Equipos de espectrometría de masas tipo MS n y MS/MS

Comparisons between the forages evaluated by maceration (Chapter 4), in association with N fertiliser application rate (Chapter 5), and ingestive mastication by dairy cows (Chapter 6) showed that the amount of CP released during comminution (by mechanical maceration or ingestive mastication) differs widely between forage species (0.2 – 13.7 g/100 g of DM). This finding agrees with previous studies (Burke, 2004; Acosta et al., 2007) and enables the acceptance of the null hypothesis that nutrient release differs between forages (section 1.2). In general, the least amount of CP released (g/100 g DM) during comminution was from plantain herbage and the greatest from lucerne (equating up to a 15-fold variation in release). Investigation into the characteristics of herbage that effect CP release during maceration, in the forage comparison study, revealed that the characteristics influencing CP release also differ between forages. There was no single common characteristic able to explain CP or fermentable CHO (measured as water-soluble carbohydrate, WSC) release across all forages. While moisture content of herbage was positively associated with CP and WSC release across forages, comparisons within forages showed a negative relationship in some forages, and positive in others. Contrary associations, like this one, have been reported elsewhere in the literature (Bryant, 1964; Lees et al., 1981; Boudon and Peyraud, 2001; Acosta et al., 2007), and attempts to determine an equation to attribute combinations of characters influencing CP release using multi-variate analysis were unsuccessful. It is suggested that the characteristics of the forages selected for this study, being a range of dicot and monocot species, were too dissimilar to elucidate the main drivers across species and future studies might consider comparing more similar forages. Therefore, under the context of the present study we cannot fully accept our second hypothesis that relationships exist between certain herbage characteristics and nutrient release. Further, those characteristics that influence nutrient release from one species cannot be confidently extrapolated to another.

Maceration of the five forages (Chapter 4), however, did indicate that species that contained the greatest concentrations of CP and NPN also tended to release the most CP during mechanical maceration. These data were combined data from the N fertiliser study (Chapter 5) and a curvilinear relationship was established between the proportion of CP release and herbage CP (R2 _{= 0.67; Figure 8.1) and note the}

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Figure 8.1 The correlation (R2) between the proportion of crude protein (CP) released and herbage CP concentration (g/ 100g DM) from five species. Equation: y = -0.0011x2 _{+ 0.0656x –}

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Release of WSCs also varied between species (range 6.2 – 20.8 g/100 g DM), but was more extensive than observed for N, where at least 85% of WSC were released during mechanical maceration, irrespective of forage species. As with N release, no single common herbage characteristic was strongly correlated with WSC release (section 4.3.5), in part because of the high release of WSC from all forages. Within forages, however, some relationships were observed. For example, WSC release was moderately positively associated with WSC concentration in lucerne, plantain and ryegrass herbage (R2 _{0.47 – 0.65,}

Table 4.7). Herbage toughness of ryegrass, and leaf strength of lucerne and plantain was positively associated with WSC release, suggesting that, at least for these species, the tougher or stronger the herbage is, the more cells will rupture during maceration resulting in greater release of WSC.

The observation in that WSC is more extensively released than CP (Chapters 4 and 5), supports findings reported elsewhere (Boudon and Peyraud, 2001; Boudon et al., 2006; Acosta et al., 2007). Those authors concluded that the relative difference in release was associated with the relative size of WSC and N molecules. Water-structural carbohydrates tend to be smaller molecules compared to large protein molecules, such as those associated with photosynthesis. The disparity in release of CP vs. WSC

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suggests that cell rupture during comminution is insufficient to allow all CP to escape. Furthermore, the immediately released CP is likely to be mainly comprised of the smaller and readily fermentable NPN molecules which would have implications on the rate of ruminal ammonia production and thus potentially NUE (Trevaskis, 2003; Hall and Huntington, 2008).

Tamminga et al. (1994) suggested a ratio of available CHO to N of ~ 5:1 for optimal microbial protein synthesis. Comparing the amount of WSC released with CP released from herbage of the five forages in this study showed that the release of CP exceeded WSC release from lucerne, but release from ryegrass and clover were below the ratio, whereas that from chicory is nearer to the optima and plantain was well above (ratio 17:1). These results may explain why the predicted (Chapter 7) rumen ammonia concentrations of dairy cows grazing lucerne and ryegrass were greater than that from cows consuming ryegrass, and similar observations from experiments feeding cows diets of lucerne and ryegrass despite the difference in total CP concentration of the diets being small (Waghorn et al, in press). Similarly, observations of very low rumen ammonia concentration and urine N concentration (Minneé et al., 2017) of cows fed plantain may also be explained by the findings of this study that demonstrated a low quantity of N released from plantain herbage compared to other forage, and the high ratio of NSC: N release.

Species differ in the amount of CP and WSC that is released from herbage during comminution. The quantity of nutrient released, the variation in the ratio of CP and WSC released, and the forms of N released (protein vs. NPN) will affect the efficiency of microbial protein synthesis, and thus NUE. Knowledge of nutrient release can aid understanding of why differences in NUE occur between forage that have similar concentration of CP in herbage.