- Top of page
- Materials and methods
This study evaluated the effect of two fibrolytic enzyme products, applied at baling, on the chemical composition and digestibility of alfalfa hay. Three replicate bales of alfalfa hay (82% dry matter) were produced with the application of one of five treatments including an untreated control and one of two fibrolytic enzyme products (DYC and ECO), either applied alone or in combination with a ferulic acid esterase-producing bacterial additive. The enzyme products were applied on the basis of endoglucanase activity. The neutral detergent fibre (NDF) concentration and accumulated temperature after storage of hay produced using DYC- or ECO-based treatments were greater (P < 0·05) than untreated hay, except for hay bales produced using ECO alone. Bales produced using ECO-based treatments had a greater (P < 0·05) in vitro NDF digestibility compared with untreated bales. The application of fibrolytic enzymes at baling may potentially improve NDF digestibility without negatively affecting chemical composition or increasing aerobic deterioration. However, the effects of fibrolytic enzymes varied depending on the product applied. Combining ferulic acid esterase-producing bacterial additives with fibrolytic enzymes did not improve the nutritive value of hay after storage.
- Top of page
- Materials and methods
Feed costs have a major influence on the efficiency and profitability of livestock production systems. Because these costs are expected to increase for the foreseeable future, the importance of feed nutritive value is unlikely to diminish (Archer et al., 1999; Finneran et al., 2012). Fibre digestibility is a primary limiting factor in livestock production efficiency, with often <60% of dietary fibre being digested by the animal (Beauchemin et al., 2003; Beauchemin and Holtshausen, 2010). Therefore, improvements in the fibre digestibility of the forage portion of an animal's diet would likely result in an increase in the efficiency and sustainability of the production system.
The use of fibrolytic enzyme products, primarily consisting of cellulases and xylanases, in ruminant diets could potentially improve fibre digestibility and subsequently increase the intake of digestible energy (Beauchemin et al., 2003). Many studies have reported improvements in the digestibility of fibre after application of fibrolytic enzymes to hay (Beauchemin et al., 1995; Eun and Beauchemin, 2007). However, the effect of fibrolytic enzyme treatment varies depending on a range of factors, including forage type, maturity and dry-matter (DM) content (Feng et al., 1996; Pinos-Rodríguez et al., 2002; Dean et al., 2008), enzyme composition and application rate (Colombatto et al., 2003; Elghandour et al., 2013), and the period of interaction between forage and enzyme (Krueger et al., 2008b). Most studies investigating the application of fibrolytic enzymes, designed for ruminant use, to forage have applied the treatment prior to feeding. However, the application of fibrolytic enzymes to a forage prior to preservation, such as at the time of baling, may provide an opportunity for a longer duration of interaction between the enzyme and the forage. In addition, a more consistent application and, subsequently, a more consistent forage nutritive value may be achieved when enzymes are applied at baling, rather than application just prior to feeding (Adesogan, 2005).
The use of lactic acid bacterial additives in hay production has been the focus of a limited number of studies, which previously reported either minor or no effects (Emanuele et al., 1992; Bass et al., 2012). However, inoculants that produce ferulic acid esterase in addition to organic acids commonly associated with forage preservation have recently become commercially available (Muck, 2010). Ferulic acid esterase can break the linkages between lignin and the cell wall carbohydrates of forages, and subsequently, can increase its digestibility and nutritive value (Krueger et al., 2008a; Addah et al., 2012). Furthermore, this may increase the availability of utilizable substrate for lactic acid bacteria present on the hay and potentially improve the preservation conditions for the forage through production of antifungal organic acids such as acetic acid and propionic acid. These potential increases in fibre digestibility, forage nutritive value and the availability of substrate for epiphytic bacteria during hay storage from ferulic acid esterase activity may be enhanced when fibrolytic enzymes are applied in combination with a third-generation inoculant.
The objective of this study was to determine the effect of two fibrolytic enzyme products produced for ruminants, applied at baling alone or in combination with a ferulic acid esterase-producing bacterial additive, on the subsequent chemical composition and digestibility of the alfalfa hay.
- Top of page
- Materials and methods
The mean (standard deviation) DM concentration of untreated bales after 90 days of storage was 884 (16·7) g kg−1 and did not differ (P > 0·05) from bales produced using either DYC alone, DYC with 11GFT, ECO alone or ECO with 11GFT that had DM concentrations of 866 (6·5), 877 (21·5), 885 (12·6) and 852 (4·9) g kg−1 respectively. The NDF concentration of hay produced using DYC- or ECO-based treatments was greater (P < 0·05) than untreated hay, except for hay bales produced using ECO alone, which did not differ from untreated bales (P > 0·05; Table 3). The ADF concentration of hay produced using ECO-based treatments was greater (P < 0·05) than untreated hay and was greater (P < 0·05) than bales produced using DYC-based treatments when averaged across inoculant treatments. Hay produced using ECO with 11GFT had a greater (P < 0·05) NDF and ADF concentration compared with hay produced with ECO alone, when control was omitted from the analysis.
Table 3. Chemical composition, heating indices and microbial counts of alfalfa hay treated with fibrolytic enzymes and inoculant.
|s.e.m. d|| ||0·16||15·9||8·8||1·2||9·3||3·3||0·988||0·399||0·466||0·568||101·8|
|Control omitted (P-values)|
|Enz. x inoc.|| ||0·181||0·026||0·088||0·001||0·483||0·146||0·223||0·202||0·756||0·045||0·005|
The WSC concentration of untreated bales was lower (P < 0·01) than bales produced using DYC with 11GFT or ECO applied alone and was greater (P > 0·01) than bales produced using ECO with 11GFT. The CP concentration of bales produced using ECO with 11GFT tended to be greater (P = 0·065) than untreated bales, while bales produced using ECO-based treatments had a greater (P < 0·05) CP concentration than DYC-based treatments when averaged across inoculant treatments. In addition, the use of 11GFT with enzyme products tended (P = 0·051) to increase the CP concentration of hay compared with hay produced with enzyme products alone. The ash concentration and pH of untreated bales did not differ (P > 0·05) from hay produced using enzyme-based treatments. No effect of treatment was observed for lactic acid bacteria, mould, yeast or total bacteria enumerations; however, the total bacteria enumerations of bales produced using ECO with 11GFT tended to be greater (P = 0·056) than bales made using ECO alone. However, hay produced using 11GFT tended (P = 0·075) to have a greater lactic acid bacteria population than hay produced using an enzyme product alone. In addition, on average, hay produced using DYC-based treatments tended to have greater (P = 0·074) mould populations than hay made with ECO-based treatments.
Hay produced using enzyme treatments had a greater (P < 0·001) accumulated temperature above ambient after 50 days compared with untreated hay, with the exception of hay produced using ECO alone, which did not differ (P > 0·05) from the untreated hay. The use of 11GFT with ECO increased (P < 0·001) the aerobic deterioration of hay bales produced compared with using the ECO product alone.
In addition, no effect of treatment was observed (P > 0·05) on the lactic acid, propionic acid and butyric acid concentration of hay bales (Table 4); however, bales produced using DYC alone had a greater ammonia concentration than bales produced using ECO alone. In addition, on average, the acetic acid concentration of bales produced using DYC tended to have a greater (P < 0·09) acetic acid and total fermentation product concentration than ECO-based bales.
Table 4. Storage fermentation characteristics of alfalfa hay treated with enzymes and inoculant.
|Control omitted (P-values)|
|Enz. x inoc.|| ||0·171||0·532||0·462||0·861||0·502||0·002|
The in vitro DMD of hay was unaffected (P > 0·05) by treatment after 24-h incubation (Table 5). However, after 48-h incubation, untreated hay and hay produced using ECO applied alone had greater (P < 0·05) DMD than ECO applied in combination with 11GFT. In general, the use of 11GFT in combination with ECO resulted in a lower (P < 0·05) DMD compared with ECO applied alone. The in vitro NDFD of untreated hay was lower (P < 0·01) than all enzyme-treated hay after 24-h incubation, whereas only ECO-based treatments had greater (P < 0·05) NDF digestibility than untreated bales after 48-h incubation. Similarly, the ADFD of ECO-based bales was greater than untreated bales after 24-h incubation, whereas no effect of treatment (P > 0·05) was observed on ADFD of hay after 48-h digestion. The NDF and ADF digestibility of hay bales produced with 11GFT did not differ (P > 0·05) from bales produced with enzyme alone. The in vitro true DMD of untreated hay and hay produced using ECO alone was greater (P < 0·01) than bales produced using DYC alone or ECO with 11GFT. No effect of treatment was observed on gas production until 12 h of incubation, after which ECO applied alone and untreated bales had a greater (P < 0·05) gas production than bales produced with ECO in combination with 11GFT.
Table 5. In vitro digestibility of DM and fibre and total gas production of alfalfa hay treated with fibrolytic enzymes and inoculant.
|Enzyme||Inoculant||DMDa||NDFDa||ADFDa||IVTDMDa||Total gas productionb|
|24 h||48 h||24 h||48 h||24 h||48 h||48 h||3 h||6 h||12 h||18 h||24 h||48 h|
|P- value|| ||0·467||0·011||0·004||0·023||0·064||0·198||0·004||0·279||0·118||0·007||0·007||0·014||0·071|
|Control omitted (P-values)|
|Enz. x inoc.|| ||0·757||0·039||0·494||0·539||0·735||0·901||0·003||0·128||0·010||0·001||0·001||0·007||0·011|
- Top of page
- Materials and methods
The mean (standard deviation) DM concentration at baling of 818 (2·25; n = 15) g kg−1 for all alfalfa hay bales produced was within the acceptable range of values for the target DM concentration of 820 g kg−1. The NDF and ADF concentrations (440 and 366 g kg−1 DM respectively) of untreated hay sampled on the day of baling were typical of alfalfa harvested at a mid-stage of physiological maturity (NRC, 2001). These fibre concentrations are slightly high compared with other values (370–438 and 208–322 g kg−1 DM respectively) reported for other studies on alfalfa hay of a similar DM content by Pinos-Rodríguez et al. (2002), Coblentz and Bertram (2012) and Coblentz and Muck (2012). Furthermore, the CP concentration (160 g kg−1 DM) of hay at baling in the present study was lower than values (168–208 g kg−1 DM) reported by these studies.
Storage of untreated bales for 90 days resulted in an increase in NDF and ADF concentrations. Coblentz et al. (2013) reported that increases in concentrations of inert components of hay during storage reflect a loss of other components of the forage, such as non-structural carbohydrates, which are lost to the atmosphere through oxidation by undesirable microorganisms. In addition, the lack of an increase in CP concentration after storage for untreated bales in the present study compared with hay at baling, in contrast to increases in other components, indicates losses of N potentially from leaf loss or the volatilization of ammonia during storage (Rotz and Muck, 1994).
With the exception of hay produced using ECO alone, hay produced using enzyme products generally had greater NDF concentrations than the untreated hay, consistent with an increase in bale temperature during the storage period. Similarly, Coblentz and Hoffman (2009) found that increases in the cell wall components of alfalfa hay were related to heating for large round hay bales. Heat is generated during the microbial oxidization of available components of hay during storage and is an indication of the aerobic deterioration that occurs in preserved forages. In the present study, the increase in the concentration of NDF of these bales despite a minimal or lack of effect on ash or CP concentration may indicate the occurrence of Maillard reactions in response to heating. This reaction results in the degradation of sugar and nitrogen components to a phenolic polymer, and thus results in increased NDF estimations (Van Soest and Mason, 1991). However, increased respiration due to increased availability of substrate in enzyme-treated hay for utilization by microbial communities present on the forage may have also contributed to this effect.
Several studies have reported that the use of exogenous fibrolytic enzymes applied before feeding can affect the rate of digestion of forages, but not the total forage digestibility (Colombatto et al., 2003; Krueger and Adesogan, 2008). The DMD in the present study did not differ between untreated and enzyme-treated hay after 24 h of incubation, whereas after 48 h of incubation, the DMD of enzyme-treated bales, with the exception of ECO applied alone, was lower than the untreated hay. This negative effect reflects the greater proportion of structural carbohydrates in these hays and therefore is an indirect result of the increased aerobic deterioration of hay produced with enzyme-based treatments during storage. In addition to aerobic deterioration, values of NDFD were greater for all hay bales produced using enzyme-based treatments. The increase was generally greater after 24 h than 48 h of in vitro rumen fluid incubation. This finding is in accordance with previous studies, which reported that the greatest effects of fibrolytic enzyme treatments are observed during the early stages of in vitro fermentation (12–18 h; Colombatto et al., 2003; Dean et al., 2008). Similarly, ADFD was greater in ECO-treated bales than untreated hay after 24-h incubation, but no effect was observed after 48 h.
Despite minimal or no effect of using ECO alone on the chemical composition of forage, accumulated heating during the storage period or DMD when compared to untreated hay, this treatment promoted an increase in NDFD after both 24- and 48-h incubations and an increase in ADFD after 24 h of incubation, when compared to untreated hay. The increase in NDFD and ADFD indicates that the long period of contact between ECO and the forage during storage may have allowed for increased access for rumen microorganisms to digestible components of the cell wall. Previous studies have reported that improvements in NDFD of fibrolytic enzyme-treated forages are generally only partially due to the direct breakdown of hemicellulose and cellulose to easily fermentable substrates (Nsereko et al., 2000). Therefore, enzyme application may also improve fibre digestibility through other actions, such as the alteration of the cell-wall structural matrix to allow for greater attachment of microorganisms in the rumen, or further symbiotic effects with rumen microorganisms (Nsereko et al., 2000; Colombatto et al., 2003). In the present study, the contrasting effects of DYC and ECO applied alone on the chemical composition, and DMD of alfalfa hay highlights the variation that occurs between products. These treatment application rates were standardized to apply the same rate of endoglucanase to forage, which typically accounts for the greatest effect on NDFD (Eun and Beauchemin, 2007, 2008); however, the more negative effect of DYC on the nutritive value of hay after storage indicates that other factors play a significant role in the mode of action of these products.
The positive effects of ECO applied alone were in contrast to the effects of ECO applied in combination with 11GFT. The use of 11GFT in these trials was investigated primarily to determine whether the ferulic acid esterase-producing ability of 11GFT could positively complement the fibrolytic enzyme products by increasing access to structural carbohydrates. However, while ECO applied alone had little effect on the hay nutritive value during the storage period compared with untreated bales, the large increases in contents of NDF and ADF, and the accumulated heat during storage of bales using ECO plus 11GFT indicate that use of the inoculant had a high synergistic, and generally negative, effect with the enzyme. Despite this, improvements in NDFD were still observed compared with the untreated bales. While use of 11GFT alone on alfalfa hay was not investigated in this study, previous studies have indicated that Lactobacillus-based bacterial additives by themselves have little or no effect on hay preservation and storage (Deetz et al., 1989; Emanuele et al., 1992; Bass et al., 2012). Similarly, no effects of inoculant use were seen in the present study on the conservation characteristics of hay storage, likely owing to the high DM content and relatively aerobic nature of the storage. While the mode of action of this synergistic effect should be studied further, results for the present study indicate that the use of a ferulic acid esterase-producing inoculant in unfavourable storage circumstances for alfalfa hay may represent a waste of resources in addition to potentially contributing to a negative effect on the nutritive value of the preserved hay.
In conclusion, the use of exogenous fibrolytic enzymes applied at baling to alfalfa hay improved NDFD, despite either negative or no effects on the chemical composition, DMD or aerobic stability of the forage. Furthermore, when improved NDFD was achieved with minimal alteration of chemical composition during storage, as observed when ECO was applied alone, improvements in the efficiency of the livestock production system may be achieved. Variation in mode of action can occur depending on enzyme product used, which highlights the importance of ensuring the compatibility of the product with the forage being preserved prior to use. The use of a ferulic acid esterase-producing inoculant in combination with fibrolytic enzymes either had little effect or had a negative effect on the nutritive value of hay after storage, and did not improve NDFD compared with enzyme products applied alone.