DNA barcoding approaches have been successfully applied for estimating diet composition. However, an accurate quantification in the diets of herbivores remains to be achieved. In the current study, we present a novel methodology that reveals the relationship between the actual proportions (by mass) of each herbage species in the diets and the relative proportions of the ITS2 gene sequences obtained from faecal samples to evaluate the diet composition of sheep in a meadow steppe. Nine common and twelve rare species of plants were employed for formulating six diets, along with the addition of feed supplements for improving the growth performance of sheep. Faecal samples were collected for DNA analysis over the period spanning days 7–12. A significant positive correlation (Spearman’s ρ = 0.389) was obtained between the actual proportions (by mass) of the herbage in the diet provided and the relative abundance of ITS2 sequences obtained from the faecal samples. A significant regression coefficient was found between the relative abundance of all common species and their respective herbage mass proportions. The accuracy of the relation equations, evaluated by utilizing the similarity coefficient, showed 84.69% similarity between the actual diet composition and the correct percentage. Taken together, the current study has provided empirical evidence for the accuracy and applicability of ITS2 as a DNA barcode for obtaining quantitative information about the diet composition of sheep grazing in species-rich grasslands.

In Europe, organic food must comply with specific regulations which do not include nutritional criteria. The ability of organic food to meet the nutritional needs of children is not assessed. This narrative review discusses the nutritional composition (macronutrients, micronutrients) of organic food compared to conventional products and its clinical relevance with a pediatric focus, and the health impact of these differences and of contaminants which interfere with metabolism. Other potential differences, particularly regarding the direct/indirect exposure to other contaminants in conventional food, are not addressed in this review. The composition of some organic food may differ from conventional food. Protein content was lower in cereals and eggs. A lower n-6:n-3 polyunsaturated fats (PUFA) ratio was observed in milk, meat and eggs. Long-chain PUFA and vitamin E may be higher in milk, meat, and fish, as some minerals and antioxidants (phenolic compounds, vitamin C) in fruits, vegetables, and starchy food and carotenoids in fruits and vegetables. Epidemiological studies suggest an association between organic diets and lower prevalence of childhood obesity, type 2 diabetes and metabolic syndrome, whereas the protective effect on allergy and cancer is controversial. Some organic food may have a better nutritional interest for children’s diet than conventional food. Standardized studies comparing food composition and diet in children are needed. Considering the lower toxicologic risk and the sustainability of organic food, the Committee on Nutrition encourages the use of organic food, provided such food is affordable, alongside specific baby food which are subject to strict specific EU regulations.

Free sugar intakes are currently higher than recommended for health, yet effective strategies for reducing consumption are yet to be elucidated. This work investigated the effects of different dietary recommendations for reducing free sugar intakes, on relevant outcomes, in UK adults consuming >5% total energy intake (TEI) from free sugars. Using a randomized controlled parallel-group design, 242 adults received nutrient-based (N=61), nutrient- and food-based (N=60), nutrient-, food- and food-substitution-based (N=63) or no (N=58) recommendations for reducing free sugars at a single timepoint, with effects assessed for the following 12 weeks. Primary outcomes were free sugar intakes as a percentage of TEI (%FS) and adherence to the recommendations at week 12. Secondary outcomes included TEI, diet composition, sugar-sweetened and low-calorie-sweetened food consumption and anthropometry. In Intention-to-Treat analyses adjusted for baseline measures, %FS reduced in intervention groups (%FSchange=-2.5 to -3.3%) compared to control (%FSchange=-1.2%) (smallest B=-0.573, p=0.03), with effects from week 1 until week 12, and no differences between interventions (largest B=0.352, p=0.42). No effects of intervention were found in dietary profiles, but change in %FS was associated with change in %TEI from non-sugar carbohydrate (B=0.141, p<0.01) and from protein (B=-0.171, p=0.02). Body weight was also lower at week 12 in intervention groups compared to control (B=-0.377, p<0.05), but associations with %FS were weak. Our findings demonstrate benefit from dietary recommendations for reducing free sugar intakes in UK adults. Limited advantages were found for the different dietary recommendations, but variety may offer individual choice.

Trial Registration: Clinicaltrials.gov: NCT04816955, registration date: 24.03.21.

When and what you eat can be linked to circadian preference (i.e., chronotype) and occupation (e.g., shift worker). Evening chronotypes, with a later circadian preference, tend to have meals later, distribute energy intake toward the end of the day(1), and more unhealthy eating habits than morning chronotypes(2); whereas night shift work is associated with later mealtimes and poor diet quality as a result of circadian disruption due to their work(3). What is unclear is whether chronotype influences the occupation-induced dietary patterns observed in shift workers. This study aimed to investigate associations between chronotype, temporal patterns of eating and diet composition in shift and non-shift workers. Adults from shift (SW) and non-shift (N-SW) populations were recruited. A Chrononutrition Questionnaire captured chronotype, duration of eating window (DEW), time of first eating occasion (FEO) and last eating occasion (LEO) while diet composition (energy, protein, total fat, saturated fat, carbohydrate, fibre, alcohol) was extracted from 7-day food diaries. Associations between chronotype and DEW/FEO/LEO, and between DEW/FEO/LEO and diet composition were determined by Spearman Rank Coefficients. 95 participants were enrolled (N-SW: n = 39; SW: n = 56); predominantly female (71%), morning chronotype (37%), on average 40.46 ± 15.08 years with BMI of 27.04 ± 5.77kg/m2. 84 returned food diaries. Later chronotype was positively associated with later times of FEO (N-SW: r = ,50, SW: r = ,69) and LEO (N-SW: r = ,63, SW: r = ,54) on free (non-work) days (p≤.002), and longer DEW (r = ,42) and later LEO (r = ,60) on workdays for non-shift workers (p<.01). However, there were no significant differences in diet composition by day/shift type between chronotypes across the study population. On afternoon shifts, longer DEW was associated with greater energy (r = ,60) and total fat intake (r = ,60) and later LEO with greater alcohol intake (r = ,59) (p<.05). On night shifts, a longer DEW was associated with lower alcohol intake (r=-.45, p<.05). Amongst non-shift workers, later FEO was associated with lower fibre intake on workdays (r=-.58, p<.001). Additionally, non-shift workers who were later chronotypes had later LEO, which on workdays associated with lower fibre (r=-.45) and alcohol intake (r=-.43); and on work-free days, associated with lower alcohol intake (r=-.45) (p<.05). Not surprisingly, evening chronotypes across the study population had longer and/or later eating windows on work-free days (i.e., free of constraints), as did non-shift workers on workdays, while the influence of chronotype on DEW, FEO, and LEO across shifts were less clear. Hence, for shift workers, occupation appeared to be a greater driver of temporal eating patterns than chronotype. Additionally, later eating times of evening chronotypes was not associated with negative diet composition. The exception was lower fibre intake amongst non-shift workers; but regardless of chronotype, shift workers may benefit from having a shorter and earlier DEW on afternoon shifts to minimise energy, fat, and alcohol intake.

Diaphus fulgens is a diel vertical migrant myctophid, occurring abundantly in the Kuroshio waters of the East China Sea. They are multiple batch spawners with a short interspawn period, resulting in high egg productivity. Multiple spawning would be supported by feeding during their spawning season; however, there is no information on the feeding habits of this species. The present study examined the diet composition, diel feeding periodicity and daily ration of mature adult D. fulgens collected at different periods of the day during February to March. The most prevalent item was appendicularians and their mucus houses, which dominated the stomach contents in both number and weight. Additionally, various species of copepods were predated, with a prevalence, by weight, of the large-sized Pleuromamma piseki. Diaphus fulgens feeds actively in the epipelagic layer at night and moves down to the mesopelagic layer in the early morning, and digests most of the consumed food by the late afternoon, showing a typical nocturnal feeding pattern. Based on diel changes in the stomach content index and instantaneous gastric evacuation rate estimated in this study (0.118–0.129 h−1), the daily ration was calculated to be 4.0–4.2% of body weight, equivalent to a daily caloric intake of 21.2–22.3 cal day−1. Since the caloric value to produce a batch of oocytes was estimated to be 8.2–11.5 cal day−1, on an energetic basis, D. fulgens can maintain the multiple spawning at a cost of approximately 37–54% of its daily caloric intake.

Feeding monogastric livestock in organic agriculture is challenging due to several tradeoffs between animal welfare aspects, resource efficiency, as well as ecological and social sustainability. Organic standards may even increase such conflicts, as is currently the case with upcoming new regulations regarding restrictions of feed sources for organic pigs in Europe. In order to contribute data for balancing reasons to minimize tradeoffs, we compared four different piglet diets, each targeted to reach a high protein quality by either a high proportion of soybean cake (SOY), inclusion of milk powder (MILK), fermentatively produced lysine (LYS) or conventional potato protein (POT). All diets were designed to meet the nutritional requirements of piglets in the best possible way, however they all represented different conflicts with either organic regulations or sustainability goals. In each of five consecutive runs, respectively three litters were assigned to every dietary treatment, resulting in 15 litters per treatment in total. In each litter, seven focus animals were defined. The piglets were studied from birth until 58 days of age. They were weaned at day 46 and sold from the farm at day 58. Piglets were individually weighed at an average age of 3, 21, 43, 50 and 58 days with simultaneous assessment of body condition score (BCS) and prevalence of diarrhea. Feed intake (FI) was recorded litter wise weekly, starting from week three. Feed conversion ratio (FCR) was calculated for the period after weaning. Statistical analysis was executed using linear mixed effect models. Regarding FI, FCR and daily weight gains, no treatment effect was found. Only at day 21, BCS was lower for piglets receiving POT. Prevalence of diarrhea increased after weaning for all treatments. All four tested diets led to similar weight gains and feed conversion in the piglets. Animals fed diet POT recovered better from diarrhea compared to the other treatments. A high soybean cake content or lysine supplementation in the diet was disadvantageous with regard to the occurrence of diarrhea. LYS diet led to signs of threonine deficit, indicating that lysine addition alone may not solve the issue. The addition of milk powder provided no extra benefit. In recognition of the health benefits, the use of 5% potato protein, even if it is sourced from conventional production, must still be considered as a sustainable option for feeding organic piglets. The sustainability implications are discussed in the paper.

Black scorpion fish (Scorpaena porcus) is an important species for both artisanal and industrial fisheries in the Turkish Black Sea, with 306 tonnes landed in 2017. The diet composition of this species was investigated through the examination of their stomach contents. A total of 621 S. porcus (ranging from 12.79–765.5 g) were caught by trammel net in the south-eastern Black Sea (Turkish waters) from December 2015 to November 2016. The importance of prey items in the diet of S. porcus was assessed using the Index of Relative Importance (%IRI). Overall, the dominant species was the isopod Idotea balthica (52.8%IRI) followed by decapods (38.5%IRI) and teleosts (8.7%IRI). Seasonal variations in the diets were observed, with isopods predominant in autumn (>85%IRI), teleosts in winter (>77%IRI) and decapod crustaceans (>78%IRI) in spring and summer. The modified Costello's graphical analysis evinced a specialist feeding strategy for S. porcus during winter, spring and autumn while a shift to generalistic feeding strategy was exhibited in summer by a limited number of S. porcus individuals.

The food composition of whiting, Merlangius merlangus, caught around the sea-cage fish farms off the coast of Perşembe, was analysed and compared with the diet of whiting from a control site off the coast of Fatsa (Ordu Province, south-eastern Black Sea). A total of 815 stomach samples were collected during the study, and of these, 195 (23.9%) were empty. According to the percentage of relative importance index (IRI%), pellet food (47.8%) and Annelida (25%) were the main prey groups of whiting in the sea-cage fish farms area, while unidentified teleost (85.3%) and Engraulis encrasicolus (8.2%) were dominant in the control site. The other prey groups in both areas were Crustaceans (Mysidae, Amphipoda, Upogebia sp.), Sprattus sprattus and Gobius spp. Seasonally, pellet food was the most consumed food in all seasons, but Mysidae was the first preference of whiting around the sea-cage fish farms in spring. In the control site, unidentified teleost was the first preference in all seasons, except winter, where E. encrasicolus was the first choice, followed by crustaceans and S. sprattus in winter. Bray–Curtis analysis shows that seasonally, there is no significant difference in the sea-cage fish farms, while there is significant difference in the control site, and two-dimensional nMDS of IRI% revealed a clear separation between both sites. SIMPER analysis revealed that the most contributing factors to the differences between seasons were pellet food in the sea-cage fish farms, and E. encrasicolus in the control site.

We examined the diet of pelagic juveniles of Trachurus japonicus in the surface layer in April, and subsequent demersal juveniles in the near bottom layer during May to June of the East China Sea (ECS) in 2005, 2008 and 2009. Diet composition of the pelagic juveniles showed a significant difference between 2005 and the other two years. That is, they preyed mainly on Paracalanus parvus s.l. and occasionally on Calanus sinicus in 2008 and 2009, while they fed mainly on Corycaeus affinis in 2005, partly corresponding with the between-year difference in prey densities. The demersal juveniles depended heavily on the fifth copepodites and females of C. sinicus which store lipids in the body, i.e. high-energy food for the juveniles, without a significant inter-annual difference. The markedly low occurrence of P. parvus s.l. and C. sinicus, which are considered to be energetically more favourable than C. affinis, from the stomach of the pelagic juveniles in 2005 corresponded with the lowest growth rates of the pelagic juveniles in the three years. Potentially, this resulted in the lowest observed recruitment level of the demersal juveniles for the ECS in 2005 over these three years.

The diet and feeding pattern of scyphomedusa Stomolophus meleagris (Rhizostomeae) was studied, by comparing stomach samples from different developmental stages and environmental zooplankton with the aim to determine diet composition, trophic niche breadth, selectivity and feeding overlap of this edible jellyfish species. Samplings were performed during April and December 2010 and in January 2011, in the coastal lagoon Las Guásimas (27°49′–27°54′N 110°40′–110°35′W), central Gulf of California, which consisted of zooplankton tows and jellyfish collections for stomach content. More than 39 prey items were identified in the gut contents (N = 69), from which eight taxa formed over 90% of the total. Fish eggs were considered main prey (58.6%), copepods (10.8%), veliger larvae of gastropod (13.0%) and bivalve (12.7%) were secondary prey while cirriped and decapod larvae were incidental prey (<3%). However, these proportions varied significantly between small, medium and large size classes of medusa as well as number and type of prey increasing as a function of medusa size. Values of Levin's index confirmed S. meleagris is a specialist predator and Pearre's index showed positive selection of fish eggs, gastropods, bivalves and cirripeds while selectivity was negative for copepods and appendicularians. The relative timing of these changes suggests that ontogenetic processes are closely related with shift in the diet, which indicates increasing predation pressure during development of the medusoid stage of this species, thus emphasizing their ecological importance in coastal ecosystems.

Utilization of long-chain alcohols (LCOH) as diet composition markers in horses and cattle was assessed in a study conducted with 12 mature crossbreed mares (385±47 kg BW) and six adult non-lactating cows (499±36 kg BW) of Asturiana de los Valles breed. The LCOH data were combined with alkane and long-chain fatty acid (LCFA) data to test the applicability of combining these markers to estimate diet composition. Animals were randomly divided into groups of three animals and received a daily total amount of 1.0 kg dry matter/100 kg BW of diets composed of different proportions of ryegrass (Lolium perenne) and woody species (Ulex gallii and heather). Diet composition was estimated from even-chain LCOH (C20-OH to C30-OH) combined or not with alkane (C25-C31 and C33) and/or LCFA (C22-FA to C28-FA, C30-FA, C32-FA and C34-FA) concentrations in diet components and faeces by least-squares procedures, using marker faecal concentrations uncorrected for incomplete faecal recovery (FR0) or corrected using mean recoveries across diets within animal species (FR1). Results showed large differences between plant species in their LCOH profiles, and that these markers offered additional discriminatory information to that provided by alkanes and LCFA. The LCOH markers were incompletely recovered in the faeces of both animal species. In cattle, LCOH FR tended to increase with carbon-chain length in a linear manner in both diets (P<0.001), whereas in horses overall data showed a curvilinear relationship between these variables. Combination of LCOH, LCFA and alkanes resulted in more accurate diet estimates. Correction of faecal LCOH concentrations to incomplete FR led to more accurate diet composition estimates in both animal species. Results obtained in this study suggest the usefulness of LCOH markers combined with alkanes and LCFA to estimate diet composition of horses and cattle grazing mixed grassy–woody plant communities.

An experiment was conducted to assess the potential of long-chain alcohols (LCOH), in alternative or combined with alkanes and long-chain fatty acids (LCFA), as faecal markers to estimate the diet composition of goats grazing heathland vegetation with associated improved pastures. A total of seven diets were offered across the grazing season. The diets were composed of mixtures of herb species (Lolium perenne and Trifolium repens) and woody species (Erica spp., Calluna vulgaris and Ulex gallii) in an attempt to simulate diet selection of goats on these complex vegetation areas. The diet composition was estimated using LCOH markers alone or combined with alkanes, LCFA and alkanes+LCFA, by least square optimization procedures. The data showed large differences between plant species in their LCOH profile. Generally, plant species showed higher LCOH concentrations than those of alkanes and lower than LCFA markers. Faecal recovery of LCOH was incomplete and increased in a linear manner (P < 0.001) with the carbon-chain length, and was influenced by diet composition and its digestibility. The diet composition estimates based on LCOH alone were more accurate (P < 0.05) than those using alkanes or LCFA alone. Results showed that the combination of LCOH with alkanes, LCFA and alkanes+LCFA resulted in more accurate (P < 0.05) estimates of diet composition, indicating that LCOH provided different discriminatory information to that of alkanes and LCFA, helping in the discrimination of the plant species used in this experiment. Results indicate that correction of faecal LCOH concentrations to incomplete faecal recovery is necessary to obtain more accurate estimates of diet composition. Nevertheless, it seemed that the use of a less accurate recovery correction on LCOH markers had a lower impact on the accuracy of estimates than that of alkanes and LCFA.

To investigate the potential use of n-alkanes (alkanes), long-chain alcohols (alcohols) and long-chain fatty acids (acids) for estimating the diet composition of sheep, in a feeding trial. A total of 18 sheep were assigned randomly to three different diets (diet A, diet B and diet C) containing up to eight herbage species (Leymus chinensis, Leymus dasystachys, Elymus sibiricum, Chenopodium album, Puccinellia chinampoensis, Medicago sativa, Saussurea sinuata and Bromus inermis). Faecal recoveries of alkanes, alcohols and acids were determined, and diet compositions were estimated using different combinations of alkanes, alcohols and acids. The faecal concentrations of individual alkanes, alcohols and acids were corrected using the mean recovery of the dietary treatment that the respective animal belonged to (diet recovery), or the mean recovery across all dietary treatments (general recovery). In general, diets did not affect the faecal recovery values for alkanes, alcohols and acids, and no difference in accuracy was found between diet composition estimates based on dietary recovery and general recovery. The accuracy of diet composition estimates declined as the number of dietary components increased from four to eight herbage species (P < 0.001). Better (P < 0.05) estimates of diet composition were obtained with the combinations of two or three marker types instead of alkanes alone. Moreover, results showed that excluding minor diet components from the calculations decreased (P < 0.05) the accuracy of diet composition estimates, whereas including extra non-grazed herbage species did not reduce (P > 0.05) the quality of diet composition estimates. These results confirmed the usefulness of alkanes, alcohols and acids as markers for determining complex diet composition of sheep. However, a negative impact on the accuracy of diet composition estimates, caused by missing minor diet components from the calculation of diet composition, could happen when plant wax markers are used to estimate the diet composition of free-ranging animals.

Specimens of twaite shad, Alosa fallax were sampled from commercial purse seiners and trammel netters in Izmir Bay during November and December 2007. A total of 287 prey items from 14 taxa from 208 stomachs were recorded. The primary food of twaite shad was found to be fish, especially anchovy Engraulis encrasicolus, which was the most frequent (%F = 66.11) and abundant (%N = 63.64) prey item, and also had the highest percentage by weight (%W = 81.91). Decapoda, Isopoda, Ostracoda and Copepoda (Calanus spp., Candocia armata, Temora stylifera from Calanoida and Corycaeus spp. from Cyclopodia), were recorded occasionally with low values for all indices. The study showed that in the Aegean Sea Alosa fallax is a predator of small pelagic fish, E. encrasicolus, A. boyeri, S. pilchardus, and some crustaceans. Benthopelagic P. acarne and demersal S. hepatus were first recorded in diet of twaite shad.

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 (δ13C) 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 δ13C values of n-alkanes can provide additional information in discriminating diet components of grazing animals.

The feeding habits of myctophid larvae of Symbolophorus californiensis were examined in the southern transition region of the western North Pacific where the main spawning and nursery grounds of S. californiensis are formed. This species is a key component of the pelagic ecosystems of this region, and their larvae attain one of the largest sizes among myctophids. To analyse gut contents larvae, including most life history stages after yolk-sac absorption (3.7 to 22.2 mm body length (BL)), were collected in the upper 100 m layer in 1997 and 1998. Feeding incidence was higher during the day than at night (53.1–92.3% versus 0–5.6%), and daytime feeding incidence increased gradually with larval growth. Larvae fed mainly on copepods of various developmental stages. Larvae of S. californiensis showed an ontogenetic change in their diet: larvae ≤7.9 mm BL (i.e. preflexion stage) fed mainly on copepod eggs and nauplii, while the larvae ≥8 mm BL consumed mainly calanoid copepodites such as Pseudocalanus and Paracalanus spp. In the largest size-class (16–22.2 mm BL), the furcilia stage of euphausiids was also an important prey item. There was an increase in the average prey size with growth in larvae ≤11.9 mm BL, while the number of prey eaten positively correlated with growth in larvae ≥12 mm BL. The trophic niche breadth also increased with larval growth, which would ensure a wide range of available food resources for the larger size-class larvae.

The limitations of the alkane technique in estimating the diet components of herbivores call for the introduction of new diet composition markers. Recently, long-chain alcohols (alcohols) and long-chain fatty acids (acids) have received the most attention and show great potential, when combined with alkanes, to estimate composition of complex diets. In the current study, faecal recoveries of alcohols and acids were determined in sheep in four different live weight groups fed three herbage species, either Leymus chinensis, L. dasystachys or Elymus sibiricum. Analysis of variance (ANOVA) was used to examine the effects of herbage species and live weight of sheep on faecal recoveries of individual alcohols and acids. Further, an indoor experiment with six sheep fed a diet of equal proportions, on dry matter (DM) basis, of three herbages was performed, allowing to assess the accuracy of alcohols and/or acids in combination with alkanes, to estimate diet composition. A one-sample t-test was carried out to test the accuracy of these estimates. Results of the first experiment indicated that the faecal recoveries of alcohols and acids were significantly affected by herbage species (P < 0.05). While the effects were significant or near significant for the faecal recoveries of some alcohols (C24-ol, C30-ol and C26-ol) (P ⩽ 0.05), no effect of live weight on faecal recoveries of acids was observed (P > 0.05). Therefore, adjustments based on diet-specific faecal recoveries might improve diet composition estimates. This was illustrated by the results of the second experiment. The diet composition estimated from alcohols or all combinations of alcohols with other marker types, after diet-specific correction of faecal recoveries, did not significantly differ from the actual composition (P > 0.05). However, using acids as additional markers resulted in poorer diet composition estimates. This study confirmed the utility of alcohols, combined with alkanes, as markers to estimate composition of complex diets. Although corrections based on mean faecal recoveries, average over animals and diets, resulted in some accuracy loss, results were still satisfactory and better than without recovery correction.

In a feeding trial with 24 sheep, we used the alkanes, long-chain alcohols (LCOH) or both of these plant wax markers, to estimate the diet composition of animals offered diets comprising alkane-labelled cottonseed meal (CSM) together with up to four forages. The diets used were: Diet 1 subterranean clover (Trifolium subterraneum); Diet 2 subterranean clover + phalaris (Phalaris aquatica); Diet 3 subterranean clover, phalaris + annual ryegrass (Lolium rigidum); and Diet 4 subterranean clover, phalaris, annual ryegrass + wheat straw (Triticum aestivum). Estimates of diet composition were made following correction of faecal alkane or LCOH concentrations for incomplete faecal recovery, using recovery estimates derived from individual animals, mean recoveries for a given dietary treatment or grand mean recoveries. Estimated dietary proportions of CSM and known intakes of CSM were used to estimate forage intake. The LCOH concentrations of the diet components were much higher than their alkane concentrations, especially for phalaris. Multivariate analyses showed that the discriminatory information provided by the LCOH was additional to that provided by the alkanes, and that a combination of (LCOH + alkanes) discriminated better between diet components than either class of marker alone. Faecal recoveries of LCOH increased with increasing carbon-chain length; there were no differences in recovery attributable to diet. The most accurate estimates of diet composition were obtained with the combination of (LCOH + alkanes). Estimates of diet composition based on LCOH alone were not as good as alkanes alone, due to the high correlation between the LCOH profiles of phalaris and ryegrass. Total grass content of the diet was very accurately estimated using LCOH. Diet composition estimates provided estimates of whole-diet digestibility, which did not differ from the measured values. Trends in the accuracy of forage intake estimates reflected those found with diet composition and almost two-thirds of estimates based on (LCOH + alkanes) had lower error than those found with alkanes alone. The results confirm that supplements labelled with plant wax components can be used to estimate forage intake, and also show that the LCOH are useful markers for estimating diet composition. Intakes were also computed using a combination of natural LCOH concentrations in the diet and the daily dose rate of even-chain alkanes administered by intra-ruminal device. Differences between intakes so estimated and the measured intakes were closely related to the difference in faecal recovery between the LCOH/alkane pair used to estimate intake, by an amount close to that expected on theoretical grounds. It is concluded that the use of plant wax LCOH, especially in combination with alkanes, will result in improved estimates of diet composition and intake in grazing animals.