Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep

Journal Pre-proof Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep Tiago Orlandi, Simone Stefanello, Mariana P. Mezzomo, Claudio A. Pozo, Gilberto V. Kozloski

PII:

S0377-8401(19)31095-8

DOI:

https://doi.org/10.1016/j.anifeedsci.2019.114384

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ANIFEE 114384

To appear in:

Animal Feed Science and Technology

Received Date:

9 August 2019

Revised Date:

18 December 2019

Accepted Date:

20 December 2019

Please cite this article as: Orlandi T, Stefanello S, Mezzomo MP, Pozo CA, Kozloski GV, Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep, Animal Feed Science and Technology (2019), doi: https://doi.org/10.1016/j.anifeedsci.2019.114384

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Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep

Tiago Orlandia, Simone Stefanelloa, Mariana P. Mezzomoa, Claudio A. Pozoa and Gilberto V. Kozloskia,*

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Departamento de Zootecnia, Universidade Federal de Santa Maria, Santa Maria, RS, 97105-

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900, Brazil

*Corresponding author.

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E-mail address: [email protected]

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

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Highlights

Supplementation with a tannin extract did not impact on digestibility. o Supplementation with a tannin extract decreased the urinary excretion of urea.

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o Supplementation with a tannin extract did not impact ammonia, urea or

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Abstract

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glucose net flux across splanchnic tissues.

This study was conducted to quantify the impact of the dietary inclusion of Acacia mearnsii bark extract (TA), a tannin extract, on total tract digestibility and on ammonia, urea and glucose splanchnic net flux in sheep. The trial was conducted with five Texel male sheep (48 ± 3.2 kg body weight (BW)) surgically implanted with chronic indwelling catheters into one mesenteric, portal and hepatic veins, in two 21-day periods, in a cross-over design. Diet was

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oat/ryegrass hay, offered ad libitum, plus concentrate offered three times per day at a rate of 14 g/kg BW. The concentrate, composed of soybean meal, cracked corn and wheat bran, included (Tannin) or not (Control) 20 g/kg dry matter (DM) of TA. Plasma flow and net flux through portal-drained viscera (PDV), liver and total splanchnic tissues (ST) were measured using four hourly samples, from 14:00 to 17:00, after the 12:00 meal. Total TA concentration in diet of TA treatment averaged 7.7 g/kg DM. The organic matter (OM) intake and digestibility were not affected by TA. The nitrogen (N) intake and fecal excretion, the

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estimated rumen microbial N flow to the small intestine and the efficiency of microbial protein synthesis (EMPS) were not affected whereas the digestibility of N compounds and the urinary excretion of urea-N were lower (P < 0.05) in TA treatment. There was no significant

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effect of TA on the net flux of ammonia, urea-N or glucose across PDV, liver and total ST. In conclusion, the dietary inclusion of tannin extract from Acacia meanrsii at a rate of 7.7 g/kg

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DM decreased the urinary excretion of urea with, however, no impact on ammonia, urea or

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glucose net flux across ST.

Abbreviations: ADF, acid detergent fiber; BW, body weight; CP, crude protein; DM, dry

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matter; EMPS, efficiency of microbial protein synthesis; EE, ether extract; GER, gut entry rate; MP, metabolizable protein; NPF, net plasma flow; NWF, net whole blood flow; aNDF,

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neutral detergent fiber; NDIN, neutral detergent insoluble N; OM, organic matter; PAH, p-

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aminohippurate; PF, plasma flow; PDV, portal-drained viscera; PD, purine derivatives; ST, splanchnic tissues; TA, Acacia mearnsii tannin extract.

Keywords: Acacia mearnsii; ammonia; gluconeogenesis; liver; sheep; ureagenesis

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1. Introduction Tannins are plant polyphenol compounds with the capacity to form complexes with proteins and carbohydrates, thereby reducing their degradation in the rumen (Makkar, 2003). The dietary inclusion of TA, an industrial source of tannins, has been investigated in the last few years as a feed additive with the potential to decrease the excretion of both methane and labile urinary N, and to improve the metabolizable protein (MP) supply to ruminants. For example, Carulla et al. (2005) observed that supplementing TA at a rate of 41 g/kg diet DM

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for lambs fed ryegrass increased forage intake whereas it depressed OM digestibility. However, because methane emission and urinary N excretion also decreased, energy and N retention were not affected. In previous studies by our group, when TA was infused at rates

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from 20 to 60 g/kg DM intake into the rumen of sheep fed fresh ryegrass, a linear decrease of both urinary N excretion and digestible OM intake was observed (Kozloski et al., 2012). The

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inclusion of a relatively low TA concentration (i.e. 15 g/kg DM) increased the outflow of

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amino acids from the rumen of steers fed corn silage plus concentrate (Ávila et al., 2015) and the dietary inclusion of 18 g of TA/kg DM decreased urinary N excretion and improved the outflow of amino acids from the rumen of steers fed oat forage plus concentrate without

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affecting total OM digestibility (Orlandi et al., 2015). Moreover, tannins may also change the profile of volatile fatty acid production by rumen bacteria, increasing the propionate

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proportion (Batha et al., 2009; Hassanat and Benchaar, 2013; Yang et al., 2017), the major

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glucose precursor in the liver of sheep. In fact, the impact of TA on rumen fermentation and total tract digestibility has been consistently evaluated whereas, however, no study has reported yet the impact of this tannin extract on ST metabolism. Thus, in addition of evaluating the effect of TA on feed intake and digestibility, the present study was conducted to evaluate the impact of a low dose of TA on the net ammonia, urea and glucose flux across ST of sheep. It was hypothesized that, as a consequence of an

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expected decreased ammonia and increased propionate absorption from the rumen, the sheep receiving TA would produce less urea and more glucose in the liver, and would excrete less urea in urine.

2. Material and methods

2.1. Animals, diet, experimental design and treatments

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All experimental procedures followed the guidelines of the Animal Care and Ethical Committee of the Universidade Federal de Santa Maria (Nº 008/2014). Five Texel male sheep (48 ± 3.2 kg BW) surgically implanted with chronic indwelling catheters into the portal,

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hepatic and mesenteric veins were used. Teflon catheters of 1.5 mm i.d. were used in portal and hepatic veins whereas a polyvinyl chloride catheter of 1.0 mm i.d. was used in mesenteric

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vein, all of them approximately 50 cm length. With the exception of the tip inserted into the

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vessels, catheters were covered with a silicone rubber (Huntington et al., 1989). Surgery and catheter maintenance procedures followed those described by Katz and Bergman (1969a) and Huntington et al. (1989). One of the carotids was surgically placed closer to the skin to

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provide arterial blood sampling access. Following surgery, the animals received analgesic and antibiotic treatment and were allowed a recovery period of at least four weeks before

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allocation to the trial. Subsequently they were housed in individual pens (2.5 m2) and, before

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starting the experimental period, they were adapted to the feeding and management conditions for two weeks. Diet was oat-ryegrass mixed hay offered ad libitum plus concentrate offered at a rate of 14 g/kg BW. The concentrate was composed of cracked corn (0.70), soybean meal (0.20) and defatted rice bran (0.10). The chemical composition of hay and concentrate is shown in Table 1. The experiment was conducted throughout two 21-day periods in a crossover design to test the following treatments: inclusion (Tannin) or not (Control) of 20 g of TA

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(Weibull Black, Tanac S.A., Montenegro, Brasil) per kg of concentrate DM. The concentrate was pelleted after mixing the ingredients. The TA was the same previously used and described by Kozloski et al. (2012) and contained 694 g/kg DM of total tannins. The feed was offered in three daily meals at 8:00h, 12:00h and 17:00h, and the animals had permanent access to water and a commercial mineral salt containing (g/kg): Ca: 120, P: 87, Na: 147, Mn: 1.3, Zn: 3.8, Fe: 1.8, Cu: 0.59, Co: 0.040, I: 0.080, Se: 0.015 and F: 0.87.

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2.2. Sampling and data collection From day 15 to 20 total feed, orts and feces were weighed and sampled daily. These samples were oven-dried at 55°C for at least 72 h and ground through a 1-mm screen for

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subsequent chemical analysis. Urine was collected daily during the collection period, in buckets containing 100 mL of 3.6 N H2SO4. The total volume of urine was measured and a

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sample of 10 mL was taken, diluted to 50 mL with distilled water and stored frozen (–20°C).

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Representative aliquots of orts, feces and urine samples were pooled by animal and period for analysis. Between day 18 to 21 of each experimental period and starting 60 minutes after offering the noon meal (i.e. approximately at 12:00h), the plasma flow (PF) through PDV and

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total ST was measured by downstream dilution of a primed (10 ml) followed by continuous infusion (2.0 mL/min) during four hours of a 15 g/L p-aminohippurate (PAH, pH 7.4)

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solution into the mesenteric vein. The PAH solution was previously filtered across cellulose

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filter paper (7.5 µm porosity) and sterilized in autoclave at 110°C during 20 minutes, and the infusion was performed across a 0.45 µm porosity filter using a syringe infusion pump (Cole Palmer Instrument, IL, USA). Beginning 60 minutes after starting the PAH infusion and for the remaining 3 hours of infusion period, samples of each arterial, portal and hepatic blood were simultaneously collected in heparinized syringes at 60 minutes intervals. For arterial blood sampling, a temporary catheter (22 gauge) was previously inserted in the carotid artery

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and attached to an extension with a three-way valve. Between sampling intervals, catheters were flushed with a physiological solution containing 20 IU of heparin/ml. The blood was transferred to a conical centrifuge tube containing sodium fluoride (2.5 mg/ml of blood), centrifuged (1000 × g during 20 minutes) and the plasma stored at 4°C. At the end of the infusion period aliquots of hourly plasma samples were taken for immediate PAH analysis and the remaining plasma was stored at -20ºC for subsequent analysis.

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2.3. Chemical analysis Dried and ground samples of feed, orts and feces were analyzed for DM content by oven-drying at 110°C overnight. Ash was then determined by combustion at 600°C for 3 h

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and OM by mass difference. Total N was assayed by the Kjeldahl method (Method 984.13) of AOAC (1997) and crude protein (CP) calculates as N × 6.25. The neutral detergent fiber

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(aNDF) analysis was based on the procedures described by Mertens (2002) without using

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sodium sulphite and with use of heat-stable α-amylase, and the concentration of acid detergent fiber (ADF) was analyzed according to Method 973.18 of AOAC (1997) except that the samples were weighed in polyester filter bags (porosity of 16 μm) and treated with neutral or

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acid detergent in an autoclave at 110 °C for 40 min (Senger et al., 2008). For sulphuric-acid lignin (sa) analysis, the bags containing residual ADF were treated with H2SO4 12 M for 3 h

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(Method 973.18 of AOAC (1997)). Analyses of soluble N, neutral detergent insoluble N

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(NDIN) and acid detergent insoluble N were performed according to Licitra et al. (1996). Ether extract (EE) concentration was determined in a reflux system (Ankom XT15; Ankom Technology, USA) with petroleum ether at 90oC for 60 minutes. The content of non-fiber carbohydrates (g/kg) was estimated as: OM - [(aNDF - (NDIN × 6.25)) + CP + EE]. In urine samples, urea concentration was determined by colorimetry (urease followed by salicylatehypochlorite reaction) using a commercial kit (Bioclin, MG, Brasil), and allantoin and uric

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acid concentrations were also determined by colorimetry according to Chen and Gomes (1992). Uric acid was determined after xanthine and hypoxanthine were converted to uric acid with xanthine oxidase. Thus, the uric acid values were calculated as the sum of uric acid, xanthine and hypoxanthine and, the total purine derivatives (PD) as the sum of uric acid and allantoin. The PAH analysis in plasma samples was performed daily after the infusion and collection period. For that, 1 mL of plasma was mixed with 9 mL of 50 g/L trichloroacetic acid solution, filtered (paper filter), and the filtrate was kept in a water bath at 90°C during

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120 minutes. The PAH concentration in filtrate was then determined using the sulfonaphthylene colorimetric method (Huntington, 1982). The analysis of other metabolites was performed in plasma samples which were stored frozen (-20°C). To improve the accuracy

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and precision of analysis, 1 mL of plasma was diluted with 9 mL of distilled water and 1 mL of diluted sample was used in each replicate tube. Plasma concentrations of glucose (glucose

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oxidase method) and urea (urease followed by salicylate-hypochlorite reaction) were

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determined by colorimetry using commercial kits (Bioclin, MG, Brasil), and ammonia concentration was analyzed using the phenol-hypochlorite procedure (Weatherburn, 1967). Despite this method may overestimate the concentrations of ammonia in plasma, we assumed

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individual samples.

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that treatments could be compared in the present study. All plasma analysis was performed on

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2.4. Calculations

The apparent digestibility of feed fractions was calculated as: ((intake (g/day) – fecal

excretion (g/day))/ intake (g/day). The true digestibility of OM was estimated considering that neutral detergent soluble fractions of the feces are endogenous in origin and only the aNDF fraction of feces originated from feed (Van Soest, 1994) as follows: [OM intake (g/day) – fecal aNDF (g/day)]/OM intake (g/day). The true digestibility of N compounds was estimated

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considering that neutral detergent soluble N of the feces are of endogenous origin and only the NDIN fraction of feces originated from feed (Van Soest, 1994) as follows: [N intake (g/day) – fecal NDIN (g/day)]/N intake (g/day). Microbial N flow to the small intestine was calculated from the urinary output of PD according to Chen and Gomes (1992). Plasma flow rates across the PDV and ST were calculated from downstream dilution of PAH according to Katz and Bergman (1969b): PF (L/h) = IRPAH/(VPAH – APAH), where IRPAH is PAH infusion rate (mg/h), VPAH and APAH are concentrations of PAH (mg/L) in venous (portal (PDV) or

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hepatic (ST)) and arterial plasma, respectively. The net flux (mmol/h) of metabolites (i.e. ammonia, urea and glucose) through PDV or ST was calculated as: (Vm – Am) × PF (L/h) where Vm and Am are metabolite concentration (mmol/L) in venous (portal (PDV) or hepatic

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(ST)) and arterial plasma. Because urea and ammonia are transferred across tissues through both plasma and red blood cells, ammonia concentrations are equal in whole blood and

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plasma, urea concentrations are equal in whole-blood water and plasma water (Milano et al.,

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2000) and the DM is higher in red blood cells than in plasma, the net plasma flux values (NPF) were corrected for the whole blood (NWF) using the equations of Martineau et al. (2009) as follows: i) NWF of ammonia = NPF × (1/(1 – hematocrit)) and ii) NWF of urea =

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NPF × (1/(1 – hematocrit)) × ((1 – 0.15)/(1 – 0.08)). The hematocrit averaged 0.23 ± 0.098 in the present study. The net flux of metabolites across the liver was calculated as the difference

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between ST minus PDV values. Negative values denote net tissue uptake and positive values

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indicate net release of a metabolite.

2.5. Statistical analysis Values of plasma parameters obtained at different times were averaged by animal and period for statistical analysis. Data were analyzed using the MIXED procedure of SAS (SAS, 2002) with treatment as the fixed factor and animal and period as random factors in the

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model. Significance was declared at P ≤ 0.05 and a tendency was considered when 0.05 < P ≤ 0.10.

3. Results Total TA and total tannin intakes in TA treatment averaged 12.0 and 8.3 g/day, corresponding to a dietary total TA and total tannin concentration of 7.7 and 5.3 g/kg DM, respectively. The DM, OM and aNDF intake and apparent digestibility, as well as the true

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OM digestibility and digestible OM intake were not affected by TA (Table 2). As average of both treatments, the digestible OM intake averaged 46 g/kg BW0.75, what is equivalent to approximately 681 kJ of metabolizable energy per kg BW0.75 or 1.7 of the basal metabolism

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requirement (Fox et al., 2004; Tedeschi et al., 2010). The N intake and fecal excretion, the urinary excretion of PD, the estimated rumen microbial N flow to the small intestine and the

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EMPS were not affected whereas the apparent and true digestibility of N compounds and the

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urinary excretion of urea-N were lower (P < 0.05) in TA treatment (Table 3). The CP (i.e. N × 6.25) intake was on average 200 g/day, sufficient to allow a BW gain of 400 g/day by a growing male sheep with 50 kg BW (NRC, 2007). The glucose concentration in arterial and

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portal plasma was not affected by treatments whereas the concentration tended to be lower in hepatic plasma (P = 0.07) for TA treatment. The ammonia concentration in arterial, portal and

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hepatic plasma were not affected by treatments whereas the urea-N concentration in arterial,

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portal and hepatic plasma were lower (P ≤ 0.03) for TA (Table 4). There was no significant effect of TA on the net flux of glucose, ammonia and urea-N across PDV, liver and total ST.

4. Discussion At a relatively low dose, tannins have shown the potential to decrease the excretion of both methane and labile urinary N, and to improve the MP supply to ruminants whereas, at

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higher doses they may reduce feed intake and digestibility (Makkar, 2003). The dietary concentration of TA in the present study was just slightly below the lowest dose, 8.6 g/kg DM intake, reported by Grainger et al. (2009) at which feed intake and digestibility in dairy cows were negatively impacted, but no negative effect of TA on these variables was observed in the present study. Tannins may also change the rumen bacterial metabolism so that a higher proportion of available nutrients are channeled to microbial mass synthesis and a lower proportion is converted to volatile fatty acids or methane (Makkar, 2003; Carulla et al., 2005).

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However, in the present study, the EMPS was not significantly affected by TA, which is in agreement with previous studies with sheep (Komolong et al., 2001) or cattle (Mezzomo et al., 2011) supplemented with quebracho tannin. In turn, as observed by Carulla et al. (2005),

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TA decreased the N compounds digestibility and the urinary urea N excretion. Environmentally, this is relevant because the labile urinary N shows high potential to increase

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ammonia and nitrous oxide emissions into the atmosphere (Misselbrook et al., 2005).

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A major objective of the present study was to evaluate the impact of TA on the net flux of metabolites across ST of sheep, focused particularly on ammonia and urea N, based on the assumption that the TA should decrease the ruminal degradation of feed N compounds

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and, consequently, decrease both the net PDV appearance of ammonia and the urea synthesis across the liver. Although the absolute mean values have followed these expectations, the

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differences between treatments were not consistent as to reach statistical significance. As

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average of both treatments in the present study, the net portal appearance of ammonia represented roughly 58% of apparently digested N, a value similar to that (i.e. 64%) reported by Martineau et al (2009) from a meta-analysis study. As summarized by Milano et al. (2000), hepatic ureagenesis depends on the coordinated supply of N to the ornithine cycle from two different precursors: mitochondrial ammonia and cytosolic aspartate. If free amino acids are the N-donor to aspartate via transamination reactions with glutamate then the ratio ammonia

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removal:urea-N production across the liver should be 0.50 or even lower. In our study, the ratio ammonia removal:urea-N release by liver was 0.42 indicating that free amino acids contributed partially with both amino groups for urea synthesis. The removal of urea-N by PDV was on average 60% of the urea-N produced across the liver, a value closed to that reported by Goetsh and Patil (1997) who compiled data of studies with sheep fed forage-based diets. By extrapolating to a 24 hours period, removal of urea-N by PDV represented on average 67% of the ingested N and 103% of the apparently

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digested N, values within those reported in literature (Huntington and Archibeque, 1999; Milano et al., 2000; Lapierre and Lobley, 2001; Stefanello et al., 2018). The urea produced by the liver is either excreted in urine or returned to the gut through saliva or transfer across the

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PDV and the difference between liver urea synthesis and urinary excretion represents gut entry rate (GER). The difference between total urea GER and transfer across the PDV should

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estimate saliva contribution to urea GER. By extrapolating to a 24-hour period, 43% of urea-

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N produced by the liver was excreted in urine and thus, PDV transference plus urinary excretion accounted for 103% of liver urea synthesis indicating any contribution of saliva to urea GER in sheep of the present study, what is biologically unlikely. In fact, this contribution

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would be relatively small: if one assumes a saliva production by a sheep of about 10 L/day (Guilloteau et al., 1995) and a urea concentration of 3 mmol/L (Piccione et al., 2006), the total

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urea-N excreted in saliva would be 60 mmol or 0.84 g/day, representing only 2.4% of the

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urea-N produced across the liver. These results indicate that in such a case, approximately 96% of the urea GER would be gone through the PDV whereas only 4% would occur through saliva. In a similar experiment with sheep Kraft et al. (2011) also reported values of urea PDV transference plus urinary excretion representing from 103 to 110% of liver urea synthesis. In turn, 12 to 18% of the urea produced by liver was estimated to be excreted in saliva of steers fed a high concentrate diet or alfalfa, respectively (Huntington, 1989). There is not clear

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explanation but, however, methodological constraints may be associated to the unexpected and/or discrepant results described above. For example, blood flow measurements were performed in all studies only during a few hours a day and values of urea net flux were extrapolated to a 24 hours period assuming a steady-state condition throughout this time, what is unlikely. Moreover, urinary N excretion is usually broadly variable throughout the days as at least seven consecutive days of total urine collection was recommended by Farenzena et al. (2017) to obtain reliable values of urinary N excretion in sheep trials. Six days of total urine

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collection were used in the present and in Kraft et al. (2011) studies, and only two days in the study with steers (Huntington, 1989).

Tannins have shown the potential to change the profile of volatile fatty acid produced

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by rumen bacteria increasing the propionate proportion (Batha et al., 2009; Hassanat and Benchaar, 2013; Yang et al., 2017), the major glucose precursor in the liver of ruminants

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(Brockman, 2005). The digestible OM intake was not affected by treatments and, thus, it

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would be expected increased production of glucose across the liver of sheep receiving TA. However, any significant impact of TA on glucose net flux across liver or ST was observed in the present study. Moreover, in agreement with literature, on average 0.31 of glucose

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5. Conclusion

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produced by liver was utilized by PDV of sheep (Huntington, 1999).

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The dietary inclusion of tannin extract from Acacia meanrsii at a rate of 7.7 g/kg DM decreased the urinary excretion of urea with, however, no impact on ammonia, urea or glucose net flux across ST.

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AUTHOR CONTRIBUTION

Ref: Manuscript entitled: “Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep”, submitted for publication in the

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Animal Feed Science and Technology journal.

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Tiago Orlandi performed trial conduction throughout all steps; Simone stefanello performed surgical preparation and sampling collection; Mariana Mezzomo performed sampling collection and laboratory analysis, Claudio Pozo performed writing and editing and Gilberto Vilmar Kozloski performed conceptualization, surgical preparation, sampling and data collection, data analysis, writing and editing.

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Conflict of interest

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We are submitting the paper “Impact of a tannin extract on digestibility and net flux of metabolites across splanchnic tissues of sheep”, for publication in Animal Feed

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Science and Technology. There is not any financial or personal conflict of interest

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associated to this manuscript.

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Acknowledgements

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) for scholarship support.

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Disclosure statement No potential conflict of interest was reported by the authors.

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Table 1. Chemical composition of experimental feeds. Item

Oat-ryegrass hay

Concentrate

874

875

Organic matter

921

859

Crude protein

87

Neutral detergent fibre

708

Acid detergent fibre

424

Non-fibre carbohydrates

142

Dry matter (g/kg)

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Ether extract Acid detergent lignin

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N fractions (g/kg of N): Soluble N

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Neutral detergent insoluble N

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Acid detergent insoluble N

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Composition (g/kg dry matter):

185 208 85

451 37

58

5

271

35

310

107

108

125

20

Table 2. Intake and digestibility by sheep fed oat-ryegrass hay plus concentrate without (Control) or with 20 g/kg of Acacia mearnsii bark extract (Tannin). Treatments Item

s.e.m.†

P-value

Control

Tannin

Hay

941

956

23.4

0.89

Concentrate

626

598

15.1

0.95

Total

1567

1554

34.0

0.93

Organic matter (OM)

1401

1398

Neutral detergent fibre (NDF)

805

773

Digestible OM

834

0.55

832

17.5

0.92

0.60

0.010

0.95

0.47

0.45

0.014

0.36

0.70

0.70

0.010

0.98

0.60

NDF OM true digestibility

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Standard error of means where n=5 per treatment.

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†

36.0

lP

OM

0.95

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Apparent digestibility

35.4

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Intake (g/day):

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Dry matter intake (g/day):

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Table 3. Intake, digestion and excretion of N compounds, rumen microbial N supply and efficiency (EMPS) in sheep fed oat-ryegrass hay plus concentrate without (Control) or with 20 g/kg of Acacia mearnsii bark extract (Tannin). Treatments Item

s.e.m.†

P-value

Control

Tannin

32.5

31.1

0.72

0.20

Total N

10.6

11.6

0.52

0.21

Neutral insoluble detergent N

3.0

3.3

Apparent

0.67

0.63

True

0.91

Intake (g/day)

Microbial N (g/day)

13.7

1.03

0.05

18.3

16.1

0.75

0.16

15.8

14.2

0.73

0.16

18.7

16.8

1.05

0.15

g rumen microbial N/kg digestible OM intake

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‡

0.02

Standard error of means where n=5 per treatment.

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†

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EMPS‡

0.04

0.003

17.1

Purine derivatives (mmol/day)

0.014

0.89

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Urea N (g/day)

0.96

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Urinary excretion:

0.05

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Digestibility:

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Faecal excretion (g/day):

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Table 4. Metabolites concentration (mmol/L) in plasma of sheep fed oat-ryegrass hay plus concentrate without (Control) or with 20 g/kg of Acacia mearnsii bark extract (Tannin). Treatments Item

s.e.m.†

P-value

Control

Tannin

Glucose

4.1

3.9

0.19

0.19

Ammonia

0.08

0.08

0.058

0.95

Urea N

14.2

13.3

0.62

0.01

Glucose

4.0

3.8

Ammonia

0.26

0.27

Urea N

14.1

0.07

0.07

0.07

0.050

0.92

14.5

13.4

0.43

0.03

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0.18

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0.15

4.0

Standard error of means where n=5 per treatment.

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†

13.1

lP

Urea N

0.153

0.02

4.3

Ammonia

0.33

0.65

Hepatic: Glucose

0.18

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Portal:

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Arterial:

23

Table 5. Portal and splanchnic plasma flow, and net flux of metabolites across the portaldrained viscera (PDV), liver and total splanchnic tissues (ST) of sheep fed oat-ryegrass hay plus concentrate without (Control) or with 20 g/kg of Acacia mearnsii bark extract (Tannin). Treatments

s.e.m.†

P-value

8.1

0.18

Control

Tannin

Portal

152

144

Splanchnic

167

160

11.6

0.45

Glucose

-16.3

-11.5

2.40

0.16

Ammonia N

39.8

32.4

6.30

0.25

Urea N

-69.3

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Item

-58.2

16.71

0.29

47.9

44.2

4.32

0.46

-44.3

-44.1

9.40

0.98

112.5

97.2

27.11

0.29

33.9

33.5

4.31

0.80

-4.4

-11.5

8.30

0.87

43.1

39.4

16.63

0.31

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Plasma flow (L/h):

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PDV net flux (mmol/h):

lP

Liver net flux (mmol/h): Glucose Ammonia N

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Urea N

Glucose

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ST net flux (mmol/h):

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Ammonia N Urea N

†

Standard error of means where n=5 per treatment.