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ANIMAL PRODUCTION |
Department of Animal and Range Sciences, North Dakota State University, Fargo 58105
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: digestion • forage • insulin • protein supplementation • steer
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), and research is needed to gain a greater understanding of microbial efficiency with various forage types and qualities (Lardy et al., 2004) and how it is affected by DIP and UIP supplementation.
Nutritional and physiological status can be characterized using blood metabolites and hormones and may be modified by protein supplementation (Cheema et al., 1991; Wiley et al., 1991; Sletmoen-Olson et al., 2000a). However, recent research indicates no effect of UIP supplementation on blood insulin (Kane et al., 2004; Encinias et al., 2005), NEFA (Encinias et al., 2005), or urea N (Strauch et al., 2001; Encinias et al., 2005). Additional insight regarding UIP supplementation when using various forage types and qualities and the effects on blood metabolites and hormones is needed.
Therefore, our objectives were to evaluate the effects of UIP supplementation on intake, digestion, microbial efficiency, in situ disappearance, and plasma hormones and metabolites in steers fed low-quality grass hay.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Steers on the supplement treatments were fed 1,040 g of supplement DM at 0800 daily, and chopped (10.2 cm in length) grass hay [mature, predominantly cool-season prairie hay that was harvested in northern Barnes County, North Dakota (6.0 % CP); Table 1] was offered ad libitum. Grass hay was fed immediately after supplement consumption and again at 1600 if necessary. Fresh water and trace-mineralized salt (minimum of 98 g of NaCl, 350 mg of Zn, 280 mg of Mn, 175 mg of Fe, 35 mg of Cu, 7 mg of I, and 7 mg of Co/100 g) were provided free choice.
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Chromic oxide was used as an indigestible flow marker. Eight grams of chromic oxide was weighed into gelatin capsules (Torpac Inc., Fairfield, NJ) and dosed through the ruminal cannula at 0800 and 2000 daily. Dosing began 5 d before and continued throughout the collection period. Duodenal samples were collected (approximately 200 mL) using a system that allowed for every other hour in a 24-h period to be sampled. Therefore, samples were collected at 0800, 1600, and 2400 on d 2; 0600, 1000, and 1800 on d 3; 0200, 1200, and 2000 on d 4; and 0400, 1400, and 2200 on d 5 of each collection period. Duodenal samples were composited across sampling times within steer and sampling period and stored frozen (–20°C).
On d 1 of each collection period, ruminal fluid samples were taken for pH, ammonia, VFA, and liquid dilution rate analysis. At 0600, 200 mL of Co-EDTA (fluid-phase marker; Uden et al., 1980) containing 827 mg of Co was placed in the midventral region of the rumen. Samples of whole ruminal contents (approximately 250 mL) were taken from the midventral region at –2 (immediately before Co-EDTA dosing), 0, 1, 2, 4, 8, 12, and 24 h in relation to supplementation. Ruminal pH was immediately determined with a portable pH meter and combination electrode (SA230, Orion, Cambridge, MA). The contents were strained through 4 layers of cheesecloth, and the fluid portion was acidified with 7.2 N H2SO4 (1 mL of acid/100 mL of strained ruminal fluid). The samples were stored at –20°C until analysis. The ruminal Co concentration was determined after centrifugation (18,000 x g; 20 min) using atomic absorption spectrometry (3030B, Perkin Elmer Inc., Wellesley, MA). Fluid dilution rate was calculated by regressing the natural logarithm of the Co concentrations against time (Warner and Stacy, 1968).
In situ degradation measurements were conducted on d 2 through 5 of each sampling period. Dacron bags (10 x 20 cm, 53 ± 10-µm pore size, Ankom, Fairport, NY) containing approximately 5 g of ground (2-mm screen) hay were incubated for 72, 48, 36, 24, 16, 12, 8, 4, and 0 h. At each incubation time, 3 bags containing hay and 1 blank were introduced into the rumen. All bags were removed at 2000 on d 5. In addition, 3 Dacron bags containing approximately 5 g of supplement and 1 blank were introduced at the 16-h incubation time. All bags were suspended in a mesh, nylon bag (18 x 24 cm) fitted with a zipper. The Dacron bags were sealed with a number 8 rubber stopper and two number 19 rubber bands. After incubation, all bags were removed and washed in warm tap water until the rinse water was clear. Then, the bags were dried in a forced-air oven (50°C), desiccated, weighed, and stored.
Ruminal fluid samples (3 L) were taken on d 6 of each sampling period for bacterial isolation. The samples were strained through 4 layers of cheesecloth and preserved with 25 mL of 0.15 M NaCl in 37% (vol/vol) formaldehyde/100 mL of strained ruminal fluid. The samples were stored at 10°C until the bacterial cells were harvested.
Intensive blood samples were collected on d 1 of each sampling period at –2, –0.5, 1, 2, 4, 8, 12, and 24 h with respect to supplementation. Daily blood samples were taken at 0730 (i.e., at the –0.5-h sampling time) on the remaining 4 d of the collection period. All blood samples were collected in 10-mL tubes containing EDTA as an anticoagulant. The samples were refrigerated until centrifuged (1,520 x g) at 4°C for 30 min. Plasma was decanted into 16 x 100-mm borosilicate glass tubes and stored frozen (–20°C) until analyses.
Laboratory Analyses.
Diets, orts, feces, in situ residues, and lyophilized (Genesis model 25 LL, Virtis, Gardener, NY) duodenal samples were analyzed for DM, ash, N (methods 930.15, 942.05, and 990.02, respectively; AOAC, 1990), and ADF and NDF (Robertson and Van Soest, 1981).
Bacterial cells were isolated from ruminal fluid by differential centrifugation (Merchen and Satter, 1983). Lyophilized, isolated bacterial cells and duodenal samples were analyzed for DM, ash, and N (AOAC, 1990). Purine analysis was conducted according to the method of Zinn and Owens (1986).
Fecal and duodenal samples were analyzed for Cr concentration by atomic absorption spectroscopy (air-plus-acetylene flame). Samples were prepared for analyses by the procedure of Williams et al. (1962). Dried fecal and duodenal samples were analyzed for DM, ash, and N as described above.
Intake and fecal output were determined by direct measurement. Duodenal DM flow was calculated by dividing the daily marker dose, in grams, by the marker concentration at the duodenum (g/g of DM, corrected for the percentage of the marker recovered in feces). Duodenal N flow was determined by multiplying the percentage composition of the duodenal contents by the duodenal DM flow. Duodenal bacterial N flow was estimated by multiplying the purine content of the duodenal samples by the N-purine ratio in isolated bacterial cells (Zinn and Owens, 1986).
Ruminal fluid samples were thawed at room temperature and centrifuged at 10,000 x g for 10 min. Then, the supernatant was mixed with 25% (wt/vol) metaphosphoric acid (1 mL of metaphosphoric acid/5 mL of ruminal fluid) and recentrifuged at 10,000 x g for 10 min. The supernatant was used for VFA analysis (Goetsch and Galyean, 1983). The internal standard was 2-ethylbutyric acid. Determination of VFA was by gas chromatography (Shimadzu Scientific Instruments, Columbia, MD; packed column, 140°C, N gas carrier).
The supernatant from the initial centrifugation was analyzed for Co and ammonia concentrations. The Co analysis was conducted by atomic absorption spectroscopy using an air-acetylene flame. Ammonia concentrations in the ruminal supernatant and duodenal fluid (after centrifugation at 30,000 x g for 20 min) were determined by the colorimetric procedure of Broderick and Kang (1980).
In situ rate and lag of NDF digestion were estimated using the nonlinear model of Mertens and Loften (1980). The rate of in situ CP disappearance (corrected for bacterial attachment) was calculated using the model outlined by Ørskov and McDonald (1979). This model calculates a rapidly degraded CP fraction (fraction A; assumed to be instantaneously degraded) and a slowly degraded fraction (fraction B; NRC, 1985). Degradation rates derived from this model are associated with fraction B. Computations of NDF and CP rates of digestion were conducted using nonlinear procedures (Marquardt method; SAS Inst. Inc., Cary, NC). Corrections for microbial contamination of in situ residue were made using purines as the microbial markers (Messman et al., 1992).
Plasma samples were allowed to thaw at room temperature before analysis. Plasma glucose was measured using a glucose oxidase kit (glucose, procedure number 510; Sigma Diagnostics, St. Louis, MO; within- and across-assay CV were 4.8 and 6.8%, respectively). Plasma urea N was determined using a urease-Berthelot determination (urea N, procedure number 640, Sigma Diagnostics; within- and between-assay CV were 4.5 and 2.1%, respectively). The Acyl-CoA synthetase-acyl-CoA oxidase method (NEFA C, Wako Pure Chemical Industries, Dallas TX; within- and across-assay CV were 4.3 and 7.6%, respectively) was used to measure plasma NEFA. Plasma GH was measured using an RIA previously validated for bovine plasma (Reynolds et al., 1990; within- and between-CV were 5.6 and 3.1%, respectively). Insulin was determined using an RIA procedure validated for bovine plasma (Sletmoen-Olson et al., 2000a; within- and across-assay CV were 9.6 and 8.6%, respectively).
Statistical Analysis.
Data were analyzed using the MIXED procedure (SAS Inst. Inc.). Intake, digestion, microbial efficiency, and in situ data were analyzed as a 4 x 4 Latin square (Cochran and Cox, 1957). The model included the effects of steer, sampling period, and treatment. Steer and sampling period were random effects, and treatment was the fixed effect. Ruminal fermentation data and intensive blood samples were analyzed as repeated measures within a 4 x 4 Latin square. The model contained the effects of steer, period, treatment, and sampling time. Steer x period x treatment and sampling time x treatment interactions were included in the model. Sampling time was used as the repeated effect, and steer nested within treatment was used as the subject. Contrasts were used to address specific questions. The specific contrasts were as follows: 1) control vs. the average of low, medium, and high UIP to determine if there was a protein response; 2) low vs. the average of medium and high UIP to determine if there was a UIP response; and 3) medium vs. high levels of UIP to determine if there was a response to level of UIP. Contrasts were conducted using SAS and were protected by a significant F-test (P < 0.10).
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Similarly, Sletmoen-Olson et al. (2000b) reported no differences in forage OM intake when feeding 5.8% CP mixed-grass prairie hay and similar levels of UIP to gestating beef cows during the last trimester of gestation and first 3 mo of lactation, and Encinias et al. (2005) reported no differences in forage DMI (% of BW) when evaluating lactating beef cows fed bromegrass hay (9.6% CP) and consuming energy, DIP, or UIP supplements. When feeding energy supplements with either added DIP (urea or soybean meal) or UIP (blood meal and feather meal) to lambs consuming 4.3% CP bromegrass hay, Ferrell et al. (1999) reported no differences in forage intake among treatments. In contrast, Strauch et al. (2001) supplemented UIP (157 g/d of DIP and 106 g/d of UIP for control; 155 g/d of DIP and 190 g/d of UIP for UIP treatment) to pre- and postpartum heifers grazing stockpiled fescue (11.7% CP) and reported a tendency for increased forage intake in UIP-supplemented heifers. Protein supplementation of ruminants consuming low-quality forages generally increases forage intake (Guthrie and Wagner, 1988; McCollum and Horn, 1990; Köster et al., 1996). Because control steers in our study did not have reduced forage intake compared with protein-supplemented steers, DIP levels may have been adequate, or the steers may have recycled adequate amounts of N to prevent reductions in forage intake. After observation of numerous protein supplementation studies, Ferrell et al. (1999) suggested that intake response to supplementation may be expected if forage intake without supplementation is low, but if forage intake without supplementation is relatively high, then an increase in intake response to supplementation is less likely. Total OM intake, expressed as grams per day and grams per kilogram of BW, tended (P = 0.10 and 0.13, respectively) to increase with protein supplementation, as a function of supplement offered in addition to hay.
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). There were no differences (P > 0.26) for low UIP vs. medium and high UIP supplements or medium vs. high supplements (P > 0.83) for apparent and true ruminal OM digestion. Postruminal OM digestion increased (P = 0.03) in steers fed medium and high UIP supplements compared with low UIP supplements. Total tract OM digestion (% of intake) increased (P = 0.01) in protein-supplemented steers compared with controls and was greater (P = 0.09) in medium and high UIP vs. low UIP-supplemented steers. Similarly, Bandyk et al. (2001) and Swanson et al. (2004) reported increased total tract OM disappearance with casein infusion compared with controls (low-quality hay) but reported no differences in site of casein infusion (rumen vs. abomasum). In contrast, Salisbury et al. (2004) reported no differences in ruminal, postruminal, or total tract OM digestibility (%) in wethers consuming low-quality grass hay (7.5% CP) when control (7.5% CP hay) vs. low UIP and high UIP supplements or low UIP vs. high UIP supplements were compared.
Ruminal, postruminal, and total tract NDF and ADF digestion were unaffected (P > 0.11) by treatment (Table 2). Although, there was a tendency (P = 0.11) for ruminal NDF digestion to be affected by treatment, unprotected contrasts indicate that ruminal NDF digestion increased (P = 0.03) in steers supplemented with protein and increased (P = 0.04) in medium and high vs. low UIP-supplemented steers. Increasing ruminal NDF digestion with supplemental DIP was reported by Salisbury et al. (2004) and Köster (1996); however, others (Krysl et al., 1987; Bohnert et al., 2002) have reported no differences in ruminal NDF digestion due to supplemental DIP. Mathis et al. (2000) reported no differences in total tract NDF digestion (%) when supplementing increasing amounts of DIP with Bermudagrass (8.2% CP) and bromegrass (5.9% CP) diets but reported increased total tract NDF digestion (%) when supplementing increasing amounts of DIP with forage sorghum (4.3% CP) diets. Differences in CP content of the forages and the potential for some of the digestible UIP to contribute to the ruminally available N pool via recycling of blood urea N to the rumen may have contributed to the differences in digestion (Mathis et al., 2000).
As designed, N intakes increased (P = 0.001) as dietary levels of CP increased (Table 3). Total duodenal flow of N increased (P = 0.05) in steers fed supplemental protein compared with controls. Total duodenal N flow also increased in steers supplemented with medium and high UIP vs. low UIP (P = 0.03) and in steers supplemented with high vs. medium UIP (P = 0.06). Increasing levels of N flow to the duodenum were a result of supplements containing increasing levels of UIP. Duodenal N flow from feed increased in steers supplemented with medium and high UIP vs. low UIP (P = 0.01) and in steers supplemented with high vs. medium UIP (P = 0.07) and was due to increased levels of UIP in the diet. Treatment did not (P = 0.30) cause a difference in bacterial N flow to the duodenum, indicating that DIP levels in control diets were adequate for bacterial CP synthesis or the steers were able to recycle adequate amounts of N to meet requirements of the bacteria.
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), which agrees with previous reports in which increased DIP levels increased ammonia N flow at the duodenum (Köster et al., 1996; Mathis et al., 2000; Bandyk et al., 2001). Duodenal ammonia flow also increased (P = 0.07) in steers fed medium and high UIP compared with low UIP. It is not apparent why duodenal ammonia flow was reduced in low compared with medium and high UIP treatments. Levels of DIP in the supplements were 24.8, 22.1, and 19.2% for low, medium, and high UIP, respectively. These data may suggest that ammonia absorption across the rumen wall was increased in steers on the low UIP treatment; however, increased ruminal absorption of ammonia is dependent on increased ruminal ammonia concentration and pH, and ruminal ammonia (mg/dL) was not different (P > 0.14) between UIP treatments. Additionally, pH decreased (P = 0.02) in low compared with medium and high UIP treatments (Table 4).
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). Apparent and true ruminal N digestion increased (P < 0.009) in steers fed supplemental protein. Similarly, Reed et al. (2004) reported increased apparent and true ruminal N disappearance and apparent total tract N disappearance with increasing level of field pea supplementation in 8% CP grass hay diets. Field peas were fed at 0, 0.81, 1.62, and 2.43 kg/d and provided approximately 0, 41.7, 83, and 125 g/d of UIP and 0, 148, 296, and 442 g/d of DIP, respectively (Reed et al., 2004). Köster et al. (1996) reported a quadratic increase in apparent ruminal N digestibility with the addition of DIP to low-quality hay diets. Similar to our study, negative ruminal N digestibilites also were reported by Köster et al. (1996) at low levels of DIP supplementation and are due to N recycling (Bunting et al., 1989) and bacterial N.
Postruminal N digestion as a percentage of N intake was greater (P = 0.007) in steers consuming the control diet vs. steers consuming diets containing supplemental protein, which partially resulted from decreased ruminal N digestion in controls (Table 3). Similarly, Salisbury et al. (2004) reported that postruminal N digestibility (% of intake) was nearly 2-fold greater for controls (consuming 7.5% CP hay) compared with UIP-supplemented wethers. Postruminal N digestion was not different (P > 0.60) among the protein-supplemented treatments. In addition, postruminal N digestion as a percentage of N entering the duodenum was unaffected (P = 0.21) by protein supplementation (data not shown). Again, Salisbury et al. (2004) reported that postruminal N digestibility as a percentage of N entering the duodenum did not differ between control and UIP-supplemented wethers. Total tract N digestion increased (% of intake, P = 0.001) in protein-supplemented steers compared with controls and increased (P = 0.03) in medium and high UIP- compared with low UIP-supplemented steers. Microbial efficiency also was unaffected (P = 0.97) by treatment. Salisbury et al. (2004) reported increased microbial efficiency in control compared with UIP-supplemented wethers consuming 7.5% CP hay. In contrast to Salisbury et al. (2004), Köster et al. (1996) reported a linear increase in microbial efficiency with increasing DIP supplementation of cows consuming 1.9% CP tall-grass forage. Differences in these studies are likely due to vast differences in forage CP and the resulting level of available DIP for microbial growth.
Ruminal pH was not different (P > 0.32) between control and protein-supplemented steers or medium vs. high UIP-supplemented steers (Table 4). However, pH was decreased (P = 0.02) in low UIP- vs. medium and high UIP-supplemented steers. Numerous researchers (Köster et al., 1996; Heldt et al., 1999; Mathis et al., 2000) have reported lower pH with increasing levels of DIP supplementation. Because changes in pH are a result of changes in ruminal fermentation, it is not apparent why ruminal pH was not different between control and protein-supplemented steers but different between low UIP- and medium and high UIP-supplemented steers. Low, medium, and high UIP treatments were formulated to contain similar levels of DIP.
Ruminal ammonia concentration increased (P = 0.001) in steers fed supplemental protein compared with controls, which was expected due to increased dietary DIP. There also was a treatment x time interaction (P = 0.001) for ruminal ammonia; however, this was due to a difference in magnitude and not due a change in treatment ranking; therefore, main effects of treatment are reported. There were no differences (P > 0.14) among protein supplemental treatments for ruminal ammonia concentration, which was expected because DIP intake was similar among treatments (257.9, 229.8, and 199.7 g of DIP/d for low, medium, and high, respectively).
Total VFA concentration was not affected (P = 0.86) by treatment (Table 4). However, molar proportions of acetate and butyrate were affected. There was a treatment x time interaction (P = 0.02) for molar proportion of acetate; however, this was due to a difference in magnitude rather than a change in treatment ranking. Molar proportion of acetate was increased (P = 0.05) in controls compared with supplemented steers, and molar proportion of acetate was decreased (P = 0.03) in low UIP- vs. medium and high UIP-supplemented steers. Other researchers (Köster et al., 1996; Heldt et al., 1999; Mathis et al., 2000) investigating DIP supplementation of low-quality hay diets have reported decreased molar proportions of acetate and increased molar proportions of propionate with DIP supplementation. Treatment did not (P = 0.42) affect molar proportions of propionate. There was no difference (P = 0.19) for molar proportion of butyrate between control and protein-supplemented steers; however, molar proportion of butyrate was increased (P = 0.008) in low UIP- vs. medium and high UIP-supplemented steers and was greater (P = 0.03) in high vs. medium UIP-supplemented steers.
Supplemental protein had no effect (P > 0.13) on in situ forage NDF digestion or CP degradation (Table 5). Similarly, Reed et al. (2004) reported no differences in rate of in situ forage DM, NDF, or N disappearance when supplementing increasing level of field peas. Krysl et al. (1987) reported no differences for in situ forage NDF disappearance with soybean meal and sorghum supplementation. Treatment tended (P = 0.10) to affect liquid vol (L) when protein was supplemented; increasing levels of UIP decreased (P = 0.06) liquid vol. Supplemental protein also had no affect (P > 0.22) on ruminal fluid dilution rate, fluid turnover time, or rate of fluid flow.
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). Protein supplementation increased (P = 0.03) blood glucose concentration compared with controls; medium and high UIP supplementation increased (P = 0.02) blood glucose compared with low UIP. Supplemental protein resulted in increased (P = 0.01) blood urea N compared with steers not receiving supplemental protein. Supplementation with medium and high UIP increased (P = 0.02) blood urea N compared with steers supplemented with low UIP, and supplementation with high UIP tended (P = 0.11) to increase blood urea N compared with steers supplemented with medium UIP.
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). Furthermore, supplementation with medium and high UIP increased (P = 0.003) baseline plasma insulin compared with low UIP, and supplementation with medium UIP increased (P = 0.02) plasma insulin compared with high UIP. Insulin production is stimulated by elevated levels of AA, which likely explains increased blood insulin concentrations in response to increased levels of dietary UIP. Supplementation of medium and high UIP may have increased baseline plasma insulin concentrations or caused increased preprandial release of insulin (daily blood samples were taken at 0.5 h before feeding). Similarly, Sletmoen-Olson et al. (2000a) reported an increase in plasma insulin concentrations in response to protein supplementation in both gestating and lactating beef cows and reported that cows fed a low UIP supplement generally had decreased plasma insulin concentrations compared with cows fed medium and high UIP supplements. However, Kane et al. (2004) reported no differences in insulin concentrations in cycling beef heifers, and Encinias et al. (2005) reported no differences in insulin concentrations in lactating beef cows fed low or high levels of UIP. Insulin response to UIP supplementation likely differs by age, production state, and duration of supplementation (Kane et al., 2004).
Protein supplementation did not affect (P > 0.21) blood glucose, GH, or NEFA concentration in samples taken daily 0.5 h before feeding (Table 6). Supplemental protein resulted in increased (P = 0.001) blood urea N compared with controls. Supplementation with medium and high UIP increased (P = 0.001) blood urea N concentration compared with low UIP, and supplementation with high UIP increased (P = 0.001) urea N concentration compared with medium UIP. These trends are similar to those reported by Sletmoen-Olson et al. (2000a) in gestating and lactating beef cows and were expected because blood urea N concentration is related to level of CP in the diet (Preston et al., 1965). Others also have reported increased blood urea N in UIP-supplemented beef cows (Wiley et al., 1991; Dhuyvetter et al., 1993; Alderton et al., 2000).
In summary, under the conditions in this study, supplementation with additional UIP when DIP requirements are met provided little added benefit. It appears that the supplemental protein levels used in this study (8.3 g/d of UIP and 257.9 g/d of DIP for the low UIP treatment), when fed in conjunction with 6.0% CP grass hay, were adequate to meet MP requirements. These data indicate increasing UIP supplementation when DIP and energy levels are held constant increases baseline plasma insulin concentrations; however, mechanisms and implications of these changes are not completely understood.
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Footnotes |
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2 Corresponding author: joel.caton{at}ndsu.edu
Received for publication September 10, 2006. Accepted for publication December 14, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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