Nitrogen Isotope Discrimination Research Articles

Demonstrating Prebiotic Peptide Evolution by Nitrogen Isotope Discrimination in an Autocatalytic Chemical Reaction Network

Demonstrating Prebiotic Peptide Evolution by Nitrogen Isotope Discrimination in an Autocatalytic Chemical Reaction Network

Incubation experiments using nitrogen isotope discrimination to estimate ammonia emission from amended sheep manure treatments

AbstractTwo 10-day in vitro experiments were conducted to investigate the relationship between nitrogen (N) isotope discrimination (δ15N) and ammonia (NH3) emissions from sheep manure. In Exp. 1, three different manure mixtures were set up: control (C); C mixed with lignite (C + L); and grape marc (GM), with 5, 4 and 5 replications, respectively. For C, urine and faeces were collected from sheep fed a diet of 550 g lucerne hay/kg, 400 g barley grain/kg and 50 g faba bean/kg; for C + L, urine and faeces were collected from sheep fed the C diet and 100 g ground lignite added to each incubation system at the start of the experiment; for GM, urine and faeces were collected from sheep fed a diet consisting of C diet with 200 g/kg of the diet replaced with GM. In Exp. 2, three different urine-faeces mixtures were set up: 2U:1F, 1.4U:1F and 1U:1F with urine to faeces ratios of 2:1, 1.4:1 and 1:1, respectively, each with 5 replications. Lignite in C + L led to significantly lower cumulative manure-N loss by 81 and 68% in comparison with C and GM groups, respectively (P = 0.001). Cumulative emitted manure NH3-N was lower in C + L than C and GM groups by 35 and 36%, respectively (P = 0.020). Emitted manure NH3-N was higher in 2U:1F compared to 1.4U:1F and 1U:1F by 18 and 26%, respectively (P < 0.001). This confirms the relationship between manure δ15N and cumulative NH3-N loss reported by earlier studies, which may be useful for estimating NH3 losses.

Plasticity of Plantago lanceolata L. in Adaptation to Extreme Environmental Conditions.

This study aimed at characterizing some adaptive changes in Plantago lanceolata L. exposed to harsh conditions of a desert-like environment generating physiological stress of limited water availability and exposure to strong light. It was clearly shown that the plants were capable of adapting their root system and vascular tissues to enable efficient vegetative performance. Soil analyses, as well as nitrogen isotope discrimination data show that P. lanceolata leaves in a desert-like environment had better access to nitrogen (nitrite/nitrate) and were able to fix it efficiently, as compared to the plants growing in the surrounding forest. The arbuscular mycorrhiza was also shown to be well-developed, and this was accompanied by higher bacterial frequency in the root zone, which might further stimulate plant growth. A closer look at the nitrogen content and leaf veins with a higher number of vessels and a greater vessel diameter made it possible to define the changes developed by the plants populating sandy habitats as compared with the vegetation sites located in the nearby forest. A determination of the photosynthesis parameters indicates that the photochemical apparatus in P. lanceolata inhabiting the desert areas adapted slightly to the desert-like environment and the time of day, with some changes of the reaction center (RC) size (photosystem II, PSII), while the plants' photochemical activity was at a similar level. No differences between the two groups of plants were observed in the dissipation of light energy. The exposure of plants to harsh conditions of a desert-like environment increased the water use efficiency (WUE) value in parallel with possible stimulation of the β-carboxylation pathway.

Extrusion of lupines with or without addition of reducing sugars: Effects on the formation of Maillard reaction compounds, partition of nitrogen and Nε-carboxymethyl-lysine, and performance of dairy cows

The extrusion of leguminous seeds induces the formation of Maillard reaction compounds (MRC) as a product of protein advanced glycation and oxidation, which lowers protein degradability in the rumen. However, the quantitative relationship between the parameters of pretreatment (i.e., addition of reducing sugars) and extrusion, and the formation of MRC has not been established yet. Moreover, the fate of the main stable MRC, Nε-carboxymethyl-lysine (CML), in the excretory routes has never been investigated in ruminants. We aimed to test the effects of the temperature of extrusion of white lupines with or without addition of reducing sugars on the formation of MRC, crude protein (CP) degradability in the rumen, N use efficiency for milk production (milk N/N intake), and performance of dairy cows. Two experiments with a replicated 4 × 4 Latin square design were conducted simultaneously with 16 (3 rumen-cannulated) multiparous Holstein cows to measure indicators of ruminal CP degradability (ruminal NH3 concentration, branched-chain volatile fatty acids), metabolizable protein supply (plasma essential AA concentration), N use efficiency (N isotopic discrimination), and dairy performance. In parallel, apparent total-tract digestibility of dry matter, organic matter, neutral detergent fibers, N, total Lys and CML, and partition of N and CML were measured with 4 cows in both experiments. The diets consisted on a DM basis of 20% raw or extruded lupines and 80% basal mixed ration of corn silage, silage and hay from permanent grasslands, pelleted concentrate, and a vitaminized mineral mix. Expected output temperatures of lupine extrusion were 115°C, 135°C, and 150°C, without and with the addition of reducing sugars before extrusion. The extrusion numerically reduced the in vitro ruminal CP degradability of the lupines, and consequently increased the predicted supply of CP to the small intestine. Nitrogen balance and urinary N excretion did not differ among dietary treatments in either experiment. Milk yield and N use efficiency for milk production increased with extrusion of lupines at 150°C without addition of reducing sugars compared with raw lupines. Nitrogen isotopic discrimination between dietary and animal proteins (the difference between δ15N in plasma and δ15N in the diet) were lower with lupines extruded at 150°C without and with addition of reducing sugars. Regardless of sugar addition, milk true protein yield was not affected, but milk urea concentration and fat:protein ratio were lower with lupines extruded at 150°C than with raw lupines. In the CML partition study, we observed that on average 26% of the apparently digested CML was excreted in urine, and a much lower proportion (0.63% on average) of the apparently digested CML was secreted in milk, with no differences among dietary treatments. In conclusion, we showed that the extrusion of white lupines without or with addition of reducing sugars numerically reduced enzymatic CP degradability, with limited effects on N partition, but increased milk yield and N use efficiency at the highest temperature of extrusion without addition of reducing sugars.

Tissue‐ and diet‐dependent stable carbon and nitrogen isotope discrimination: a calibration study in a captive shorebird species

In ecology, stable‐isotope ratios are widely used to determine diets of organisms and reconstruct food webs. This is usually done by analyzing the stable‐isotope ratios of nitrogen (δ15N), which increase with increasing trophic level, and those of carbon (δ13C), which correlate with the δ13C value of food source(s) and generally differ between terrestrial and marine food sources. Assimilation of food changes stable‐isotope ratios, resulting in different values between the food source and its consumer. These differences are known as isotope trophic discrimination factors and, if known, can be used to determine from the stable‐isotope ratios in the consumer's tissue what the consumer has been eating. What is often ignored is that discrimination factors can differ between consumer's food sources and also between tissue types. Therefore, we performed a controlled feeding study in red knots Calidris canutus to determine discrimination factors between different food sources and red knot tissues. We kept two groups of red knots in captivity on a stable diet, one group feeding on mudsnails and the other on Trouvit pellets, for several months, during which the birds molted their feathers. We analyzed δ13C and δ15N in both food sources and in five red knot tissues (blood cells, blood plasma and three feather types) and subsequently calculated the isotope discrimination factors. We confirmed that the discrimination factors differed between tissues, and also between diets. Our values deviated from general averages reported in reviews on a wide range of animals/birds, but were very similar to values from previous red knot and dunlin studies. We therefore think that our discrimination factors can be used in future stable isotope studies, not only on red knots, but also on other marine shorebird species and plea for careful consideration of using the right discrimination factors.Keywords: δ13C, δ15N, discrimination factor, red knot, shorebird, stable isotope

Addressing the Relationship between Leaf Nitrogen and Carbon Isotope Discrimination from the Three Levels of Community, Population and Individual.

The carbon, nitrogen and water cycles of terrestrial ecosystems are important biogeochemical cycles. Addressing the relationship of leaf nitrogen (N) and carbon isotope discrimination (Δ) will enhance the understanding of the links between these three cycles in plant leaves because Δ can reflect time-integrated leaf-level water-use efficiency (WUE) over the period when the leaf material is produced. Previous studies have paid considerable attention to the relationship. However, these studies have not effectively eliminated the interference of environmental factors, inter-species, and inter-individual differences in this relationship, so new research is necessary. To minimize these interferences, the present work explored the relationship at the three levels of community, population, and plant individual. Three patterns of positive, negative and no relationship were observed across communities, populations, and individuals, which is dependent on environmental conditions, species, and plant individuals. The results strongly suggested that there is no general pattern for the relationship between leaf N and Δ. Furthermore, the results indicated that there is often no coupling between leaf-level long-term WUE and leaf N in the metabolic process of carbon, N and water in leaves. The main reason for the lack of this relationship is that most plants do not invest large amounts of nitrogen into photosynthesis. In addition, the present study also observed that, for most plant species, leaf N was not related to photosynthetic rate, and that variations in photosynthetic rates are mainly driven by stomatal conductance.

Toxicity and Starvation Induce Major Trophic Isotope Variation in Daphnia Individuals: A Diet Switch Experiment Using Eight Phytoplankton Species of Differing Nutritional Quality.

Stable isotope values can express resource usage by organisms, but their precise interpretation is predicated using a controlled experiment-based validation process. Here, we develop a stable isotope tracking approach towards exploring resource shifts in a key primary consumer species Daphnia magna. We used a diet switch experiment and model fitting to quantify the stable carbon (δ13C) and nitrogen (δ15N) isotope turnover rates and discrimination factors for eight dietary sources of the plankton species that differ in their cellular organization (unicellular or filamentous), pigment and nutrient compositions (sterols and polyunsaturated fatty acids), and secondary metabolite production rates. We also conduct a starvation experiment. We evaluate nine tissue turnover models using Akaike's information criterion and estimate the repetitive trophic discrimination factors. Using the parameter estimates, we calculate the hourly stable isotope turnover rates. We report an exceedingly faster turnover value following dietary switching (72 to 96 h) and a measurable variation in trophic discrimination factors. The results show that toxic stress and the dietary quantity and quality induce trophic isotope variation in Daphnia individuals. This study provides insight into the physiological processes that underpin stable isotope patterns. We explicitly test multiple alternative dietary sources and fasting and discuss the parameters that are fundamental for field- and laboratory-based stable isotope studies.

Nitrogen isotope discrimination in open-pollinated and hybrid canola suggests indirect selection for enhanced ammonium utilization.

Nitrogen isotope discrimination (Δ15N) may have utility as an indicator of nitrogen use in plants. A simple Δ15N-based isotope mass balance (IMB) model has been proposed to provide estimates of efflux/influx (E/I) ratios across root plasma membranes, the proportion of inorganic nitrogen assimilation in roots (P root) and translocation of inorganic nitrogen to shoots (Ti/Tt) under steady-state conditions. We used the IMB model to investigate whether direct selection for yield in canola (Brassica napus L.) has resulted in indirect selection in traits related to nitrogen use. We selected 23 canola lines developed from 1942 to 2017, including open-pollinated (OP) lines developed prior to 2005 as well as more recent commercial hybrids (CH), and in three separate experiments grew them under hydroponic conditions in a greenhouse with either 0.5 mM ammonium, 0.5 mM nitrate, or 5 mM nitrate. Across all lines, E/I, Proot and Ti/Tt averaged 0.09±0.03, 0.82±0.05 and 0.23±0.06 in the low nitrate experiment, and 0.31±0.06, 0.71±0.07 and 0.42±0.12 in the high nitrate experiment, respectively. In contrast, in the ammonium experiment average E/I was 0.40±0.05 while Ti/Tt averaged 0.07±0.04 and Proot averaged 0.97±0.02. Although there were few consistent differences between OP and CH under nitrate nutrition, commercial hybrids were collectively better able to utilize ammonium as their sole nitrogen source, demonstrating significantly greater overall biomass and a lower Proot and a higher Ti/Tt, suggesting a somewhat greater flux of ammonium to the shoot. Average root and whole-plant Δ15N were also slightly higher in CH lines, suggesting a small increase in E/I. An increased ability to tolerate and/or utilize ammonium in modern canola hybrids may have arisen under intensive mono-cropping.

Relationship Between Nitrogen Isotopic Discrimination and the Proportion of Dietary Nitrogen Excreted in Urine by Sheep Offered Different Levels of Dietary Non-Protein Nitrogen

Urinary nitrogen (N) excretion (UN) as a proportion of N intake (NI; UN/NI) is a major determinant of N excretion from ruminants and could be predicted from the N isotopic discrimination occurring between dietary and animal proteins (Δ15N). This study investigated the usefulness of Δ15N and other plasma biomarkers to reflect changes in UN/NI from sheep offered different levels of dietary urea. Eighteen Merino rams (age, 1–2 years; live weight, 41 ± 3 kg) were allocated to three dietary N treatments for a N balance study. Treatments were control (C), control + 0.5% urea (C+0.5%), and control + 1.2% urea (C+1.2%) and designed to provide maintenance, maintenance plus an additional 15%, and maintenance plus an additional 33% NI, respectively. The urea effect term was used for one-way ANOVA and regression analysis. As NI increased, the UN and retained N (RN) increased linearly (p < 0.001), but UN/NI only increased in treatment C+1.2% compared with C (p < 0.05). Plasma Δ15N was positively and significantly correlated with UN and UN/NI (r = 0.52, p = 0.028; and r = 0.68, p = 0.002, respectively) and increased linearly (p < 0.001) with the highest values observed in C+1.2%. Urine δ15N changed linearly between C and C+1.2%, but plasma δ15N increased quadratically (p < 0.05). Plasma urea N increased in a linear way across dietary urea levels (p < 0.001). The N isotopic difference between plasma and urine (plasma δ15N–urine δ15N) of C did not vary from either of the other treatments; however, it differed between C+0.5% and C+1.2% (p < 0.05). The study confirmed the potential usefulness of plasma Δ15N to estimate UN/NI from sheep. Moreover, plasma δ15N–urine δ15N can be proposed as a new biomarker of N excretion from small ruminants. These approaches, however, need to be tested in various study conditions.

Nitrogen isotopic discrimination as a biomarker of between-cow variation in the efficiency of nitrogen utilization for milk production: A meta-analysis

Estimating the efficiency of N utilization for milk production (MNE) of individual cows at a large scale is difficult, particularly because of the cost of measuring feed intake. Nitrogen isotopic discrimination (Δ15N) between the animal (milk, plasma, or tissues) and its diet has been proposed as a biomarker of the efficiency of N utilization in a range of production systems and ruminant species. The aim of this study was to assess the ability of Δ15N to predict the between-animal variability in MNE in dairy cows using an extensive database. For this, 20 independent experiments conducted as either changeover (n = 14) or continuous (n = 6) trials were available and comprised an initial data set of 1,300 observations. Between-animal variability was defined as the variation observed among cows sharing the same contemporary group (CG; individuals from the same experimental site, sampling period, and dietary treatment). Milk N efficiency was calculated as the ratio between mean milk N (grams of N in milk per day) and mean N intake (grams of N intake per day) obtained from each sampling period, which lasted 9.0 ± 9.9 d (mean ± SD). Samples of milk (n = 604) or plasma (n = 696) and feeds (74 dietary treatments) were analyzed for natural 15N abundance (δ15N), and then the N isotopic discrimination between the animal and the dietary treatment was calculated (Δ15n = δ15Nanimal - δ15Ndiet). Data were analyzed through mixed-effect regression models considering the experiment, sampling period, and dietary treatment as random effects. In addition, repeatability estimates were calculated for each experiment to test the hypothesis of improved predictions when MNE and Δ15N measurements errors were lower. The considerable protein mobilization in early lactation artificially increased both MNE and Δ15N, leading to a positive rather than negative relationship, and this limited the implementation of this biomarker in early lactating cows. When the experimental errors of Δ15N and MNE decreased in a particular experiment (i.e., higher repeatability values), we observed a greater ability of Δ15N to predict MNE at the individual level. The predominant negative and significant correlation between Δ15N and MNE in mid- and late lactation demonstrated that on average Δ15N reflects MNE variations both across dietary treatments and between animals. The root mean squared prediction error as a percentage of average observed value was 6.8%, indicating that the model only allowed differentiation between 2 cows in terms of MNE within a CG if they differed by at least 0.112 g/g of MNE (95% confidence level), and this could represent a limitation in predicting MNE at the individual level. However, the one-way ANOVA performed to test the ability of Δ15N to differentiate within-CG the top 25% from the lowest 25% individuals in terms of MNE was significant, indicating that it is possible to distinguish extreme animals in terms of MNE from their N isotopic signature, which could be useful to group animals for precision feeding.

Post-anthesis Relationships Between Nitrogen Isotope Discrimination and Yield of Spring Wheat Under Different Nitrogen Levels.

Wheat grain yield and nitrogen (N) content are influenced by the amount of N remobilized to the grain, together with pre-anthesis and post-anthesis N uptake. Isotopic techniques in farmed areas may provide insight into the mechanism underlying the N cycle. 15N-labeled urea was applied to microplots within five different fertilized treatments 0 kg ha–1 (N1), 52.5 kg ha–1 (N2), 105 kg ha–1 (N3), 157.5 kg ha–1 (N4), and 210 kg ha–1 (N5) of a long-term field trial (2003–2021) in a rainfed wheat field in the semi-arid loess Plateau, China, to determine post-anthesis N uptake and remobilization into the grain, as well as the variability of 15N enrichment in aboveground parts. Total N uptake was between 7.88 and 29.27 kg ha–1 for straw and 41.85 and 95.27 kg ha–1 for grain. In comparison to N1, N fertilization increased straw and grain N uptake by 73.1 and 56.1%, respectively. Nitrogen use efficiency (NUE) and harvest index were altered by N application rates. The average NUE at maturity was 19.9% in 2020 and 20.01% in 2021; however, it was usually higher under the control and low N conditions. The amount of 15N excess increased as the N rate increased: N5 had the highest 15N excess at the maturity stage in the upper (2.28 ± 0.36%), the middle (1.77 ± 0.28%), and the lower portion (1.68 ± 1.01%). Compared to N1, N fertilization (N2–N5) increased 15N excess in the various shoot portions by 50, 38, and 35% at maturity for upper, middle, and lower portions, respectively. At maturity, the 15N excess remobilized to the grain under N1–N5 was between 5 and 8%. Our findings revealed that N had a significant impact on yield and N isotope discrimination in spring wheat that these two parameters can interact, and that future research on the relationship between yield and N isotope discrimination in spring wheat should take these factors into account.

Breeding for salt tolerance in wheat: The contribution of carbon isotopic signatures

Use of low-quality water for supplemental irrigation is expected to become soon a common practice in the Mediterranean area, where durum wheat is the main cultivated cereal. Breeding for salt stress tolerance may contribute to the improvement of wheat resilience to irrigation with brackish water. Various traits can be considered as indicators of salt stress tolerance, which include agronomical and physiological criteria. However, the complexity of salinity tolerance mechanisms, the G × E interaction and the lack of correlation between controlled and open field conditions causes uncertainty in the selection process. The present review highlights the main advantages and limitations of different agronomical and physiological traits used in screening for salt stress tolerance in wheat. Special focus is given to carbon and nitrogen isotope discrimination, that remains a bottleneck in breeding for salt stress tolerance. The use of different statistical tools to analyse data related to salt stress tolerance is also discussed in this review.

The effect of sheep genetic merit and feed allowance on nitrogen partitioning and isotopic discrimination

Animal nitrogen (N) partitioning is a key parameter for profitability and sustainability of ruminant production systems, which may be predicted from N isotopic discrimination or fractionation (Δ15N). Both animal genetics and feeding level may interact and impact on N partitioning. Therefore, this study aimed to assess the interactive effects of genetic merit (G) and feed allowance (F) on N partitioning and Δ15N in sheep. The sheep were drawn from two levels of G (high G vs. low G; based on New Zealand Sheep Improvement Limited (http://www.sil.co.nz/) dual (wool and meat) growth index) and allocated to two levels of F (1.7 (high F) vs. 1.1 (low F) times Metabolisable Energy requirement for maintenance) treatments. Twenty-four Coopworth rams were divided into four equal groups for a N balance study: high G × high F, high G × low F, low G × high F, and low G × low F. The main factors (G and F) and the interaction term were used for 2-way ANOVA and regression analysis. Higher F led to higher N excretions (urinary N (UN); faecal N (FN); manure N), retained N, N use efficiency (NUE), and urinary purine derivatives excretion (P < 0.05). On the other hand, higher UN/N intake, and plasma Δ15N were observed with the lower F (P < 0.05). Higher G led to increased UN, FN, manure N, apparent N digestibility, and urinary purine derivatives excretion (P < 0.05). Higher F only increased UN in high G sheep, with no effect on low G sheep (P < 0.05). Regression analysis results demonstrated potential to use plasma Δ15N to reflect the effects of G and F on NUE and UN/N intake. Further research is urged to study interactive effects of genetic and feeding level on sheep N partitioning.

Tissue-specific Isotopic Incorporation Turnover Rates and Trophic Discrimination Factors in the Freshwater Shrimp Macrobrachium borellii (Crustacea: Decapoda: Palaemonidae).

The interpretation of isotopic data in ecology requires knowledge about two factors: turnover rate and the trophic discrimination factor, which have not been well described in freshwater shrimps. We performed a 142-day diet shift experiment on 174 individuals of the omnivorous shrimp Macrobrachium borellii, measured their growth, and temporally serially sampled muscle and hepatopancreas tissue to quantify carbon and nitrogen incorporation rates and isotope discrimination factors. Shrimps were fed with artificial diets (δ13C = -26.1‰, δ15N= 2.1‰) for 45 days in attempt to standardize the shrimps' initial δ13C and δ15N values for subsequent experiments. Shrimps were then fed with another artificial diet (δ13C = -16.1‰, δ15N = 15.8‰) and the change in δ13C and δ15N was observed for a period of 97 days. The trophic discrimination factor (∆) for δ13C was significantly higher in hepatopancreas (0.7 ± 0.36‰) than in muscle (-0.1 ± 0.83‰); however, the opposite was the case for δ15N (1.7 ± 0.43‰ and 3.6 ± 0.42‰, respectively). In the hepatopancreas the mean residence time (τ) of 13C was 26.3 ± 4.3 days compared to a residence time of 16.6 ± 5.51 days for δ15N, whereas the τ in muscle was 75.8 ± 25 days for δ13C and 40 ± 25 days for δ15N. The rate of incorporation of carbon into muscle was higher than that predicted by allometric equations relating isotopic incorporation rate to body mass that was developed previously for invertebrates. Our results support ranges of traditional trophic discrimination factor values observed in muscles samples of different taxa (∆15N around 3‒3.5‰ and ∆13C around 0‒1‰), but our work provides evidence that these traditionally used values may vary in other tissues, as we found that in the hepatopancreas ∆15N is around 1.7‰.

The origin of N isotopic discrimination and its relationship with feed efficiency in fattening yearling bulls is diet-dependent.

Nitrogen (N) isotopic discrimination (i.e. the difference in natural 15N abundance between the animal proteins and the diet; Δ15N) is known to correlate with N use efficiency (NUE) and feed conversion efficiency (FCE) in ruminants. However, results from the literature are not always consistent across studies, likely due to isotopic discrimination pathways that may differ with the nature of diets. The objective of the present study was to assess at which level, from rumen to tissues, Δ15N originates and becomes related to NUE and FCE in fattening yearling bulls when they are fed two contrasted diets. Twenty-four Charolais yearling bulls were randomly divided into two groups and fed during 8 months, from weaning to slaughter, either 1) a high starch diet based on corn silage supplying a balanced N to energy ratio at the rumen level (starch) or 2) a high fiber diet based on grass silage supplying an excess of rumen degradable N (fiber). All animals were slaughtered and samples of different digestive pools (ruminal, duodenal, ileal and fecal contents), animal tissues (duodenum, liver and muscle), blood and urine were collected for each animal. Ruminal content was further used to isolate liquid-associated bacteria (LAB), protozoa and free ammonia, while plasma proteins were obtained from blood. All samples along with feed were analyzed for their N isotopic composition. For both diets, the digestive contribution (i.e. the N isotopic discrimination occurring before absorption) to the Δ15N observed in animal tissues accounted for 65 ± 11%, leaving only one third to the contribution of post-absorptive metabolism. Concerning the Δ15N in digestive pools, the majority of these changes occurred in the rumen (av. Δ15N = 2.12 ± 0.66‰), with only minor 15N enrichments thereafter (av. Δ15N = 2.24 ± 0.41‰), highlighting the key role of the rumen on N isotopic discrimination. A strong, significant overall relationship (n = 24) between Δ15N and FCE or NUE was found when using any post-absorptive metabolic pool (duodenum, liver, or muscle tissues, or plasma proteins; 0.52 < r < 0.73; P ≤ 0.01), probably as these pools reflect both digestive and post-absorptive metabolic phenomena. Fiber diet compared to starch diet had a lower feed efficiency and promoted higher (P ≤ 0.05) Δ15N values across all post-absorptive metabolic pools and some digestive pools (ruminal, duodenal, and ileal contents). The within-diet relationship (n = 12) between Δ15N and feed efficiency was not as strong and consistent as the overall relationship, with contrasted responses between the two diets for specific pools (diet x pool interaction; P ≤ 0.01). Our results highlight the contrasted use of N at the rumen level between the two experimental diets and suggests the need for different equations to predict FCE or NUE from Δ15N according to the type of diet. In conclusion, rumen digestion and associated microbial activity can play an important role on N isotopic discrimination so rumen effect related to diet may interfere with the relationship between Δ15N and feed efficiency in fattening yearling bulls.

Biochemical and mesophyll diffusional limits to photosynthesis are determined by prey and root nutrient uptake in the carnivorous pitcher plant Nepenthes × ventrata.

Carnivorous plants can enhance photosynthetic efficiency in response to prey nutrient uptake, but the underlying mechanisms of increased photosynthesis are largely unknown. Here we investigated photosynthesis in the pitcher plant Nepenthes × ventrata in response to different prey-derived and root mineral nutrition to reveal photosynthetic constrains. Nutrient-stressed plants were irrigated with full inorganic solution or fed with four different insects: wasps, ants, beetles or flies. Full dissection of photosynthetic traits was achieved by means of gas exchange, chlorophyll fluorescence and immunodetection of photosynthesis-related proteins. Leaf biochemical and anatomical parameters together with mineral composition, nitrogen and carbon isotopic discrimination of leaves and insects were also analysed. Mesophyll diffusion was the major photosynthetic limitation for nutrient-stressed Nepenthes × ventrata, while biochemistry was the major photosynthetic limitation after nutrient application. The better nutrient status of insect-fed and root-fertilized treatments increased chlorophyll, pigment-protein complexes and Rubisco content. As a result, both photochemical and carboxylation potential were enhanced, increasing carbon assimilation. Different nutrient application affected growth, and root-fertilized treatment led to the investment of more biomass in leaves instead of pitchers. The study resolved a 35-year-old hypothesis that carnivorous plants increase photosynthetic assimilation via the investment of prey-derived nitrogen in the photosynthetic apparatus. The equilibrium between biochemical and mesophyll limitations of photosynthesis is strongly affected by the nutrient treatment.

Effects of hydrolysable tannin-treated grass silage on milk yield and composition, nitrogen partitioning and nitrogen isotopic discrimination in lactating dairy cows

The objective of this study was to evaluate the effects of oak tannin extract (OTE) added in forage before ensiling on dairy cows fed at 92% of their digestible protein requirements. Six multiparous lactating Holstein cows were used in a crossover design (two treatments × two periods). The control treatment (CON) was based on a diet including 50% of grass silage, whereas the experimental treatment (TAN) included grass silage sprayed with OTE (26 g/kg DM) just before baling. Milk yield (on average 24 kg fat protein corrected milk per day) was not affected, but both milk and rumen fatty acids profiles were impacted by OTE. Nitrogen intake (415 g N per cow per day) and nitrogen use efficiency (NUE; 0.25 on average) were not affected, but a shift from urine (−8% of N intake relatively to control, P = 0.06) to faecal N (+5%; P = 0.004) was observed with the TAN diet (P ≤ 0.05). Nitrogen apparent digestibility was thus reduced for TAN (−3%; P ≤ 0.05). The effect of OTE on ruminal and milk FA profiles suggests an impact on rumen microbiota. Nitrogen isotopic discrimination between animal proteins and diet (Δ15N) was evaluated as a proxy for NUE. While no differences in NUE were observed across diets, a lower Δ15N of plasma proteins was found when comparing TAN v. CON diets. This finding supports the concept that Δ15N would mainly sign the N partitioning at the metabolic level rather than the overall NUE, with the latter also being impacted by digestive processes. Our results agree with a N shift from urine to faeces, and this strategy can thus be adopted to decrease the environmental impact of ruminant protein feeding.

Nitrogen partitioning and isotopic discrimination are affected by age and dietary protein content in growing lambs

It is difficult to separate an age-dependent fall in nitrogen use efficiency (NUE; N balance/N intake) in growing ruminants from a progressively decrease in animal protein requirements over time. This study examined the effect of dietary protein content on N partitioning, digestibility and N isotopic discrimination between the animal and its diet (Δ15Nanimal-diet) evaluated at two different fattening periods (early v. late). Twenty-four male Romane lambs (age: 19 ± 4.0 days; BW: 8.3 ± 1.39 kg) were equally allocated to three dietary CP treatments (15%, 17% and 20% CP on a DM basis). Lambs were reared with their mothers until weaning, thereafter housed in individual pens until slaughter (45 kg BW). During the post-weaning period, lambs were allocated twice (early fattening (30 days post-weaning) and late fattening (60 days post-weaning)) to metabolic cages for digestibility and N balance study. When diet CP content increased, the average daily gain of lambs increased (P < 0.05) while the age at slaughter decreased (P = 0.01), but no effect was observed on feed efficiency (P > 0.10). Diet CP content had limited effect on lamb carcass traits. Higher fibre digestibility was observed at the early v. late fattening period (P < 0.001). The N intake and the urinary N excretion increased when diet CP content increased (P < 0.001) and when shifting from early to late fattening period (P < 0.001). Faecal N excretion (P = 0.14) and N balance (P > 0.10) were not affected by diet CP content. Nitrogen digestibility increased (P < 0.001) as the diet CP content increased and on average it was greater at late v. early fattening period (P = 0.02). The NUE decreased (P = 0.001) as the diet CP content increased and as the lamb became older (P < 0.001). However, the age-dependent fall in NUE observed was lower at high v. low dietary CP content (CP × age interaction; P = 0.04). The Δ15Nanimal-diet was positively correlated (P < 0.05) with N intake (r = 0.59), excretion of faecal N (r = 0.41), urinary N (r = 0.69) and total manure N (r = 0.64), while negatively correlated with NUE (r = −0.57). Overall, the experiment showed NUE was lower in older lambs and when lambs were fed high diet CP content, and that Δ15Nanimal-diet was a useful indicator not only for NUE but also for urinary N excretion, which is a major environmental pollution factor on farm.

Amino acid isotope discrimination factors for a carnivore: physiological insightsfrom leopard sharks and their diet.

Stable isotopes are important ecological tools, because the carbon and nitrogen isotopic composition of consumer tissue reflects the diet. Measurements of isotopes of individual amino acids can disentangle the effects of consumer physiology from spatiotemporal variation in dietary isotopic values. However, this approach requires knowledge of assimilation patterns of dietary amino acids. We reared leopard sharks (Triakis semifasciata) on diets of squid (Loligo opalescens; 1250days; control sharks) or squid then tilapia (Oreochromis sp.; switched at 565days; experimental sharks) to evaluate consumer-diet discrimination factors for amino acids in muscle tissue. We found that control sharks exhibited lower nitrogen isotope discrimination factors (∆15N) than most previous consumer studies, potentially because of urea recycling. Control sharks also had large carbon isotope discrimination factors (∆13C) for three essential amino acids, suggesting microbial contributions or fractionation upon assimilation. Compared to controls, experimental sharks exhibited higher ∆13C values for four amino acids and ∆15N values for seven amino acids, corresponding with differences between diets in δ13C and δ15N values. This suggests that not all amino acids in experimental sharks had reached steady state, contrary to the conclusion of a bulk isotope study of these sharks. Our results imply that (1) the magnitude of a shift in dietary δ13C and δ15N values temporarily influences the appearance of discrimination factors; (2) slow turnover of amino acid isotopes in elasmobranch muscle precludes inferences about seasonal dietary changes; (3) elasmobranch discrimination factors for amino acids may be affected by urea recycling and microbial contributions of amino acids.

Host feeding ecology and trophic position significantly influence isotopic discrimination between a generalist ectoparasite and its hosts: Implications for parasite-host trophic studies

Despite being one of the most prevalent forms of consumerism in ecological communities, parasitism has largely been excluded from food-web models. Stable isotope analysis of consumers and their diets has been widely used in the study of food webs for decades. However, the amount of information regarding parasite stable isotope ecology is limited, restricting the ability of ecologists to use stable isotope analysis to study parasites in food webs. This study took advantage of distinct differences in the feeding ecology and trophic position of different species of fish known to host the same common micropredatory gnathiid isopod to study the effects of host stable isotope ecology on that of the associated micropredator. Blood engorged juvenile gnathiids were in most cases indistinguishable from their hosts' blood, but significant isotope discrimination was observed for adults. Males were generally lower in δ13C and δ15N than host blood whereas host-specific isotopic discrimination for females varied among the different host species. Model predictions indicated that there is a significant effect of host blood isotope ratios on the rate of carbon and nitrogen isotopic discrimination between gnathiids and their host’s blood. As such, general differences in the feeding ecology and trophic positions of the different host species were reflected in their associated gnathiids, indicating that stable isotope analysis of gnathiids can provide significant details concerning previous hosts. The results presented herein have significant implications for how stable isotopes may be used as a tool to study the trophic dynamics and feeding ecology of gnathiids.