On-line version ISSN 2221-4062
S. Afr. j. anim. sci. vol.39 n.5 Pretoria Jan. 2009
E. Raffrenato#; P.J. van Soest; M.E. van Amburgh
Department of Animal Science, Cornell University, Ithaca - NY, USA
The objective of this work was to study the effects of both Klason lignin (KL) and acid detergent lignin (ADL) on in vitro neutral detergent fibre (NDF) digestion (IVNDFd) in an effort to assess if acid labile phenolic compounds affect the rate of degradation (kd). Eighty five forages (lucerne, maize silages and grasses) were analyzed for NDF, ADL, KL and IVNDFd (6, 12, 24, 30, 36, 48, and 96 h fermentations were used for kd estimations). Correlations were estimated among lignin types (KL vs . ADL), lignin and extent of IVNDFd, and lignin type and NDF kd and tested for significance. Within and among all forage types, the correlation between ADL and KL was in general positive when on NDF basis and high and positive when on DM basis (0.77 to 0.90). Within and among all forages, only ADL was consistently negatively correlated with IVNDFd at all time points (-0.54 to -0.94). Correlations among forages for NDF kd and lignin type were not consistent. Among all forages, KL was negatively correlated with IVNDFd and NDF kd. The correlation between IVNDFd and ADL increased as fermentation length increased among all forages. However, the correlation of KL and IVNDFd was greatest up to 48 h of fermentation suggesting that the soluble phenolics affected both the rate and extent of IVNDFd. Unlike ADL, KL disappeared during IVNDFd, and in most forages there was a high negative correlation associated with the difference between KL and ADL (ΔL) and IVNDFd, except for brown midrib hybrids maize and early cut grasses that had a different behaviour. Among forages, a one unit increase in ΔL corresponded to an average 18% decrease in the kd demonstrating that the greater the difference between KL and ADL the lower the rate of digestion.
Keywords: Acid detergent lignin, Klason, digestibility
The rate (kd) and extent of NDF digestion is important because it impacts on the energy available from fibre, the passage of particles out of the rumen and the physical fill of the rumen (Mertens, 1993). The kd and the extent of digestion of NDF are affected by lignin and the impact of KL and ADL, however, the difference among these two measures for NDF digestion remains obscure. Since KL appears to represent less polymerized phenolic compounds, the potential to negatively impact the ND solubles by either dilution or hindering digestion appears to be a possible hypothesis.
The lignin methods differentiate the phenolic acids primarily by acid solubility. The two lignin methods of interest are Klason lignin (KL) (Theander & Westerlund, 1986) and the acid-detergent lignin (ADL) (Van Soest et al ., 1991). The KL results in higher values and represents total phenolic acids and some acid dispersible phenolics while ADL represents more polymerized phenolics that appear to behave in a more nutritionally uniform manner and impact the extent of NDF digestion in anaerobic conditions.
Correlation coefficients observed between KL and ADL and digestibility did not indicate any advantage of either method in predicting rate or extent of digestion (Jung et al. , 1997). Fukushima & Hatfield (2004) presented contrasting results for the relationship between in vitro NDF digestibility (IVNDFd) and KL with low correlations between the two measures and even lower correlations between ADL and IVNDFd.
Acid detergent lignin has been related to the ultimate extent of NDF digestion in the study of Chandler et al. (1980) who fermented waste residues for 60 or 90 days in methane fermenters. However, previous work has suggested that soluble phenolic-carbohydrate complexes are released during fibre degradation and might affect microbial degradation and subsequent energy yield by diluting the neutral detergent solubles (Gaillard & Richards, 1975; Lowry et al. , 1994), thereby implicating un-polymerized phenolics and not just ADL in digestibility. Since the ND soluble fraction of forage material contains the greatest energy content, anything that dilutes this fraction will reduce the energy content of the forage.
The objective of this work was to study the relationship between KL and ADL with in vitro NDF digestion in an effort to assess if acid labile phenolic compounds might affect the rate (kd) of NDF degradation.
Materials and Methods
Eighty-five forages of various species and stages of maturity were harvested, dried in a forced air oven at 60 ºC for 24 h and then ground through a 1 mm screen in a Wiley Mill (Thomas Scientific, Swedesboro NJ). The samples consisted of different maturities (early vegetative to boot stage) of lucerne, silages and grasses (Lolium,Phleum pratense,Dactylis glomerata L, Panicum coloratum L) and varieties of maize silage (standard hybrids and brown midrib hybrids (BMR, which are low lignin hybrids with a visible marker)) were analyzed for NDF (Mertens, 2002), ADL (Van Soest, 1973), KL (Theander & Westerlund, 1986) and IVNDFd. In vitro NDF fermentations were conducted in Erlenmeyer flasks following the procedures of Goering & Van Soest (1970) on samples ground through a 1 mm sieve. After digestion, residues were analyzed for NDF with 0.250 mL amylase (3 000 U/mL, Megazyme International, Ireland) without sodium sulphite and filtered in crucibles fitted with Whatman 934-AH glass microfibre filters (Whatman International Ltd., Maidstone, UK). Residues were also analyzed for ADL and KL using the modification of glass filters in the crucible as a filtering aid. Residues from 6, 12, 24, 30, 36, 48, and 96 h fermentations were used for kd estimations (Van Amburgh et al. , 2003).
For evaluation of the relationships, correlations were estimated simple Pearson correlations for all forage types between lignin measures, lignin and extent of IVNDFd, and lignin type and IVNDFd kd and tested for significance (P <0.05).
Results and Discussion
The chemical compositions and the in vitro digestibility values of the forages used in the study are shown in Table 1. The highest NDF digestibility was for lucerne at six hours, for the grasses harvested during the early vegetative stage, at 48 hours and for the BMR at 96 hours. Correlations among lignin types and extent (IVNDFd) and rate of digestion (kd) were all statistically significant (P <0.05). Within and among all forage types, the correlations between ADL and KL were high and positive, except for the BMR maize and the early cut grasses. Furthermore, correlations on dry matter basis were larger than on NDF basis for all forages. This shows how the neutral detergent solution might disperse some of the soluble phenolics. Temperate, vegetative grasses had KL measures exceeding 15% and ADL values less than 2%.
Among all forages, the correlation between KL and IVNDFd was greater early in the fermentation period, whereas the correlation between IVNDFd and ADL increased as fermentation length increased, which is consistent with previous findings (Chandler et al ., 1980). Within and among all forages, both KL and ADL were negatively correlated with IVNDFd at any time point of digestion (Table 2). This suggests a two-fold effect of lignin types on both the extent and rate of NDF digestion. Since KL and ADL are positively correlated in most plants, the observation that KL impacts digestion is not extraordinary, except that the KL is dispersed and less recoverable as digestion progresses. This suggests that soluble phenolics or unpolymerized phenolics are potentially attached to some carbohydrate moieties resulting in less digestion or create some steric hindrance or resistance creating a barrier for bacterial enzyme activity.
Among all forages, KL was negatively correlated with IVNDFd and kd, and this was particularly true for conventional maize silages and mature grasses and less for BMR maize, early cut grasses and lucerne. The difference between the KL and ADL (ΔL) and extent and rate of digestion did not behave similarly among forages, especially in BMR maize and early cut grasses. Figure 1 shows the different behaviour of BMR and early cut grasses. This further supports the presence of dispersible lignin or acid-labile phenolic acids that impacted microbial degradation consistent with the concept of steric hindrance resulting in diverse correlations with the respective kd. The data further suggest that BMR maize, although harvested at maturity, behaves like an immature plant with respect to lignin polymerization and acid-labile phenolic acids.
Overall, these data clearly indicate that both lignin types impact the rate of digestion and imply that as NDF digestion occurs, acid-labile phenolic acids have the ability to modulate the rate of digestion of temperate forages and it appears that these phenolics would be recovered in the ND soluble, reducing the energy available from microbial digestion.
The data presented demonstrates several key factors related to forage digestion by ruminal microbes and the need for improved methods of forage and fibre analyses. Methods for measuring NDF digestibility and lignin need to include steps to improve recovery such as the use of specific filter papers. This enhances our ability to describe the behaviour of fibre degradation in a more quantitative and descriptive manner which will allow both plant breeders and nutritionists to make better decisions about genetic selection and forage selection and feeding behaviour for specific agronomic conditions. The factors within a plant that impact digestibility are different among plant varieties and need to be considered when predicting forage digestion and energy yield.
Chandler, J.A., Jewell, W.J., Gossett, J.M., Van Soest, P.J. & Robertson, J.B., 1980. Predicting methane fermentation biodegradability. J. Biotech. Bioengin. Symp. No. 10, John Wiley & Sons. pp. 93-107. [ Links ]
Fukushima, R.S. & Hatfield, R.D., 2004. Comparison of the acetyl bromide spectrophotometeric method with other analytical lignin methods for determining lignin concentration in forage samples. J. Agric. Food Chem. 52, 3713-3720. [ Links ]
Gaillard , B.D.E. & Richards, G.N., 1975. Presence of soluble lignin-carbohydrate complexes in the bovine rumen. Carbohydr. Res. 42, 135-145. [ Links ]
Goering, H.K. & Van Soest, P.J., 1970. Forage and Fiber Analysis. USDA Agric. Handb. 379. U.S. Gov. Print. Office, Washington, D.C., USA. [ Links ]
Jung, H.G., Mertens, D.R. & Payne, A.J., 1997. Correlation of acid detergent lignin and Klason lignin with digestibility of forage dry matter and neutral detergent fiber. J. Dairy Sci. 80, 1622-1628. [ Links ]
Lowry, J.B., Conlan, L.L., Schlink, A.C. & McSweeney, C.S., 1994. Acid detergent dispersible lignin in tropical grasses. J. Sci. Food Agric. 65, 41-49. [ Links ]
Mertens, D.R., 1993. Chap. 21. In: Forage Cell Wall Structure and Digestibility. Eds Jung, H.G., Buxton, D.R., Hatfield, R.D. & Ralph, J., Amer. Soc. Agron., Madison, W.I., USA. [ Links ]
Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds using refluxing in beakers or crucibles: collaborative study. J. AOAC Int. 85, 1217-1240. [ Links ]
Theander, O. & Westerlund, E.A., 1986. Studies on dietary fiber. 3. Improved procedures for analysis of dietary fiber. J. Agric. Food Chem. 34, 330-336. [ Links ]
Van Amburgh, M.E., Van Soest, P.J., Robertson, J.B. & Knaus, W.F., 2003. Corn silage neutral fiber: refining a mathematical approach for in vitro rates of digestion. Proc. Cornell Nutr. Conf. pp. 99-108. [ Links ]
Van Soest, P.J. 1973. Collaborative study of acid-detergent fiber and lignin. J. Off. Anal. Chem. 56, 781-784. [ Links ]
Van Soest, P.J., Robertson, J.B., & Lewis, B.A., 1991. Symposium: Carbohydrate methodology, metabolism, and nutritional implications in dairy cattle. J. Dairy Sci. 74, 3583-3597. [ Links ]