Role of ammonia and biogenic amines in intake of grass silage by ruminants

In Northern- and Western-Europe, grass silage is a major component in winter feeding rations for ruminants. The intake of ensiled grass is often lower than the intake of hay or the fresh grass of similar digestibility. This intake depression is attributed to the fermentation products present in the silage. These include organic acids (lactic acid, volatile fatty acids) and N-containing fermentation products (ammonia (NH 3 ) and amines). The impact of N-containing fermentation products, notably amines, in lowering silage intake is suggested, because of their physiological activity in intermediary metabolism.Chapter 2 reviews the impact of fermentation in silage on silage dry matter intake and the controlling mechanisms involved. More extensively fermented silage shows lower intake when compared with hay or fresh forage from the same sward, especially when high amounts of volatile fatty acids and NH 3 are present. According to literature, this lower intake is mainly due to impaired palatability and earlier satiation induced by chemostatic control mechanisms at rumen level or in the intermediary metabolism. With silage rations, rumen fill is not a factor controlling intake.Supra-physiological doses of individual fermentation products (acids and NH 3 ) added to the diet, or intra-ruminally infused depress intake. No intake depression is observed at physiological doses. Only few studies have been carried out on the impact of amines on the control of silage intake.In Chapter 3 the type and amounts of amines formed in grass silage in relation to the rate of fermentation were investigated. Silages were made from direct cut and wilted perennial ryegrass in laboratory silos. Differences in the rate of fermentation were induced by addition of molasses, cell wall degrading enzymes and inoculation with lactic acid bacteria. Fermentation was restricted by formic acid, while the influence of enterobacteria on the formation of amines was determined by inoculation with a strain identified as belonging to the species Enterobacter sakazakii. This strain was isolated from silage. The total amine content of fresh grass was low (0. 1 - 0. 2 g/kg DM). The silages, of good quality, contained considerable amounts of amines, ranging from 0. 1 g/kg DM in the wilted and formic acid treated silages to 7.4 g/kg DM in a low DM, untreated silage. Irrespective of the total amount of amines in the silage, tyramine, cadaverine, putrescine and histamine were, in descending order, the principal biogenic amines present. The amines were mainly formed during the first 10 d of fermentation, and formation was highest in silages with a slow acidification rate. In this study, no additional amines were formed upon inoculation with the enterobacterium. The amount of amines in the silages correlated with amounts of NH 3 , indicating amine formation being related to overall protein degradation.The amines predominantly present in silage (Chapter 3), and NH 3 were subsequently tested for their influence on silage intake in sheep (Chapter 4) and dairy cows (Chapter 5). A mixture of amines, similar in composition to the amines found in silage at a total rate of 2.8 g/kg DM and NH 3 (2.9 g/kg DM) were added to a formic acid treated silage of good quality. These treatments were compared to the unsupplemented silage and to the same grass ensiled untreated. In the sheep and dairy cows, intake of the untreated silage was lower than the formic acid treated silage. The lower daily intake mainly resulted from the lower intake during the principal meal after feeding, due to palatability effects and earlier satiation. Addition of NH3 altered neither daily intake nor intake behaviour in sheep and cows. The amines added, tended to depress daily intake in the sheep, but not in cows. The slightly lower intake resulted from lower intake rates during both the principal meal and the subsequent small meals. The depressed intake rate was attributed to reduced palatability as indicated by the depressed initial intake rate during the principal meal. In sheep, no treatment effects were observed on total rumen pool size, DM and neutral-detergent fibre content, and rumen motility. These variables were not measured in cows. Furthermore, NH 3 and amines did not alter rumen pH, NH 3 and volatile fatty acid concentrations in the sheep and cows. Amine addition, however, increased slightly the amount of rumen fluid in sheep, which was concomitant with the lower osmolality in the rumen. This suggested a diluting effect of amines, which was also observed to a slight extent in cows. For all treatments, in sheep as well as in cows, only traces of amines were present in the rumen.From these studies, was concluded that at the added concentrations, NH 3 and amines are not a direct cause of lower silage intake. The combination of amines, however, tended to have a negative effect on palatability.In Chapter 6, the degradation of histamine, tyramine, cadaverine and putrescine in the rumen contents was studied in vitro . A solution containing a mixture of the amines previously mentioned, was added to rumen content taken from sheep. These sheep were adapted to either a silage diet containing high amounts of amines, low amounts of amines, or to hay containing no amines. Subsequently, the rumen contents to which the amines had been added were incubated for 5 h. Amine addition increased NH 3 concentration in the rumen contents. The highest rate of NH 3 production was found in rumen contents of sheep adapted to high amounts of amines in the diet. Addition of amines did not affect gas production. After 5 h a part of the added amines was degraded in all types of rumen contents. Highest amine degradation took place in rumen content of sheep adapted to the amine rich diet. The amount of amines degraded agreed stoichiometrically with the amount of NH 3 produced, indicating that amines in the rumen are degraded by deamination. In general, the breakdown was highest for histamine, followed by tyramine, putrescine and cadaverine. These results indicate that in animals adapted to grass silage with high concentrations of biogenic amines, the accumulation of amines in the rumen is prevented by an increase of the amine-degrading capacity of the rumen microbiota.In Chapter 7, a comparison was made between effects of addition of amines (4.9 g/kg DM) and addition of a combination of amines (2.7 g/kg DM), NH 3 (3.0 g/kg DM) and y- aminobutyric acid (GABA; 5.0 g/kg DM) on intake in sheep. These products were again added to a formic acid treated silage of good quality. The combination of amines alone did not alter daily intake when compared to the unsupplemented control silage. The combination of amines, NH 3 and GABA slightly increased intake. The amines, however lowered slightly the intake rate of the principal meal and the average intake rate of the small meals. Nevertheless, rumen NH 3 content was the highest after the principal meal for the silage supplemented with the combination of amines, NH 3 and GABA. No effects of amines or the mixture of amines, NH 3 and GABA were observed on rumen pH, osmolality, rumen pool size and liquid content or flow rate. It is concluded that amines alone or amines combined with NH 3 and GABA in concentrations normally found in mediumto poor-quality silages do not affect intake by direct effects on chemostatic regulation. A slight negative effect of a-mines on silage palatability, however, cannot be excluded.Bearing in mind the adaptation of the rumen microorganisms to degrade dietary amines reported in Chapter 6, it is conceivable that during the first days of feeding an amine rich silage the amines accumulate in the rumen. They may than pass the rumen and cause a direct effect on intake through their activity in intermediary metabolism. In Chapter 8, this direct effect of amines was studied during a diet change-over experiment with a silage having a low amine content (1.4 g/kg DM) to the same silage supplemented with 7.2 9 amines/kg DM. In addition, the effect of preconditioning on the acute effect during the diet change-over was tested by previously feeding a silage containing 5.2 g naturally formed amines/kg DM. The first 4 d after the change-over, amines tended to lower daily intake and depressed significantly the intake during the principal meal. Preconditioning lowered this acute effect of amines. After 14 d, the sheep were adapted to the dietary amines. The rate of amine degradation in the rumen was increased, resulting in only traces of amines being present in the rumen. The daily pattern of intake, however, remained different with lower intake during the first 5 h after feeding. This confirmed the negative effect of amines on palatability.In Chapter 9 the microbiological background of the formation of biogenic amines in silage is discussed. Based on literature, it is likely that enterobacteria and heterofermentative lactic acid bacteria are responsible for the formation of amines in medium- to good-quality silages.This thesis allows to conclude that after adaptation, neither biogenic amines nor ammonia directly reduce intake by effects on chemostatic regulating mechanisms. However, a slight negative effect of amines on palatability is probable. So, amines only slightly influence intake pattern during the day, but without lowering total daily intake. The amines in silage, thus, appear to be one of the factors that has a slight, or possibly in extreme situations, a more pronounced negative effect on taste. Other factors with effect on intake include negative palatability by pH and acetic acid, or disbalance in nutrient supply at rumen or liver level. Although a possible negative effect on intake of each these factors is difficult to demonstrate and to quantify, together by cumulative effects they are likely to reduce silage intake.