Evaluation of n-alkanes and their carbon isotope enrichments (d13C) as diet composition markers

Plant cuticular n-alkanes have been successfully used as markers to estimate diet composition and intake of grazing herbivores. However, additional markers may be required under grazing conditions in botanically diverse vegetation. This study was conducted to describe the n-alkane profiles and the carbon isotope enrichment of n-alkanes of common plant species from the Mid Rift Valley rangelands of Ethiopia, and evaluate their potential use as nutritional markers. A total of 23 plant species were collected and analysed for long-chain n-alkanes ranging from heptacosane to hexatriacontane (C27 to C36), as well as their carbon isotopic ratio (13C/12C). The analysis was conducted by gas chromatography/combustion isotope ratio mass spectrometry following saponification, extraction and purification. The isotopic composition of the n-alkanes is reported in the delta notation (d13C) relative to the Vienna Pee Dee Belemnite standard. The dominant n-alkanes in the species were C31 (mean ± s.d., 283 ± 246 mg/kg dry matter) and C33 (149 ± 98 mg/kg dry matter). The carbon isotopic enrichment of the n-alkanes ranged from -19.37‰ to -37.40‰. Principal component analysis was used to examine interspecies differences based on n-alkane profiles and the carbon isotopic enrichments of individual n-alkanes. Large variability among the pasture species was observed. The first three principal components explained most of the interspecies variances. Comparison of the principal component scores using orthogonal procrustes rotation indicated that about 0.84 of the interspecies variances explained by the two types of data sets were independent of each other, suggesting that the use of a combination of the two markers can improve diet composition estimations. It was concluded that, while the n-alkane profile of the pasture species remains a useful marker for use in the study region, the d13C values of n-alkanes can provide additional information in discriminating diet components of grazing animal

Nutrition of grazing cattle in the Mid Rift Valley of Ethiopia: use of an improved n-alkane method to estimate nutrient intake

Nutrient intake is an important factor that determines the performance of production animals. In free ranging animals, direct measurement of nutrient intake is difficult to conduct, and it is frequently estimated indirectly by the aid of markers. The aim of this thesis was to investigate the potential of using cuticular n-alkanes and their carbon isotope enrichments (δ13C) as markers to study the nutritional ecology of grazing animals under tropical conditions. In addition, this improved method was used to determine the seasonal patterns of nutrient intake and diet composition of grazing cattle in the Mid Rift Valley grasslands of Ethiopia. The first focus of the thesis was to quantify the interspecies variability in the n-alkane profile and δ13C values of alkanes among commonly available pasture species in the Mid Rift Valley of Ethiopia. The analysis showed that the variability is sufficiently large to allow n-alkane and their δ13C values to be used as diet composition markers, with a combined use of the two increasing the discriminatory power. Faecal recovery of dosed and natural alkanes in cattle consuming low-quality tropical roughages was investigated in an indoor balance study. The recovery of synthetic alkanes dosed in the form of molasses boluses was considerably higher than adjacent natural odd-chain alkanes, and correction appears necessary when intake is estimated with the double n-alkane method. The next focus of the thesis was to generate information on the nutritive value of pasture species and nutritional status of grazing cattle in the region. Large variability was observed in the nutritive value and methane production potential of pasture species as evaluated in vitro, with scope for selection of genotypes with high nutritive value and low methane production potential for a sustainable pastureland management. The nutritional status of grazing cattle measured using a combination of n-alkanes, their δ13C values and visual observations showed that diet composition and nutrient intake of the animals is highly dependent on rainfall patterns, with a cyclic positive (wet period) and negative (dry period) energy and nutrient balance observed over the grazing seasons. Energy intake was more limiting than crude protein for body weight gain in most of the grazing seasons. While mature and non-producing animals appeared to tolerate nutritional restriction in the dry period and regain lost body condition in the following wet periods, young animals before the age of puberty may need supplementary feeding. Furthermore, concentrate supplementation of finishing animals needs to coincide with the onset of the wet season to take advantage of compensatory growth. In conclusion, the n-alkanes method coupled with isotope enrichment in n-alkanes and visual observations as used in the present study can provide realistic nutritional data for free-ranging cattle which correlates well with changes in body conditions. </p

Using n-alkanes and their carbon isotope enrichments (d13C) to estimate the botanical composition of pasture mixes from the Mid Rift Valley grasslands of Ethiopia

The present study was conducted to assess the accuracy of n-alkanes and their carbon isotope enrichment to estimate the diet composition of herbivores in the Mid Rift Valley rangelands of Ethiopia. Five common grass species which are abundantly available in the area were selected, from which several composite grass mixtures were prepared containing all the five species in varying proportions (from 0.05 to 0.35). The n-alkane profile and n-alkane isotopic ratio (13C/12C) of each grass species and composite grass mixtures were determined by gas chromatography/combustion isotope ratio mass spectrometry. The botanical composition of the composite mixtures was estimated using the n-alkane profile and n-alkane 13C/12C-ratio of individual species using least squares optimization and linear programming procedures and compared to the actual botanical mixture composition. Three alternative scenarios (inclusion of an additional 0, 5, or 10 of species in addition to those that made up the mixes) and two options where additional botanical species were included were simulated. There was close alignment between estimated and measured botanical compositions with significant relationships (P <0.001). The percentage contribution of the species in the simulated pasture mixtures was accurately estimated when the five species making up the grass mixes were used as the only inputs in the calculation. However, when additional botanical species were introduced, the accuracy declined with a significant increase (P = 0.003) in the mean square error of the prediction. The type of species in the extra inputs did not influence the results. In all scenarios, the combined use of n-alkanes and their d13C values improved the linear relationship and reduced (P = 0.002) the mean square error between estimated and measured botanical compositions. The best fit equation (R2 = 0.996, P <0.001) was obtained when n-alkanes and their d13Cvalues were used together and no extra species was included as input in the calculation. The present study shows that a high degree of accuracy can be obtained in estimating the botanical composition of grass mixtures using n-alkanes composition and n-alkanes 13C/12C-ratio. It is important to increase the number of markers used or limit the number of potential diet components to improve the quality of prediction

Estimation of feed intake and digestibility in cattle consuming quality tropical roughage diets using molasses-based n-alkane boluses

A feeding experiment was conducted to measure the faecal recovery rates of n-alkanes and to evaluate molasses-based alkane boluses for feed intake and digestibility estimations in cattle consuming low-quality tropical roughages. The experiment was performed in a cross-over design with four experimental diets, four 21-day feeding runs and eight bulls. The animals received a measured amount of the experimental diets that resulted in little refusal throughout the experiment. After seven days of adaptation, the animals were dosed with molasses-based alkane boluses (each containing 200 g C32 and 150 g C36) twice daily at 07:00 and 18:00 h. Concurrent with the alkane dosing, faecal spot samples were taken twice daily until the end of each run. In addition, total faecal collections were performed over the last 5 days of each run. The mean faecal recovery rate of both natural and dosed n-alkanes ranged between 0.61 and 0.86, with the recovery showing an upward trend with increasing carbon-chain length. The recovery rate of dosed alkanes was considerably higher than that of adjacent odd-chain alkanes. Whilst diets did not differ (P=0.23) in the recovery of even-chain n-alkanes, an effect of diet (P=0.01) was observed in the recovery of odd-chain n-alkanes. The faecal concentration of dosed alkanes reached equilibrium 3.30 days into the alkane dosing. On the assumption of similar faecal recovery of adjacent n-alkanes, intake was underestimated by 12% (