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Intake, digestibility and nitrogen utilization in cattle fed tropical forage and supplemented with protein in the rumen, abomasum, or both

Intake, digestibility and nitrogen utilization in cattle fed tropical forage and supplemented... Background: There is little information in the tropics with regard the comparative understanding of how an increased nitrogen supply in the rumen or in the intestines affects efficiency of nitrogen utilization in cattle. This study evaluated the effects of supplementation with nitrogenous compounds in the rumen, abomasum, or both on intake, digestibility and the characteristics of nitrogen utilization in cattle fed tropical forage. Four rumen- and abomasum-fistulated Nellore bulls (227 ± 11 kg) were used. Four treatments were evaluated: control, ruminal supplementation (230 g/d of supplemental protein in the rumen), abomasal supplementation (230 g/d of supplemental protein in the abomasum), and ruminal and abomasal supplementation (115 g/d protein in both the rumen and the abomasum). The basal forage diet consisted of Tifton 85 hay with a crude protein (CP) level of 78.4 g/kg dry matter. Casein was used as a supplement. The experiment was conducted using a 4 × 4 Latin square. Results: There were no differences between the treatments (P > 0.10) with regard to forage intake. The intake and total digestibility of CP increased (P < 0.01) with supplementation. The nitrogen balance in the body increased (P < 0.01) and muscle protein mobilization decreased (P < 0.01) with supplementation, regardless of the supplementation site. The efficiency of nitrogen utilization did not differ among the treatments (P > 0.10). Conclusions: The supplementation of cattle fed tropical forage with protein in the rumen, abomasum, or both similarly increased the nitrogen accretion in animal, which reflects improvements on nitrogen status in animal body. Keywords: Casein, Nitrogen balance, Rumen ammonia nitrogen, Supplementation, 3-methylhistidine Background associated to cell wall, which decreases the degradability of It has been established that rumen degradable protein forage crude protein and contributes for the low RAN (RDP) constitutes the most important supplement for cat- concentrations [5]. Negative NBR emphasize the import- tle fed low-quality forages. In studies conducted under ance of available nitrogen in the rumen through urea tropical conditions, low concentrations of rumen ammonia recycling, which is a byproduct of the nitrogen that is nitrogen (RAN) have been associated with negative esti- absorbed or mobilized from endogenous sources. Recycled mates of nitrogen balance in the rumen (NBR), which nitrogen may contribute significantly to the supply of ni- might increase the mobilization of body proteins to sustain trogen in the rumen [4, 6]. rumen microbial growth [1–4]. Particularly for tropical Once the requirements of the first limiting factor (RDP) grasses, a great portion of total nitrogen can be found have been met, supplying rumen undegradable protein (RUP) could improve the supply of metabolizable protein and decrease the proportion of nitrogen compounds that * Correspondence: detmann@ufv.br 1 is recycled to the rumen, thereby increasing the availability Department of Animal Science, Universidade Federal de Viçosa, Av. P.H. Rolfs, s/n°, Viçosa, Minas Gerais 36570-900, Brazil of nitrogen for anabolic purposes [7] and reducing the Full list of author information is available at the end of the article © 2016 Rufino et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 2 of 10 mobilization of endogenous protein [2, 3]. However, supplemental protein (pure casein; Labsynth, Diadema, RUP supplementation would be a less efficient means SP, Brazil). This protein source was used as the RDP and to maintain the level of RAN compared with a direct RUP supplement because of its high-protein content, it is supply of RDP [8]. Recent studies conducted under readily degraded in the rumen and/or digested in the tropical conditions have shown that protein supple- small intestine, and to avoid confounding effects by using ments for ruminants may directly affect the efficiency different protein sources in the rumen and abomasum. of conversion of metabolizable protein into net protein The experiment consisted of four 24-day experimental [2, 3]. However, there is little information in the tropics periods. An 8-day interval was applied between experi- with regard the comparative understanding of how an mental periods to reduce the residual effects of the treat- increasednitrogensupply inthe rumenor in the intes- ments. The first 14 d of each period were used for tines affects efficiency of nitrogen utilization in cattle. treatment adaptation. Prior to the experiment, the ani- In this sense, basic research using pure protein sources, mals were adapted to the experimental conditions and such as casein, could be helpful to understand the true basal forage for 14 d. effects of protein supplementation on animal metabol- The total supplement was separated into two portions ism and the utilization efficiency of nitrogen from RDP of equal weight and supplied to the animals when the for- or RUP in cattle fed tropical forages. age was offered (0600h and 1800h). The ruminal supple- The objective of the current study was to evaluate the ment was packaged in paper bags and placed directly into effects of supplementation with protein in the rumen, the rumen of the animals. The casein for the abomasal abomasum, or both on intake, digestibility, the rumen supplementation was diluted in saline solution (NaCl, 9 g/ dynamics of fibrous compounds, and the efficiency of ni- L). The lids of the abomasal cannulas were fitted with ap- trogen utilization in cattle fed tropical forage. proximately 15 cm of polyethylene tubing to form external valves. The supplement was infused into the abomasum Methods through these valves. th This experiment was carried out at the Department of Ani- The samples were collected between the 15 and th mal Science of the Universidade Federal de Viçosa, Viçosa, 24 d of each experimental period. The forage sup- th th Brazil. All surgical and animal care procedures were plied from the 15 to 18 d and the orts obtained th th conducted according to the regulations of the Brazilian from the 16 to 19 d were used to measure the National Council on the Control of Animal Experimen- voluntary intake. tation (CONCEA). Fecal grab samples were taken from the rectum of the th th Four rumen- and abomasum-fistulated Nellore bulls animals between the 16 and 19 d of each experimen- th with an initial average body weight (BW) of 227 ± 11 kg tal period according to the following schedule: 16 day th th were used in this experiment. The animals were kept in – 0600h and 1400h, 17 day – 0800h and 1600h, 18 th individual stalls (2 by 5 m) with concrete floors covered day – 1000h to 1800h, 19 day – 1200h and 2000h. with a rubber layer and equipped with individual feeders Samples of abomasal digesta were simultaneously col- and water dispensers. The animals had unrestricted ac- lected with the fecal samples. These samples were oven- cess to mineral mix. dried (60 °C) and processed in a knife mill (1- and 2- The basal forage consisted of Tifton 85 (Cynodon sp.) mm; Model 4, Thomas Wiley Co., Swedesboro, NJ). hay, which had an average crude protein (CP) content of Total urine collection was performed on the 20th day of 78.4 g/kg dry matter (DM). The forage was provided ad each experimental period. Collecting funnels were at- libitum daily at 0600h and 1800h, allowing approxi- tached to the animals to direct the urine into polyethylene mately 100 g/kg in orts. flasks that were kept cool in a polystyrene cooler with ice. This study evaluated the following treatments: control The collections began at 0600h and lasted 24 h. At the (no supplementation); ruminal supplementation, with a end of the collection period, the urine was measured, and daily supply of 230 g of supplemental CP in the rumen; two 50-mL aliquots were collected for analyses. The first abomasal supplementation, with a daily supply of 230 g aliquot was used to assess nitrogen, urea, and creatinine of supplemental CP in the abomasum; and ruminal and contents. The second aliquot was used to quantify the abomasal supplementation, with a total daily supply of content of 3-methyl histidine (3-MH). 230 g of supplemental CP (115 g of CP in both the abo- On the 21st day, blood samples were taken from the an- masum and the rumen). imals at 0600h, 1200h, 1800h and 2400h. directly from the The amount of supplement (230 g of CP/d) accounted jugular vein using vacuum tubes with either coagulation for approximately 35 % of the daily CP requirements, accelerator gel (BD Vacutainer®, SST II Advance, Franklin 55 % of the RDP daily requirements, or 100 % of the RUP Lakes, NJ) or coagulation inhibitor gel (BD Vacutainer® daily requirements of a 250-kg Zebu bull with a weight K2, Franklin Lakes, NJ). The samples collected with gain of 0.5 kg/d [9]. Casein was used as the source of coagulation accelerator were centrifuged (2,700 × g for Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 3 of 10 20 min) to separate the serum, and their urea and creatin- Table 1 Chemical composition of hay and casein ine levels were evaluated. The samples obtained with co- Item Hay Casein agulation inhibitor were refrigerated (4 °C). At the end Dry matter (DM), g/kg as fed 907 ± 8.2 892 of the collection period, the samples were combined for Organic matter, g/kg DM 933 ± 0.6 973 each animal to assess the concentration of free amino Crude protein, g/kg DM 78 ± 0.3 899 acids (AA). NDFap , g/kg DM 730 ± 7.8 - Rumen content samples were also collected at 0600h, st NDIP , g/kg crude protein 360 ± 78 - 1200h, 1800h and 2400h during the 21 d of each ex- perimental period to isolate microorganisms using the Lignin, g/kg DM 58 ± 0.7 - technique described by Cecava et al. [10]. In addition, iNDF , g/kg DM 391 ± 5.2 - ruminal aliquots were taken to evaluate pH, and concen- a NDFap NDF assayed with a heat-stable alpha-amylase and corrected for trations of RAN and volatile fatty acids (VFA; acetate, contaminant ash and protein; NDIP neutral detergent insoluble protein; iNDF indigestible neutral detergent fiber propionate, and butyrate). These samples were manually Mean ± standard error collected at the liquid–solid interface of the rumen mat, filtered through a triple cheesecloth layer, and subjected Fecal excretion and abomasal flow were estimated by to pH evaluation (potentiometer TEC-3P-MP, Tecnal, using the indigestible NDF (iNDF) as internal marker. The Piracicaba, SP, Brazil). Next, a 40-mL aliquot was sepa- samples of hay, orts, feces, abomasal digesta, and ruminal rated, fixed with 1 mL of H SO (1:1), and frozen (−20 °C) contents, processed by passing through a 2-mm screen 2 4 for RAN concentration analysis. A second 20-mL aliquot sieve, were evaluated with regard to iNDF content using was fixed with 5 mL of a meta-phosphoric acid solution F57 bags (Ankom Technology Corp., Macedon, NY) and (250 g/L) and kept at −20 °C for subsequent assessment of an in situ incubation procedure for 288 h (method the VFA concentration. INCT-CA no. F-008/1) [11]. Importantly, the supple- The rumen evacuation procedure was performed on the ment infused into the abomasum was not considered in nd th 22 and 24 d to quantify the resident mass and the rates the ruminal digestibility and outflow calculation; rather, of passage and degradation of the fibrous material. The it was only used to calculate the intestinal digestibility rumen content was removed at 1000h (4 h after the morn- coefficients. ing feeding) and 0600 (before the morning feeding) on the The rates of intake and ruminal passage of NDF were aforementioned days, respectively. The collected material estimated by the ratio of NDF intake and abomasum flow was packed in a polyethylene container and weighed. The on the rumen mass of NDF, respectively. The degradation material was stirred by hand and an aliquot of approxi- rate of NDF was obtained as the difference between the mately 50 g/kg was removed. The remaining material was rates of intake and passage [12]. returned to the rumen of the animals. The samples The RAN concentration was quantified using the colori- were oven-dried (60 °C) and processed in a knife mill metric technique described by Detmann et al. [11] (method (1- and 2-mm). INCT-CA no. N-006/1). The concentrations obtained at Subsequently, the samples of hay, orts, feces, abomasal different sampling times were combined for each animal digesta, and ruminal contents (the samples collected from and period in order to obtain a single value that repre- ruminal evacuation) were pooled per animal and experi- sented the average daily RAN concentration. The rumen mental period. pH values were combined in a similar way. Chemical analyses were performed on the samples that The VFA concentration was evaluated on pooled rumen were processed to pass through a 1-mm sieve. The con- fluid samples composed of proportional sample volumes tents of DM (method INCT-CA no. G-003/1), organic for each collection (per animal and period) and evaluated matter (OM; method INCT-CA no. M-001/1), CP (Kjel- using HPLC (Shimadzu chromatograph, Model SPD-10A dahl procedure; method INCT-CA no. N-001/1), neutral VP) with a reverse phase column (using a mobile phase of detergent fiber corrected for ash and protein (NDFap; orthophosphoric acid in water, 10 mL/L) and a UV de- using a heat-stable α-amylase, omitting sodium sulfite and tector at a wavelength of 210 nm. correcting for residual ash and protein; method INCT-CA The samples of ruminal microorganisms were analyzed no. F-002/1), neutral detergent insoluble protein (method for CP, as described for feed samples, and for purine INCT-CA no. N-002/1), and acid detergent lignin (method bases [13]. The purine bases were used to assess the mi- INCT-CA no. F-005/1) were quantified according to the crobial concentrations in the abomasal digesta based on standard analytical procedures of the Brazilian National the N :N ratio in rumen microorganisms. RNA total Institute of Science and Technology in Animal Science The urine samples were analyzed for nitrogen content (INCT-CA; Table 1) [11]. The casein samples were evalu- as described for the CP analysis of the feed samples. The ated for DM, OM, and CP contents according the methods urea and creatinine concentrations in the urine and blood described above (Table 1). serum were evaluated using the enzymatic-colorimetric Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 4 of 10 (K047, Bioclin Co., Belo Horizonte, MG, Brazil) and alka- Table 2 Effects of supplementation with protein in different sites on voluntary intake in cattle fed tropical forage line picrate (K016, Bioclin Co., Belo Horizonte, MG, Brazil) methods, respectively. The 3-MH in urine and the Supplementation site total free AA in blood were obtained using the HPLC Item Control R A R + A SEM P-value techniques described by Jones et al. [14] and Pitta et al. kg/d [15], respectively. DM 5.05 5.60 5.13 5.12 0.48 0.229 The urea nitrogen filtered in the kidneys and the frac- DMF 5.05 5.35 4.88 4.87 0.48 0.307 tional excretion of urea nitrogen were calculated from OM 4.72 5.25 4.81 4.80 0.45 0.226 the following equations: b a a a CP 0.403 0.648 0.611 0.613 0.040 <0.001 UEC NDFap 3.65 3.87 3.54 3.54 0.33 0.348 UNFK ¼  SUN ð1Þ SC iNDF 1.93 2.05 1.87 1.84 0.19 0.222 b a b ab EUN DOM 2.22 2.60 2.31 2.40 0.22 0.060 FEUN ¼ ð2Þ UNFK DNDF 1.94 2.01 1.84 1.87 0.17 0.362 g/kg body weight where UNFK is the urea nitrogen filtered in the kidneys DM 20.4 22.4 20.8 20.7 1.2 0.263 (g/d), UEC is the urinary excretion of creatinine (g/d), SC is the average concentration of serum creatinine OM 19.1 21.0 19.5 19.4 1.1 0.260 (mg/dL), SUN is the average concentration of serum NDFap 14.8 15.5 14.3 14.3 0.8 0.337 urea nitrogen (mg/dL), FEUN is the fractional excretion iNDF 7.8 8.2 7.6 7.4 0.5 0.273 of urea nitrogen (g/g), and EUN is the urinary excretion a, b within a row, means without a common superscript differ (P < 0.10) of urea nitrogen (g/d). DM dry matter; DMF dry matter from forage; OM organic matter; CP crude protein; NDFap neutral detergent fiber assayed with a heat-stable alpha- The experiment was carried out and analyzed according amylase and corrected for contaminant ash and protein; iNDF indigestible to a 4 × 4 Latin square design balanced for residual effects neutral detergent fiber; DOM digested organic matter; DNDF digested neutral with four treatments (fixed effect), four animals (random detergent fiber Control = without supplementation; R = ruminal supplementation; effect), and four experimental periods (random effect). A = abomasal supplementation All of the statistical procedures were carried out using the MIXED procedure of SAS 9.3. Due to the high prob- ability of type II error, we adopted α = 0.10. When neces- sary, the treatment means were compared using protected Fisher’s least significant difference. The data from one purine base concentration in a microbial sample and one Table 3 Effects of supplementation with protein in different sites creatinine concentration in a blood sample were lost dur- on total, ruminal, and intestinal digestibilities (g/g) in cattle fed ing the analysis. tropical forage Supplementation site Results Item Control R A R + A SEM P-value There were no differences among treatments with regard Total to the intake of DM (P > 0.22), forage (P > 0.30), OM OM 0.468 0.495 0.485 0.502 0.017 0.142 (P > 0.22), neutral detergent fiber (NDF; P > 0.34), iNDF b a a CP 0.475 0.656 0.639 0.664ª 0.023 <0.001 (P > 0.22), and digested NDF (P > 0.36) (Table 2). NDFap 0.531 0.519 0.523 0.532 0.019 0.898 The CP intake increased with supplementation (P < 0.01), but no differences were found between the supplementa- Ruminal tion sites (P > 0.10). The mean CP intakes were 0.403 kg/d OM 0.300 0.324 0.277 0.284 0.031 0.265 and 0.624 kg/d for the control and supplementation treat- b a c b CP −0.221 0.123 −0.987 −0.284 0.139 <0.001 ments, respectively (Table 2). Rumen supplementation in- c bc a ab NDFap 0.502 0.512 0.549 0.528 0.024 0.041 creased the intake of digested OM (DOM) compared with Intestinal that of the control and with that of the abomasal supple- d c a b CP 0.561 0.607 0.772 0.718 0.026 <0.001 mentation treatments (P < 0.10). An intermediate value of a, b, c, d DOM intake was observed for the rumen/abomasal sup- within a row, means without a common superscript differ (P < 0.10) Control = without supplementation; R = ruminal supplementation; plementation (Table 2). A = abomasal supplementation The results of the total digestibility coefficient of CP OM, organic matter; CP, crude protein; NDFap, neutral detergent fiber assayed with a heat-stable alpha-amylase and corrected for contaminant ash were similar to those observed for the CP intake (Table 3). and protein Supplementation increased total digestibility of CP from g Ruminal and intestinal digestibilities were calculated as the fraction of the 0.475 g/g to 0.653 g/g on average. No differences were mass that entered the digestion site Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 5 of 10 found among treatments for the total digestibility of OM Table 4 Effects of supplementation with protein in different sites on the resident mass of fiber in the rumen, the fractional (P > 0.14) and NDF (P > 0.89) (Table 2). rates of NDF rumen dynamics, and on the characteristics of No differences were observed among treatments with re- ruminal fermentation in cattle fed tropical forage gard to the ruminal digestibility of OM (P > 0.26; Table 3). Treatments The rumen digestibility of CP was higher (P <0.10) with Item C R A R + A SEM P-value ruminal supplementation than for the other treatments and was lowest for the abomasal supplementation. Ruminal CP Resident mass in the rumen digestibility was intermediate from the control and supple- NDF, g/kg BW 10.7 10.0 11.8 9.6 1.3 0.337 mentation in the rumen and abomasum. Importantly, the iNDF, g/kg BW 8.4 8.0 9.2 8.3 1.0 0.821 CP rumen digestibility was positive in the ruminal supple- NDF rumen dynamics mentation group and negative for the other treatments ki, /h 0.060 0.071 0.052 0.063 0.008 0.238 (Table 3). kp, /h 0.029 0.034 0.023 0.030 0.003 0.149 Abomasal and ruminal/abomasal supplementation re- sulted in higher NDF rumen digestibility compared with kd, /h 0.030 0.037 0.029 0.034 0.005 0.338 the control (P < 0.10), with an intermediate value observed Ruminal characteristics for ruminal supplementation (Table 3). b a b ab RAN, mg/dL 4.15 13.66 5.56 8.22 1.91 0.078 The CP intestinal digestibility differed across all of the pH 6.83 6.75 6.79 6.72 0.12 0.914 treatments (P < 0.10). The following treatments are pre- VFA, mmol/dL 5.321 5.396 5.731 5.940 0.279 0.256 sented in descending order: abomasal supplementation, Acetate, mol/mol 73.71 73.40 75.25 74.06 0.87 0.489 ruminal/abomasum supplementation, rumen supplemen- tation, and control (Table 3). Propionate, mol/mol 18.76 19.08 17.74 19.32 0.79 0.559 Treatments did not affect ruminal NDF (P > 0.33) and Butyrate, mol/mol 7.53 7.53 7.01 6.69 0.32 0.230 iNDF (P > 0.82) mass; their mean values were 10.5 and A:P 3.96 3.86 4.29 3.84 0.22 0.501 8.5 g/kg BW, respectively. In addition, no differences were a, b within a row, means without a common superscript differ (P < 0.10) observed among treatments with regard to the rates of in- Control = without supplementation; R = ruminal supplementation; A = abomasal supplementation take (P > 0.23), degradation (P > 0.33), and passage of NDF NDF neutral detergent fiber; iNDF indigestible neutral detergent fiber; ki, kp (P > 0.14) (Table 4). and kd rates of intake, passage and degradation; RAN rumen ammonia The ruminal pH did not vary across treatments nitrogen; VFA volatile fatty acids; A:P acetate to propionate ratio (P > 0.91), and an average value of 6.78 was observed. However, higher RAN concentrations were found for the supplementation compared with the control treatment, rumen supplementation group compared with the control regardless of the supplementation site (P < 0.10; Table 5). and the abomasum supplementation groups (P <0.10). However, in spite of the great numerical differences be- These latter groups did not differ from each other tween control and supplemented treatments, no differences (P > 0.10). Rumen/abomasal supplementation resulted in the efficiency of nitrogen utilization were observed in an intermediate RAN concentration (Table 4). among the treatments (P > 0.16). There were no differences among the treatments re- The highest estimate of NBR was observed with rumen gard to the VFA concentration (P > 0.25), the average supplementation (P < 0.10), followed by ruminal/abomasal value of which was 5.60 mmol/dL. In addition, the supplementation, abomasum supplementation, and the acetate (P > 0.48), propionate (P > 0.55), and butyrate control. No differences were observed between the latter (P > 0.23) molar ratios, as well as the acetate:propionate two treatments (P > 0.10). Importantly, only ruminal sup- ratio (P > 0.50), did not differ across treatments (Table 4). plementation resulted in positive NBR (Table 5). The SUN concentration (Table 5) was higher with rumi- The treatments affected the amount of CP digested in nal supplementation compared with ruminal/abomasum the intestines (PDI; P < 0.01). Larger amounts of PDI supplementation and control (P < 0.10). The average SUN were observed in the abomasal supplementation group concentration obtained with abomasal supplementation compared with the ruminal/abomasal supplementation occupied an intermediate position compared with the and the control treatments. The ruminal supplementa- other supplementation types. No differences were found tion group held an intermediate position between these across the treatments with regard to the concentration of groups (Table 5). AA in the blood (P >0.48; Table 5). The control treatment exhibited greater urinary excre- Nitrogen intake and urinary excretion increased with tion of 3-MH compared with the supplement treatments protein supplementation, regardless of the supplementa- (P < 0.10), which did not differ from each other (P > 0.10). tion site (P < 0.10). However, the nitrogen fecal excretion The urinary excretion of urea nitrogen was higher for did not vary among the treatments (P > 0.61). Similarly, the the supplementation groups than for the control group apparent nitrogen balance (NB) increased with nitrogen (P < 0.10). In addition, ruminal and ruminal/abomasal Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 6 of 10 Table 5 Effects of supplementation with protein in different sites on the characteristics of nitrogen utilization in the animals in cattle fed tropical forage Supplementation site Item Control R A R + A SEM P-value c a ab b SUN, mg/dL 7.29 17.16 13.46 13.39 1.30 0.029 BAA, μmol/mg creatinine 113 123 129 119 12 0.485 b a a a Nitrogen intake, g/d 64.5 103.8 97.8 98.0 6.4 <0.001 Fecal nitrogen, g/d 34.0 36.0 35.5 33.4 4.0 0.611 b a a a Urinary nitrogen, g/d 26.3 48.5 40.4 46.3 4.2 0.010 b a a a Nitrogen balance, g/d 4.1 19.3 22.0 18.3 6.6 0.062 c a c b NBR, g/d −14.4 12.2 −23.2 −2.0 7.5 0.005 ENU g retained nitrogen/g of ingested nitrogen 0.04 0.18 0.22 0.18 0.05 0.165 g retained nitrogen /g of nitrogen absorbed in the intestines 0.07 0.27 0.35 0.27 0.04 0.402 c bc a b PDI, g/d 280 347 535 416 45 0.003 a b b b 3-MH, mg/g of creatinine 50.8 20.6 29.6 31.0 13.1 0.077 b a a UEUN, g/d 13.4 28.9 25.9ª 30.1 2.9 0.002 b a b a UFK, g/d 42.9 ± 6.5 60.9 ± 5.6 43.9 ± 5.6 55.0 ± 5.6 — 0.062 FUEUN, g/g 0.35 ± 0.09 0.48 ± 0.08 0.63 ± 0.08 0.55 ± 0.08 — 0.121 NMIC, g/d 53.1 ± 13.0 61.2 ± 11.1 61.9 ± 13.0 58.8 ± 11.1 — 0.932 a, b, c within a row, means without a commom superscript differ (P < 0.10) SUN serum urea nitrogen; BAA amino acids in blood; NBR nitrogen balance in the rumen; ENU efficiency of nitrogen utilization; PDI protein digested in the intestine; 3-MH urinary excretion of 3-methyl histidine; UEUN urinary excretion of urea nitrogen; UFK urea nitrogen filtered in the kidneys; FUEUN fractional excretion of urea nitrogen; NMIC ruminal production of microbial nitrogen compounds Control = without supplementation; R = ruminal supplementation; A = abomasal supplementation supplementation increased the urea filtered in the kid- nitrogenous compounds in the rumen [1, 17–19] or in neys (P < 0.10) compared with the other treatments. the abomasum [6, 8, 20]. Supplementation did not alter the fractional excretion The lack of changes on forage and NDF intake in the of urea nitrogen (P > 0.12). However, supplementation response to nitrogen supplementation might be associ- resulted in a 60 % higher fractional excretion of urea ni- ated with the content of CP in the basal forage (Table 1). trogen compared with the control (Table 5). The ruminal The previously cited authors studied forages with CP con- synthesis of microbial nitrogenous compounds did not dif- tents that were typically below 60 g/kg DM. The average fer among the treatments (P > 0.93), with an average value dietary CP in the control treatment, calculated as the ratio of 58.7 g/day (Table 5). between CP and DM intake, was 79.8 g/kg. Nitrogen does not positively affect voluntary forage intake when the diet- ary CP is above 70–80 g/kg DM [3, 21]. Discussion The stimulation of low-quality forage intake via nitro- Nitrogen supplementation for animals fed low-quality for- gen supplementation is usually associated with a reduc- age favors the growth of fibrolytic bacteria and increases tion in the physical constraints to intake [16, 22]. In this the ruminal degradation and voluntary intake of fiber, as context, nitrogen supplementation could increase the well as the energy extraction from forage fiber [16]. Spe- degradation rate of NDF, which concomitantly increases cifically, supplementation with nitrogenous compounds in the passage rate of non-degraded and indigestible fiber the abomasum of animals fed low-quality forage could from the rumen, increasing forage intake [15]. stimulate intake via nitrogen recycling, albeit with less in- However, supplementation did not alter the ruminal dy- tensity compared with rumen supplementation [6, 8]. namics of NDF (Table 4). This result might be also due to However, positive effects on the voluntary intake of forage the CP content of the basal forage, which was above the were not observed for any supplementation site in the minimum level required to support fibrolytic activity in the present study (Table 2). rumen [23]. When the removal of fiber residues from the The results obtained here differ from those reported rumen is limited, animals can increase their rumen volume by several authors who found increases in the voluntary to accommodate a greater mass of resident fiber [24]. With intake of low-quality forage in cattle supplemented with this adaptation, and keeping the passage and fractional Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 7 of 10 degradation rates constant, the animal is able to increase intestinal digestibility coefficient was 0.93 g/g. Therefore, the amount of degraded fiber. However, no effect was casein supplied in the rumen should result in a higher observed with regard to the rumen fiber mass (Table 4). total digestion compared to casein supplied in the aboma- Therefore, considering the results regarding voluntary sum, which would explain the greater intake of digested intake and fiber dynamics, it can be stated that the CP OM in the animals that received ruminal supplementation content of the basal forage was adequate to support ru- (Table 2). minal function. The most prominent effects of supplementation in the Although supplementation increased the NDF digest- present study were observed on the metabolism and the ibility in the rumen, the observed effect was small, which nitrogen accretion in animal body, as other authors have resulted in no differences among treatments with regard shown in tropical conditions [2–4, 28]. NDF intake and total digestibility, and VFA concentra- The main effect of supplementation can be associated tion (Tables 2, 3, and 4). The stimulation observed with with an increase in NB (Table 5), which would represent supplementation most likely resulted from some im- an increase in weight gain. Although similar amounts of provement in the availability of RAN (Table 4). The protein were supplied to the supplemented animals, the “protein effect” can explain the low rumen digestibility pathways through which these supplements affect protein coefficient with rumen supplementation. This result cor- accretion in the body seem differ because the way in responds to an increase in the competition for essential which supplemental nitrogen is utilized could depend on substrates between fibrolytic and non-fibrolytic species the supplementation site (i.e., the rumen or the intestines). when the supplements contain true protein [25]. This Due to the high degradability of casein in the rumen, “protein effect” can occur simultaneously with the nitro- little of the ruminal protein supplement would reach the gen stimulation of fibrolytic activity in low-quality forage intestines to be digested. Considering that the ruminal [26]. Therefore, the similarity between control and rumi- degradation coefficient is 0.80 g/g [27] and the intestinal nal supplementation treatments regarding the ruminal digestibility coefficient was 0.93 g/g, the additional pro- digestibility of NDF may be a result of a counterbalan- tein mass (230 g CP/d) should provide only 42.8 g of cing of the nitrogen stimulus and the “protein effect.” additional PDI. This result explains the small increase in This theory also explains the intermediate position PDI that was observed with ruminal supplementation achieved by the ruminal/abomasum supplementation (Table 5). Considering the efficiency of utilization of the group (Table 3). absorbed nitrogen (0.27 g/g; Table 5), that additional The only between-treatment difference found with re- PDI should increase NB by approximately 1.8 g/d, which gard to the total digestion was the CP digestibility. Specif- is equivalent to approximately 11 % of the increase in ically, these values were higher in the supplemented NB in relation to the control treatment. Therefore, the animals compared with the non-supplemented animals probable escape of casein to the small intestine cannot (Table 3). This result occurred as a result of the increased explain the effects of ruminal casein supplementation on CP intake provided by supplementation (Table 2) because, NB. It should be emphasize that there was no effect of for the dietary non-fibrous components, such as the CP supplementation on ruminal synthesis of microbial ni- derived from casein, the apparent digestibility coefficient trogen (Table 5) and an increase in metabolizable pro- is proportional to the intake [21]. A similar result was ob- tein supply from microbial protein did not occur. served for the intestinal digestibility of CP, which was The most prominent effect of the ruminal supplementa- especially high when the casein was infused into the abo- tion was the increase in RAN concentration (Table 4), masum (Table 3). The assessment of the amount of appar- which means an overall improvement in nitrogen avail- ently digested CP in the intestines using the Lucas test ability in the animal gastrointestinal tract and also for the approach revealed a true intestinal digestibility of CP of animal metabolism. The RAN concentration observed in 0.931 g/g [CI(β) :0.838 ≤ β ≤ 1.024]. Considering that non-supplemented animals (4.15 mg/dL) was below that 0.90 there was no difference among the treatments with regard reported by Detmann et al. [22] for an equilibrium be- to the fecal excretion of nitrogen (Table 5), this estimate tween the inflow and outflow of nitrogen in the rumen suggests that the protein supplement was almost com- of animals fed tropical forage (approximately 8.4 mg pletely digested in the intestines. of RAN/dL), which resulted in negative NBR values The intake of DOM increased with supplementation (Table 5). in the rumen. However, no between-treatment differ- The overall effects of nitrogen availability on ruminant ences were found with regard to the intake of digested metabolism have been associated with a better adequate NDF (Table 2). The increased intake of DOM is there- protein or nitrogen status [7, 29]. Theoretically, the ni- fore attributed solely to the effects of the non-fibrous trogen status defines the availability of different nitro- portion of the diet. Casein is estimated to have a ruminal genous compounds in both quantity and quality for all digestion greater than 0.80 g/g [27], whereas its required physiological functions in animal metabolism, Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 8 of 10 including the functions associated with the metabolism nitrogen that is recycled through rumen wall seems to of other compounds such as energy compounds [7]. be relatively constant [17]. Therefore, there will be lesser Taking into account the theoretical concept of nitrogen nitrogen accretion in the animal body under low nitro- status, it could be realized that the nitrogenous com- gen contents in the diet because a greater percentage of pounds would be used in different metabolic functions the ingested nitrogen will be directed to recycling and, following an order of priority to the animal: survival, as a consequence, a lower percentage of nitrogen will be maintenance, and production [7]. Thereby, a positive ni- available for production [7]. trogen accretion in the animal body or products only will The pattern here observed for NBR can be used to give take place after supplying the higher priority demands for some support for the assumptions about metabolic prior- nitrogenous compounds. ities of nitrogen. Several estimates of negative NBR has One of the possible high-priority metabolic functions been obtained in experiments carried out in the tropics, is the recycling of nitrogen to the gastrointestinal tract. and the main factor to influence that is the nitrogen avail- Such a statement seems to be plausible because a con- ability in the diet [7]. This pattern highlights that nitrogen tinuous supplying on nitrogen for microbial growth in flow to abomasum can be greater than nitrogen intake in the rumen must be seen as a strategy for animal survival several occasions. In these cases, there is a more signifi- [21, 30]. Under a nitrogen deficiency, the animal is able cant dependency on recycling events to provide adequate to decrease urinary nitrogen excretion and increase the nitrogen supplying to the rumen. Then, the animal will fraction of dietary nitrogen that is recycled to the rumen decrease the efficiency of utilization of metabolizable pro- [4, 31]. In fact, despite the absence of a significant differ- tein for gain (decreased anabolism as supported by the ence (P > 0.12), the fractional excretion of urea nitrogen lower numerical efficiency of nitrogen utilization, Table 5) in the control treatment was approximately 71 % of that and sometimes also increase the breakdown of muscle observed in the ruminal supplementation treatment protein to supply the nitrogen demands of higher priority (Table 5). This result indicates that a smaller percentage (increased catabolism as supported by greater 3-MH ex- of the circulating urea was eliminated in the urine, and cretion, Table 5). more prominent fraction was directed for reuse (e.g., for The availability of RAN increased with rumen supple- recycling) when supplement was not provided. mentation (Table 4), making the NBR positive, reflecting When nitrogen deficiency becomes more severe, the improvement in nitrogen status in the animal body. In this animal can increase the myofibrillar protein mobilization way, there was an increase in the accretion of absorbed ni- to sustain the mass of recycling nitrogen [4, 32]. This trogen (Table 5). Costa et al. [2] observed similar pattern was observed in this study through urinary excretion of by providing nitrogen compounds that were highly de- 3-MH, which was decreased when supplement was pro- gradable in the rumen to cattle grazing tropical forage vided (Table 5). (99 g CP/kg DM). The 3-MH is an AA formed from the methylation of On the other hand, the direct effect of abomasal sup- histidine after its inclusion in the muscle proteins (i.e., plementation was based on the increased availability of actin and myosin). When muscle proteins are degraded, AA absorbed in the small intestine, which can be seen the 3-MH cannot be reused for protein synthesis and is by the greater amount of PDI compared with the control excreted in the urine [33]. Thus, the 3-MH urinary (Table 5). This metabolizable protein supply can be dir- excretion is a marker for the degradation of muscle pro- ectly incorporated into tissue, thereby increasing the NB tein that, in turn, can be used for other physiological or (Table 5). Importantly, the efficiency of utilization of metabolic functions, such as the supply of higher prior- absorbed nitrogen and NB were similar for ruminal and ity nitrogen demands. The control treatment therefore abomasal supplementation groups (Table 5). increased the mobilization of muscle protein by ap- The largest mass of available AA in the small intestine proximately 146 % compared with the ruminal supple- directly increases the nitrogen status in the animal me- mentation treatment (Table 5). Similarly, Pitta et al. tabolism by supplying the requirements for tissue syn- [15] found a reduction in the plasma concentration of thesis and providing nitrogen to the higher demand 3-MH by providing a protein supplement to sheep fed events without major mobilizations of muscle protein. low-quality forage. The reduction of 3-MH excretion observed in the ab- Importantly, 3-MH urinary excretion was strongly and omasal supplementation group indirectly confirmed this negatively correlated with SUN (r = −0.892; P < 0.01) and effect (Table 5). RAN (r = −0.693; P < 0.09) concentrations, which indi- According to Bandyk et al. [8] and Wichersham et al. cates that muscle protein mobilization is negatively asso- [6, 20], supplementation with protein sources that are ciated with nitrogen availability or nitrogen status. not degradable in the rumen can increase the supply of Considering a normal feeding situation, without any RAN through nitrogen recycling, although less efficiently prominent dietary nitrogen deficiency, the amount of than supplementation with RDP sources. The increased Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 9 of 10 SUN concentration, which is mediated by the greater Therefore, even if the source of RUP is inexpensive, the availability of nitrogen in the intestines, favors this in- most appropriate formulas would depend on the profile of crease in recycling, as was observed in the present study digestible AA in the small intestine. (Table 5). Consequently, supplementation with RUP sources are An increased SUN concentration can increase the differ- only feasible or logical after the beneficial effects of RDP ence between the concentrations in the blood and rumen had been explored, thereby providing additional per- and increases urea transfer [20]. This result should in- formance gains through direct supply of metabolizable crease the pool of RAN [6, 20]. Wickersham et al. [20] protein [8, 20]. found that the proportion of microbial nitrogen produced from recycled nitrogen increased from 0.31 to 0.58 g/g Conclusion when cattle fed low-quality forage were supplemented The supplementation of cattle fed tropical forage with with nitrogenous compounds in the small intestine. protein in the rumen, abomasum, or both can increase The RAN concentration after abomasal supplementa- the retention of nitrogen in animal. However, the meta- tion was increased by only 1.41 mg/dL over the control bolic pathways involved in improving nitrogen accretion treatment, and this increase was not significant (P >0.10). differ between supplementation sites. The improvement This result contradicts the studies described above. How- obtained with rumen supplementation seems based on ever, abomasal supplementation decreased the rumen di- a direct increase in dietary nitrogen availability and sta- gestibility of CP and, consequently, the NBR (Tables 3 and tus. Moreover, the improvement obtained with aboma- 5). Considering that the supplemental nitrogen was not sum supplementation results from an increased supply used to estimate ruminal digestibility when supplement of metabolizable protein. was supplied in the abomasum and that forage intake was Abbreviations constant among treatments (Table 2), the only likely cause 3-MH: urinary excretion of 3-methyl histidine; AA: amino acids; BW: body for the decreased NBR under abomasal supplementation weight; CP: crude protein; DM: dry matter; DMF: dry matter from forage; DNDF: digested neutral detergent fiber; DOM: digested organic matter; is an increase in the amount of nitrogen coming from iNDF: indigestible neutral detergent fiber; ki kp and kd: rates of intake, recycling. It can therefore be inferred that abomasal passage and degradation of NDF; NB: nitrogen balance; NBR: nitrogen supplementation increased the pool of available nitro- balance in the rumen; NDF: neutral detergent fiber; NDFap: neutral detergent fiber assayed with a heat-stable alpha-amylase and corrected for gen in the rumen, although less efficiently than did ru- contaminant ash and protein; OM: organic matter; PDI: protein digested in minal supplementation. the intestine; RAN: rumen ammonia nitrogen; RDP: rumen degradable In general, the effects of ruminal/abomasal supplemen- protein; RUP: rumen undegradable protein; SUN: serum urea nitrogen; VFA: volatile fatty acids. tation were between those of ruminal supplementation and those of abomasal supplementation. The intermediate Competing interests RAN concentration (Table 4) and PDI (Table 5) provided The authors declare that they have no competing interests. animals with the previously discussed effects of body ni- Authors’ contribution trogen retention, providing NB that was similar to the ex- ED, conceived the study, performed the statistical analysis, contributed to clusive rumen or abomasum supplementation (Table 5). draft the manuscript, and coordinate the research group. LMAR, EDB, DIG and WLSR, carried out the experimental trial, performed the chemical analysis, From the results obtained in the present study, it can and helped to draft the manuscript. SCVF and MFP, helped to draft the be stated that nitrogen supplementation in the rumen or manuscript. All authors read and approved the final manuscript. abomasum should show similar effects but as a result of Acknowledgments different ways to improve nitrogen status in the animal The authors thank the Conselho Nacional de Pesquisa e Desenvolvimento metabolism. In theory, these mechanisms provide a pos- Científico (CNPq), the Fundação de Amparo à Pesquisa de Minas Gerais sibility for choosing between supplementation with RDP (FAPEMIG), and the INCT Ciência Animal for financial support. or RUP. Author details Consequently, additional factors should be considered Department of Animal Science, Universidade Federal de Viçosa, Av. P.H. with regard to the most suitable production system. The Rolfs, s/n°, Viçosa, Minas Gerais 36570-900, Brazil. Department of Animal Science, Universidade Federal Rural da Amazônia, Campus de Parauapebas, costs involved in using RDP sources are almost always C.P. 3017, Bairro Cidade Nova, Parauapebas, Pará 68515-970, Brazil. lower than those of RUP. Considering that the primary mechanism for increasing nitrogen retention in the body Received: 26 June 2015 Accepted: 11 February 2016 using RDP is an overall improvement in dietary nitrogen availability, the use of non-protein nitrogen sources, such References as urea, could reduce costs compared with the use of 1. Sampaio CB, Detmann E, Paulino MF, Valadares Filho SC, Souza MA, RUP. In addition, the responses to RUP supplementation Lazzarini I, et al. Intake and digestibility in cattle fed low-quality tropical forage and supplemented with nitrogenous compounds. Trop Anim Health for increased metabolizable protein supply depend on AA Prod. 2010;42:1471–9. doi:10.1007/s11250-010-9581-7. profile of the protein source, which must be compatible 2. Costa VAC, Detmann E, Paulino MF, Valadares Filho SC, Henriques LT, with the profile required by the animal anabolism. Carvalho IPC. Total and partial digestibility and nitrogen balance in grazing Rufino et al. 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Liv Sci. 2009;126: Submit your next manuscript to BioMed Central 136–46. doi:10.1016/j.livsci.2009.06.013. 17. Marini JC, Van Amburgh ME. Nitrogen metabolism and recycling in Holstein and we will help you at every step: heifers. J Anim Sci. 2003;81:545–52. • We accept pre-submission inquiries 18. Lazzarini I, Detmann E, Sampaio CB, Paulino MF, Valadares Filho SC, Souza MA, et al. Dinâmicas de trânsito e degradação da fibra em detergente • Our selector tool helps you to find the most relevant journal neutro em bovinos alimentados com forragem tropical de baixa qualidade • We provide round the clock customer support e compostos nitrogenados. Arq Bras Med Vet Zootec. 2009;61:635–47. • Convenient online submission doi:10.1590/S0102-09352009000300017. 19. Figueiras JF, Detmann E, Paulino MF, Valente TNP, Valadares Filho SC, • Thorough peer review Lazzarini I. Intake and digestibility in cattle under grazing during dry season • Inclusion in PubMed and all major indexing services supplemented with nitrogenous compounds. Rev Bras Zootec. 2010;39: • Maximum visibility for your research 1303–12. doi:10.1590/S1516-35982010000600020. 20. Wickersham TA, Titgemeyer EC, Cochran RC, Wickersham EE. Effect of Submit your manuscript at undegradable intake protein supplementation on urea kinetics and www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science and Biotechnology Springer Journals

Intake, digestibility and nitrogen utilization in cattle fed tropical forage and supplemented with protein in the rumen, abomasum, or both

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Springer Journals
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Copyright © 2016 by Rufino et al.
Subject
Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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2049-1891
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10.1186/s40104-016-0069-9
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26900467
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Abstract

Background: There is little information in the tropics with regard the comparative understanding of how an increased nitrogen supply in the rumen or in the intestines affects efficiency of nitrogen utilization in cattle. This study evaluated the effects of supplementation with nitrogenous compounds in the rumen, abomasum, or both on intake, digestibility and the characteristics of nitrogen utilization in cattle fed tropical forage. Four rumen- and abomasum-fistulated Nellore bulls (227 ± 11 kg) were used. Four treatments were evaluated: control, ruminal supplementation (230 g/d of supplemental protein in the rumen), abomasal supplementation (230 g/d of supplemental protein in the abomasum), and ruminal and abomasal supplementation (115 g/d protein in both the rumen and the abomasum). The basal forage diet consisted of Tifton 85 hay with a crude protein (CP) level of 78.4 g/kg dry matter. Casein was used as a supplement. The experiment was conducted using a 4 × 4 Latin square. Results: There were no differences between the treatments (P > 0.10) with regard to forage intake. The intake and total digestibility of CP increased (P < 0.01) with supplementation. The nitrogen balance in the body increased (P < 0.01) and muscle protein mobilization decreased (P < 0.01) with supplementation, regardless of the supplementation site. The efficiency of nitrogen utilization did not differ among the treatments (P > 0.10). Conclusions: The supplementation of cattle fed tropical forage with protein in the rumen, abomasum, or both similarly increased the nitrogen accretion in animal, which reflects improvements on nitrogen status in animal body. Keywords: Casein, Nitrogen balance, Rumen ammonia nitrogen, Supplementation, 3-methylhistidine Background associated to cell wall, which decreases the degradability of It has been established that rumen degradable protein forage crude protein and contributes for the low RAN (RDP) constitutes the most important supplement for cat- concentrations [5]. Negative NBR emphasize the import- tle fed low-quality forages. In studies conducted under ance of available nitrogen in the rumen through urea tropical conditions, low concentrations of rumen ammonia recycling, which is a byproduct of the nitrogen that is nitrogen (RAN) have been associated with negative esti- absorbed or mobilized from endogenous sources. Recycled mates of nitrogen balance in the rumen (NBR), which nitrogen may contribute significantly to the supply of ni- might increase the mobilization of body proteins to sustain trogen in the rumen [4, 6]. rumen microbial growth [1–4]. Particularly for tropical Once the requirements of the first limiting factor (RDP) grasses, a great portion of total nitrogen can be found have been met, supplying rumen undegradable protein (RUP) could improve the supply of metabolizable protein and decrease the proportion of nitrogen compounds that * Correspondence: detmann@ufv.br 1 is recycled to the rumen, thereby increasing the availability Department of Animal Science, Universidade Federal de Viçosa, Av. P.H. Rolfs, s/n°, Viçosa, Minas Gerais 36570-900, Brazil of nitrogen for anabolic purposes [7] and reducing the Full list of author information is available at the end of the article © 2016 Rufino et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 2 of 10 mobilization of endogenous protein [2, 3]. However, supplemental protein (pure casein; Labsynth, Diadema, RUP supplementation would be a less efficient means SP, Brazil). This protein source was used as the RDP and to maintain the level of RAN compared with a direct RUP supplement because of its high-protein content, it is supply of RDP [8]. Recent studies conducted under readily degraded in the rumen and/or digested in the tropical conditions have shown that protein supple- small intestine, and to avoid confounding effects by using ments for ruminants may directly affect the efficiency different protein sources in the rumen and abomasum. of conversion of metabolizable protein into net protein The experiment consisted of four 24-day experimental [2, 3]. However, there is little information in the tropics periods. An 8-day interval was applied between experi- with regard the comparative understanding of how an mental periods to reduce the residual effects of the treat- increasednitrogensupply inthe rumenor in the intes- ments. The first 14 d of each period were used for tines affects efficiency of nitrogen utilization in cattle. treatment adaptation. Prior to the experiment, the ani- In this sense, basic research using pure protein sources, mals were adapted to the experimental conditions and such as casein, could be helpful to understand the true basal forage for 14 d. effects of protein supplementation on animal metabol- The total supplement was separated into two portions ism and the utilization efficiency of nitrogen from RDP of equal weight and supplied to the animals when the for- or RUP in cattle fed tropical forages. age was offered (0600h and 1800h). The ruminal supple- The objective of the current study was to evaluate the ment was packaged in paper bags and placed directly into effects of supplementation with protein in the rumen, the rumen of the animals. The casein for the abomasal abomasum, or both on intake, digestibility, the rumen supplementation was diluted in saline solution (NaCl, 9 g/ dynamics of fibrous compounds, and the efficiency of ni- L). The lids of the abomasal cannulas were fitted with ap- trogen utilization in cattle fed tropical forage. proximately 15 cm of polyethylene tubing to form external valves. The supplement was infused into the abomasum Methods through these valves. th This experiment was carried out at the Department of Ani- The samples were collected between the 15 and th mal Science of the Universidade Federal de Viçosa, Viçosa, 24 d of each experimental period. The forage sup- th th Brazil. All surgical and animal care procedures were plied from the 15 to 18 d and the orts obtained th th conducted according to the regulations of the Brazilian from the 16 to 19 d were used to measure the National Council on the Control of Animal Experimen- voluntary intake. tation (CONCEA). Fecal grab samples were taken from the rectum of the th th Four rumen- and abomasum-fistulated Nellore bulls animals between the 16 and 19 d of each experimen- th with an initial average body weight (BW) of 227 ± 11 kg tal period according to the following schedule: 16 day th th were used in this experiment. The animals were kept in – 0600h and 1400h, 17 day – 0800h and 1600h, 18 th individual stalls (2 by 5 m) with concrete floors covered day – 1000h to 1800h, 19 day – 1200h and 2000h. with a rubber layer and equipped with individual feeders Samples of abomasal digesta were simultaneously col- and water dispensers. The animals had unrestricted ac- lected with the fecal samples. These samples were oven- cess to mineral mix. dried (60 °C) and processed in a knife mill (1- and 2- The basal forage consisted of Tifton 85 (Cynodon sp.) mm; Model 4, Thomas Wiley Co., Swedesboro, NJ). hay, which had an average crude protein (CP) content of Total urine collection was performed on the 20th day of 78.4 g/kg dry matter (DM). The forage was provided ad each experimental period. Collecting funnels were at- libitum daily at 0600h and 1800h, allowing approxi- tached to the animals to direct the urine into polyethylene mately 100 g/kg in orts. flasks that were kept cool in a polystyrene cooler with ice. This study evaluated the following treatments: control The collections began at 0600h and lasted 24 h. At the (no supplementation); ruminal supplementation, with a end of the collection period, the urine was measured, and daily supply of 230 g of supplemental CP in the rumen; two 50-mL aliquots were collected for analyses. The first abomasal supplementation, with a daily supply of 230 g aliquot was used to assess nitrogen, urea, and creatinine of supplemental CP in the abomasum; and ruminal and contents. The second aliquot was used to quantify the abomasal supplementation, with a total daily supply of content of 3-methyl histidine (3-MH). 230 g of supplemental CP (115 g of CP in both the abo- On the 21st day, blood samples were taken from the an- masum and the rumen). imals at 0600h, 1200h, 1800h and 2400h. directly from the The amount of supplement (230 g of CP/d) accounted jugular vein using vacuum tubes with either coagulation for approximately 35 % of the daily CP requirements, accelerator gel (BD Vacutainer®, SST II Advance, Franklin 55 % of the RDP daily requirements, or 100 % of the RUP Lakes, NJ) or coagulation inhibitor gel (BD Vacutainer® daily requirements of a 250-kg Zebu bull with a weight K2, Franklin Lakes, NJ). The samples collected with gain of 0.5 kg/d [9]. Casein was used as the source of coagulation accelerator were centrifuged (2,700 × g for Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 3 of 10 20 min) to separate the serum, and their urea and creatin- Table 1 Chemical composition of hay and casein ine levels were evaluated. The samples obtained with co- Item Hay Casein agulation inhibitor were refrigerated (4 °C). At the end Dry matter (DM), g/kg as fed 907 ± 8.2 892 of the collection period, the samples were combined for Organic matter, g/kg DM 933 ± 0.6 973 each animal to assess the concentration of free amino Crude protein, g/kg DM 78 ± 0.3 899 acids (AA). NDFap , g/kg DM 730 ± 7.8 - Rumen content samples were also collected at 0600h, st NDIP , g/kg crude protein 360 ± 78 - 1200h, 1800h and 2400h during the 21 d of each ex- perimental period to isolate microorganisms using the Lignin, g/kg DM 58 ± 0.7 - technique described by Cecava et al. [10]. In addition, iNDF , g/kg DM 391 ± 5.2 - ruminal aliquots were taken to evaluate pH, and concen- a NDFap NDF assayed with a heat-stable alpha-amylase and corrected for trations of RAN and volatile fatty acids (VFA; acetate, contaminant ash and protein; NDIP neutral detergent insoluble protein; iNDF indigestible neutral detergent fiber propionate, and butyrate). These samples were manually Mean ± standard error collected at the liquid–solid interface of the rumen mat, filtered through a triple cheesecloth layer, and subjected Fecal excretion and abomasal flow were estimated by to pH evaluation (potentiometer TEC-3P-MP, Tecnal, using the indigestible NDF (iNDF) as internal marker. The Piracicaba, SP, Brazil). Next, a 40-mL aliquot was sepa- samples of hay, orts, feces, abomasal digesta, and ruminal rated, fixed with 1 mL of H SO (1:1), and frozen (−20 °C) contents, processed by passing through a 2-mm screen 2 4 for RAN concentration analysis. A second 20-mL aliquot sieve, were evaluated with regard to iNDF content using was fixed with 5 mL of a meta-phosphoric acid solution F57 bags (Ankom Technology Corp., Macedon, NY) and (250 g/L) and kept at −20 °C for subsequent assessment of an in situ incubation procedure for 288 h (method the VFA concentration. INCT-CA no. F-008/1) [11]. Importantly, the supple- The rumen evacuation procedure was performed on the ment infused into the abomasum was not considered in nd th 22 and 24 d to quantify the resident mass and the rates the ruminal digestibility and outflow calculation; rather, of passage and degradation of the fibrous material. The it was only used to calculate the intestinal digestibility rumen content was removed at 1000h (4 h after the morn- coefficients. ing feeding) and 0600 (before the morning feeding) on the The rates of intake and ruminal passage of NDF were aforementioned days, respectively. The collected material estimated by the ratio of NDF intake and abomasum flow was packed in a polyethylene container and weighed. The on the rumen mass of NDF, respectively. The degradation material was stirred by hand and an aliquot of approxi- rate of NDF was obtained as the difference between the mately 50 g/kg was removed. The remaining material was rates of intake and passage [12]. returned to the rumen of the animals. The samples The RAN concentration was quantified using the colori- were oven-dried (60 °C) and processed in a knife mill metric technique described by Detmann et al. [11] (method (1- and 2-mm). INCT-CA no. N-006/1). The concentrations obtained at Subsequently, the samples of hay, orts, feces, abomasal different sampling times were combined for each animal digesta, and ruminal contents (the samples collected from and period in order to obtain a single value that repre- ruminal evacuation) were pooled per animal and experi- sented the average daily RAN concentration. The rumen mental period. pH values were combined in a similar way. Chemical analyses were performed on the samples that The VFA concentration was evaluated on pooled rumen were processed to pass through a 1-mm sieve. The con- fluid samples composed of proportional sample volumes tents of DM (method INCT-CA no. G-003/1), organic for each collection (per animal and period) and evaluated matter (OM; method INCT-CA no. M-001/1), CP (Kjel- using HPLC (Shimadzu chromatograph, Model SPD-10A dahl procedure; method INCT-CA no. N-001/1), neutral VP) with a reverse phase column (using a mobile phase of detergent fiber corrected for ash and protein (NDFap; orthophosphoric acid in water, 10 mL/L) and a UV de- using a heat-stable α-amylase, omitting sodium sulfite and tector at a wavelength of 210 nm. correcting for residual ash and protein; method INCT-CA The samples of ruminal microorganisms were analyzed no. F-002/1), neutral detergent insoluble protein (method for CP, as described for feed samples, and for purine INCT-CA no. N-002/1), and acid detergent lignin (method bases [13]. The purine bases were used to assess the mi- INCT-CA no. F-005/1) were quantified according to the crobial concentrations in the abomasal digesta based on standard analytical procedures of the Brazilian National the N :N ratio in rumen microorganisms. RNA total Institute of Science and Technology in Animal Science The urine samples were analyzed for nitrogen content (INCT-CA; Table 1) [11]. The casein samples were evalu- as described for the CP analysis of the feed samples. The ated for DM, OM, and CP contents according the methods urea and creatinine concentrations in the urine and blood described above (Table 1). serum were evaluated using the enzymatic-colorimetric Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 4 of 10 (K047, Bioclin Co., Belo Horizonte, MG, Brazil) and alka- Table 2 Effects of supplementation with protein in different sites on voluntary intake in cattle fed tropical forage line picrate (K016, Bioclin Co., Belo Horizonte, MG, Brazil) methods, respectively. The 3-MH in urine and the Supplementation site total free AA in blood were obtained using the HPLC Item Control R A R + A SEM P-value techniques described by Jones et al. [14] and Pitta et al. kg/d [15], respectively. DM 5.05 5.60 5.13 5.12 0.48 0.229 The urea nitrogen filtered in the kidneys and the frac- DMF 5.05 5.35 4.88 4.87 0.48 0.307 tional excretion of urea nitrogen were calculated from OM 4.72 5.25 4.81 4.80 0.45 0.226 the following equations: b a a a CP 0.403 0.648 0.611 0.613 0.040 <0.001 UEC NDFap 3.65 3.87 3.54 3.54 0.33 0.348 UNFK ¼  SUN ð1Þ SC iNDF 1.93 2.05 1.87 1.84 0.19 0.222 b a b ab EUN DOM 2.22 2.60 2.31 2.40 0.22 0.060 FEUN ¼ ð2Þ UNFK DNDF 1.94 2.01 1.84 1.87 0.17 0.362 g/kg body weight where UNFK is the urea nitrogen filtered in the kidneys DM 20.4 22.4 20.8 20.7 1.2 0.263 (g/d), UEC is the urinary excretion of creatinine (g/d), SC is the average concentration of serum creatinine OM 19.1 21.0 19.5 19.4 1.1 0.260 (mg/dL), SUN is the average concentration of serum NDFap 14.8 15.5 14.3 14.3 0.8 0.337 urea nitrogen (mg/dL), FEUN is the fractional excretion iNDF 7.8 8.2 7.6 7.4 0.5 0.273 of urea nitrogen (g/g), and EUN is the urinary excretion a, b within a row, means without a common superscript differ (P < 0.10) of urea nitrogen (g/d). DM dry matter; DMF dry matter from forage; OM organic matter; CP crude protein; NDFap neutral detergent fiber assayed with a heat-stable alpha- The experiment was carried out and analyzed according amylase and corrected for contaminant ash and protein; iNDF indigestible to a 4 × 4 Latin square design balanced for residual effects neutral detergent fiber; DOM digested organic matter; DNDF digested neutral with four treatments (fixed effect), four animals (random detergent fiber Control = without supplementation; R = ruminal supplementation; effect), and four experimental periods (random effect). A = abomasal supplementation All of the statistical procedures were carried out using the MIXED procedure of SAS 9.3. Due to the high prob- ability of type II error, we adopted α = 0.10. When neces- sary, the treatment means were compared using protected Fisher’s least significant difference. The data from one purine base concentration in a microbial sample and one Table 3 Effects of supplementation with protein in different sites creatinine concentration in a blood sample were lost dur- on total, ruminal, and intestinal digestibilities (g/g) in cattle fed ing the analysis. tropical forage Supplementation site Results Item Control R A R + A SEM P-value There were no differences among treatments with regard Total to the intake of DM (P > 0.22), forage (P > 0.30), OM OM 0.468 0.495 0.485 0.502 0.017 0.142 (P > 0.22), neutral detergent fiber (NDF; P > 0.34), iNDF b a a CP 0.475 0.656 0.639 0.664ª 0.023 <0.001 (P > 0.22), and digested NDF (P > 0.36) (Table 2). NDFap 0.531 0.519 0.523 0.532 0.019 0.898 The CP intake increased with supplementation (P < 0.01), but no differences were found between the supplementa- Ruminal tion sites (P > 0.10). The mean CP intakes were 0.403 kg/d OM 0.300 0.324 0.277 0.284 0.031 0.265 and 0.624 kg/d for the control and supplementation treat- b a c b CP −0.221 0.123 −0.987 −0.284 0.139 <0.001 ments, respectively (Table 2). Rumen supplementation in- c bc a ab NDFap 0.502 0.512 0.549 0.528 0.024 0.041 creased the intake of digested OM (DOM) compared with Intestinal that of the control and with that of the abomasal supple- d c a b CP 0.561 0.607 0.772 0.718 0.026 <0.001 mentation treatments (P < 0.10). An intermediate value of a, b, c, d DOM intake was observed for the rumen/abomasal sup- within a row, means without a common superscript differ (P < 0.10) Control = without supplementation; R = ruminal supplementation; plementation (Table 2). A = abomasal supplementation The results of the total digestibility coefficient of CP OM, organic matter; CP, crude protein; NDFap, neutral detergent fiber assayed with a heat-stable alpha-amylase and corrected for contaminant ash were similar to those observed for the CP intake (Table 3). and protein Supplementation increased total digestibility of CP from g Ruminal and intestinal digestibilities were calculated as the fraction of the 0.475 g/g to 0.653 g/g on average. No differences were mass that entered the digestion site Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 5 of 10 found among treatments for the total digestibility of OM Table 4 Effects of supplementation with protein in different sites on the resident mass of fiber in the rumen, the fractional (P > 0.14) and NDF (P > 0.89) (Table 2). rates of NDF rumen dynamics, and on the characteristics of No differences were observed among treatments with re- ruminal fermentation in cattle fed tropical forage gard to the ruminal digestibility of OM (P > 0.26; Table 3). Treatments The rumen digestibility of CP was higher (P <0.10) with Item C R A R + A SEM P-value ruminal supplementation than for the other treatments and was lowest for the abomasal supplementation. Ruminal CP Resident mass in the rumen digestibility was intermediate from the control and supple- NDF, g/kg BW 10.7 10.0 11.8 9.6 1.3 0.337 mentation in the rumen and abomasum. Importantly, the iNDF, g/kg BW 8.4 8.0 9.2 8.3 1.0 0.821 CP rumen digestibility was positive in the ruminal supple- NDF rumen dynamics mentation group and negative for the other treatments ki, /h 0.060 0.071 0.052 0.063 0.008 0.238 (Table 3). kp, /h 0.029 0.034 0.023 0.030 0.003 0.149 Abomasal and ruminal/abomasal supplementation re- sulted in higher NDF rumen digestibility compared with kd, /h 0.030 0.037 0.029 0.034 0.005 0.338 the control (P < 0.10), with an intermediate value observed Ruminal characteristics for ruminal supplementation (Table 3). b a b ab RAN, mg/dL 4.15 13.66 5.56 8.22 1.91 0.078 The CP intestinal digestibility differed across all of the pH 6.83 6.75 6.79 6.72 0.12 0.914 treatments (P < 0.10). The following treatments are pre- VFA, mmol/dL 5.321 5.396 5.731 5.940 0.279 0.256 sented in descending order: abomasal supplementation, Acetate, mol/mol 73.71 73.40 75.25 74.06 0.87 0.489 ruminal/abomasum supplementation, rumen supplemen- tation, and control (Table 3). Propionate, mol/mol 18.76 19.08 17.74 19.32 0.79 0.559 Treatments did not affect ruminal NDF (P > 0.33) and Butyrate, mol/mol 7.53 7.53 7.01 6.69 0.32 0.230 iNDF (P > 0.82) mass; their mean values were 10.5 and A:P 3.96 3.86 4.29 3.84 0.22 0.501 8.5 g/kg BW, respectively. In addition, no differences were a, b within a row, means without a common superscript differ (P < 0.10) observed among treatments with regard to the rates of in- Control = without supplementation; R = ruminal supplementation; A = abomasal supplementation take (P > 0.23), degradation (P > 0.33), and passage of NDF NDF neutral detergent fiber; iNDF indigestible neutral detergent fiber; ki, kp (P > 0.14) (Table 4). and kd rates of intake, passage and degradation; RAN rumen ammonia The ruminal pH did not vary across treatments nitrogen; VFA volatile fatty acids; A:P acetate to propionate ratio (P > 0.91), and an average value of 6.78 was observed. However, higher RAN concentrations were found for the supplementation compared with the control treatment, rumen supplementation group compared with the control regardless of the supplementation site (P < 0.10; Table 5). and the abomasum supplementation groups (P <0.10). However, in spite of the great numerical differences be- These latter groups did not differ from each other tween control and supplemented treatments, no differences (P > 0.10). Rumen/abomasal supplementation resulted in the efficiency of nitrogen utilization were observed in an intermediate RAN concentration (Table 4). among the treatments (P > 0.16). There were no differences among the treatments re- The highest estimate of NBR was observed with rumen gard to the VFA concentration (P > 0.25), the average supplementation (P < 0.10), followed by ruminal/abomasal value of which was 5.60 mmol/dL. In addition, the supplementation, abomasum supplementation, and the acetate (P > 0.48), propionate (P > 0.55), and butyrate control. No differences were observed between the latter (P > 0.23) molar ratios, as well as the acetate:propionate two treatments (P > 0.10). Importantly, only ruminal sup- ratio (P > 0.50), did not differ across treatments (Table 4). plementation resulted in positive NBR (Table 5). The SUN concentration (Table 5) was higher with rumi- The treatments affected the amount of CP digested in nal supplementation compared with ruminal/abomasum the intestines (PDI; P < 0.01). Larger amounts of PDI supplementation and control (P < 0.10). The average SUN were observed in the abomasal supplementation group concentration obtained with abomasal supplementation compared with the ruminal/abomasal supplementation occupied an intermediate position compared with the and the control treatments. The ruminal supplementa- other supplementation types. No differences were found tion group held an intermediate position between these across the treatments with regard to the concentration of groups (Table 5). AA in the blood (P >0.48; Table 5). The control treatment exhibited greater urinary excre- Nitrogen intake and urinary excretion increased with tion of 3-MH compared with the supplement treatments protein supplementation, regardless of the supplementa- (P < 0.10), which did not differ from each other (P > 0.10). tion site (P < 0.10). However, the nitrogen fecal excretion The urinary excretion of urea nitrogen was higher for did not vary among the treatments (P > 0.61). Similarly, the the supplementation groups than for the control group apparent nitrogen balance (NB) increased with nitrogen (P < 0.10). In addition, ruminal and ruminal/abomasal Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 6 of 10 Table 5 Effects of supplementation with protein in different sites on the characteristics of nitrogen utilization in the animals in cattle fed tropical forage Supplementation site Item Control R A R + A SEM P-value c a ab b SUN, mg/dL 7.29 17.16 13.46 13.39 1.30 0.029 BAA, μmol/mg creatinine 113 123 129 119 12 0.485 b a a a Nitrogen intake, g/d 64.5 103.8 97.8 98.0 6.4 <0.001 Fecal nitrogen, g/d 34.0 36.0 35.5 33.4 4.0 0.611 b a a a Urinary nitrogen, g/d 26.3 48.5 40.4 46.3 4.2 0.010 b a a a Nitrogen balance, g/d 4.1 19.3 22.0 18.3 6.6 0.062 c a c b NBR, g/d −14.4 12.2 −23.2 −2.0 7.5 0.005 ENU g retained nitrogen/g of ingested nitrogen 0.04 0.18 0.22 0.18 0.05 0.165 g retained nitrogen /g of nitrogen absorbed in the intestines 0.07 0.27 0.35 0.27 0.04 0.402 c bc a b PDI, g/d 280 347 535 416 45 0.003 a b b b 3-MH, mg/g of creatinine 50.8 20.6 29.6 31.0 13.1 0.077 b a a UEUN, g/d 13.4 28.9 25.9ª 30.1 2.9 0.002 b a b a UFK, g/d 42.9 ± 6.5 60.9 ± 5.6 43.9 ± 5.6 55.0 ± 5.6 — 0.062 FUEUN, g/g 0.35 ± 0.09 0.48 ± 0.08 0.63 ± 0.08 0.55 ± 0.08 — 0.121 NMIC, g/d 53.1 ± 13.0 61.2 ± 11.1 61.9 ± 13.0 58.8 ± 11.1 — 0.932 a, b, c within a row, means without a commom superscript differ (P < 0.10) SUN serum urea nitrogen; BAA amino acids in blood; NBR nitrogen balance in the rumen; ENU efficiency of nitrogen utilization; PDI protein digested in the intestine; 3-MH urinary excretion of 3-methyl histidine; UEUN urinary excretion of urea nitrogen; UFK urea nitrogen filtered in the kidneys; FUEUN fractional excretion of urea nitrogen; NMIC ruminal production of microbial nitrogen compounds Control = without supplementation; R = ruminal supplementation; A = abomasal supplementation supplementation increased the urea filtered in the kid- nitrogenous compounds in the rumen [1, 17–19] or in neys (P < 0.10) compared with the other treatments. the abomasum [6, 8, 20]. Supplementation did not alter the fractional excretion The lack of changes on forage and NDF intake in the of urea nitrogen (P > 0.12). However, supplementation response to nitrogen supplementation might be associ- resulted in a 60 % higher fractional excretion of urea ni- ated with the content of CP in the basal forage (Table 1). trogen compared with the control (Table 5). The ruminal The previously cited authors studied forages with CP con- synthesis of microbial nitrogenous compounds did not dif- tents that were typically below 60 g/kg DM. The average fer among the treatments (P > 0.93), with an average value dietary CP in the control treatment, calculated as the ratio of 58.7 g/day (Table 5). between CP and DM intake, was 79.8 g/kg. Nitrogen does not positively affect voluntary forage intake when the diet- ary CP is above 70–80 g/kg DM [3, 21]. Discussion The stimulation of low-quality forage intake via nitro- Nitrogen supplementation for animals fed low-quality for- gen supplementation is usually associated with a reduc- age favors the growth of fibrolytic bacteria and increases tion in the physical constraints to intake [16, 22]. In this the ruminal degradation and voluntary intake of fiber, as context, nitrogen supplementation could increase the well as the energy extraction from forage fiber [16]. Spe- degradation rate of NDF, which concomitantly increases cifically, supplementation with nitrogenous compounds in the passage rate of non-degraded and indigestible fiber the abomasum of animals fed low-quality forage could from the rumen, increasing forage intake [15]. stimulate intake via nitrogen recycling, albeit with less in- However, supplementation did not alter the ruminal dy- tensity compared with rumen supplementation [6, 8]. namics of NDF (Table 4). This result might be also due to However, positive effects on the voluntary intake of forage the CP content of the basal forage, which was above the were not observed for any supplementation site in the minimum level required to support fibrolytic activity in the present study (Table 2). rumen [23]. When the removal of fiber residues from the The results obtained here differ from those reported rumen is limited, animals can increase their rumen volume by several authors who found increases in the voluntary to accommodate a greater mass of resident fiber [24]. With intake of low-quality forage in cattle supplemented with this adaptation, and keeping the passage and fractional Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 7 of 10 degradation rates constant, the animal is able to increase intestinal digestibility coefficient was 0.93 g/g. Therefore, the amount of degraded fiber. However, no effect was casein supplied in the rumen should result in a higher observed with regard to the rumen fiber mass (Table 4). total digestion compared to casein supplied in the aboma- Therefore, considering the results regarding voluntary sum, which would explain the greater intake of digested intake and fiber dynamics, it can be stated that the CP OM in the animals that received ruminal supplementation content of the basal forage was adequate to support ru- (Table 2). minal function. The most prominent effects of supplementation in the Although supplementation increased the NDF digest- present study were observed on the metabolism and the ibility in the rumen, the observed effect was small, which nitrogen accretion in animal body, as other authors have resulted in no differences among treatments with regard shown in tropical conditions [2–4, 28]. NDF intake and total digestibility, and VFA concentra- The main effect of supplementation can be associated tion (Tables 2, 3, and 4). The stimulation observed with with an increase in NB (Table 5), which would represent supplementation most likely resulted from some im- an increase in weight gain. Although similar amounts of provement in the availability of RAN (Table 4). The protein were supplied to the supplemented animals, the “protein effect” can explain the low rumen digestibility pathways through which these supplements affect protein coefficient with rumen supplementation. This result cor- accretion in the body seem differ because the way in responds to an increase in the competition for essential which supplemental nitrogen is utilized could depend on substrates between fibrolytic and non-fibrolytic species the supplementation site (i.e., the rumen or the intestines). when the supplements contain true protein [25]. This Due to the high degradability of casein in the rumen, “protein effect” can occur simultaneously with the nitro- little of the ruminal protein supplement would reach the gen stimulation of fibrolytic activity in low-quality forage intestines to be digested. Considering that the ruminal [26]. Therefore, the similarity between control and rumi- degradation coefficient is 0.80 g/g [27] and the intestinal nal supplementation treatments regarding the ruminal digestibility coefficient was 0.93 g/g, the additional pro- digestibility of NDF may be a result of a counterbalan- tein mass (230 g CP/d) should provide only 42.8 g of cing of the nitrogen stimulus and the “protein effect.” additional PDI. This result explains the small increase in This theory also explains the intermediate position PDI that was observed with ruminal supplementation achieved by the ruminal/abomasum supplementation (Table 5). Considering the efficiency of utilization of the group (Table 3). absorbed nitrogen (0.27 g/g; Table 5), that additional The only between-treatment difference found with re- PDI should increase NB by approximately 1.8 g/d, which gard to the total digestion was the CP digestibility. Specif- is equivalent to approximately 11 % of the increase in ically, these values were higher in the supplemented NB in relation to the control treatment. Therefore, the animals compared with the non-supplemented animals probable escape of casein to the small intestine cannot (Table 3). This result occurred as a result of the increased explain the effects of ruminal casein supplementation on CP intake provided by supplementation (Table 2) because, NB. It should be emphasize that there was no effect of for the dietary non-fibrous components, such as the CP supplementation on ruminal synthesis of microbial ni- derived from casein, the apparent digestibility coefficient trogen (Table 5) and an increase in metabolizable pro- is proportional to the intake [21]. A similar result was ob- tein supply from microbial protein did not occur. served for the intestinal digestibility of CP, which was The most prominent effect of the ruminal supplementa- especially high when the casein was infused into the abo- tion was the increase in RAN concentration (Table 4), masum (Table 3). The assessment of the amount of appar- which means an overall improvement in nitrogen avail- ently digested CP in the intestines using the Lucas test ability in the animal gastrointestinal tract and also for the approach revealed a true intestinal digestibility of CP of animal metabolism. The RAN concentration observed in 0.931 g/g [CI(β) :0.838 ≤ β ≤ 1.024]. Considering that non-supplemented animals (4.15 mg/dL) was below that 0.90 there was no difference among the treatments with regard reported by Detmann et al. [22] for an equilibrium be- to the fecal excretion of nitrogen (Table 5), this estimate tween the inflow and outflow of nitrogen in the rumen suggests that the protein supplement was almost com- of animals fed tropical forage (approximately 8.4 mg pletely digested in the intestines. of RAN/dL), which resulted in negative NBR values The intake of DOM increased with supplementation (Table 5). in the rumen. However, no between-treatment differ- The overall effects of nitrogen availability on ruminant ences were found with regard to the intake of digested metabolism have been associated with a better adequate NDF (Table 2). The increased intake of DOM is there- protein or nitrogen status [7, 29]. Theoretically, the ni- fore attributed solely to the effects of the non-fibrous trogen status defines the availability of different nitro- portion of the diet. Casein is estimated to have a ruminal genous compounds in both quantity and quality for all digestion greater than 0.80 g/g [27], whereas its required physiological functions in animal metabolism, Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 8 of 10 including the functions associated with the metabolism nitrogen that is recycled through rumen wall seems to of other compounds such as energy compounds [7]. be relatively constant [17]. Therefore, there will be lesser Taking into account the theoretical concept of nitrogen nitrogen accretion in the animal body under low nitro- status, it could be realized that the nitrogenous com- gen contents in the diet because a greater percentage of pounds would be used in different metabolic functions the ingested nitrogen will be directed to recycling and, following an order of priority to the animal: survival, as a consequence, a lower percentage of nitrogen will be maintenance, and production [7]. Thereby, a positive ni- available for production [7]. trogen accretion in the animal body or products only will The pattern here observed for NBR can be used to give take place after supplying the higher priority demands for some support for the assumptions about metabolic prior- nitrogenous compounds. ities of nitrogen. Several estimates of negative NBR has One of the possible high-priority metabolic functions been obtained in experiments carried out in the tropics, is the recycling of nitrogen to the gastrointestinal tract. and the main factor to influence that is the nitrogen avail- Such a statement seems to be plausible because a con- ability in the diet [7]. This pattern highlights that nitrogen tinuous supplying on nitrogen for microbial growth in flow to abomasum can be greater than nitrogen intake in the rumen must be seen as a strategy for animal survival several occasions. In these cases, there is a more signifi- [21, 30]. Under a nitrogen deficiency, the animal is able cant dependency on recycling events to provide adequate to decrease urinary nitrogen excretion and increase the nitrogen supplying to the rumen. Then, the animal will fraction of dietary nitrogen that is recycled to the rumen decrease the efficiency of utilization of metabolizable pro- [4, 31]. In fact, despite the absence of a significant differ- tein for gain (decreased anabolism as supported by the ence (P > 0.12), the fractional excretion of urea nitrogen lower numerical efficiency of nitrogen utilization, Table 5) in the control treatment was approximately 71 % of that and sometimes also increase the breakdown of muscle observed in the ruminal supplementation treatment protein to supply the nitrogen demands of higher priority (Table 5). This result indicates that a smaller percentage (increased catabolism as supported by greater 3-MH ex- of the circulating urea was eliminated in the urine, and cretion, Table 5). more prominent fraction was directed for reuse (e.g., for The availability of RAN increased with rumen supple- recycling) when supplement was not provided. mentation (Table 4), making the NBR positive, reflecting When nitrogen deficiency becomes more severe, the improvement in nitrogen status in the animal body. In this animal can increase the myofibrillar protein mobilization way, there was an increase in the accretion of absorbed ni- to sustain the mass of recycling nitrogen [4, 32]. This trogen (Table 5). Costa et al. [2] observed similar pattern was observed in this study through urinary excretion of by providing nitrogen compounds that were highly de- 3-MH, which was decreased when supplement was pro- gradable in the rumen to cattle grazing tropical forage vided (Table 5). (99 g CP/kg DM). The 3-MH is an AA formed from the methylation of On the other hand, the direct effect of abomasal sup- histidine after its inclusion in the muscle proteins (i.e., plementation was based on the increased availability of actin and myosin). When muscle proteins are degraded, AA absorbed in the small intestine, which can be seen the 3-MH cannot be reused for protein synthesis and is by the greater amount of PDI compared with the control excreted in the urine [33]. Thus, the 3-MH urinary (Table 5). This metabolizable protein supply can be dir- excretion is a marker for the degradation of muscle pro- ectly incorporated into tissue, thereby increasing the NB tein that, in turn, can be used for other physiological or (Table 5). Importantly, the efficiency of utilization of metabolic functions, such as the supply of higher prior- absorbed nitrogen and NB were similar for ruminal and ity nitrogen demands. The control treatment therefore abomasal supplementation groups (Table 5). increased the mobilization of muscle protein by ap- The largest mass of available AA in the small intestine proximately 146 % compared with the ruminal supple- directly increases the nitrogen status in the animal me- mentation treatment (Table 5). Similarly, Pitta et al. tabolism by supplying the requirements for tissue syn- [15] found a reduction in the plasma concentration of thesis and providing nitrogen to the higher demand 3-MH by providing a protein supplement to sheep fed events without major mobilizations of muscle protein. low-quality forage. The reduction of 3-MH excretion observed in the ab- Importantly, 3-MH urinary excretion was strongly and omasal supplementation group indirectly confirmed this negatively correlated with SUN (r = −0.892; P < 0.01) and effect (Table 5). RAN (r = −0.693; P < 0.09) concentrations, which indi- According to Bandyk et al. [8] and Wichersham et al. cates that muscle protein mobilization is negatively asso- [6, 20], supplementation with protein sources that are ciated with nitrogen availability or nitrogen status. not degradable in the rumen can increase the supply of Considering a normal feeding situation, without any RAN through nitrogen recycling, although less efficiently prominent dietary nitrogen deficiency, the amount of than supplementation with RDP sources. The increased Rufino et al. Journal of Animal Science and Biotechnology (2016) 7:11 Page 9 of 10 SUN concentration, which is mediated by the greater Therefore, even if the source of RUP is inexpensive, the availability of nitrogen in the intestines, favors this in- most appropriate formulas would depend on the profile of crease in recycling, as was observed in the present study digestible AA in the small intestine. (Table 5). Consequently, supplementation with RUP sources are An increased SUN concentration can increase the differ- only feasible or logical after the beneficial effects of RDP ence between the concentrations in the blood and rumen had been explored, thereby providing additional per- and increases urea transfer [20]. This result should in- formance gains through direct supply of metabolizable crease the pool of RAN [6, 20]. Wickersham et al. [20] protein [8, 20]. found that the proportion of microbial nitrogen produced from recycled nitrogen increased from 0.31 to 0.58 g/g Conclusion when cattle fed low-quality forage were supplemented The supplementation of cattle fed tropical forage with with nitrogenous compounds in the small intestine. protein in the rumen, abomasum, or both can increase The RAN concentration after abomasal supplementa- the retention of nitrogen in animal. However, the meta- tion was increased by only 1.41 mg/dL over the control bolic pathways involved in improving nitrogen accretion treatment, and this increase was not significant (P >0.10). differ between supplementation sites. The improvement This result contradicts the studies described above. How- obtained with rumen supplementation seems based on ever, abomasal supplementation decreased the rumen di- a direct increase in dietary nitrogen availability and sta- gestibility of CP and, consequently, the NBR (Tables 3 and tus. Moreover, the improvement obtained with aboma- 5). Considering that the supplemental nitrogen was not sum supplementation results from an increased supply used to estimate ruminal digestibility when supplement of metabolizable protein. was supplied in the abomasum and that forage intake was Abbreviations constant among treatments (Table 2), the only likely cause 3-MH: urinary excretion of 3-methyl histidine; AA: amino acids; BW: body for the decreased NBR under abomasal supplementation weight; CP: crude protein; DM: dry matter; DMF: dry matter from forage; DNDF: digested neutral detergent fiber; DOM: digested organic matter; is an increase in the amount of nitrogen coming from iNDF: indigestible neutral detergent fiber; ki kp and kd: rates of intake, recycling. It can therefore be inferred that abomasal passage and degradation of NDF; NB: nitrogen balance; NBR: nitrogen supplementation increased the pool of available nitro- balance in the rumen; NDF: neutral detergent fiber; NDFap: neutral detergent fiber assayed with a heat-stable alpha-amylase and corrected for gen in the rumen, although less efficiently than did ru- contaminant ash and protein; OM: organic matter; PDI: protein digested in minal supplementation. the intestine; RAN: rumen ammonia nitrogen; RDP: rumen degradable In general, the effects of ruminal/abomasal supplemen- protein; RUP: rumen undegradable protein; SUN: serum urea nitrogen; VFA: volatile fatty acids. tation were between those of ruminal supplementation and those of abomasal supplementation. The intermediate Competing interests RAN concentration (Table 4) and PDI (Table 5) provided The authors declare that they have no competing interests. animals with the previously discussed effects of body ni- Authors’ contribution trogen retention, providing NB that was similar to the ex- ED, conceived the study, performed the statistical analysis, contributed to clusive rumen or abomasum supplementation (Table 5). draft the manuscript, and coordinate the research group. LMAR, EDB, DIG and WLSR, carried out the experimental trial, performed the chemical analysis, From the results obtained in the present study, it can and helped to draft the manuscript. SCVF and MFP, helped to draft the be stated that nitrogen supplementation in the rumen or manuscript. All authors read and approved the final manuscript. abomasum should show similar effects but as a result of Acknowledgments different ways to improve nitrogen status in the animal The authors thank the Conselho Nacional de Pesquisa e Desenvolvimento metabolism. In theory, these mechanisms provide a pos- Científico (CNPq), the Fundação de Amparo à Pesquisa de Minas Gerais sibility for choosing between supplementation with RDP (FAPEMIG), and the INCT Ciência Animal for financial support. or RUP. Author details Consequently, additional factors should be considered Department of Animal Science, Universidade Federal de Viçosa, Av. P.H. with regard to the most suitable production system. The Rolfs, s/n°, Viçosa, Minas Gerais 36570-900, Brazil. Department of Animal Science, Universidade Federal Rural da Amazônia, Campus de Parauapebas, costs involved in using RDP sources are almost always C.P. 3017, Bairro Cidade Nova, Parauapebas, Pará 68515-970, Brazil. lower than those of RUP. 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Intake and digestibility in cattle under grazing during dry season • Inclusion in PubMed and all major indexing services supplemented with nitrogenous compounds. Rev Bras Zootec. 2010;39: • Maximum visibility for your research 1303–12. doi:10.1590/S1516-35982010000600020. 20. Wickersham TA, Titgemeyer EC, Cochran RC, Wickersham EE. Effect of Submit your manuscript at undegradable intake protein supplementation on urea kinetics and www.biomedcentral.com/submit

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