Evaluation of volatile fatty acid stoichiometry models to predict enteric methane production in lactating Holstein cows

Introduction Dietary supplements of sunflower oil (SO) in combination with marine lipids (fish oil or marine algae; MA) are known to increase the concentration of potentially beneficial bioactive lipids, including cis-9, trans-11 conjugated linoleic acid in ovine milk (Toral et al. 2010a).Changes in milk fat composition are related to diet-induced alterations in ruminal biohydrogenation (BH) of unsaturated fatty acids (FA) and associated ruminal microbiota.Inclusion of SO with fish oil also promotes the accumulation of oxygenated 18-carbon saturated FA in ruminal digesta in sheep (Toral et al. 2010b).However, the bacterial species responsible for the formation and accumulation of keto and hydroxyl FA in the rumen are not well characterised.In the current study, ruminal fluid from lactating ewes fed a diet supplemented with SO plus incremental amounts of MA was used to investigate the association between the formation and accumulation of 18carbon saturated keto FA and the variations in the rumen bacterial community. Materials and methodsTwenty-five lactating Assaf ewes were allocated to 5 experimental treatments: a total mixed ration (TMR) without lipid supplementation (Control diet) or supplemented with 25 g SO/kg DM plus 0, 8, 16 or 24 g MA/kg DM (SO, SOMA 1 , SOMA 2 and SOMA 3 diets, respectively).After 28 days on treatments, and 3 hours after removing the morning meal, samples of ruminal fluid were collected from each animal using a stomach tube.Aliquots of ruminal fluid for DNA extraction and FA determination were immediately frozen at -80ºC, freeze-dried and stored at -80ºC.Ruminal fluid FA composition was determined by GC and HPLC (Toral et al., 2010b).Microbial DNA extraction and terminal restriction fragment length polymorphism (T-RFLP) analysis of the total bacterial community were conducted as reported previously (Belenguer et al. 2010).Data were analysed by one-way ANOVA, with the MIXED procedure of the SAS software package Version 9.1 (SAS Inst.Inc., Cary, NC, USA).Results Four 18-carbon keto acids were detected in ruminal fluid (9-, 10-, 13-and 15-O-18:0).Relative abundance of all 18-carbon keto acids was low in ruminal fluid for the Control and SO treatments.However, inclusion of MA in the diet increased the accumulation of 10-O-18:0 in ruminal fluid of up to 3% of total FA (Figure 1A).The T-RFLP analysis revealed that certain fragments (T-RF) compatible with Quinella-like bacteria were present at a higher relative frequency in ruminal fluid of sheep on SOMA treatments after HhaI (98 bp) or MspI (150 plus 268 bp) digestions (Figure 1B).A concomitant accumulation of 10-O-18:0 and increased proportion of Quinella-like bacteria in ruminal fluid was also observed in ewes fed diets containing SO plus fish oil (Belenguer et al. 2010; Toral et al. 2010b).

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