The use of exogenous enzymes and essential oils to enhance ruminal feed degradation and reduce methane production in vitro

Abstract

Improving production efficiency and reducing the environmental impact of ruminant production systems would enhance the sustainability and global competitiveness of the livestock sector. Therefore, increasingly more emphasis is being put on climate warming threats and the intensification of livestock production systems in modern livestock practices. Greater awareness is placed on improving the utilization of conventional and non-conventional feedstuffs. In addition, the potential improvements that pure and mixtures of exogenous enzymes alone and in combination with essential oils (EOs) might have on rumen fermentation characteristics and reduction of enteric methane need to be studied in ruminants. In this regard identifying effective supplementation programmes for specific livestock rations is a step in the right direction. This study assessed several feed additives in order to produce the most promising ratio between methane emissions and degradable organic matter. The first experiment in this study aimed to determine the effect of treating three substrates with graded levels of cellulase PLUS (cellulase), xylanase PLUS (xylanase) and a mixture (1 : 1 wt wt-1 mixture of cellulase and xylanase) on in vitro gas production (ivGP). The substrates were pre-treated for 24 hours with these three exogenous fibrolytic enzymes (EFEs) at five levels of application, namely 0, 0.5, 1, 1.5 and 2 ml g-1 DM, in order to identify the optimum level of application that improves ruminal degradation of these substrates. In the first experiment, the addition of EFEs showed a tendency towards significantly improved ivGP throughout the incubation period and across all three substrates (P < 0.05). The optimal dose was determined as 1.5 ml g-1 DM and this was used in the subsequent in vitro experiments. This experiment was followed by evaluating the influence of exogenous enzyme blends (fibrolytic and proteolytic) on ivGP, in vitro organic matter degradability (ivOMD) and in vitro methane production (ivCH4). A factorial treatment combination of fibrolytic enzymes, namely control; xylanase at 1.5 ml g-1 DM (xylanase); cellulase at 1.5 ml g-1 DM (cellulase); and a 1 : 1 cellulase-xylanase mixture at 1.5 ml g-1 DM (mixture) and protease enzyme (0 and 0.5 ml g-1 DM substrate (protease)) were assessed using two substrates, that is, Eragrostis curvula and sunflower oilcake meal. The second experiment complemented the findings of the first experiment in that fibrolytic enzymes may generally increase ivGP. However, the addition of protease improved only ivGP of sunflower oilcake meal (P < 0.05). Fibrolytic enzymes improved ivOMD of sunflower oilcake meal, whilst the addition of protease improved the ivOMD of Eragrostis curvula (P < 0.05). The addition of fibrolytic enzymes increased ivCH4, but not throughout the incubation period (P < 0.05). The addition of protease reduced ivCH4 production of Eragrostis curvula at some incubation intervals (P < 0.05). In contrast, the addition of fibrolytic enzyme increased ivCH4 ivGP-1 and ivCH4 ivOMD-1 for Eragrostis curvula substrate (P < 0.05). The final experiment, namely the influence of various EOs in combination with fibrolytic enzymes on ivGP, ivCH4 and ivOMD after 48 h of incubation of Eragrostis curvula, was investigated. The treatments included control with no enzyme and no EOs (T1); a 1 : 1 cellulase-xylanase mixture at 1.5 ml g-1 DM (enzyme) (T2); and a combination of this enzyme treatment and the EOs (Next Enhance®) (a commercial product that has 43 : 3.85 wt wt-1 of cinnamaldehyde and diallyldisulphide + diallyltrisulphide) (T3); cinnamon (T4); garlic (T5); and a blend of cinnamon and garlic oil (T6). These treatments were assessed in an ivGP study using Eragrostis curvula hay as a substrate. The third experiment also indicated that the addition of fibrolytic enzyme could increase ivGP and ivCH4 and to some extent showed a tendency to increase the ivOMD (P < 0.05) of the Eragrostis curvula substrate. Among the EOs evaluated, garlic and a combination of CinnGar with enzyme reduced ivGP compared with the enzyme treatment alone (P < 0.05). Furthermore, all of the EOs in combination with the enzyme treatment showed a tendency to reduce enteric methane production during the early stages of the incubation period compared with the enzyme treatment alone (P < 0.08). CinnGar in combination with enzyme reduced ivCH4 throughout the incubation period (P < 0.05). Furthermore, a clear tendency was noted for the reduction in ivCH4 and ivGP ivOMD-1 for the garlic and CinnGar treatments (P < 0.095). Based on the results of this study, in vivo evaluation of the mixture enzyme treatment in combination with the CinnGar treatment is recommended to verify the repeatability of the results. Further studies on the use of these exogenous enzyme and EOs combinations under different conditions (rumen fluid donors, ruminal pH, substrate, dose levels, etc.) should also be conducted in order to attain a more informed recommendation on their efficacy. Following successful screenings, the best additives should be validated in in vivo trials.