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Mechanisms of CH4 production in the rumen
Most of the enteric CH4 produced by ruminants has its origin in the rumen (~90% vs. 10% at mean in the large intestine) (Murray et al., 1976). Microbial digestion of feed components in the rumen under anaerobic conditions results in the production of volatile fatty acids (VFA), mainly acetate, propionate and butyrate used by the animal as source of energy, and the production of gases (CO2 and CH4) eliminated through eructation (Murray et al., 1976; Boadi et al., 2004). Rumen microbes find the energy they need in the form of ATP through dehydrogenation reactions releasing hydrogen (H2) in the rumen. H2 produced is used by methanogenic archaea, a microbial group distinct from Eubacteria, to reduce CO2 into CH4 (Martin et al., 2010) according to the equation: CO2 + 4H2 → CH4 + 2 H2O. Methanogenesis is essential for an optimal performance of the rumen (McAllister and Newbold, 2008) by avoiding H2 accumulation which would lead to inhibition of dehydrogenase activity and cessation of fermentation (McAllister and Newbold, 2008). The amount of H2 produced in the rumen is highly dependent on composition of basal diet, nature of forage, chemical composition, on the organization of the microbial ecosystem, and on the residence time of feeds in the rumen. The production of VFA is not equivalent in terms of H2 output. For instance, the formation of propionate (propionogenesis) consumes H2 whereas the formation of acetate and butyrate results in a net release of H2 (Bannink et al., 2011). The quantity of CH4 produced per unit of fermented feed is proportional (CH4 = 0.45 acetate – 0.275 propionate + 0.40 butyrate) to the pattern of the VFA produced (Moss et al., 2000). Therefore, a lower acetate and butyrate production results in lower CH4 emission (and vice versa) while propionate formation serves as a competitive pathway for H2 use in the rumen.
Though VFA are the main driving variables for excess H2 and methanogenis, other factors of minor importance exist (Figure 1): the use of amino acids as nitrogen (N) source for microbial biomass synthesis results in net production of H2, whereas that of ammonia (NH3) induces a net consumption H2 (Bannink et al., 2011). Biohydrogenation of long chain unsaturated FA and some chemical compounds (e.g. sulphate, nitrate, ferric iron, manganese and fumarate) though quickly reduced and exhaust (McAllister and Newbold, 2008; van Zijderveld, 2011; Jeyanathan et al., 2014) also serve as alternative H2 sink.
Measurement of Methane Emission from Dairy Cattle
To properly evaluate mitigation strategies, it must be possible to quantify cattle emissions under diverse conditions. Various techniques used to measure CH4 emissions in vivo have been reported in literature. These include the use of gold standard reference method (respiration chambers; (Frankenfield, 2010)), SF6 tracer technique (Storm et al., 2012) and the Green-Feed (C-lock Inc., USA) system (Hammond et al., 2013) and the novel approaches such as laser CH4 detector (Chagunda, 2013). Their strengths and weaknesses have been reported by Storm et al. (2012) and Chagunda (2013). Generally, the high costs involved and the complexity of these techniques limit their applicability on a large scale (Table 1). Also it is important to examine critically the measurement methods used when evaluating mitigation dietary strategies.
Lipid metabolism in the mammary gland
In ruminants, FA secreted into milk have double origins: they are derived from either preformed FA from peripheral circulation (from adipose tissue mobilization or dietary origin) or FA synthesis in mammary secretory epithelial cells also known as de novo synthesis (Shingfield et al., 2010; Bauman et al., 2011). Mammary epithelial cells use acetate and β-hydroxybutyrate as substrates for de novo synthesis of even saturated FA (from 4 to 16 carbons) (Månsson, 2008). All of C4:0 to C12:0, about 95% of C14:0 and about half of C16:0 secreted in milk emanate from de novo FA synthesis whereas the remaining C16:0 and longer chain fatty acids (≥ 18 atoms of carbon) are derived from peripheral circulation (Chilliard et al., 2000).
The triglycerides (TAG) are transported to the mammary gland into chylomicrons and very low-density lipoproteins (VLDL) through the blood (Shingfield et al., 2010). Within the capillary wall in the mammary gland, the enzyme lipoprotein lipase hydrolyses the VLDL TAG into glycerol and NEFA which are taken up by the mammary epithelial cell (Bauman et al., 2011). Saturated FA entering into mammary epithelial cells can be desaturated by stearyol-CoA desaturase (delta-9 desaturase) (Palmquist et al., 2005). Stearoyl and palmitoyl-CoA are the preferred substrates for delta-9 desaturase but C18:0 is the predominant precursor with OA as the corresponding product (Bernard et al., 2008). Also this desaturation is the principal source of cis-9 trans-11 Congugated LA (Rumenic acid, RA) in milk (Mosley et al., 2006).
Furthermore, endogenous chain elongation of propionyl-CoA as precursor leads to the formation of C5:0, C7:0, C9:0, and C11:0 in milk and these add up to the odd-chain FA C13:0, C15:0 and C17:0 transferred from the duodenum (Fievez et al., 2012). These odd-chain FA can further be desaturated by delta-9 desaturase, but only the conversion of C17:0 to cis-9 C17:1 seems quantitatively important (Fievez et al., 2012).
Thus the different pathways (de novo FA synthesis, uptake from circulation of long chain FA and desaturation) contribute to the FA pool of milk (Chilliard et al., 2000).
Relationships between milk fatty acid profiles and CH4 production in the rumen
Chilliard et al. (2009) were the first authors to predict CH4 emissions (g/d) using individual milk FA concentrations. These authors relied on 32 paired observations derived from 8 dairy cows, used according to 4 x 4 Latin square design, and receiving 4 dietary treatments which consisted of a maize silage-based control diet and 3 diets supplemented with linseed under different forms (whole crude seed, extruded seed and oil) to evaluate relations between milk FA and measured CH4 emissions. Stepwise multiple regression analysis was performed to describe CH4 emissions from a combination of dietary intake components, milk yield and composition, and milk FA concentrations. Two high predictive equations were proposed and containing milk FA concentrations (cis-9 C14:1, C16:0, trans-16+cis-14 C18:1, and C18:2n-6) and forage intake (Table 2). The forage intake estimates the part of the organic matter fermented in the rumen that leads to the acetate-CH4 pathway. The milk C16:0 is a main de novo synthesised FA (acetate being the precursor). Moreover, trans-16+cis-14C18:1 is an intermediate of LA ruminal biohydrogenation, and C18:2n-6 is the main dietary FA of maize silage. However, the contribution of cis-9 C14:1 was rather unexpected (Chilliard et al., 2009). A second equation (equation 2, Table 2) was thus developed excluding the latter two FA, and this slightly decreased the R2 value of the prediction compared with the first equation (0.953 to 0.931).
Although Chilliard et al. (2009) concluded that the predictive equations established in their study are valid only for diets supplemented with linseed, Moate et al. (2014) using data from dairy cows that were fed grape marc (rich in PUFA; LA) supported the findings of Chilliard et al. (2009). They found a strong negative correlation (R²=0.63; equation 9 Table 2) between CH4 emissions and the concentration of milk total C18 (Moate et al., 2014). A combination of the data from Chilliard et al. (2009) and that of Moate et al. (2014) improved the R² value of total C18 (R² = 0.81). Moate et al. (2014) concluded that the findings of Chilliard et al. (2009) can as well be extended to diets containing grape marc a feedstuff rich in LA instead of LNA as occurs in linseed.
Mohammed et al. (2011) studied the relationships between CH4 emissions and milk FA concentrations from an experiment with 16 lactating Holstein cows offered a control diet with calcium salts of palm oil, and diets supplemented with sunflower seed, linseed and canola seed during four 28-day periods used according to a 4 x 4 Latin square design. Oilseeds were added to diets to provide 33g added fat /kg DM. Different predictive equations were developed from only milk FA concentrations (equation 4, Table 2), milk FA combined with rumen endodiniomorph concentrations (equation 5, Table 2) and milk FA combined with DM intake, and rumen endodiniomorph concentrations, (equation 6, Table 2). The authors found a negative relationship between CH4 and cis-9 C17:1, cis-11 C18:1 and sum of trans-C18:1 while a positive relationship was found between CH4 and trans, trans CLA and anteiso C15:0. The negative relationship between cis-9 C17:1 and CH4 were expected as it is a product from the desaturation of C17:0 (Microbial origin) whose precursor is propionate and propionogenesis is negatively related to CH4 production (Fievez et al., 2003). The inclusion of cis-11 C18:1, sum of trans-C18:1 and trans, trans CLA in the equation can also be ascribed to biohydrogenation intermediates of PUFA.
Coding of experiments
First, each publication was coded as “reference” with unique whole numbers from 1 to 15. In the case where a publication presented more than one experiment, a number was (1, 2, etc…) added in order to properly differentiate them.
A second level of encoding was to classify experiments into two categories according to lipid and non-lipid supplementation. The group “lipid” (Nexp = 11, Nt =24) consisted of experiments testing effects of various fatty acids (lauric acid, myristic acid, stearic acid) and fatty acid sources (algal meal, linseed, coconut oil, soya bean oil, sunflower seeds, flax seeds, cotton seeds and canola seeds ) on CH4 emission. The group “non-lipid” (Nexp = 6, Nt =17) consisted of experiments testing essential oils (Origanum vulgare leaves), chemical additives (monensin), yeast (Saccharomyces cerevisiae), and forage type (maize silage and grass silage). One experiment that tested grape marc containing both lipid and non-lipid compounds was included in both categories.
Experiments were further coded according to the classes of lipid and non-lipid supplements. The group “lipid-saturated fatty acids” (Nexp = 5, Nt =14) tested lauric acid, myristic acid, stearic acid, coconut oil, and a combination of lauric acid, myristic and linseed oil. The group “lipid-unsaturated fatty acids” (Nexp = 7, Nt =22) supplemented algal meal, linseed, soya bean oil, sunflower seeds, flax seeds, cotton seeds, canola seeds and grape marc. One experiment that tested a mixture of both saturated (lauric acid, myristic acid) and unsaturated fatty acid (linseed oil) in equal proportions was classified under both groups. Experiments under “non-lipid chemical” (Nexp = 3, Nt =7) tested chemical compounds (monensin and grape marc) whereas the group “non-lipid other” (Nexp = 3, Nt =10) consisted of experiments testing essential oils (Origanum vulgare leaves), yeast (Saccharomyces cerevisiae), and type of forage (maize silage and grass silage).
Meta-analysis
The data created was subsequently exported to Minitab (version 15) for analysis. In order to explore the relationships between CH4 and milk fatty acids, various approaches were taken. Three units of CH4 measurements (CH4 g/d, CH4 g/kg DMI and CH4 g/kg milk) were chosen for this. Firstly, global correlation was calculated using all treatments irrespective of the experiment. Secondly, correlations that were significant (P < 0.05) and with an absolute value of 0.4 and above (|-0.4| ≤ r ≤ |-1|) where kept for further analysis. Descriptive statistics of the remaining data were obtained and when the number of treatments is below 10, the data was discarded. Thirdly, with the remaining data, scatter plot diagrams with CH4 on the ordinate and individual milk FA concentration (% of milk FA) on abscissa were made. From the scatter plot, one experiment (Moate et al., 2013) was excluded as it presented a high leverage effect and then the above steps were repeated.
Table of contents :
OVERVIEW OF INRA
I. Background information on INRA
II. Missions of INRA
III. Clermont-Ferrand-Theix-Lyon Research Centre
IV. Herbivores Joint Research Unit
V. The team AGL
VI. The team DIMA
ABSTRACT
RÉSUMÉ
1. INTRODUCTION
2 LITERATURE REVIEW
2.1 Mechanisms of CH4 production in the rumen
2.2 Measurement of Methane Emission from Dairy Cattle
2.3 Lipid metabolism in the rumen
2.4 Lipid metabolism in the mammary gland
2.5 Relationships between milk fatty acid profiles and CH4 production in the rumen .
2.6 Objectives of Internship
3 MATERIALS AND METHODS
3.1 Selection of publications
3.2 Creating the data base
3.3 Data entry and verification
3.4 Coding of experiments
3.5 Meta-analysis
4 RESULTS
5 DISCUSSION
5.1 Milk branched – chain fatty acids (BCFA)
5.2 Milk C16:0
5.3 Intermediates of rumen biohydrogenation
6 CONCLUSION
Publications used for the meta-analysis
APPENDIX
