USE OF NATURAL ADDITIVES AS FEED SUPPLEMENTS TO REDUCE THE METHANE EMISSION FROM RUMINANTS

Since the ban of antibiotics as growth promoting feed additives by the European Union in 2006, plant extracts and plant secondary metabolites have been considered as an alternative to manipulate the rumen. The term ‘plant secondary metabolite’ is used to describe a vast array of chemical compounds in plants that are not involved in the biochemical processes of plant growth and reproduction. These have been studied and used in medicines and preservation of foods for a substantial time. The technology to assess methane emission in vivo is very slow, expensive, labour intensive and unsuitable for large-scale individual animal measurements. For this reason, a great deal of research has been conducted using in vitro simulation technique, which estimates the emission in vivo. However, the in vitro systems are often disapproved because of their short term duration (i.e. 24 hrs) and the lack of adaptation (they are conducted using rumen fluid which had not been exposed to the presence of the investigated compounds before). Based on this argument, this thesis aims to examine different aspects, regarding systems and procedures, used to investigate rumen fermentation and methane emission from ruminants, as follows: 1) Screening of different natural additives using a batch culture system; 2) Verify, through a batch culture system, the effect of the subministration in vivo of some natural additives in order to adapt the rumen fluid used as inoculum; 3) Asses the effect of one natural extract using a long-term continuous culture system; 4) Study the consequences on dairy products after the subministration of natural additives to lactating dairy cows. The first contribution aimed to explored effects of four pure plant extracts (allyl-sulfyde, cinnamaldehyde, eugenol, and limonene) and one synthetic compound (monensin), on in vitro rumen fermentation and methane (CH4) production of a commercial diet for dairy cows, using a batch culture system, equipped with devices for automated gas venting at fixed pressure. Two dosages were tested: 3 or 30 mg/g of diet for the plant extracts; 0.015 or 0.030 mg/g of diet for Monensin. All the additives were ineffective at the low dosage. The high dosage of limonene caused a marked depression of neutral detergent fiber degradability (NDFd) (-68%) and true dry matter degradability (TDMd) (-14%) compared to the control; less marked reductions were noted for high dosage of eugenol (-15% and -4%, for NDFd and TDMd, respectively) and of monensin (-16% and -3%, for NDFd and TDMd, respectively). No effects emerged for other additives on NDFd and TDMd, irrespective by the dosage. Compared to the control, high dosage of allyl sulfide, cinnamaldheyde, eugenol, limonene and monensin significantly reduced gas production (ml/g DM; -16%, -12%, -9%, -38%, -12%, respectively). In vitro CH4 production was significantly reduced only by high dosage of allyl sulfide, cinnamaldheyde, limonene and monensin (-32%, -12%, -43%, -18%, respectively, compared to the control). Only high dosage of allyl sulfide, and limonene significantly reduce CH4 proportion (-18%, -12% respectively, compared to the control). The most promising results were observed for the high dosage of cinnamaldehyde, that reduced in vitro CH4 production without compromising degradability and VFA production. Based on the results of the first trial, cinnamaldehyde, limonene and allyl-sulfide have been selected as the most effective in the reduction of methane emission, but also preserving the feed degradability and rumen functionality. The three compounds have been tested following a different procedure, indeed, the second trial aim was to evaluate the effect of rumen fluid which has not been adapted (NAF) and rumen fluid that has been adapted (AF) to the presence of the extract in vivo, before the in vitro tests. Therefore, four dry cows were fed diets with and without the pure compounds, according to a 4×4 Latin Square design, and the collected rumen fluids were used as in vitro inocula with or without the addition of the 3 compounds during the incubation. Cinnamaldehyde, limonene and allyl-sulfide have been tested for in vitro gas production kinetics and end products of fermentation. In general, the results confirmed the first additive performed, however, regarding the adaptation in vivo of the rumen fluid, the use of AF did not affect any fermentation parameter compared to NAF. However, adding an in vitro diet with allyl-sulfide, the magnitude of the effects of this pure compound tended to be greater when incubated with AF compared to NAF. These results suggest that the administration of pure compounds to the cows can affect the rumen microbial activity and the response of in vitro experiments. The third trial examined another category of plant secondary metabolites, flavonoids, which have recently gained interest because of their wide range of biological activities, but also because of their antimicrobial properties. Previously, it has been hypothesised that an extract of liquorice, rich in prenylated isoflavonoids and particularly glabridin, might potentially improve the efficiency of nitrogen utilisation and reduce methane production in the rumen. The experiment was carried out using a different in vitro system, the long-term rumen simulation technique (RuSimTec), which maintained the vitality and functionality of the rumen microbiome for long time and adapt the rumen fluid to the presence of the extract. The results obtained showed that when liquorice extract is added at 1 g/L, ammonia production decreased (-51%; P<0.001) without affecting the overall fermentation process. When added at 2 g/L, decreases in ammonia production (-77%; P<0.001), methane (-27%; P=0.039) and total VFA production (-15%; P=0.003) were observed. These effects in fermentation were probably related to decreases in protozoa numbers, a less diverse bacteria population as well as changes in the structure of both the bacterial and archaeal communities. The inclusion of an isoflavonoid-rich extract from liquorice in the diet may potentially improve the efficiency of the feed utilisation by ruminants. Many studies have investigated the use of the plant secondary metabolites in vitro, but very limited research have examined the consequences may derived from feeding dairy cows with the additives. In particular, the focus of the fourth contribution is the use of garlic and garlic second metabolite as feed additives, recently proposed as modulators to reduce the enteric methane emissions of ruminants. Since there is no evidence available, this study aimed to investigate the influence of garlic and allyl sulfide on dry matter intake (DMI), productive performances, milk coagulation properties, cheese yield, milk and cheese sensory profiles, and rheological characteristics. Four dairy cows were fed a total mixed ration either receiving 0 g/d (control) or supplemented with 100 or 400 g/d of garlic cloves or 2 g/d of Allyl sulfide in 4 consecutive experimental periods in a 4×4 Latin square design. Each experimental period consisted of 7 d of transition and 14 d of treatment. Milk samples were collected from each cow for chemical analysis and cheese-making. The organoleptic properties of the milk and 63 d ripened cheeses were assessed by a panel of 7 trained sensory evaluators. The experimental treatments had no effects on DMI, milk yield, feed efficiency (milk yield/DMI), milk coagulation properties, nutrient recovery and cheese yield. Garlic-like aroma, taste and flavour of milk and cheese were significantly influenced by the treatments, particularly the highest dose of garlic cloves, and we found close exponential relationships between milk and cheese for garlic-like aroma (R2 = 0.87) and garlic-like flavour (R2 =0.79). Allyl sulfide and 400 g/d of garlic cloves resulted in lower pH, shear force and shear work of ripened cheeses compared with the other treatments. Garlic cloves and Allyl sulfide had opposite effects on cheese colour indices. In conclusion, the addiction of 400 g/d of garlic to the feed of lactating dairy cows highly influences the sensory and rheological characteristics of cheese.