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Effects of Feeding Oxidized Fat With or Without Dietary Antioxidants on Nutrient Digestibility, Microbial Nitrogen, and Fatty Acid Metabolism

      Abstract

      A dual-effluent continuous culture system was used to investigate, in a 2 × 2 factorial design, the effect of feeding a fresh (FF) or oxidized (OF) blend of unsaturated fats (33% fish oil, 33% corn oil, 26% soybean oil, and 7% inedible tallow) when supplemented with a blend of antioxidants (AO; Agrado Plus, Novus International Inc.; Agrado Plus is a trademark of Novus International Inc. and is registered in the United States and other countries) on nutrient digestibility, bacterial protein synthesis, and fatty acid metabolism. Twice a day for 10 d, 12 fermenters were fed a diet that consisted of 52% forage and 48% grain mixture that contained 3% (dry matter basis) FF or OF, with or without AO. The OF contained a higher concentration of peroxides (215 vs. 3.5 mEq/kg), and a lower concentration of unsaturated fatty acids than the FF. Feeding OF reduced nitrogen digestibility, microbial nitrogen yield, and efficiency (expressed as kilograms of dry matter digested) and increased the outflow of saturated fatty acids in the effluent when compared with feeding FF. Adding AO improved total carbohydrate, neutral, and acid detergent fiber digestibilities and the amount of digested feed nitrogen converted to microbial nitrogen across the types of fats. From this study, we concluded that feeding OF reduced microbial nitrogen and increased the outflow of saturated fatty acids. Feeding AO improved fiber digestibility by rumen microorganisms, regardless of the type of fat.

      Key words

      Introduction

      In the body of an animal there is a natural balance between the formation of free radicals during the normal metabolism of the cells and the endogenous antioxidant capacity of the animal that prevents free radicals from accumulating and harming the cells. However, the levels of free radicals can exceed the antioxidant capacity of the animal, leading to oxidative stress (
      • Miller J.K.
      • Brezeinska-Slebodizinska E.
      Oxidative stress, antioxidants, and animal function.
      ;
      • Weiss W.P.
      Requirements of fat-soluble vitamins for dairy cows: A review.
      ). High-producing dairy cows are prone to oxidative stress, and the situation can be exacerbated under certain environmental, physiological, and dietary conditions (
      • Bernabucci U.
      • Ronchi B.
      • Lacetera N.
      • Nardone A.
      Markers of oxidative status in plasma and erythrocytes of transition cows during hot season.
      , 
      • Bernabucci U.
      • Ronchi B.
      • Nardone A.
      Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows.
      ;
      • Castillo C.
      • Hernandez J.
      • Bravo A.
      • Lopez-Alonso M.
      • Pereira V.
      • Benedito J.L.
      Oxidative status during late pregnancy and early lactation in dairy cows.
      ;
      • Lohrke B.
      • Viergutz T.
      • Kanitz W.
      • Losand B.
      • Weiss D.G.
      • Simko M.
      Short communication: Hydroperoxides in circulating lipids from dairy cows: Implications for bioactivity of endogenous-oxidized lipids.
      ). Generation of free radicals during peroxidation of essential fatty acids in the lipid membranes can damage cells and can impair the production and health status of the animal (
      • Miller J.K.
      • Brezeinska-Slebodizinska E.
      Oxidative stress, antioxidants, and animal function.
      ). The impact of free radicals and oxidative stress is unknown in ruminal microorganisms. Dietary lipids such as supplemental fat, oilseeds, and distillers grains, if not stabilized, can be significant contributors to the load of free radicals in the animal (
      • Andrews J.
      • Vazquez-Anon M.
      • Bowman G.
      Fat stability and preservation of fatty acids with AGRADO® antioxidant in feed ingredients used in ruminant rations.
      ). Decreased performance, increased gut turnover, and a compromised immune response have been reported in production animals fed oxidized fat (OF;
      • Cabel M.C.
      • Waldroup P.W.
      • Shermer W.D.
      • Calabotta D.F.
      Effects of ethoxyquin feed preservative and peroxide level on broiler performance.
      ;
      • Dibner J.J.
      • Kitchell M.L.
      • Atwell C.A.
      • Ivey F.J.
      The effect of dietary ingredients and age on the microscopic structure of the gastrointestinal tract in poultry.
      ). Inclusion of dietary antioxidants (AO) ameliorates these negative effects by scavenging peroxides and reducing the peroxidation of fatty acids (
      • Frankel E.N.
      Antioxidants. Lipid Oxidation.
      ).
      In a compilation of feedlot trials, feeding 150 mg/kg of ethoxyquin in the form of Agrado Plus (Novus International Inc., St. Louis, MO; Agrado Plus is registered in the United States and other countries) was found to reduce the incidence of liver abscess and improve BW gain across studies (
      • Vázquez-Añón M.
      • Scott F.
      • Miller B.
      • Peters T.
      Evaluation of the effects of dietary antioxidant (Agrado) on feedlot performance and carcass characteristics.
      , 
      • Vázquez-Añón M.
      • Scott F.
      • Miller B.
      • Peters T.
      Evaluation of the incidence of liver abscess in feedlot cattle fed a dietary antioxidant (AGRADO).
      ). In dairy cattle, feeding 50 mg/kg of ethoxyquin (
      • Smith J.L.
      • Sheffield L.G.
      • Saylor D.
      Impact of ethoxyquin on productivity of dairy cattle.
      ) improved milk yield and efficiency as well as OM digestibility, suggesting an anti-oxidant effect on rumen fermentation. The objective of this study was to evaluate the effect of feeding fresh fat (FF) or OF with or without dietary AO on nutrient digestibility, fatty acid metabolism, and bacterial protein synthesis by using continuous culture fermenters.

      Materials and Methods

      A lactating dairy ration was formulated to support 40 kg/d of milk production with a predicted DMI of 24 kg/d. Dietary ingredients and nutrient composition are shown in Table 1 and 2. The diet consisted of 52% forage and 48% concentrate mixture that contained 3% experimental fat on a DM basis. The experimental fat consisted of a blend of nonstabilized unsaturated fats that contained 33% fish oil, 33% corn oil, 26% soybean oil, and 7% inedible tallow. Half of the experimental fat was oxidized (OF) by bubbling air through the fat at 92°C for 24 h to achieve a peroxide value of 215 mEq/kg (method Cd 12-57;
      AOCS
      Official Methods and Recommended Practices of the AOCS.
      ). The study consisted of 4 treatments: 1) fresh nonoxidized fat (FF) added to the diet at 3%; 2) FF added to the diet at 3% plus 200 mg/kg of dietary AO (FF + AO); 3) OF added to the diet at 3%; 4) OF added to the diet at 3% plus 200 mg/kg of dietary AO (OF + AO). The dietary AO consisted of a liquid blend of ethoxyquin and tertiary-butyl-hydroquinone and was added to the experimental fat just prior to mixing of the diets at a rate of 200 mg/kg of DM of the final diet. The diets were stored at 0°C between feedings and allowed to come to room temperature prior to feeding. Peroxide values and changes in fatty acid profile were used to assess the quality and stability of the experimental fats prior to adding the AO.
      Table 1Ingredient and nutrient composition of the basal diet
      ItemAmount, % (DM basis)
      Alfalfa balage4.56
      Corn silage28.12
      Mixed grass hay19
      Soybean meal (44%)15.58
      Corn gluten meal0.66
      Soyhulls3.8
      Flaked barley5.22
      Steam-flaked corn17.48
      Fresh fat
      Fresh fat was added to the fresh fat (FF) and fresh fat plus antioxidant (FF + AO) treatment diets.
      0 or 3
      Oxidized fat
      Oxidized fat was added to the oxidized fat (OF) and oxidized fat plus antioxidant (OF + AO) treatment diets.
      0 or 3
      Dietary antioxidant
      Dietary antioxidant was added to FF + AO and OF + AO treatment diets in the form of Agrado Plus (Novus International, St. Louis, MO).
      0 or 0.02
      Urea0.66
      Magnesium oxide0.01
      Dicalcium phosphate0.28
      Sodium bicarbonate0.97
      Limestone0.28
      Trace minerals and salts0.19
      Vitamin A, D, E mix0.11
      Vitamin E0.06
      CP18.6
      Soluble protein, % of CP34.4
      NDF28.3
      ADF18.1
      NSC
      Includes starch + sugar.
      31.6
      Starch25.7
      Sugar5.9
      Ether extract5.5
      Ash6.1
      NFC
      Calculated NFC.
      41.7
      1 Fresh fat was added to the fresh fat (FF) and fresh fat plus antioxidant (FF + AO) treatment diets.
      2 Oxidized fat was added to the oxidized fat (OF) and oxidized fat plus antioxidant (OF + AO) treatment diets.
      3 Dietary antioxidant was added to FF + AO and OF + AO treatment diets in the form of Agrado Plus (Novus International, St. Louis, MO).
      4 Includes starch + sugar.
      5 Calculated NFC.
      Table 2Effect of feeding fresh (FF) or oxidized fat (OF) in the presence and absence of antioxidants (AO) on the daily amounts of fatty acids fed to continuous cultures
      Item, mg/dTreatment
      Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      FFOFFF + AOOF + AO
      12:04444
      14:055899352
      15:08888
      16:0675632660690
      16:1106104111114
      18:0152141146155
      18:1851793836865
      18:21,7391,5911,7251,703
      18:3229209227224
      20:024222425
      20:49798
      21:020152017
      22:018161619
      20:51248812599
      24:0127207
      22:670446951
      Other fatty acids405342340440
      Unsaturated fatty acids
      Unsaturated fatty acids included 14:1, 16:1, 18:1-isomers, 18:2, 18:3, 22:1, 20:5, and 22:6.
      3,1362,8413,1073,072
      Saturated fatty acids
      Saturated fatty acids included 12:0, 14:0, 15:0, 16:0, 18:0, 20:0, 21:0, 22:0, and 24:0.
      1,0139701,0341,020
      1 Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      2 Unsaturated fatty acids included 14:1, 16:1, 18:1-isomers, 18:2, 18:3, 22:1, 20:5, and 22:6.
      3 Saturated fatty acids included 12:0, 14:0, 15:0, 16:0, 18:0, 20:0, 21:0, 22:0, and 24:0.

      Continuous Culture System

      A 12-unit dual-effluent continuous culture system, as described by
      • Hoover W.H.
      • Crooker B.A.
      • Sniffen C.J.
      Effects of differential solid-liquid removal rates on protozoa numbers in continuous cultures of rumen contents.
      , was used in this study. Ruminal inoculum was obtained from 2 rumen-cannulated lactating Holstein cows. Ruminal inoculum was pooled before inoculating the 1,164-mL fermenters. Fermenters were fed the experimental diets (ground to pass a 4-mm sieve) for 10 d in 2 equal feedings at 12-h intervals (Table 1). All treatments were fermented in triplicate for 10 d in continuous cultures. Continuous culture conditions were defined to represent average in vivo flow rates as follows: liquid dilution rate, 12%/h; solids retention time, 24 h; feed intake, 100 g of DM/d; fermentation temperature, 39°C. The pH was recorded at 0.5-h intervals. The dual-flow continuous culture technique, under the conditions defined herein, was established by
      • Hannah S.M.
      • Stern M.D.
      • Ehle F.R.
      Evaluation of a dual flow continuous culture system for estimating bacterial fermentation in vivo of mixed diets containing various soya bean products.
      as a valid method of simulating in vivo rumen fermentation. It represents a practical and appropriate alternative to in vivo methods.
      The artificial saliva of
      • Weller R.A.
      • Pilgrim A.F.
      Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system.
      was continuously infused at a rate to provide the 12%/h liquid flow for fermentation periods of 10 d. The first 7 d were for equilibration. During the last 3 d, the effluents were collected and a 1-L sample was composited and saved for analysis. After the effluent was collected on d 10, the contents of the fermenters were stirred vigorously prior to being allowed to settle to dislodge some of the solids associated with microbes. The upper fluid layer was used for collection of microbes as described by
      • Lean I.J.
      • Miller Webster T.K.
      • Hoover W.
      • Chalupa W.
      • Sniffen C.J.
      • Evans E.
      • Block E.
      • Rabiee A.R.
      Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters.
      .

      Chemical Analysis

      Feed DM was determined by oven-drying at 100°C for 24 h. Effluent DM was determined by centrifuging a 34- to 40-g sample of effluent at 30,000 × g for 45 min as described by
      • Lean I.J.
      • Miller Webster T.K.
      • Hoover W.
      • Chalupa W.
      • Sniffen C.J.
      • Evans E.
      • Block E.
      • Rabiee A.R.
      Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters.
      . For digestibility determination, DM digested and OM digested were corrected for microbial DM and OM. Determination of NDF and ADF contents in the feed was as described by

      Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS, USDA, Washington, DC.

      and
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      , and in continuous culture effluents as described by
      • Crawford Jr., R.J.
      • Shriver B.J.
      • Varga G.A.
      • Hoover W.H.
      Buffer requirements formaintenance of pH during fermentation of individual feeds in continuous culture.
      . Total nitrogen in feed and effluents, and bacterial, ammonia, and ether extraction were determined according to
      AOAC
      Official Methods of Analysis.
      . Analysis of VFA was performed in accordance with the gas chromatographic separation procedure described by
      • Lean I.J.
      • Miller Webster T.K.
      • Hoover W.
      • Chalupa W.
      • Sniffen C.J.
      • Evans E.
      • Block E.
      • Rabiee A.R.
      Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters.
      . Effluent and bacterial concentrations of purines were determined by the procedures of
      • Zinn R.A.
      • Owens F.N.
      A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis.
      to partition effluent nitrogen flow into microbial and dietary fractions and to calculate DM and OM. The sugars and starches of the feeds and effluents were determined by the procedure of

      Smith, D. 1969. Removing and analyzing total nonstructural carbohydrates from plant tissue. Wisc. Agric. Exp. Stn. Res. Rep. 41. Univ. Wisconsin, Madison.

      , except that ferricyanide was used to detect reducing sugars.
      Fermenter outflow samples were freeze-dried and converted to methyl esters in sodium methoxide-methanolic HCl as described by
      • Kramer J.K.G.
      • Fellner V.
      • Dugan M.E.R.
      • Sauer F.D.
      • Mossoba M.M.
      • Yurawecz M.P.
      Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids.
      . Analysis of fermenter outflow fatty acids was done on a high-performance gas chromatograph (HP5890A GC, Agilent Technologies, Inc., Santa Clara, CA) equipped with a flame-ionization detector and a 30 m × 0.25 mm (0.2 μm film) Supelco 2380 fused-silica capillary column. The injector and detector temperatures were held at 250 and 260°C, respectively. The carrier gas was He (20 cm/s) with an inlet pressure of 104 kPa. The column temperature was programmed for 140°C for 3 min, then increased to 220°C at 2°C/min, and held at 220°C for 2 min. Peaks were quantified by comparison with an internal standard (17:0), which was added prior to methylation.

      Statistical Analysis

      Data were analyzed as a completely randomized design by ANOVA with the GLM procedure of SAS (
      SAS Institute
      SAS User's Guide: Statistics.
      ). Main effects of type of fat and presence of dietary AO were tested as a 2 × 2 factorial arrangement. Significance differences were declared at P < 0.05 and P > 0.05, but P ≤ 0.10 were considered trends.

      Results

      Nutrient Digestibility

      Digestion coefficients are shown in Table 3. Digestion of CP was reduced (P = 0.01) by OF compared with FF. The rates of CP digestibility observed in this study were high but within the range reported for other continuous culture fermenters fed a variety of diets (
      • Bargo F.
      • Varga G.
      • Muller L.D.
      • Kolver E.S.
      Pasture intake and substitution rate effects on nutrient digestion and nitrogen metabolism during continuous culture fermentation.
      ;
      • Lean I.J.
      • Miller Webster T.K.
      • Hoover W.
      • Chalupa W.
      • Sniffen C.J.
      • Evans E.
      • Block E.
      • Rabiee A.R.
      Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters.
      ). One possible explanation for the high digestibility may lie in the method used to arrive at microbial nitrogen flow. Altering feed conditions increased the variability of RNA concentrations within the mixed rumen microbial population (
      • Smith R.H.
      • McAllan A.B.
      Some factors influencing the chemical composition of mixed rumen bacterial.
      ). Such variability in the RNA nitrogen content of the bacterial cells could have resulted in an overestimation of bacterial nitrogen flow and in inflated protein digestion. Digestion of NDF was not affected by fat source, whereas AO significantly increased NDF digestion (P = 0.02) when added to either the FF or the OF diet. Digestion of ADF was numerically improved in the OF diet (P = 0.08) compared with that of the FF diet and, as with NDF digestion, addition of AO increased ADF digestion (P = 0.04) when added to either fat source. Digestion of DM, OM, and NSC was not affected by the treatments. However, digestion of total carbohydrates (in g/d) was increased (P = 0.05) by AO regardless of fat source, mostly by improving NDF digestibility. Production rates (in mmol/d) and molar ratios of VFA, along with average fermentation pH, were not affected by fat source in the presence or absence of AO, with the exception of butyrate (Table 4). Fermenters fed OF diets had higher butyrate production (53 vs. 59 ± 2.01; P = 0.02) and molar ratios (13.8 vs. 14.8 ± 0.46; P = 0.02) than those fed FF diets.
      Table 3Effect on digestion coefficients for DM, OM, CP, fiber, and NSC of feeding fresh (FF) or oxidized fat (OF) in the presence and absence of antioxidant (AO) to continuous culture fermenters
      Digestion, %Treatment
      Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      P-value
      FFOFFF + AOOF + AOSEFatAOFat × AO
      DM67.469.869.570.92.000.490.560.86
      OM61.061.865.063.11.770.760.170.47
      CP97.987.598.593.02.350.010.220.33
      NDF35.740.14645.22.640.510.020.35
      ADF44.050.050.954.02.230.080.040.53
      NSC
      Includes sugar and starch.
      69.570.769.469.01.020.720.430.45
      Total carbohydrates,
      Grams of NDF + grams of NSC digested per day.
      g/d
      32.233.534.834.90.890.470.050.534
      1 Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      2 Includes sugar and starch.
      3 Grams of NDF + grams of NSC digested per day.
      Table 4Effects of treatments on volatile fatty acid (VFA) production, molar ratios, and average daily pH in the fermenter
      ItemTreatment
      Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      P-value
      FFOFFF + AOOF + AOSEFatAOFat × AO
      Total VFA, mmol/d3853973963867.920.870.990.22
      molar %
      Acetic acid53.35252.4540.930.850.570.17
      Propionic acid28.428.829.126.91.170.460.620.3
      Isobutyric acid0.790.740.690.740.030.990.140.16
      Butyric acid13.814.813.2150.460.020.610.39
      mmol/d
      Acetic acid2052062072094.10.760.610.99
      Propionic acid1091141151046.10.620.70.22
      Isobutyric acid3.132.72.90.080.930.040.19
      Butyric acid535952582.010.020.660.97
      Average pH6.296.146.156.180.050.270.320.1
      1 Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.

      Nitrogen Metabolism

      Table 5 shows the effects of the treatments on nitrogen partitioning and on microbial efficiency and composition. Microbial nitrogen yield was less for the OF than the FF diet (P = 0.03), but bypass nitrogen was greater (P = 0.01) for the OF diet, due in part to the lower protein digestibility. This resulted in higher NAN for the OF treatment (P = 0.01). The addition of AO caused a numerical (P = 0.09) improvement in microbial nitrogen in both fat treatments. As a result, the FF + AO treatment produced more microbial nitrogen than any other treatment. Addition of AO numerically reduced ammonia levels (P = 0.06) for both fat sources.
      Table 5Effect of feeding fresh (FF) or oxidized fat (OF) in the presence or absence of antioxidants (AO) to continuous culture fermenters on nitrogen partitioning, microbial growth, efficiency, and composition
      ItemTreatment
      Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      P-value
      FFOFFF + AOOF + AOSEFatAOFat × AO
      CP digested, %97.987.598.5932.350.010.220.34
      NAN, g/d2.592.692.682.690.010.010.010.10
      Ammonia N, mg/dL18.9317.9817.5816.620.610.160.060.99
      Bypass N, g/d0.070.410.050.230.080.010.210.31
      Microbial N, g/d2.522.282.632.470.080.030.090.66
      Efficiencies
       Microbial N, g/kg of DM digested37.432.738.134.81.530.030.390.63
       Microbial N, g/kg of OM digested43.839.243.141.81.570.100.540.32
       Microbial N, g/kg of total carbohydrate digested78.468.275.7713.660.080.990.48
       Feed N,
      Digested feed N converted to microbial N (%).
      %
      79.778.481.3810.670.260.010.49
       Total VFA,
      VFA = volatile fatty acids.
      mol/kg of carbohydrate digested
      12.0111.8811.3811.080.330.530.060.8
       Total VFA, mol/kg of microbial N1531751501586.380.050.160.3
      Bacterial composition
       Nitrogen, %9.899.709.689.480.080.050.020.81
       Ash, %8.7610.009.0313.711.810.130.330.40
       RNA-N, % of total N10.311.0010.0910.470.360.240.490.81
      1 Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      2 Digested feed N converted to microbial N (%).
      3 VFA = volatile fatty acids.
      Because of the low carbohydrate digested (in g/d) and the overall high yield of microbial nitrogen, efficiencies were high across all treatments. Compared with OF, however, FF resulted in higher microbial nitrogen per unit of digested DM (P = 0.03), with numerical improvements in OM (P = 0.1) and total carbohydrates (P = 0.08). The significant improvement in microbial nitrogen per unit of digested DM with FF diets was the result of a significant improvement in microbial nitrogen yield, with a concomitant slight numerical reduction in DM digested. Incorporation of feed nitrogen into microbial nitrogen was greater (P = 0.01) in the presence of AO, regardless of fat source. The nitrogen content of the microbes was reduced with the AO (P = 0.02) and OF (P = 0.02) diets, suggesting changes in the microbial population when feeding AO and OF (Table 5).

      Fatty Acid Metabolism

      Oxidation of the experimental fat by bubbling air during heating oxidized the fat, as reflected by the higher levels of peroxides in the OF compared with the FF (215 vs. 3.5 mEq of peroxides/kg of fat) and the lower supply of unsaturated fatty acids in the OF and OF + AO final diets (Table 2).
      The outflow of fatty acids in the effluent varied with AO and type of fat as described in Table 6. Feeding FF diets resulted in lower outflows of 16:0 (P = 0.01), 18:0 (P = 0.01), and 24:0 (P = 0.01), with a trend for 22:0 (P = 0.06), and resulted in higher outflows of 20:5 (P = 0.04) and other fatty acids (P = 0.01), with a trend for trans-18:1 (P = 0.07), compared with when OF diets were fed. In general, feeding FF diets reduced the out-flow of saturated fatty acids (P = 0.01), with a trend for improvement in the outflow of unsaturated fatty acids (P = 0.09), when compared with OF diets. The presence of AO in the diets tended to reduce the outflow of 18:3 (P = 0.08) and increase that of 24:0 (P = 0.02) across the types of fats as described in Table 6. A significant AO × type of fat interaction was observed only for 22:6. The mechanism by which AO reduced 22:6 in the OF diets, but not in the FF diets, is unclear and might require further research.
      Table 6Effect of feeding fresh (FF) or oxidized fat (OF) in the presence or absence of antioxidant (AO) to continuous cultures of mixed ruminal microbes on the daily outflow of fatty acids in the effluent
      Item, mg/dTreatment
      Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      P-value
      FFOFFF + AOOF + AOSEFatAOFat × AO
      16:0677705665726130.010.770.25
      16:16053615860.380.620.81
      18:0193299199273260.010.710.56
      Trans-18:11,4081,2351,4481,367610.070.190.47
      Cis-18:1380395394427170.210.210.61
      18:2340340329354200.540.940.56
      20:02020192110.240.340.18
      18:36364515260.790.080.97
      Cis-9, trans-11 18:2 conjugated linoleic acid1814141040.360.260.98
      22:01922212310.060.140.63
      20:54022372540.010.900.48
      24:0161717200.50.010.020.08
      22:62634321540.260.100.01
      Other fatty acids846769802811140.050.930.02
      Unsaturated fatty acids
      Unsaturated fatty acids included 14:1, 16:1, isomers-18:1, 18:2, 18:3, CLA, 22:1, 20:5, and 22:6.
      1,9641,7741,9821,890730.090.380.52
      Saturated fatty acids
      Saturated fatty acids included 12:0, 14:0, 15:0, 16:0, 18:0, 20:0, 21:0, 22:0, and 24:0.
      9881,1859831,130270.010.310.38
      1 Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.
      2 Unsaturated fatty acids included 14:1, 16:1, isomers-18:1, 18:2, 18:3, CLA, 22:1, 20:5, and 22:6.
      3 Saturated fatty acids included 12:0, 14:0, 15:0, 16:0, 18:0, 20:0, 21:0, 22:0, and 24:0.

      Discussion

      The microorganisms present in the continuous culture fermenters responded differently to the 2 types of fat. Feeding OF reduced protein digestibility and microbial nitrogen yield, content, and efficiency (expressed as DM digested) when compared with feeding FF. In prokaryotic and eukaryotic organisms, oxidative stress occurs when there is an undesirable accumulation of free radicals in the cells, resulting in disrupted cellular membrane integrity, DNA replication, and life span of the cells (
      • Scandalios J.G.
      Oxidative stress: Molecular perception and transduction of signals triggering antioxidant gene defenses.
      ). Ruminal microorganisms are predominantly anaerobes, with a less-developed antioxidant capacity than aerobe organisms (
      • Brioukhanov A.L.
      • Netrusov A.I.
      Catalase and superoxide dismutase: Distribution, properties, and physiological role in cells of strict anaerobes.
      ). It is possible that the amount of peroxides in the OF diet would be enough to create oxidative stress in the ruminal microorganism, compromising their proteolytic activity and optimal growth.
      Changes in the output of fatty acids in the effluent with type of fat might reflect changes in microorganism fatty acid metabolism. Feeding OF increased the out-flow of saturated fatty acids such as 16:0 and 18:0 and reduced the outflow of unsaturated FA such as 20:5, with a trend for trans-18:1, compared with feeding FF. A lower outflow of trans-18:1 and a higher outflow of 18:0 would imply more complete conversion of these fatty acids to 18:0 when feeding OF diets. The higher biohydrogenation with OF diets was further substantiated by the higher outflow of saturated fatty acids. Changes in bacterial composition, lower CP digestibility, lower microbial nitrogen yield, higher concentration of butyrate, lower trans 18:1 content, and higher 16:0 and 18:0 contents in the outflow from fermenters fed OF would reflect changes in the microbial population toward butyrate-producing microorganisms with biohydrogenation activity and low proteolytic activity. It is possible that those rumen microorganisms capable of taking the biohydrogenation process to completion were less susceptible to OF.
      The presence of AO in the diet was effective in both the FF and OF diets, causing several positive responses. Antioxidants mostly improved fiber and carbohydrate digestibilities, with modest improvements in microbial nitrogen yield or changes in VFA and pH, regardless of the degree of oxidation of the fat. Improvements in fiber digestibility with no alteration in VFA or pH with AO supplementation would suggest microbial metabolic changes favoring cellulolytic activity. According to
      • Hino T.
      • Andoh N.
      • Ohgi H.
      Effects of β-carotene and α-tocopherol on rumen bacteria in the utilization of long-chain fatty acids and cellulose.
      , feeding AO such as α-tocopherol and β-carotene alleviates the negative effect of a high level of unsaturated fat supplementation on microbial growth, cellulose digestion, and fatty acid utilization by the rumen microflora, similarly to the present study. The mechanism by which AO compounds ameliorate the toxic effect of excessive unsaturated fatty acids has not been well depicted and might vary with the AO compound and type of fat. The AO fed in the current study are highly lipophylic quinoline compounds. Under an aqueous solution such as rumen fluid, these compounds stay within the lipid moieties, protecting the fatty acids from further peroxidation (
      • Frankel E.N.
      Antioxidants. Lipid Oxidation.
      ) and perhaps reducing the toxic effect of unsaturated fatty acids.
      The potential role of AO in the biohydrogenation process in the rumen cannot be ruled out. In the current study, AO supplementation tended to reduce the out-flow of 18:3, suggesting increased 18:3 biohydrogenation. Recent studies (
      • Bell J.A.
      • Griinari J.M.
      • Kennelly J.J.
      Effect of safflower oil, flaxseed oil, monensin, and vitamin E on concentration of conjugated linoleic acid in bovine milk fat.
      ;
      • Pottier J.
      • Focant M.
      • Debier C.
      • De Buysser G.
      • Goffe C.
      • Mignolet E.
      • Froidmont E.
      • Larondelle Y.
      Effect of dietary vitamin E on rumen biohydrogenation pathways and milk fat depression in dairy cows fed high-fat diets.
      ) have linked supplementation of AO such as tocopherol with a lower concentration of trans-10 18:1 in milk and reduced milk fat depression. In our current study, no changes were observed in conjugated linoleic acid or total trans 18:1 with AO supplementation; however, the concentrations of the different trans isomers were not evaluated. The understanding of the role of AO in the biohydrogenation process requires further research.

      Conclusions

      Oxidized fat reduced microbial protein metabolism, as measured by lower digestion of CP, lower microbial nitrogen yield, and increased biohydrogenation, by increasing the outflow of saturated fatty acids and reducing trans-18:1 when compared with FF. Feeding AO improved the utilization of both OF and FF diets by increasing the fiber and carbohydrate digestibilities and efficiency of use of feed nitrogen for microbial nitrogen. Feeding AO might alleviate the negative effect of feeding unsaturated fatty acids to rumen microbes, regardless of the level of oxidation.

      Acknowledgments

      The fermentation analysis was conducted at the Rumen Profiling Laboratory at West Virginia University. The authors appreciate the collaboration of T. Webster and W. Hoover in conducting the fermenter study and J. Andrews for oxidizing the fat.

      Supplementary data

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