In vitro study of pomegranate by-products on methane production

Methane eructated from ruminants is considered to be one of the most important contributors to global warming, imposing an environmental burden that cannot be ignored. Meanwhile, it represents a loss of 2% to 15% of the gross energy intake (Johnson and Johnson 1995). Animal nutritionists have been studying manipulation of the rumen microbial ecosystem to reduce methane emission without the adverse effects on rumen function. There is a need to identify feed additives to modify ruminal fermentation characteristics and increase the efficiency of feed utilization, thereby inhibiting the ruminal methanogenesis. In recent years, essential oils (Benchaar 2007), plant secondary metabolites such as condensed tannins and saponins (Pen et al., 2006; Bhatta etal, 2009) and dietary lipids (Dohme et al., 2001) have arisen as attractive rumen modifiers to improve rumen microbial metabolism as well as inhibit methane production in ruminants. Particularly, Woodward et al. (2006) showed a positive effect of fish oil (FO) on reducing CH4 emissions in a short-term study, whereas no reduction was observed for a longer-term study. The positive effects of tannin-rich plant extracts on methane emission and methanogens population in vitro as well as in vivo have been examined (Patra et al., 2006; Patra and Saxena, 2010; Becker et al., 2014). The objective of this study was to evaluate the effect of pomegranate by-products (pomegranate oil and peel) on the in vitro rumen fermentation with respect to methane emissions. Basic substrate was contained of 600 g/kg forage and 400 g/kg concentrate. Pomegranate oil was administered in doses of 2, 4, and 6% of substrate DM in the first trial and in the second trial powder peel pomegranate was added in doses of 2.5, 5, and 7.5% of substrate DM. Syringes were inoculated with 40 ml of a rumen fluid–buffer mix (10 ml rumen fluid and 30 ml buffer) and were incubated at 39°C. The gas production was measured at different incubation times and total gas production, CO2, hydrogen, and methane production were determined after 24h of incubation. Data were analyzed by GLM procedure of SAS (2001) software. The results of the first experiment showed that the addition of pomegranate oil to substrate did not affect total gas production, digestibility parameters (b and c), metabolizable energy (ME) and organic materails dry (OMD). Methane production was decreased by addition of 6% of pomegranate oil (P< 0.05), but not by lower levels of oil. Lipids significantly decreased CH4 emission in the short-term study (−27%), but this effect was not observed after 11 wk of lipid supplementation. On the other hand, several in vitro trials have shown that FO reduced CH4 methanogenesis (Fievez et al. 2003; Patra and Yu 2013). Saturated medium chain fatty acids, C10-C14, also lead to methane reduction. At ruminal temperature, an increasing chain length of medium chain fatty acids seems to reduce their efficiency in inhibiting methanogens and methane formation due to lower solubility (Bucher et al. 2008; Patra 2012). Beauchemin et al. (2008) reviewed the practical application of lipids to reduce methanogenesis. Oil supplementation to diet decreased methane emission by 80% in vitro (Fievez et al. 2003) and about 25% in vivo (Machmu¨ller et al. 2000). The toxic effects of certain oils on rumen protozoa contributed to reduce methane production (Machmu¨ller et al., 1998). The addition of canola oil at 0%, 3.5% or 7% to the diets of sheep reduced the number of rumen protozoa by 88–97% (Machmu¨ller et al. 1998). The detrimental impact of unsaturated fatty acids has also been reported (Henderson1973). Machmulleur et al. (1998) observed coconut oil as more effective inhibitor followed by rapeseed, sunflower seed, and linseed oil. Coconut oil comprises medium chain fatty acids. Dong et al. (1997) compared canola oil to coconut oil and demonstrated coconut oil as more effective methane inhibitor. Coconut oil controlled rumen methanogens by changing the metabolic activity and composition (Machmu¨ller et al., 2003). The inclusion of sunflower oil to the diet of cattle resulted in 22% decrease of methane emissions (McGinn et al., 2004). In the second experiment, adding of 5 and 7.5% of pomegranate peel to substrate DM reduced (P <0.05) total gas production after 24, 72, and 96 h of incubation. Metabolizable energy, b fraction, and OMD digestibility reduced by pomegranate peel powder. Because these parameters dependent the total gas production. Methane production was reduced by adding pomegranate peel to diet, but no difference was observed between methane produced by diet contained different doses of pomegranate peel. However, a direct effect of condensed tannins on rumen methanogens cannot totally be excluded (Field et al. 1989). Furthermore, tannins decrease the degradation of nutrients in the rumen, which then may be degraded in the hindgut. This could have contributed to a lower methane emission, because hindgut fermentation differs from ruminal fermentation by resulting in a lower methane production per unit of fermented nutrients (Fievez, et al. 1999). A meta-analysis of in vivo experiments with tannins by Jayanegara et al. (2012) reported a relatively close relationship between dietary tannin concentration and CH4 production per unit of digestible OM. According to Goel and Makkar (2012), the antimethanogenic effect of tannins depends on the dietary concentration and is positively related to the number of hydroxyl groups in their structure. These authors concluded that hydrolyzable tannins tend to act by directly inhibiting rumen methanogens, whereas the effect of condensed tannins on CH4 production is more through inhibition of fiber digestion. They also pointed out that more animal research is needed with these compounds to establish their antimethanogenic effect. Hydrolysable tannins are hydrolyzed in the rumen and some could be toxic (Lowry et al., 1996; McSweeney et al. 2003). Addition of pomegranate oil and pomegranate peel powder to base substrate did not have a significant effect on digestibility in in vitro conditions, but reduce the production of methane gas as a harmful greenhouse gas in the atmosphere, that would loss significant portion of the energy of the feed. Therefore, the use of pomegranate oil and pomegranate peel in the present experiment as inexpensive edible compounds not only have no negative effects on feeding ruminants, but also a good alternative to ionophors and other antigens.