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Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro
Bioresource Technology 101 (2010) 8469–8472
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro N.A. Krueger a, R.C. Anderson a,*, L.O. Tedeschi b, T.R. Callaway a, T.S. Edrington a, D.J. Nisbet a a
United States Department of Agriculture, Agriculture Research Service, Southern Plains Agriculture Research Center, Food and Feed Safety research Unit, 2881 F&B Road, College Station, TX 77845, USA b Department of Animal Sciences, Texas A&M University, 2471 TAMU, College Station, TX 77843-2471, USA
a r t i c l e
i n f o
Article history: Received 4 February 2010 Received in revised form 21 May 2010 Accepted 2 June 2010 Available online 26 June 2010 Keywords: Biohydrogenation Fermentation Fiber digestibility Glycerol
a b s t r a c t Ruminant-derived foods contain high proportions of saturated fats as a result of ruminal biohydrogenation that rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. The objective of this study was to evaluate the effects of glycerol on ruminal free-fatty acid (FFA) production rates and in vitro fermentation kinetics of alfalfa hay. In vitro incubations demonstrated 48% and 77% reductions in rates of FFA accumulation in incubations supplemented with 2% and 20% glycerol as compared to controls. In vitro incubations with alfalfa hay demonstrated that increasing levels of glycerol did not affect NDF digestibility of the hay. Additionally, increasing amounts of glycerol decreased the acetate to propionate ratio in the rumen. These results suggest that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. Published by Elsevier Ltd.
1. Introduction Ruminant-derived foods contain high proportions of saturated fats due to an intense biohydrogenation that occurs in the rumen, saturating and thus limiting the availability of free unsaturated fatty acids for assimilation (Harfoot and Hazelwood, 1987). Strategies to enrich ruminant-derived foods with unsaturated fatty acids are desired as they are considered beneficial for good human health. Lipolysis is pre-requisite for biohydrogenation because saturase enzymes act only on free-fatty acids (FFAs). Lipolysis releases fatty acids from the glycerol backbone of triacylglycerols, galactolipids, or phospholipids via hydrolysis processes that occur in the rumen (Fievez et al., 2007). Despite considerable research on ruminal biohydrogenation, there are only a few reports on the purification and properties of lipase enzymes from rumen bacteria and those have reported almost exclusively on enzymes from Anaerovibrio lipolytica (Henderson, 1970, 1971; Henderson and Hodgkiss, 1973; Hobson and Summers, 1966; Prins et al., 1975). Results from these studies indicated that A. lipolytica may produce two lipases, the more active form being produced extracellularly and the less active form being cell associated (Hobson and Summers, 1966). The lipases exhibit pH optima of 7.4 and activity is inhibited by ZnCl2, HgCl2 and high chloride concentrations (Henderson, 1970, 1971). The production of lipase by A. lipolytica was influenced by the bacterium’s growth rate, with maximal production occurring * Corresponding author. Tel.: +1 979 260 9317; fax: +1 979 260 9332. E-mail address: [email protected] (R.C. Anderson). 0960-8524/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.biortech.2010.06.010
during the logarithmic growth phase, and that it was constitutively produced in A. lipolytica strain L 1741. Other than these reports, little is known regarding factors affecting expression of this activity. Considering that the hydrolysis product, glycerol, can be utilized as a nutrient for the growth of rumen, lipase-producing bacteria such as A. lypolytica (Krueger et al., unpublished data), we hypothesized that excessive concentrations of glycerol may affect lipase activity. Glycerol is a by-product from the production of biodiesel and has been evaluated as a potential feed ingredient for livestock in various stages of production (Cerrate et al., 2006; Donkin and Doane, 2007; Kerley, 2007; Schroder and Sudekum 1999). Research has shown that feeding glycerol up to a rate of 10% of the daily dietary dry matter has no effect on feed intake or performance in finishing beef cattle or lactating dairy cows (Kerley 2007; Schroder and Sudekum 1999). The objectives of this study were to evaluate two levels of glycerol and their potential inhibitory effect against ruminal lipolysis by mixed rumen microbes and to determine the effects of feeding various amounts of glycerol on fermentation kinetics of alfalfa hay.
2. Methods 2.1. Experiment 1: Potential inhibitory effects of glycerol Ruminal contents (fluid and solids) were obtained from a nonlactating cannulated dairy cow receiving a diet consisting of 50% feedlot grain-type ration and 50% dried-distillers grains. Freshly
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collected ruminal contents were gently mixed with a spoon while under CO2 and 5 g were anaerobically (CO2) dispensed into triplicate sets of 18 150 mm straight walled tubes each containing 10% olive oil and one of the three levels of glycerol, 0%, 2%, or 20%. This experiment was conducted twice, with each experimental run conducted on separate days. Tubes were sealed with #1 butyl rubber stoppers and subsequently incubated at 39 °C . After 24 h incubation, the amount of accumulated FFA was measured colorimetrically using the method of Kwon and Rhee (1986) with slight modifications as follows. Tubes were unsealed, samples were acidified with 500 ll of concentrated HCl and 10 ml of isooctane was added. The isooctane/rumen content mixture was vortexed for 60 s to extract FFA. The isooctane layer was pulled off and placed into a clean 13 100 mm screw top glass tube. Samples were dried to total dryness under nitrogen gas. Dried samples were re-suspended in 5 ml of isooctane. A 200 ll subsample was placed into a clean 16 150 mm borosilicate glass tube containing 1800 ll of isooctane and 2 ml of copper acetate/pyridine solution (25 g Cu acetate per 500 ml of water adjusted to pH 6.1 with pyridine). The mixture was vortexed for 90 s and the absorbance of the isooctane layer was read at 715 nm against a standard curve of oleic acid (0.10 lmol/ml). 2.2. Experiment 2: Effects of feeding various amounts of glycerol on fermentation kinetics of alfalfa hay A completely randomized design with an one way treatment arrangement (five levels of glycerol) was used. Ground (2 mm) samples of alfalfa hay (0.12–0.20 g) supplemented with 0%, 5%, 10%, 20%, or 40% glycerol were inoculated (n = 3) with cheese-cloth filtered (4 layers) bovine rumen fluid, obtained from a ruminallycannulated donor cow consuming ad libitum bermudagrass hay, and subsequently incubated for 48 h in 125 ml Wheaton bottles. Incubations were conducted as previously described in detail (Tedeschi et al., 2009) with slight modifications. Briefly, because the maximum amount of fermentable substrate that can be used in the aforementioned gas production system, has been standardized to 0.2 g, additions of alfalfa had to be correspondingly reduced in incubations containing increasing amounts of added glycerol, a fermentable substrate, to keep the final amount of total fermentable substrate to 0.2 g. Gas production was measured using a computerized gas monitoring apparatus according to Tedeschi et al. (2008a,b). After 48 h incubation, gas production filtrate was collected and analyzed for volatile fatty acids (VFAs) according to Salanitro and Muirhead (1975).
mixed rumen microbes (Fig. 1). Results from in vitro incubations of ruminal contents with two levels of glycerol partially supported our hypothesis that glycerol may inhibit lipolysis as evidenced by 48% and 77% reductions (P < 0.05) in FFA accumulation in incubations supplemented with 2% or 20% added glycerol, respectively, as compared to controls (5.06 ± 0.06 lmol FFA/mL per h). These results provided evidence that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. Considering that extruded oils provide about 15% protection and that Ca-protected fats sources and whole oil seeds provide about 40% protection to ruminal fats, as determined by in vitro assays (Gulati et al., 1997), feeding glycerol, has the potential to make an immediate impact on enhancement of beef meat quality. In experiment 2, various levels of glycerol were evaluated for their effect on anaerobic fermentation kinetics, NDF digestibility, and VFA concentration. The results of the fermentation kinetic are presented in Table 1. The fast and slow pools were assumed to be glycerol and fiber pools, respectively. Gas fermentation in the first (faster) pool increased linearly (P < 0.001; from 8.87 to 18.4 ± 0.81 ml) as the amount of glycerol increased. Additionally, higher levels of glycerol resulted in a quadratic decrease (P = 0.012; from 0.18 to 0.12 ± 0.02 1/h) of the first pool fractional rate of fermentation (kd). However, the fractional rate of fermentation of glycerol was slower at 20% and 40% compared to 0 (control), 5, and 10% of glycerol (Table 1). Even though gas production in the second (slower) pool decreased quadratically (P = 0.009; from 9.03 to 4.27 ± 0.46 ml), there was no clear relationship when glycerol levels were between 0% and 20%. Glycerol consistently decreased (from 0.06 to 0.03 ± 0.004/h) the fractional fermentation rate in a linear fashion (P = 0.002). The NDF digestibility, determined after 48 h incubation, did not differ (P = 0.53) among the treatments which is consistent with the linear (P = 0.036) decline of the gas production in the fiber fraction as glycerol levels increased. Additionally, this is also consistent with previous the research which shows that feeding levels of glycerol up to 20% of the total ration does not have any effect on nutrient digestibility or animal performance (Donkin and Doane, 2007; Kerley, 2007; Schroder and Sudekum 1999). Increasing amounts of glycerol had a linear decrease on acetate (P = 0.002) accumulation (Table 2), which is consistent with other published research (Remond et al., 1993; Wang et al., 2009). There was a linear (P = 0.049) and a quadratic (P = 0.011) effect of glyc-
2.3. Statistical analysis In experiment 1, FFA accumulations were normalized between experiment runs by expressing results as a percentage of the controls and analyzed for treatment differences using a general analysis of variance with an LSD separation of the means. In experiment 2, the logistic 2-pool model was used to obtain the gas fermentation parameters (Tedeschi et al., 2008b). The parameters of the logistic 2-pool model were compared among the treatments. The PROC IML of SAS was used to generate the orthogonal coefficients for unequally assigned levels of the treatments. Treatment differences were evaluated using the PROC GLIMMIX procedures of SAS. Comparisons of Least Square means between treatments were performed using Tukey’s post hoc test. 3. Results and discussion In experiment 1, two levels of glycerol (2% and 20%) were tested for their potential inhibitory effect against ruminal lipolysis from
Fig. 1. Effects of feeding various levels of glycerol on free-fatty acid accumulation in vitro.
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N.A. Krueger et al. / Bioresource Technology 101 (2010) 8469–8472 Table 1 Effects of various levels of glycerol on the fermentation kinetics of alfalfa hay in vitro in experiment 2. Glycerol (% of diet, DM)
NDFD1 Model parameters2 Fast pool gas production (ml) Fast pool fermentation rate (h 1) Lag time, h Slow pool gas production (ml) Slow pool fermentation rate (h 1) a,b,c 1 2 3
SEM
Control
5
10
20
40
45.73a
53.48a
51.20a
50.15a
48.40a
a
a
8.87 0.18a 2.02a 8.69a 0.06a
11.69 0.17a 1.70a 9.03a 0.04b
a,b
b,c
12.08 0.18a 2.39a 9.00a 0.04b
L 5.17
c
15.24 0.14b 2.25a 7.28a 0.04b
P-value contrasts3
18.36 0.12b 3.66b 4.27b 0.03b
0.81 0.01 0.32 0.46 0.004
Q 0.5689
<0.001 0.070 0.098 0.036 0.002
0.6054 0.002 0.012 0.692 0.009 0.028
Distinct superscripts within a row indicates statistical difference at 0.05 using Tukey’s test. Neutral detergent fiber digestibility, % of NDF. A logistic 2-pool model was used. The fast and slow pools were assumed to be glycerol and fiber pools, respectively. L is linear, Q is quadratic.
Table 2 Effects of various levels of glycerol on volatile fatty acid concentrations in vitro in experiment 2. VFA, lmol/ml
Acetate Propionate Iso-butyrate Butyrate Lactate Iso-valerate Valerate Succinate A:P ratio a,b,c
Glycerol (% of diet, DM)
SEM
Control
5
10
20
40
97.3a 31.7a,b 0.24a 13.9a 0.14a 5.2a 7.5a 1.5a 2.6a
53.1b 19.8b 0.84a 7.5a 0.05a 2.7b 3.7b 0.81b 2.7a
53.7b 23.8b 0.50a 8.9a 0.13a 2.7b 4.6b 0.71b 2.3a,b
53.3b 29.5b 0.39a 9.3a 0.04a 2.7b 4.5b 0.75b 1.8b,c
51.6b 45.2a 0.8a 13.8a 0.07a 2.8b 5.2b 0.78b 1.2c
8.89 4.85 0.22 1.74 0.05 0.44 0.91 0.11 0.16
P-value contrasts Linear
Quadratic
0.002 0.049 0.216 0.188 0.379 0.001 0.008 <0.001 0.652
0.057 0.011 0.680 0.251 0.581 0.058 0.747 0.020 <0.001
Distinct subscripts within a row indicates statistical difference at 0.05 using Tukey’s test.
erol on propionate accumulation due primarily to the high accumulation observed in incubations supplemented with 40% glycerol, which while not significantly different from controls was higher than in all other glycerol-supplemented incubations (Table 2). Consequently, there was a quadratic effect (P < 0.0001) of glycerol supplementation on the acetate to propionate ratio (Table 2). Butyrate accumulations were unaffected by glycerol supplementation (Table 2). These results indicated that glycerol supplementation greater than 20% may negatively affect digestion of the more fibrous fraction of the feed (NDF) and this is supported by our finding that slow pool fermentation rates were also decreased by glycerol supplementation (Table 1) which may thus have altered the VFA profile. Lactate and succinate, which typically do not accumulate to appreciable levels in rumen incubations, were measured in small amounts in our incubations and likely represent electron sinks during the extended incubation (Table 2). Potential effects of glycerol on products of amino acid fermentation were less clear as accumulations of iso-valerate and valerate were decreased by glycerol supplementation but accumulations of iso-butyrate were unaffected (Table 2). Therefore, utilizing glycerol as a feed ingredient in cattle diets could potentially inhibit bacterial fat degradation and may promote ruminal passage of the total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. The retention of glycerol within the rumen will depend on the absorption as well as the microbial fermentation and fluid passage rate of this compound. The observed effects of glycerol on ruminal VFA accumulations (Donkin and Doane 2007; Kijora et al., 1998) suggest that glycerol is not absorbed so fast as to preclude appreciable microbial fermentation. These finding suggests that long-term feeding of glycerol may ultimately select and enrich the populations of glycerol-fermenting microbes such as Megasphaera elsdenii and Selenomonas rumiantium.
4. Conclusion Results from this study demonstrated that utilizing glycerol at an inclusion rate of up to 20% decreased the rate of free-fatty acid accumulation. Additionally, glycerol addition decreased fermentation rate and, when added at levels less than 20%, appeared to have no negative effect on NDF digestibility. Results from this study suggest that utilizing glycerol as a short-term feed ingredient in cattle diets can potentially inhibit bacterial fat degradation. Further research is needed to determine if such a strategy may ultimately promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products.
References Cerrate, S., Yan, F., Wang, Z., Coto, C., Sacakli, P., Waldroup, P.W., 2006. Evaluation of glycerine from biodiesel production as a feed ingredient for broilers. J. Poultry Sci. 5, 1001–1007. Donkin, S.S., Doane, P., 2007. Gylcerol as a feed ingredient in dairy rations. Tri State Dairy Conference. pp. 97-103. Fievez, V., Vlaeminck, B., Jenkins, T., Enjalbert, F., Doreau, M., 2007. Assessing rumen biohydrogenation and its manipulation in vivo, in vitro, in situ. Eur. J. Lipid Sci. Technol. 109, 740–756. Gulati, S.K., Ashes, J.R., Scott, T.W., 1997. In vitro assessment of fat supplements for ruminants. Anim. Feed Sci. Technol. 64, 127–132. Harfoot, C.G., Hazelwood, G.P., 1987. Lipid metabolism in the rumen. In: Hobson, P.N., Stewart, C.S. (Eds.), The Ruminal Microbial Ecosystem. Blackie Academic and Professional, London, pp. 382–419. Henderson, C., 1970. The lipases produced by Anaerovibrio lipolytica in continuous culture. Biochem J. 199, 5–6. Henderson, C., 1971. A study on the lipase produced by Anaerovibrio lipolytica, a rumen bacteria. J. Gen. Microbiol. 65, 81–89. Henderson, C., Hodgkiss, W., 1973. An electron microscopic study of Anaerovibrio lipolytica (strain 5 S) and its lipolytic enzyme. J. Gen. Microbiol. 76, 389– 393.
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Hobson, P.N., Summers, R., 1966. The continuous culture of anaerobic bacteria. J. Gen. Microbiol. 47, 53–65. Kerley, M.C. 2007. Could glycerin – a biodiesel byproduct – be used as a cattle feed? Sci. Daily. http://www.sciencedaily.com/releases/2007/05/070525090245.htm (accessed 9.10.2008). Kijora, C., Bergner, H., Gotz, K.P., Bartlet, J., Szakacs, J., Sommer, A., 1998. Research note: investigation on the metabolism of glycerol in the rumen of bulls. Arch. Tierernahr. 51, 314–348. Kwon, D.Y., Rhee, J.S., 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Amer. Oil Chem. Soc. 63, 89–92. Prins, R.A., Lankhorst, A., van der Meer, P., van Nevel, C.J., 1975. Some characteristics of Anaerovibrio lipolytica, a rumen lipolytic organism. Ant. Van Leewonhoek. 41, 1–11. Remond, B., Souday, E., Jouany, J.P., 1993. In vitro and in vivo fermentation of glycerol by rumen microbes. Anim. Feed Sci. Technol. 41, 121–132. Salanitro, J.P., Muirhead, P.A., 1975. Quantitative method for the gas chromatographic analysis of short-chain monocarboxylic and dicarboxylic acids in fermentation media. Appl. Microbiol. 29, 374–381.
Schroder, A., Sudekum, H.-K., 1999. Glycerol as a by-product of the biodiesel production in diets for ruminants. 10th International Rapseed Congress. Canberra, Australia. http://www.regional.org.au/au/gcirc/1/241.htm (accessed 5.29.2009). Tedeschi, L.O., Konohoff, P.J., Karges, K., Gibson, M.L., 2009. Effects of chemical composition variation on the dynamics of ruminal fermentation and biological value of corn milling (co) products. J. Dairy. Sci. 92, 401–413. Tedeschi, L.O., Schofield, P., Pell, A.N. 2008a. Determining feed quality for ruminants using in vitro gas production technique. Building an anaerobic fermentation chamber.In: The 4th Workshop on Modeling in Ruminant Nutrition. Application of the Gas Production Technique, Juiz de Fora, Brazil. p. 16. Tedeschi, L.O., Schofield, P., Pell, A.N. 2008b. Dertermining feed quality for ruminants using in vitro gas production technique. Evaluating different models to assess gas production measurements. In: The 4th Workshop of Modeling in Ruminant Nutrition:Application of the Gas Production Technique, Juiz de For a, MG, Brazil. p.15. Wang, C., Lui, Q., Huo, W.J., Yang, W.Z., Dong, K.H., Huang, X.Y., Guo, G., 2009. Effects of feeding glycerol on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers. Livestock Sci. 121, 15–20.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro N.A. Krueger a, R.C. Anderson a,*, L.O. Tedeschi b, T.R. Callaway a, T.S. Edrington a, D.J. Nisbet a a
United States Department of Agriculture, Agriculture Research Service, Southern Plains Agriculture Research Center, Food and Feed Safety research Unit, 2881 F&B Road, College Station, TX 77845, USA b Department of Animal Sciences, Texas A&M University, 2471 TAMU, College Station, TX 77843-2471, USA
a r t i c l e
i n f o
Article history: Received 4 February 2010 Received in revised form 21 May 2010 Accepted 2 June 2010 Available online 26 June 2010 Keywords: Biohydrogenation Fermentation Fiber digestibility Glycerol
a b s t r a c t Ruminant-derived foods contain high proportions of saturated fats as a result of ruminal biohydrogenation that rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. The objective of this study was to evaluate the effects of glycerol on ruminal free-fatty acid (FFA) production rates and in vitro fermentation kinetics of alfalfa hay. In vitro incubations demonstrated 48% and 77% reductions in rates of FFA accumulation in incubations supplemented with 2% and 20% glycerol as compared to controls. In vitro incubations with alfalfa hay demonstrated that increasing levels of glycerol did not affect NDF digestibility of the hay. Additionally, increasing amounts of glycerol decreased the acetate to propionate ratio in the rumen. These results suggest that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. Published by Elsevier Ltd.
1. Introduction Ruminant-derived foods contain high proportions of saturated fats due to an intense biohydrogenation that occurs in the rumen, saturating and thus limiting the availability of free unsaturated fatty acids for assimilation (Harfoot and Hazelwood, 1987). Strategies to enrich ruminant-derived foods with unsaturated fatty acids are desired as they are considered beneficial for good human health. Lipolysis is pre-requisite for biohydrogenation because saturase enzymes act only on free-fatty acids (FFAs). Lipolysis releases fatty acids from the glycerol backbone of triacylglycerols, galactolipids, or phospholipids via hydrolysis processes that occur in the rumen (Fievez et al., 2007). Despite considerable research on ruminal biohydrogenation, there are only a few reports on the purification and properties of lipase enzymes from rumen bacteria and those have reported almost exclusively on enzymes from Anaerovibrio lipolytica (Henderson, 1970, 1971; Henderson and Hodgkiss, 1973; Hobson and Summers, 1966; Prins et al., 1975). Results from these studies indicated that A. lipolytica may produce two lipases, the more active form being produced extracellularly and the less active form being cell associated (Hobson and Summers, 1966). The lipases exhibit pH optima of 7.4 and activity is inhibited by ZnCl2, HgCl2 and high chloride concentrations (Henderson, 1970, 1971). The production of lipase by A. lipolytica was influenced by the bacterium’s growth rate, with maximal production occurring * Corresponding author. Tel.: +1 979 260 9317; fax: +1 979 260 9332. E-mail address: [email protected] (R.C. Anderson). 0960-8524/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.biortech.2010.06.010
during the logarithmic growth phase, and that it was constitutively produced in A. lipolytica strain L 1741. Other than these reports, little is known regarding factors affecting expression of this activity. Considering that the hydrolysis product, glycerol, can be utilized as a nutrient for the growth of rumen, lipase-producing bacteria such as A. lypolytica (Krueger et al., unpublished data), we hypothesized that excessive concentrations of glycerol may affect lipase activity. Glycerol is a by-product from the production of biodiesel and has been evaluated as a potential feed ingredient for livestock in various stages of production (Cerrate et al., 2006; Donkin and Doane, 2007; Kerley, 2007; Schroder and Sudekum 1999). Research has shown that feeding glycerol up to a rate of 10% of the daily dietary dry matter has no effect on feed intake or performance in finishing beef cattle or lactating dairy cows (Kerley 2007; Schroder and Sudekum 1999). The objectives of this study were to evaluate two levels of glycerol and their potential inhibitory effect against ruminal lipolysis by mixed rumen microbes and to determine the effects of feeding various amounts of glycerol on fermentation kinetics of alfalfa hay.
2. Methods 2.1. Experiment 1: Potential inhibitory effects of glycerol Ruminal contents (fluid and solids) were obtained from a nonlactating cannulated dairy cow receiving a diet consisting of 50% feedlot grain-type ration and 50% dried-distillers grains. Freshly
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N.A. Krueger et al. / Bioresource Technology 101 (2010) 8469–8472
collected ruminal contents were gently mixed with a spoon while under CO2 and 5 g were anaerobically (CO2) dispensed into triplicate sets of 18 150 mm straight walled tubes each containing 10% olive oil and one of the three levels of glycerol, 0%, 2%, or 20%. This experiment was conducted twice, with each experimental run conducted on separate days. Tubes were sealed with #1 butyl rubber stoppers and subsequently incubated at 39 °C . After 24 h incubation, the amount of accumulated FFA was measured colorimetrically using the method of Kwon and Rhee (1986) with slight modifications as follows. Tubes were unsealed, samples were acidified with 500 ll of concentrated HCl and 10 ml of isooctane was added. The isooctane/rumen content mixture was vortexed for 60 s to extract FFA. The isooctane layer was pulled off and placed into a clean 13 100 mm screw top glass tube. Samples were dried to total dryness under nitrogen gas. Dried samples were re-suspended in 5 ml of isooctane. A 200 ll subsample was placed into a clean 16 150 mm borosilicate glass tube containing 1800 ll of isooctane and 2 ml of copper acetate/pyridine solution (25 g Cu acetate per 500 ml of water adjusted to pH 6.1 with pyridine). The mixture was vortexed for 90 s and the absorbance of the isooctane layer was read at 715 nm against a standard curve of oleic acid (0.10 lmol/ml). 2.2. Experiment 2: Effects of feeding various amounts of glycerol on fermentation kinetics of alfalfa hay A completely randomized design with an one way treatment arrangement (five levels of glycerol) was used. Ground (2 mm) samples of alfalfa hay (0.12–0.20 g) supplemented with 0%, 5%, 10%, 20%, or 40% glycerol were inoculated (n = 3) with cheese-cloth filtered (4 layers) bovine rumen fluid, obtained from a ruminallycannulated donor cow consuming ad libitum bermudagrass hay, and subsequently incubated for 48 h in 125 ml Wheaton bottles. Incubations were conducted as previously described in detail (Tedeschi et al., 2009) with slight modifications. Briefly, because the maximum amount of fermentable substrate that can be used in the aforementioned gas production system, has been standardized to 0.2 g, additions of alfalfa had to be correspondingly reduced in incubations containing increasing amounts of added glycerol, a fermentable substrate, to keep the final amount of total fermentable substrate to 0.2 g. Gas production was measured using a computerized gas monitoring apparatus according to Tedeschi et al. (2008a,b). After 48 h incubation, gas production filtrate was collected and analyzed for volatile fatty acids (VFAs) according to Salanitro and Muirhead (1975).
mixed rumen microbes (Fig. 1). Results from in vitro incubations of ruminal contents with two levels of glycerol partially supported our hypothesis that glycerol may inhibit lipolysis as evidenced by 48% and 77% reductions (P < 0.05) in FFA accumulation in incubations supplemented with 2% or 20% added glycerol, respectively, as compared to controls (5.06 ± 0.06 lmol FFA/mL per h). These results provided evidence that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. Considering that extruded oils provide about 15% protection and that Ca-protected fats sources and whole oil seeds provide about 40% protection to ruminal fats, as determined by in vitro assays (Gulati et al., 1997), feeding glycerol, has the potential to make an immediate impact on enhancement of beef meat quality. In experiment 2, various levels of glycerol were evaluated for their effect on anaerobic fermentation kinetics, NDF digestibility, and VFA concentration. The results of the fermentation kinetic are presented in Table 1. The fast and slow pools were assumed to be glycerol and fiber pools, respectively. Gas fermentation in the first (faster) pool increased linearly (P < 0.001; from 8.87 to 18.4 ± 0.81 ml) as the amount of glycerol increased. Additionally, higher levels of glycerol resulted in a quadratic decrease (P = 0.012; from 0.18 to 0.12 ± 0.02 1/h) of the first pool fractional rate of fermentation (kd). However, the fractional rate of fermentation of glycerol was slower at 20% and 40% compared to 0 (control), 5, and 10% of glycerol (Table 1). Even though gas production in the second (slower) pool decreased quadratically (P = 0.009; from 9.03 to 4.27 ± 0.46 ml), there was no clear relationship when glycerol levels were between 0% and 20%. Glycerol consistently decreased (from 0.06 to 0.03 ± 0.004/h) the fractional fermentation rate in a linear fashion (P = 0.002). The NDF digestibility, determined after 48 h incubation, did not differ (P = 0.53) among the treatments which is consistent with the linear (P = 0.036) decline of the gas production in the fiber fraction as glycerol levels increased. Additionally, this is also consistent with previous the research which shows that feeding levels of glycerol up to 20% of the total ration does not have any effect on nutrient digestibility or animal performance (Donkin and Doane, 2007; Kerley, 2007; Schroder and Sudekum 1999). Increasing amounts of glycerol had a linear decrease on acetate (P = 0.002) accumulation (Table 2), which is consistent with other published research (Remond et al., 1993; Wang et al., 2009). There was a linear (P = 0.049) and a quadratic (P = 0.011) effect of glyc-
2.3. Statistical analysis In experiment 1, FFA accumulations were normalized between experiment runs by expressing results as a percentage of the controls and analyzed for treatment differences using a general analysis of variance with an LSD separation of the means. In experiment 2, the logistic 2-pool model was used to obtain the gas fermentation parameters (Tedeschi et al., 2008b). The parameters of the logistic 2-pool model were compared among the treatments. The PROC IML of SAS was used to generate the orthogonal coefficients for unequally assigned levels of the treatments. Treatment differences were evaluated using the PROC GLIMMIX procedures of SAS. Comparisons of Least Square means between treatments were performed using Tukey’s post hoc test. 3. Results and discussion In experiment 1, two levels of glycerol (2% and 20%) were tested for their potential inhibitory effect against ruminal lipolysis from
Fig. 1. Effects of feeding various levels of glycerol on free-fatty acid accumulation in vitro.
8471
N.A. Krueger et al. / Bioresource Technology 101 (2010) 8469–8472 Table 1 Effects of various levels of glycerol on the fermentation kinetics of alfalfa hay in vitro in experiment 2. Glycerol (% of diet, DM)
NDFD1 Model parameters2 Fast pool gas production (ml) Fast pool fermentation rate (h 1) Lag time, h Slow pool gas production (ml) Slow pool fermentation rate (h 1) a,b,c 1 2 3
SEM
Control
5
10
20
40
45.73a
53.48a
51.20a
50.15a
48.40a
a
a
8.87 0.18a 2.02a 8.69a 0.06a
11.69 0.17a 1.70a 9.03a 0.04b
a,b
b,c
12.08 0.18a 2.39a 9.00a 0.04b
L 5.17
c
15.24 0.14b 2.25a 7.28a 0.04b
P-value contrasts3
18.36 0.12b 3.66b 4.27b 0.03b
0.81 0.01 0.32 0.46 0.004
Q 0.5689
<0.001 0.070 0.098 0.036 0.002
0.6054 0.002 0.012 0.692 0.009 0.028
Distinct superscripts within a row indicates statistical difference at 0.05 using Tukey’s test. Neutral detergent fiber digestibility, % of NDF. A logistic 2-pool model was used. The fast and slow pools were assumed to be glycerol and fiber pools, respectively. L is linear, Q is quadratic.
Table 2 Effects of various levels of glycerol on volatile fatty acid concentrations in vitro in experiment 2. VFA, lmol/ml
Acetate Propionate Iso-butyrate Butyrate Lactate Iso-valerate Valerate Succinate A:P ratio a,b,c
Glycerol (% of diet, DM)
SEM
Control
5
10
20
40
97.3a 31.7a,b 0.24a 13.9a 0.14a 5.2a 7.5a 1.5a 2.6a
53.1b 19.8b 0.84a 7.5a 0.05a 2.7b 3.7b 0.81b 2.7a
53.7b 23.8b 0.50a 8.9a 0.13a 2.7b 4.6b 0.71b 2.3a,b
53.3b 29.5b 0.39a 9.3a 0.04a 2.7b 4.5b 0.75b 1.8b,c
51.6b 45.2a 0.8a 13.8a 0.07a 2.8b 5.2b 0.78b 1.2c
8.89 4.85 0.22 1.74 0.05 0.44 0.91 0.11 0.16
P-value contrasts Linear
Quadratic
0.002 0.049 0.216 0.188 0.379 0.001 0.008 <0.001 0.652
0.057 0.011 0.680 0.251 0.581 0.058 0.747 0.020 <0.001
Distinct subscripts within a row indicates statistical difference at 0.05 using Tukey’s test.
erol on propionate accumulation due primarily to the high accumulation observed in incubations supplemented with 40% glycerol, which while not significantly different from controls was higher than in all other glycerol-supplemented incubations (Table 2). Consequently, there was a quadratic effect (P < 0.0001) of glycerol supplementation on the acetate to propionate ratio (Table 2). Butyrate accumulations were unaffected by glycerol supplementation (Table 2). These results indicated that glycerol supplementation greater than 20% may negatively affect digestion of the more fibrous fraction of the feed (NDF) and this is supported by our finding that slow pool fermentation rates were also decreased by glycerol supplementation (Table 1) which may thus have altered the VFA profile. Lactate and succinate, which typically do not accumulate to appreciable levels in rumen incubations, were measured in small amounts in our incubations and likely represent electron sinks during the extended incubation (Table 2). Potential effects of glycerol on products of amino acid fermentation were less clear as accumulations of iso-valerate and valerate were decreased by glycerol supplementation but accumulations of iso-butyrate were unaffected (Table 2). Therefore, utilizing glycerol as a feed ingredient in cattle diets could potentially inhibit bacterial fat degradation and may promote ruminal passage of the total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products. The retention of glycerol within the rumen will depend on the absorption as well as the microbial fermentation and fluid passage rate of this compound. The observed effects of glycerol on ruminal VFA accumulations (Donkin and Doane 2007; Kijora et al., 1998) suggest that glycerol is not absorbed so fast as to preclude appreciable microbial fermentation. These finding suggests that long-term feeding of glycerol may ultimately select and enrich the populations of glycerol-fermenting microbes such as Megasphaera elsdenii and Selenomonas rumiantium.
4. Conclusion Results from this study demonstrated that utilizing glycerol at an inclusion rate of up to 20% decreased the rate of free-fatty acid accumulation. Additionally, glycerol addition decreased fermentation rate and, when added at levels less than 20%, appeared to have no negative effect on NDF digestibility. Results from this study suggest that utilizing glycerol as a short-term feed ingredient in cattle diets can potentially inhibit bacterial fat degradation. Further research is needed to determine if such a strategy may ultimately promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products.
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