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ANIMAL NUTRITION |
* Departament de Ciència Animal i dels Aliments and
and
Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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0.105) by diet. The 30:70 diet resulted in faster (P = 0.045) fluid passage rate and decreased (P = 0.015) ammonia N concentration compared with the 12:88 diet, but no differences (P 0.244) were detected in ruminal pH and total VFA concentration between diets. The rate of degradation and the effective degradability of N in protein supplements was greater with the 30:70 diet for peas (P 
0.008) and lupin seeds (P 0.02), and in the 12:88 diet for sunflower meal (P 0.06). Degradation of NDF of alfalfa hay was low with both diets (18.5 and 23.7 % for 12:88 and 30:70, respectively); however, the rate and extent of DM and NDF degradation were greater (P 0.016) with the 30:70 diet, suggesting a higher cellulolytic activity. Total VFA concentration and the proportion of propionate increased (P 0.035), and the acetate proportion decreased (P = 0.021) with age. Average pH, ammonia N concentration, and passage rates were not affected (P 0.168) by age. Degradation rate and effective degradability of N of sunflower meal, peas, lupin seeds, and of DM of alfalfa hay increased (P 0.08) with age, but degradation kinetics of NDF of alfalfa hay was not affected (P 0.249). The increase in the rate and extent of N degradation with age would suggest an increase in proteolytic activity, and the changes in the fermentation pattern may reflect an increase in amylolytic activity caused mainly by an increase in the gross intake of nonstructural carbohydrates and by adaptation of ruminal microflora after long exposure to these nutrients.
Key Words: Growing Cattle • Protein Degradation • Ruminal Fermentation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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; AFRC, 1993; , 1996) use protein degradation values mostly estimated in situ and obtained in adult animals fed diets with a higher forage-to-concentrate ratio than that commonly used in intensive beef production systems. Recent reviews of in situ procedures for estimating protein degradability have emphasized that the diet fed to cannulated animals, especially the forage-to-concentrate ratio, alters estimations of the rate and extent of protein degradation (Huntington and Givens, 1995; Vanzant et al., 1998; Broderick and Cochran, 2000). Moreover, protein degradability values estimated in mature calves are not applicable to young weaned calves because of an effect of age and rumen development on ruminal degradability (Lallès and Poncet, 1990; Vazquez-Añón et al., 1993). Evidence of this ruminal development is the increase in microbial protein reaching the duodenum with age (Quigley et al., 1985; Devant et al., 2000). Devant et al. (2000) also observed changes in the fermentation profile and DM degradation of straw in heifers fed high-concentrate diets from 100 to 230 kg BW, indicating that changes continue to occur long after weaning. It is likely that these changes also affect protein degradation in the rumen, but information on how the rate and extent of degradation vary as ruminants grow is very limited. The objective of this study was to describe the changes in ruminal fermentation and protein degradation of plant protein supplements in growing Holstein heifers from 80 to 250 kg BW fed high-concentrate diets with different forage-to-concentrate ratios.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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One week after surgery, animals were weaned and assigned randomly to one of two experimental diets (Table 1). Diets were isoenergetic (2.75 Mcal of ME/kg of DM) and isonitrogenous (15.1% CP; DM basis) and formulated to meet requirements for animals growing at 0.9 kg/d (NRC, 1996). Diets were designed to differ in forage-to-concentrate ratio (12:88 vs. 30:70), NDF content (21.6 vs. 27.9%; DM basis), and the source of forage NDF (barley straw vs. dehydrated alfalfa). Mean lengths of barley straw and dehydrated alfalfa were 4.1 and 2.8 cm, respectively. Subsequently, diets will be referred to as 12:88 and 30:70. Because dehydrated alfalfa is rich in protein, soybean hulls also were included in the 30:70 diet to maintain the CP content at 15%. Diets were offered once daily (0800) as a total mixed ration. Heifers were housed individually in outdoor paved and covered pens (2.0 m x 1.4 m) equipped with individual feed bunks and with ad libitum access to feed and water.
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Four plant protein supplements were incubated in situ from d 8 to 14 to estimate effective ruminal degradability (ED) of N. Protein supplements were solvent-extracted soybean meal (SBM: 49.1% CP, 12.1% NDF; DM basis), solvent-extracted sunflower meal (SFM: 33.8% CP, 37.8% NDF; DM basis), peas (23.0% CP, 8.3% NDF; DM basis), and lupin seeds (34.4% CP, 23.9% NDF; DM basis). Samples were ground to pass a 3-mm screen (hammer mill; P. Prats S.A., Sabadell, Spain). Alfalfa hay (19.8% CP, 29.9% NDF; DM basis) also was incubated at the same time to estimate ruminal degradation of DM and NDF. Plant protein sources (1.5 g, except 1 g for SBM; as-fed basis) and alfalfa hay (2 g; as-fed basis) were weighed into 5 cm x 10 cm nylon bags made from nitrogen-free polyester (Ankom Technology Co., Fairport, NY) with a mean pore size of 50 ± 15 µm and heat-sealed (Dea Lun Co., Ltd, Taiwan). Protein supplements were incubated for 2, 4, 8, 12, 24, 36, 48, and 72 h, whereas alfalfa hay was incubated for 2, 4, 8, 12, 24, 48, 72, and 96 h. Bags were placed at the same time and removed sequentially. One empty bag for each hour and diet was included and used as a blank in N analysis. This sequence was repeated twice within each period, and the two sequences were treated as replications for statistical analyses. Bags were removed and washed with cold water in a washing machine (three cycles of 5 min), and dried at 103°C in a forced-air oven for 24 h. Alfalfa hay residues were dried at 60°C over 72 h. Before ruminal incubation, bags were soaked in warm water for 30 min. Washing losses (time 0) were estimated by soaking the bags for 30 min in warm water and then washed as the incubated bags. Bag residues of protein supplements were analyzed for DM and N, and alfalfa hay residues were analyzed for DM and NDF.
On d 15, 30 min before the morning feeding, heifers were dosed intraruminally with chromium mordanted SBM (22.5 g/kg of DMI, representing 1.2 g of Cr/kg of DMI) and Co EDTA (1 g/kg of DMI) in a 50-mL aqueous solution (Udén et al., 1980) to estimate ruminal particulate and fluid passage rates. A 500-mL sample of ruminal contents was collected with a vacuum pump from different locations in the rumen before the morning feeding and at 0.5, 1, 2, 4, 8, 12, 16, and 24 h after feeding and squeezed through two layers of cheesecloth. After sampling, extra ruminal contents were returned to the rumen. The pH of the ruminal fluid was measured immediately and three subsamples were taken. First, a 4-mL subsample of strained fluid was acidified with 4 mL of 0.2 N HCl and frozen. Samples were later thawed in the refrigerator (12 h at 4°C), then centrifuged at 25,000 x g for 20 min, and the supernatant fraction was analyzed for NH3 N by spectrophotometry (Chaney and Marbach, 1962). Second, based on the method of Jouany (1982), 1 mL of a solution made up of 0.2% (wt/wt) mercuric chloride (to impede microbial growth), 2% (vol/vol) orthophosphoric acid, and 0.2% (wt/wt) 4-methylvaleric (internal standard) in distilled water was added to 4 mL of strained ruminal fluid, and the mixture was frozen. Volatile fatty acids were analyzed with a polyethylene glycol TPA-treated capillary column (BP21, SGE Europe Ltd., Milton Keynes, U.K.) by gas chromatography using a model G1530A (6890) gas chromatograph (Hewlett Packard GmbH Chemische Analysentechnik, Waldbronn, Germany). Third, a 30-mL sample of strained ruminal fluid was frozen and stored. Samples were later centrifuged at 25,000 x g for 20 min before being analyzed for Co concentration by atomic absorption spectrophotometry (Udén et al., 1980). Samples at 0.5 and 1 h after feeding were only used for Co determination.
For Cr analysis, ruminal particulate samples were collected from d 15 to 17 before the morning feeding and at 4 and 12 h after feeding. Samples were dried at 103°C over 48 h, and Cr concentration was determined according to the procedure of Le Du and Penning (1982) by atomic absorption spectrophotometry. Before marker administration, a sample of ruminal contents was collected to determine the background concentrations of Co and Cr.
Feeds, refusals, and supplements were analyzed for DM (24 h at 103°C) and ash (4 h at 550°C). Nitrogen content was determined using the Kjeldahl procedure (AOAC, 1990), ether extract was analyzed according to AOAC methods (1990), and NDF according to Van Soest et al. (1991) using sodium sulfite and 
-amylase.
Calculations
Ruminal fluid pH, NH3 N, and VFA measures after feeding were averaged across time by calculating the area under the ruminal data vs. time curve and dividing by total time (Pitt and Pell, 1997). Ruminal fluid and particulate passage rates (kp) were calculated as the slope of the regression line of the natural logarithm of Co or Cr concentration vs. time, respectively (Faichney, 1975). Coefficients of determination for ruminal passage rates averaged 0.94 for Co and 0.89 for Cr. Values for ruminal disappearance of N and NDF vs. time were fitted to the exponential equation of Ørskov and McDonald (1979) and Dhanoa (1988), respectively. Effective degradation was calculated using the following equation:
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where a, b, c, and kp are the soluble fraction, insoluble but potentially degradable fraction, rate of degradation, and rate of passage, respectively. Coefficients a, b, and c were obtained from the exponential equations of the NLIN procedure of SAS (SAS Inst., Inc., Cary, NC), and kp was obtained from the external marker used to estimate ruminal particulate passage rate.
Statistical Analyses
Data were analyzed using PROC MIXED (Littell et al., 1996) of SAS (SAS Inst., Inc., Cary, NC) for repeated measures. The model contained the effect of diet, period, and their interaction as fixed effects. Animal was used as a random effect, and period was the repeated factor. For the ruminal data collected at different times after feeding, the period x time after feeding interaction was the repeated factor. The period represented the heifers’ age in weeks.
The REML was used to estimate the variance components, and the Satterthwaite method was used to approximate the degrees of freedom. For each analyzed variable, data were subjected to four covariance structures: variance components, compound symmetric, one-band unstructured, and autoregressive of order one. The covariance structure that yielded the smaller Akaike and Schwarz’s Bayesian criterion based on their –2 res log likelihood was considered to provide the best fit. Standard errors presented in tables correspond to the maximum SE of the diet and period with the lowest number of observations. Regression analysis was conducted using the REG procedure of SAS, and the CORR procedure was used to determine the correlation between variables. Effects were considered significant at P 0.05.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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. Mean BW of heifers across diets was 86.5 ± 3.7, 163.4 ± 17.6, and 258.5 ± 25.7 kg at 13, 27, and 41 wk of age, respectively. There were no statistically significant interactions between age and diet for any variable; therefore, main effects are presented and discussed separately.
Diet Effect
Intake of DM, OM, CP, and NDF, expressed as kg/d or as g/BW0.75, did not differ statistically between diets (Table 2). Thus, the difference in forage-to-concentrate ratio was not sufficient to make the difference in NDF intake statistically significant, and in spite of the differences in ingredient composition, the two diets resulted in similar intake and growth rate.
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). No differences were detected for postprandial pH evolution (Figure 1A). Despite the high levels of concentrate, no clinical signs of acidosis were observed in any animal. These results agree with Devant et al. (2000, 2001), who recorded similar pH data with heifers fed high-concentrate diets. Beauchemin et al. (2003) observed that when steers were changed from a high-forage to a high-concentrate diet, the risk of acidosis decreased as time progressed. Therefore, it is likely that animals receiving the same high-concentrate diet from weaning were probably adapted to the high-concentrate intake and the risk of acidosis was minimized.
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) were not affected by diet, nor were the molar proportions of propionate, butyrate, and valerate. In contrast, the molar proportion of acetate tended to increase (P = 0.098) and branched-chain VFA (BCVFA) tended to decrease (P = 0.093) in the 30:70 compared with the 12:88 diet (Table 3).
Ruminal NH3 N concentration was higher (P = 0.015) in the 12:88 diet (Table 3), but average concentration in both diets was above the level suggested to maximize microbial protein synthesis (5 mg/100 mL; Satter and Slyter, 1974). A time after feeding x diet interaction was detected for postprandial evolution of NH3 N as a result of greater diurnal fluctuation in NH3 N concentration for heifers consuming the 12:88 diet (Figure 1C; P = 0.003). The rapid postprandial decrease in NH3 N in the 12:88 diet can be explained by a rapid incorporation of NH3 N into microbial protein due to a higher availability of energy from nonstructural carbohydrates immediately after feeding, as observed by Bourquin et al. (1994a).
Particulate passage rate was not affected by diet (average of 6.99 ± 0.78 %/h) and was similar to the results reported by Devant et al. (2001) in similar experimental conditions, but greater than the average reported by Owens and Goetsch (1986) for high-concentrate diets. Fluid passage rate was greater (P = 0.045) in the 30:70 diet and was similar to the averages reported by Owens and Goetsch (1986) for a high-concentrate diet and high intake. This higher passage rate may be due to an increase in mastication and salivation caused by the higher forage content in the 30:70 diet than a physical constraint in ruminal volume (Cole et al., 1976). The higher fluid passage rate observed in the 30:70 diet could be responsible for the lower NH3 N concentration observed in this diet resulting from a greater escape of NH3 N to the abomasum (Varga and Prigge, 1982), together with the lower protein intake noted with this diet (P = 0.11). A negative correlation was observed between fluid passage rate and NH3 N concentration (r = –0.52; P < 0.05).
Bourquin et al. (1994b) observed that dietary effects on in situ degradation were visible on the rate and extent of degradation but not on the soluble or potentially degradable fractions of the incubated feedstuff. In the present experiment, diet did not affect the soluble and potentially degradable fractions of N of plant protein supplements (Table 4), except for soluble fraction of SBM (P = 0.04). Rate of degradation of N was higher in the 30:70 diet for peas and lupin seeds (P 0.008), and tended to be higher in the 12:88 diet for SFM (P = 0.06). Effective degradability of N was greater in the 30:70 diet for peas and lupin seed (P 0.02) and in the 12:88 diet for SFM (P = 0.02). Effective degradability of NDF of alfalfa hay was very low in both diets (Table 5), indicating low cellulolytic activity. Because average pH was 6.37, the low cellulolytic activity can likely be attributed to a substrate effect rather than to pH inhibition (Mould and Ørskov, 1983). In both diets, pH was below 6 for less than 12 h per day, and this decrease in pH should not have seriously inhibited NDF degradation (Calsamiglia et al., 2002). Alfalfa hay had a greater rate and extent of DM (P = 0.016 and 0.001, respectively) and NDF (P = 0.006 and 0.014, respectively) degradation in the 30:70 vs. the 12:88 diet. This response cannot be attributed to a pH effect, as both diets had similar trends for ruminal pH, but rather to a greater cellulolytic fermentation (Hoover, 1986). A basal diet containing alfalfa could account for a more specific and efficient microflora adapted to the structure and composition of alfalfa cell wall, as Akin (1979) reported that the type of bacteria associated with fiber digestion varies among forages. This is supported by the trend of greater acetate proportion observed when this diet was fed.
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, mean values of nitrogen ED of protein supplements were lower than those reported in current beef feeding systems, probably because degradation values used by these systems usually have been obtained in animals fed a basal diet with a higher proportion of forage than that used in the present experiment. The decreased pH typically observed in high-concentrate diets also could account for a decrease in proteolysis (Erfle et al., 1982; Shiver et al., 1986) and cellulolysis (Hoover, 1986). Ganev et al. (1979) observed that, as plant proteins are protected by a cellulose structure, protein degradation is greater in high-forage diets because cellulolytic activity is greater in the ruminal conditions achieved with these diets, facilitating access to protein and, as a consequence, its degradation. However, the low degradability of NDF of alfalfa hay and the small difference between diets were not sufficient to cause a clear effect of forage-to-concentrate ratio on protein degradation.
Age Effect
As expected, the intake of DM, OM, CP, and NDF, expressed as kg/d, increased (Table 2; P < 0.001) with age. Intake of DM was strongly related to BW (DMI = 1.01 + 0.024 BW; r2 = 0.92; P < 0.001). In contrast, the effect of age disappeared when the intake was expressed as g/kg BW0.75. Although the numerical increase in NDF intake was not significant, the NDF content of the refusals decreased (Table 2; P = 0.001) with age in both diets (Figure 2), indicating that heifers selected against forage at 13 wk of age, were not selective at 27 wk, and selected for forages at 41 wk. This may reflect an effect of BW on intake behavior and nutrient selection (Demment and Greenwood, 1988).
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; P < 0.001). At 13 wk of age, pH recovered to the prefeeding pH sooner after feeding and the lowest and highest pH values tended to be higher at this age (Table 3; P = 0.13 and 0.08, respectively).
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, who observed that male Holstein calves weaned at 4 wk of age reached adult VFA concentration 2 wk after weaning. France and Siddons (1993) observed that ruminal VFA concentration reflects the balance between the rate of production and the rate of removal. The majority of VFA produced are removed by absorption through the ruminal wall, and a smaller proportion passes to the omasum. In the present experiment, ruminal conditions of pH did not seem to impair ruminal absorption of VFA, and the fluid passage rate did not vary with age, so the production rate may have increased. At each age, diurnal variation of total VFA concentration fluctuated concurrently with ruminal pH, peaking at the time in which pH started to increase and also exhibiting a time after feeding x age interaction (Figure 3B; P = 0.09). Total VFA concentration accounted for 66.7% of the variance in ruminal pH (P < 0.001). Molar proportion of acetate decreased (P = 0.02) and propionate increased (P = 0.001) with age, causing a decrease (P = 0.03) in the acetate-to-propionate ratio from 3.6 to 1.7, at wk 13 and 41, respectively. The acetate:propionate:butyrate molar proportions changed from 63:18:13 at wk 13 to 52:33:11 at wk 41, indicating an increase in amylolytic fermentation related to cellulolytic fermentation. The VFA proportions observed at wk 41 agreed with the proportions observed by Rogers and Davis (1982) in cows fed a high-concentrate diet. The acetate-to-propionate ratio at 13 wk of age (3.6) was higher than reported by other authors for weaned calves fed high-concentrate diets (Anderson et al., 1987; Vazquez-Añón et al., 1993; Devant et al., 2000). This high acetate-to-propionate ratio at 13 wk could be due to a low propionate production caused by a high average and minimum pH (6.50 and 5.95, respectively). At this pH, cellulolytic bacterial populations have adequate conditions for growth, and propionate production is low (Sutton, 1981). With age, the increasingly high gross intake of nonstructural carbohydrates accounts for the change to an amylolytic pattern and the substantial increase in propionate. The concentration of BCVFA also decreased with age (Table 3), probably due to a lower deamination of AA because the amylolytic bacteria prefer peptides and AA as a source of N (Russell et al., 1983). Proteolysis is not impaired but it stops before AA deamination. Ruminal NH3 N concentration was not affected by age and averaged 9.3 ± 2.5 mg/100 mL (Table 3). Diurnal variation of NH3 N concentration followed the same trend as pH at each age, but no time after feeding x age interaction was detected (Figure 3C). Passage rates of fluid and particulate fractions were not affected by age, and averaged 10.41 ± 1.13 and 6.99 ± 1.47 %/h, respectively (Table 3).
The soluble and the insoluble but potentially degradable fractions of N of protein supplements were not affected by age (Table 4). The rate of degradation and the ED of N increased (P < 0.05) with age in all supplements, except for SBM. On average, the ED of N was 7.7% greater at wk 41 than at wk 13. Dry matter ED of alfalfa hay also increased (P = 0.02) with age, although NDF degradation was not affected. Using similar conditions, Devant et al. (2000) observed that degradability of barley straw increased with age, which corresponded to an increase in the proportion of acetate in the rumen, although no effect on N degradability was observed.
The lack of effect of age on NDF degradation would confirm that the change in ruminal fermentation was toward a more amylolytic than cellulolytic activity, causing this low extent of NDF degradation observed in high-concentrate diets. Changes in ruminal fermentation end products observed from wk 13 to 41 suggest a higher amylolytic activity acquired with age, probably due to an increase in the gross intake of nonstructural carbohydrates and to an adaptation of ruminal microflora to these nutrients. This higher amylolytic activity, not associated with low pH, could be the main factor responsible for the increase in the degradation rate and ED of N of plant protein supplements because proteolytic ruminal microorganisms tend to be amylolytic rather than cellulolytic (Siddons and Paradine, 1981).
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: Edifici V, Facultat de Veterinaria (phone: +34-93-581-2815; fax: +34-93-581-1494; e-mail: Alfred.Ferret{at}uab.es).
Received for publication July 15, 2004. Accepted for publication April 1, 2005.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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