2001).

Sample Collection and Laboratory Analyses. Each experimental period was 31 d long and included a 21-d adaptation to the diet and a 10-d voluntary intake determination. The animals were weighed at the beginning and at the end of each adaptation and treatment period before the 0800 feeding on 2 consecutive days. Diets were offered for ad libitum intake with a 10% refusal level throughout each experimental period. The daily amount of the diet distributed was based on the refusal level of the previous collection day. The feed refusals were weighed before each 1600 feeding, and a 5% sample was collected. Feed refusal samples were placed in forced-air ovens at 60°C immediately after collection, dried to a constant weight, and pooled per animal at each treatment period. Concentrate, corn silage, and feed refusals were ground to pass a 1-mm screen (Cyclotec 1.093, FOSS TECATOR AB, Höganäs, Sweden) before DM, OM, and Kjeldahl N (AOAC, 1990) analyses were conducted.

Calculations and Statistical Analyses. Difference between the amounts of DM, OM, and N offered and those refused determined the daily voluntary intake of the diets. Average BW measured during the voluntary intake determination was used to express the DMI relative to metabolic BW. The ADG was estimated on the whole experimental period (31 d). Data were analyzed as a replicated 3 x 3 Latin square using the MIXED procedure (SAS Inst. Inc., Cary, NC), with OEB level and period as fixed factors and bull as a random factor. Least squares means were obtained for each treatment, and linear and quadratic orthogonal polynomial contrasts on equally spaced treatments were used to evaluate responses. Differences among treatments were considered significant when P < 0.05, whereas when P > 0.05 but P < 0.10, differences were considered to indicate a trend toward a significant effect.

Trial 2

Animals and Diets. Six double-muscled Belgian Blue bulls (304 ± 12 kg of initial BW; 443 ± 12 kg of final BW), fitted with a ruminal cannula (67 mm i.d.) and a T-type cannula at the proximal duodenum, were individually penned (1.5 x 2.5 m). Fresh water was available at all times. Bulls received the same diets as described previously (Table 1) in 2 equal meals at 0800 and 2000 at an intake level of 85 g of DM/kg of BW^0.75 to decrease the variation associated with feed refusals.

Digestibility was measured using chromic oxide (Cr₂O₃, VWR International Europe BVBA, Leuven, Belgium) as a digestibility marker. From 5 d before the sample collection and continually thereafter, 3.5 g of Cr₂O₃/kg of DMI was administrated to each bull twice daily, just before each meal, intraruminally via gelatin capsules. Bulls were weighed at the beginning and at the end of each treatment period before the 0800 feeding on 2 consecutive days. Feed offered was adjusted to the metabolic BW of each animal and fixed during each period.

Experimental Protocol and Samples Collection. The 6 bulls were allocated to 3 treatment periods in a replicated 3 x 3 Latin square, separated by a 21-d rest period. The treatment period lasted 15 d and included digesta (duodenal and fecal), urine, blood, and ruminal fluid collections. Refusals were collected daily before the morning feeding, weighed, and dried at 60°C. Samples of each diet were obtained each time diets were mixed, ground to pass a 1-mm screen (Cyclotec 1.093, FOSS TECATOR AB), and pooled by period on an equal weight basis. Urine was collected during the first 10 d of each treatment period with an adaptation of the apparatus of Veenhuizen et al. (1984). During the collection, urine was acidified with 2 M H₂SO₄ to pH 3 at 0900, 1700, and 2100 to avoid N losses. Total urine collection was weighed every morning at 0900, sampled, and frozen at –0°C. The majority of the fecal output was collected simultaneously into a container placed underneath each animal and was sampled daily at 0830 and frozen at –20°C.

On d 11, central venous catheters (polyurethane aliphatic No.67262J17, Plastimed, Le Plessis-Bouchard, France) were inserted into 1 of the external jugular veins of each bull after an i.v. injection of analgesic (xylazine 2%, 0.08 mL/100 kg of BW). On d 12, blood samples (20 mL) were slowly collected into 2 heparinized tubes (143 IU/10 mL; Vacutainer Systems, Becton Dickinson Benelux S. A., Erembodegem, Belgium) every 2 h during 24 h. Blood was centrifuged at 1,200 x g for 15 min immediately after sampling. Plasma was removed and frozen at –20°C until further analyses.

Samples of duodenal digesta were collected on d 13 and 14 to cover 12 h by 1.5-h steps. After removal of the cap of the duodenal cannula, accumulated digesta was discarded, and, when the flow appeared normal, 400 mL of duodenal contents was collected. For each collection, 100 g of duodenal contents was immediately used to extract liquid-associated bacteria according to the method described by Poncet and Rémond (2002). The remainder of each duodenal sample was frozen at –20°C. At the end of each treatment period, rumen digesta was sampled 0, 1, 2, 3, 4, 6, 8, 10, and 12 h after the morning meal.

Laboratory Analyses. Concentrate, corn silage, and feed refusals pooled per animal at each treatment period were ground to pass a 1-mm screen (Cyclotec 1.093, FOSS TECATOR AB) before DM, OM, ether extract, Kjeldahl N (AOAC, 1990), NDF, and ADF (Van Soest et al., 1991) analyses were conducted. The NDF was determined without use of sodium sulfite but with the addition of heat-stable amylase (Termamyl 120, NOVO Nordisk, Bagsvaerd, Denmark) for the last 30 min of boiling. An aliquot of these samples was ground to pass a 0.5-mm screen for the total starch determination (Faisant et al., 1995). After freeze-drying and grinding (1 mm), DM, OM, N, NDF, and Cr₂O₃ (François et al., 1978) were measured in feces pooled per animal and per period on an equal weight basis. Urinary N was also analyzed (AOAC, 1990).

After thawing, plasma urea-N (PUN) was measured according to Berthelot with a commercial kit (Ecoline S+, DiaSys GmbH, Holzheim, Germany).

The pH of ruminal fluid was measured (Microprocessor pH-meter, pH 320/SET, WTW, Weilheim, Germany) immediately after sampling and filtration (Edestahl, Analysensieb, Retsch, Haan, Germany, aperture 250 µm). Supernatant from centrifugation at 12,000 x g for 12 min was frozen at –20°C after H₂SO₄ acidification to pH 3, until NH₃-N and VFA determinations. The NH₃-N concentration was determined by steam distillation with MgO using a 2,300 Kjeltec Analyser Unit (FOSS TECA-TOR AB). After thawing, ruminal fluid samples were centrifuged at 12,000 x g for 12 min, and an aliquot was diluted (1:1) with 2-methylvalerate (2 mg/mL; VWR International Europe BVBA) as internal standard. These subsamples were filtered through a 0.45-µm membrane (Acrodisc syringe filters, Pall Corporation, Ann Arbor, MI) before VFA determination by HPLC. Chromatographic system consisted of a liquid chromatograph (Alliance 2690, Waters, Milford, MA) equipped with a prepacked Aminex HPX-87H strong cation-exchange resin column (BioRad Labs., Richmond, CA) in combination with a UV detector (Waters 486). The mobile phase was 3 mM H₂SO₄ with 5% acetonitrile pumped at 0.6 mL/min. The UV absorbance was measured at 210 nm. Integration was performed with Millenium 32 software (Waters).

Duodenal samples were freeze-dried, ground to pass a 1-mm screen, and pooled per animal and period before DM, OM, N, NH₃-N, NDF, and Cr₂O₃ determinations on the whole digesta. A small portion was ground to pass a 0.5-mm screen for the starch and purine determinations. Purine content was measured with the method of Zinn and Owens (1986) adapted for the hydrolysis of nucleotides by perchloric acid (Makkar and Becker, 1999) and for the wash solution used for washing the precipitate of free purines with silver nitrate (Obispo and Dehority, 1999) to improve purine recovery. Duodenal bacteria samples were freeze-dried, ground with a coffee grinder and pooled per animal and period before N, OM, and purine content determinations.

Calculations and Statistical Analyses. Nutrient flows at the duodenum and fecal output were calculated by dividing the amount of Cr₂O₃ dosed by the concentration of Cr₂ O₃ in the duodenal or fecal sample. The proportion of microbial N flowing at the duodenum was calculated by dividing the duodenal purine:N ratio by the corresponding purine:N ratio in duodenal microbes. The efficiency of microbial protein synthesis was calculated by dividing duodenal microbial-N flow by the quantity of OM apparently or truly digested in the rumen. The OM truly digested in the rumen was obtained by the subtracting of duodenal microbial OM flow from the total OM flow. Endogenous N was assumed to be 2.3 g/ kg of DMI for the calculation of endogenous N flowing to the duodenum (Ouellet et al., 2002). It was assumed that the treatments did not affect this value. The endogenous N flow was subtracted from the duodenal nonmicrobial N to estimate the undegraded dietary N flowing to the duodenum. Nitrogen balance across the forestomach was estimated by the difference between N flowing at the duodenum and N ingested, whereas the net N balance across the forestomach was calculated by subtracting the duodenal endogenous N flow estimated according to Ouellet et al. (2002) from N balance.

Statistical analyses on N balance, total tract and ruminal digestibility, and microbial protein synthesis were conducted using the MIXED procedure (SAS Inst. Inc.). Data were analyzed as a replicated 3 x 3 Latin square, with OEB level and period as fixed factors and bull as a random factor. Least squares means were obtained for each treatment, and linear and quadratic orthogonal polynomial contrasts on equally spaced treatments were used to evaluate responses. For data collected over time within each sampling period (ruminal pH and concentrations of NH₃ and VFA and PUN), a repeated-measures-in-time analysis was performed using the MIXED procedure of SAS. The smallest value of the Akaike’s information criteria was used to select the most appropriate covariance structure. Effects in the model included bull, period, treatment, time, and treatment x time interaction. When treatment x time interactions were significant, variables were analyzed within time periods. Differences among treatments were considered significant when P < 0.05, whereas when P > 0.05 but P < 0.10, differences were considered to indicate a trend toward a significant effect.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Trial 1

Effects of the OEB levels on voluntary DM, OM, and N intakes are summarized in Table 2. Rumen-degradable protein balance had a linear effect on DM (P = 0.02) and OM intake (P = 0.02). Intake was lower for diets characterized by an OEB deficit. Because CP contents in the diet were different (Table 1), voluntary N intake was highly influenced by treatments (P < 0.001). Average daily gain increased (P = 0.02) linearly with the OEB level of the diet.

Ruminal Fermentation Characteristics and Plasma Urea N. There was no interaction between treatment and time (P = 0.71) in ruminal pH (Figure 1). The average ruminal pH (Table 3) decreased only from the lowest OEB level (–3.7 g of OEB/kg of DM) to the medium OEB level (–.2 g of OEB/kg of DM). There was no interaction between OEB level and time (P = 0.17) in total VFA concentration in the rumen (Figure 2). For individual VFA, the treatment x time interaction was only significant for ruminal valerate. This interaction was largely due to differences in magnitude of responses at specific time points (data not shown). Total VFA concentration mean increased linearly (P = 0.006) as the rumen-degradable protein supply increased and resulted from a significant linear increase of the ruminal acetate and propionate concentration. Moreover, OEB tended to have a quadratic effect on rumen butyrate. There was an effect of time and interaction between time and level of OEB in the diet (P < 0.001) in ruminal NH₃-N (Figure 3). Temporal pattern of ruminal NH₃-N concentration was similar, but peak concentrations were influenced by OEB. At 1, 2, 3, and 4 h after the meal distribution, ruminal NH₃-N levels were significantly different among the treatments (Figure 3). There was a time effect and treatment x time interaction (P < 0.001) in PUN (Figure 4). Plasma urea-N was low in bulls receiving the –23.7 g of OEB/kg of DM diet (range of 1.5 to 2.4 mg/100 mL over time) and increased linearly (P < 0.001) as the OEB value of the diet increased (Figure 4; ranges over time of 4.3 to 6.0 and 6.0 to 9.9 mg/100 mL, respectively, for –9.2 and 5.3 g of OEB/kg of DM diets).

View larger version (7K): [in this window] [in a new window]   Figure 1. Ruminal pH in bulls fed diets with a rumen-degradable protein balance (OEB) of –23.7 (), –9.2 (), or 5.3 g of OEB/kg of DM (). The cross on the x-axis indicates feeding time. Error bars indicate SEM for n = 6.

View larger version (8K): [in this window] [in a new window]   Figure 2. Ruminal total VFA concentrations in bulls fed diets with a rumen-degradable protein balance (OEB) of –23.7 (), –9.2 (), or 5.3 g of OEB/kg of DM (). The cross on the x-axis indicates feeding time. Error bars indicate SEM for n = 6.

View larger version (8K): [in this window] [in a new window]   Figure 3. Ruminal NH3-N concentrations in bulls fed diets with a rumen-degradable protein balance (OEB) of –3.7 (), –9.2 (), or 5.3 g of OEB/kg of DM (). The cross on the x-axis indicates feeding time. Error bars indicate SEM for n = 6.

View larger version (6K): [in this window] [in a new window]   Figure 4. Plasma urea-N in bulls fed diets with a rumen-degradable protein balance (OEB) of –23.7 (), –9.2 (), or 5.3 g of OEB/kg of DM (). The cross on the x-axis indicates feeding time. Error bars indicate SEM for n = 12.

N Balance. Results of N balance are shown in Table 5. Fecal N output was not found to differ and reached on average 50.9 g/d. On the other hand, urinary N output increased linearly (P < 0.001) with OEB level. Absolute amounts of N retained and relative amounts expressed as a percentage of N digested were significantly influenced by treatments (Table 5). Nitrogen retention relative to the N intake was not affected by dietary OEB level and reached on average 35.4% of ingested N.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
According to the Dutch protein system (Tamminga et al., 1994), the optimal OEB value of a diet is close to zero and corresponds to a rumen-degradable N:FOM ratio equal to 25 g of N/kg of FOM, which reflects a well-balanced availability of energy and N to rumen microbes. However, the Dutch protein system tolerates small negative values in growing cattle, because of N recycling. This lack of OEB is only allowed in animals weighing more than 250 kg when the DVE intake exceeds the DVE requirement (Van Vliet et al., 1994), because the N from the surplus of absorbed AA can be recycled to the rumen (Hvelplund and Madsen, 1990). According to Fiems et al. (1999a), this tolerable lack of OEB contains a safety margin, mainly because the criterion based on the BW is too restrictive and the need of N available in the rumen is probably overestimated in the Dutch protein system. Therefore, feeding double-muscled Belgian Blue bulls with diets providing DVE amounts strictly equivalent to their requirements and characterized by a negative OEB value was proposed to minimize N excretion without modifying growth performances and N retention.

In situations of ad libitum intake, voluntary DMI could be highly affected by RDP level in the diet (Peyraud et al., 1997). The objective of the first trial was to investigate the effect of an OEB deficit on double-muscled Belgian Blue bull voluntary intake. Rumen-degradable protein balance had a linear effect on DMI. According to the experimental design, 6 observations per treatment were made, and animal effect was well taken into account by the use of the Latin square design. However, due to the short duration of each experimental period and the potential for treatment effects on gut fill, the ADG observations have to be considered with caution. Dry matter intake reached on average 84.1 g of DM/kg of BW^0.75 and was slightly greater than voluntary DMI measured by Fiems et al. (1999b) on 123 double-muscled Belgian Blue bulls fed corn silage-based diets, which reached 78.2 g of DM/kg of BW^0.75. Dry matter and OM voluntary intake decreased linearly with decreasing OEB (Table 2). Therefore, the linear decrease in ADG observed when decreasing the OEB level of the diet was related to these reductions of intake. The effect of decreasing OEB level on intake and growth confirms the need for a minimum threshold of dietary N (Ketelaars and Tolkamp, 1992).

Although intake level was restricted, DMI measured during the metabolism trial was close (i.e., 95%) to ad libitum intake levels measured in trial 1. Increased loss of N in urine is the most common effect observed with increasing CP or RDP in diets (Hristov et al., 2004). With dairy cows, de Boer et al. (2002) demonstrated the importance of OEB value in decreasing N losses; decreasing OEB from 1,000 to 0 g/d resulted in a linear decrease in urinary N excretion. Indeed, decreasing the OEB from a positive value to zero contributes to limiting the N excess in the rumen, which is excreted as urea in the urine. In this experiment, the daily OEB intake reached –62, –62, and 36 g/d, and decreasing OEB value of the diet was also associated with an important linear decrease in urinary N excretion. Reducing the OEB value from 5.3 g/kg of DM to –9.2 g/kg of DM and –23.7 g/kg of DM was associated with reductions of 26.5 and 48.8% of the urinary N outputs. Feeding a diet with a negative OEB value means that there was a shortage of RDP to sustain the microbial protein synthesis. Therefore, to supply bacteria with its need for N, N recycling in the rumen was likely enhanced, and N excretion in urine was decreased (Marini and Van Amburgh, 2003). Although N excretion decreased greatly with the negative OEB diet, absolute amounts of N retained by the bulls were significantly affected by the level of OEB in the diets (Table 5). Nitrogen retained daily by the bulls decreased linearly as the dietary OEB level was decreased. Nitrogen retention measured with –23.7 and –9.2 g of OEB/kg of DM diets represented, respectively, 72 and 90% of the N retained by the bulls fed the 5.3 g of OEB/kg of DM diet. Indeed, 51.4% of the incremental supply of N was excreted between the lowest and the medium OEB level, and between the medium and the greatest OEB level, 74.6% of the additional N intake was excreted.

In this experiment, N retained by the bulls could depend on the amount of digestible proteins and its AA composition and on the energy available for the protein accretion in the body. Total tract and ruminal digestibility of OM, NDF, and starch were not negatively influenced by OEB deficit (Table 4). Total tract and ruminal digestibility measured in this experiment were consistent with those generally measured on double-muscled Belgian Blue bulls (Fiems et al., 1997; Valkeners et al., 2006). According to these results, the fiber and starch degradation activities of the rumen microbes were not affected by a lack of ruminally available N, meaning that the energy availability for animal maintenance and growth were the same for the 3 diets. Nevertheless, total VFA concentration decreased linearly as the dietary OEB level was decreased. For the –23.7 g of OEB/kg of DM diet, the decrease of total VFA concentration was associated with an increase in mean ruminal pH of 0.1 unit (Table 3) and was mainly due to a significant decrease in acetate concentration. Ruminal concentration of VFA is the net result of production and absorption (Owens and Goetsch, 1988), and the rate of absorption increases as ruminal pH decreases (Dijkstra et al., 1993). Moreover, the ruminal pH effect on the rate of VFA absorption increases with increasing VFA chain length (Rémond et al., 1996). However, as the average ruminal pH increased with the lowest OEB diet, the lowest VFA concentration measured was probably the result of a lower VFA production rather than a greater VFA absorption through the rumen epithelium.

On the other hand, level of OEB in the diet had no significant effect on NAN and microbial N flows at the duodenum (P = 0.36 and 0.37, respectively). However, NAN and microbial N flows measured with the 5.3 g of OEB/kg of DM diet were 8.8 and 11.3% numerically greater compared with those measured with the negative OEB diets.

Ruminal NH₃-N concentration was highly influenced by the level of OEB in the diet (Table 3; Figure 3). Decreasing CP concentration in the diet or degradation of dietary N usually results in decreased NH₃-N concentrations in the rumen (Milton et al., 1997; Peyraud et al., 1997; Hristov et al., 2004; Ipharraguerre et al., 2005). In growing double-muscled Belgian Blue bulls, Valkeners (2005) found positive correlations between the OEB value of a diet and the ruminal NH₃-N concentration either measured 1 h after its ingestion or on average over 12 h after its ingestion (R² = 0.83 and 0.74, respectively). Concentration of NH₃-N in the rumen is a function of the rate of ruminal N degradation, concentration of rumen-degraded N above microbial needs, and the amount of dietary energy available to the ruminal microorganisms. The decrease in rumen-available N from urea in the diet with the –23.7 and –9.2 g of OEB/kg of DM diets resulted in a decrease of 80 and 47% in the ruminal NH₃-N concentration compared with the 5.3 g of OEB/kg of DM diet.

Plasma urea-N has repeatedly been shown to correlate positively to the level of dietary CP (Marini and Van Amburgh, 2003), RDP (Davidson et al., 2003), or OEB (Fiems et al., 1999a). Johnson and Preston (1995) found that increasing the CP level from 10 to 16% in steer diets by 2% steps caused a 1- to 3-mg increase in PUN concentrations between each CP level. Their findings are in agreement with our results, because the average PUN increased by 3.1 mg/100 mL between –23.7 and –9.2 g of OEB/kg of DM diets and by 2.9 mg/100 mL between –9.2 and 5.3 g of OEB/kg of DM diets. In our experiment, the PUN concentration was closely related to the ruminal NH₃-N evolutions. With the positive OEB diet, a large production of NH₃-N in the rumen occurred after the degradation of the urea included in the diet. It is very likely that large amounts of NH₃ were lost from the rumen fluid by absorption through the rumen epithelium and transferred by portal blood to the liver to produce urea. Our results showed that the net N balance across the forestomach amounted to –10.5 g/d for the positive OEB diet. According to our results, the maximum NH₃-N concentration in the rumen was followed 3 h later by the maximum urea-N concentration in the plasma. This delay between these 2 peaks was in agreement with those observed by Gustafsson and Palmquist (1993). With the 2 negative OEB diets, PUN variations throughout the 12 h after the meal were small, and even nonexistent (Figure 4). These PUN evolutions were consistent with the low ruminal NH₃-N concentrations measured with the bulls fed these diets. Moreover, the net N balance across the forestomach amounted to 0.6 and 17.8 g of N/d for –9.2 and –23.7 g of OEB/kg of DM diets, respectively.

Amounts of N apparently digested in the rumen increased in response to increments in RDP input (Table 5) and were largely negative for bulls fed the –23.7 and –9.2 g of OEB/kg of DM diets. It seems that more N was transferred from the blood to the rumen of bulls fed diets characterized by an OEB deficit to sustain microbial growth and fermentation. This observation is supported by studies with Holstein heifers (Marini and Van Amburgh, 2003) and dairy cows (Al-Dehneh et al., 1997), in which it was estimated that the amount and proportion of endogenous urea N used by ruminal microbes increased when the availability of feed N in the rumen, but not of energy, became increasingly deficient. Moreover, N balance across the forestomach amounted to 33.5 and 14.8 g/d for the bulls fed the –23.7 and –9.2 g of OEB/ kg of DM diets, respectively. True ruminal digestibility of N measured with the experimental diets decreased (P = 0.02) in response to the decrease of the OEB level of the diet. These values were close to their respective theoretical ruminal N degradability for –9.2 and 5.3 g of OEB/ kg of DM diets, but represented less than 90% of the theoretical value for –23.7 g of OEB/kg of DM diets. The measured efficiency of the microbial synthesis was lower than the value used in the Dutch protein system when the FOM corresponded to the predicted ones. Consequently, the calculated OEB values of the diets were greater than the values based on the chemical and tabular values (Table 1). Total tract apparent digestibility of N was significantly different among the treatments (Table 5). This response can be attributed to significant increase of N intake and to the increased N degradation and losses of NH₃ from the rumen with bulls fed the 5.3 g of OEB/kg of DM diet. Additionally, the suggested larger incorporation of endogenous N into microbial protein synthesized in the rumen of bulls fed the 2 diets with an OEB deficit might have contributed to explain these differences.

With ruminants fed very low N diets, the percentage of urea in the urine can be very low; urea-N represented only 17.9% of the urinary N excretion in Marini and Van Amburgh (2003) with a 1.45% N diet. These low excretions of urea-N are related to a lower urea production and a very efficient recycling into the gut; thus, N excreted in the urine would mainly consist of compounds such as creatinine and creatine, metabolic products of purine metabolism (allantoin, uric acid, xanthine, and hypoxanthine), and small amounts of other more complex products of protein degradation and would represent the minimal urinary N excretion that cattle can achieve. The results of Marini and Van Amburgh (2003) showed that N intake had little or no effect on the urinary nonurea fraction. According to Ørskov and MacLeod (1982), the minimal urinary N excretion of steers (350 kg of BW) fed N-free diet by intragastric infusion could be estimated at 0.295 g of N/kg of BW^0.75. In our experiment, this minimal N excretion reached 24.9, 24.9, and 25.0 g of N/d for bulls fed, respectively, the –23.7, –9.2, and 5.3 g of OEB/kg of DM diets and represented 89, 62, and 46% of the observed urinary N excretion. For the lowest OEB level, urinary N is close to the N excretion measured when a N-free diet (Ørskov and MacLeod, 1982) is fed, which indicates that urinary N probably could not be reduced more, and this could explain why N retention measured with this diet is lower than with the positive OEB diet. However, because there is no rumen fermentation to sustain with animals fed by intragastric infusion, N recycling into the gut is reduced, and N excretion overestimated. Conversely, products of microbial nucleic acid degradation would not be present in urine of these animals and, therefore, would lead to a decrease in their minimal urinary N excretion. It would be very interesting, in our trial, to measure urea-N excreted in the urine to determine if bulls fed the diet with the greatest OEB deficit have achieved their minimal urinary N excretion.

Feeding diets characterized by an adequate DVE supply and a OEB close to –10 g/kg of DM could be a feeding strategy to reduce N losses from the farm with little effect on the animal performance and voluntary intake. Reduced OEB may reduce N excretion in the environment but may also result in decreased N retention.

	Footnotes

 
¹ The research was funded by Direction Générale de l’Agriculture, Ministère de la Région Wallonne, Belgium.

² We gratefully acknowledge SCAM (Seilles, Belgium) for providing all the feed concentrate used in this research.

³ We would like to express our sincere thanks to M.-G. Brouckaert, L. Givron, and S. Streel for their assistance with sample collection and analysis associated with this project and to H. Lapierre (Dairy and Swine Research and Development Center, Agriculture and Agri-Food Canada, Lennoxville, Quebec) for help with the preparation of the manuscript.

⁴ Corresponding author: beckers.y{at}fsagx.ac.be

Received for publication May 8, 2007. Accepted for publication November 16, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


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