Phytochemical profile and determination of cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity of aqueous and ethanolic extracts of Pseudobombax marginatum (A. St.-Hil.) A. Robyns.

Pseudobombax marginatum, popularly known as "embiratanha," is widely used by traditional communities as anti-inflammatory and analgesic agent. This study aimed to determine the phytochemical profile as well as cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity attributed to exposure to aqueous (AqEx) and ethanolic (EtEx) extracts of embiratanha bark. Phytochemical screening was conducted using thin-layer chromatography (TLC). Cell viability was analyzed using MTT assay with human mammary gland adenocarcinoma (MDA-MB-231) and macrophage (J774A.1) cell lines, exposed to concentrations of 12.5, 25, 50, or 100 µg/ml of either extract. For acute oral toxicity, comet assay and micronucleus (MN) tests, a single dose of 2,000 mg/kg of either extract was administered orally to Wistar rats. TLC analysis identified classes of metabolites in the extracts, including cinnamic acid derivatives, flavonoids, hydrolyzable tannins, condensed tannins, coumarins, and terpenes/steroids. In the cytotoxicity assay, the varying concentrations of extracts derived from embiratanha induced no significant alterations in the viability of MDA-MB-231 cells. The lowest concentration of EtEx significantly increased macrophage J774A.1 viability. However, the higher concentrations of AqEx markedly lowered macrophage J774A.1 viability. Animals exhibited no toxicity in the parameters analyzed in acute oral toxicity, comet assay, and MN tests. Further, EtEx promoted a significant reduction in DNA damage index and DNA damage frequency utilizing the comet assay, while the group treated with AqEx exhibited no marked differences. Thus, data demonstrated that AqEx or EtEx of embiratanha may be considered safe at a dose of 2,000 mg/kg orgally under our experimental conditions tested.

Comparative Study of Condensed and Hydrolysable Tannins during the Early Stages of Zebrafish Development.

In this study, we present data on the effects of condensed tannins (CTs) and hydrolysable tannins (HTs), polyphenols extracted from plants, at different concentrations on zebrafish development to identify the range of concentrations with toxic effects. Zebrafish embryos were exposed to CTs and HTs at two different concentration ranges (5.0-20.0 µgL-1 and 5.0-20.0 mgL-1) for 72 h. The toxicity parameters were observed up to 72 h of treatment. The uptake of CTs and HTs by the zebrafish larvae was assessed via HPLC analysis. A qRT-PCR analysis was performed to evaluate the expressions of genes cd63, zhe1, and klf4, involved in the hatching process of zebrafish. CTs and HTs at 5.0, 10.0, and 20.0 µgL-1 were not toxic. On the contrary, at 5.0, 10.0, and 20.0 mgL-1, HTs induced a delay in hatching starting from 48 h of treatment, while CTs showed a delay in hatching mainly at 48 h. The analysis of gene expression showed a downregulation in the group exposed to HTs, confirming the hatching data. We believe that this study is important for defining the optimal doses of CTs and HTs to be employed in different application fields such as the chemical industry, the animal feed industry, and medical science.

Effect of Acacia mearnsii forage or tannin extract on rumen dry matter and crude protein degradation.

This study investigated rumen degradation kinetics of dry matter (DM) and crude protein (CP) in compound feed with different tannin extract inclusions and Acacia mearnsi forage (AMF) relative to dairy feeds (perennial ryegrass+white clover mixture pasture, maize silage, lucerne hay and Themeda triandra hay). The compound feed had 0.75%, 1.5% and 3% tannins extract inclusions while the control was a commercial compound feed. Triplicates of each feed per incubation period were incubated in two fistulated Jersey cows for 0, 6, 24, 48, 72, 96 and 120 h, resulting in six replicates per feed. Tannin extract inclusions in compound feed only affected (p < 0.05) the (a) fraction, degradation rate and potential degradability (PD) for DM degradation, and affected (p < 0.05) the (a) and (b) fractions, as well as PD for CP degradation. The (a) fraction and degradation rate for DM degradation changed linearly (p < 0.05). The (a) fraction, PD and effective degradability (ED) of DM degradation changed quadratically (p < 0.05). Except for the degradation rate, the feed type affected (p < 0.05) the degradation parameters in both DM and CP degradations. For DM and CP degradations, (a) fraction was similarly the least in Themeda triandra hay and AMF but similarly the highest in maize silage, perennial ryegrass+white clover mixture pasture and lucerne hay. The (b) fraction was the least in AMF for both DM and CP degradations but the highest for pasture's DM degradation and similarly the highest in maize silage, lucerne hay and T. triandra hay for CP degradation. The PD was the least in AMF for both DM and CP degradations and similarly the highest in pasture DM but similarly the highest in perennial ryegrass+white clover mixture pasture, maize silage and lucerne hay for CP degradation. Furthermore, the ED was the lowest in AMF and the highest for perrenial ryegrass + white clover mixture pasture for DM degradation and same trend was observed for CP degradation whereby perrenial ryegrass + white clover mixture pasture, maize silage and lucerne had the highest ED. Digestible undegraded protein was the highest in AMF and similarly the least in dairy feeds. Tannin source inclusion in ruminant diets should be moderate to prevent rumen DM or CP degradation limitation.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Phenotypic Variation in Phytochemical Defense of Trembling Aspen in Western North America: Genetics, Development, and Geography.

Trembling aspen (Populus tremuloides) is arguably the most important deciduous tree species in the Intermountain West of North America. There, as elsewhere in its range, aspen exhibits remarkable genetic variation in observable traits such as morphology and phenology. In contrast to Great Lakes populations, however, relatively little is known about phytochemical variation in western aspen. This survey of phytochemistry in western aspen was undertaken to assess how chemical expression varies among genotypes, cytotypes (diploid vs. triploid), and populations, and in response to development and mammalian browsing. We measured levels of foliar nitrogen, salicinoid phenolic glycosides (SPGs) and condensed tannins (CTs), as those constituents influence organismal interactions and ecosystem processes. Results revealed striking genotypic variation and considerable population variation, but minimal cytotype variation, in phytochemistry of western aspen. Levels of SPGs and nitrogen declined, whereas levels of CTs increased, with tree age. Browsed ramets had much higher levels of SPGs, and lower levels of CTs, than unbrowsed ramets of the same genotype. We then evaluated how composite chemical profiles of western aspen differ from those of Great Lakes aspen (assessed in earlier research). Interestingly, mature western aspen trees maintain much higher levels of SPGs, and lower levels of CTs, than Great Lakes aspen. Phenotypic variation in chemical composition of aspen - a foundation species - in the Intermountain West likely has important consequences for organismal interactions and forest ecosystem dynamics. Moreover, those consequences likely play out over spatial and temporal scales somewhat differently than have been documented for Great Lakes aspen.

Alkaline oxidization can increase the in vitro antiparasitic activity of proanthocyanidin-rich plant extracts against Ascarissuum.

Proanthocyanidins (PAs) are a class of plant specialized metabolites with well-documented bioactivities such as antiparasitic effects. However, little is known about how the modification of PAs influences their bioactivity. The objective of this study was to investigate a wide range of PA-containing plant samples to determine if extracts containing PAs modified by oxidation had altered antiparasitic activities, compared to the original extracts that had not been modified in alkaline conditions. We extracted and analyzed samples from 61 proanthocyanidin-rich plants. The extracts were then oxidized under alkaline conditions. We used these non-oxidized and oxidized proanthocyanidin-rich extracts to conduct a detailed analysis of direct antiparasitic effects against the intestinal parasite Ascaris suum in vitro. These tests showed that the proanthocyanidin-rich extracts had antiparasitic activity. Modification of these extracts significantly increased the antiparasitic activity for the majority the extracts, suggesting that the oxidation procedure enhanced the bioactivity of the samples. Some samples that showed no antiparasitic activity before oxidation showed very high activity after the oxidation. High levels of other polyphenols in the extracts, such as flavonoids, was found to be associated with increased antiparasitic activity following oxidation. Thus, our in vitro screening opens up the opportunity for future research to better understand the mechanism of action how alkaline treatment of PA-rich plant extracts increases their biological activity and potential as novel anthelmintics.

Digestibility and nitrogen balance of goats on high and low protein rations supplemented with a commercial tannin feed-additive.

Despite the increasing importance of goat production in response to high demand for their products and their relative robustness to environmental stressors, and in contrast to other ruminant species, little data is available on how tannin extract feeding affects their feed intake, nutrient digestion and nitrogen (N) metabolism. Therefore, a trial in Oman investigated the respective variables by using a commercial tannin feed additive. In a 4 (treatments) x 3 (periods) x 2 (animals) Youden square, two weaned Batinah bucks each were fed a high or low protein diet of Rhodes grass hay and crushed barley grain, with or without the addition of a chestnut and quebracho tannin extract at 2 g/kg metabolic weight. Feed offered, feed refused and faeces and urine excreted were quantified to determine diet digestibility, total N excretion, N retention and rumen microbial protein synthesis (MPS). Due to their young age and low live weight, feed intake of goats was relatively low. Crude protein level and tannin addition had no statistically significant effect on dry matter (DM) and N intake, DM digestibility, N excretion in faeces and urine, as well as MPS. In consequence, no benefit of tannin feeding could be confirmed for the goats' N retention, irrespective of diet composition. These results indicate, on one hand, an effective neutralisation of the tested tannin extract along the gastrointestinal tract of goats, but on the other hand, that stimulation of MPS or N retention by tannins cannot be evidenced when diet components are present that simultaneously release energy and protein, as is the case with barley.

The effects of feeding dried browse leaves on rumen ammonia levels, methanogens and protozoa amplification of sheep in the Coastal Savannah of Ghana.

Tanniferous browse leaves are reported to inhibit methanogens and protozoa activity in the rumen, thus contributing to a reduction of methane emission. This study evaluated the influence of feeding dried browse leaves to sheep on rumen ammonia concentration, the base pair at which protozoa and methanogens were amplified and double stranded DNA concentration (dsDNA) from rumen fluid and faeces. The eight treatments were urea treated rice straw, Albizzia lebbek (AL), Moringa oleifera (MO), Millettia thonningii (MT), AL + MO, AL + MT, AL + MO + MT and MO + MT. After feeding 32 ram lambs for 3 months, one ram lamb on each of the eight experimental diets was randomly selected and slaughtered to obtain rumen fluid. Genomic DNAs were extracted from methanogen and protozoa strains obtained from rumen liquor and from faecal matter of sheep. Rumen ammonia was determined using spectrophotometer. Methanogens and protozoa from rumen fluid and faeces were amplified at 1100 base pair, 200-1100 base pair, 320-1100 base pair and 200-750 base pair respectively. Rumen ammonia concentration, dsDNA from rumen fluid and faeces ranged from 14.51 to 23.01 mg/dl, 65 to 900 µg/ml and 100 to 950 µg/g respectively. The rumen ammonia concentration met the requirements for efficient growth of microbes. The presence of methanogens and protozoa in the rumen fluid and in the faeces indicated that dried browse diets were able to inhibit the growth of both protozoa and methanogens in the rumen by eliminating them, and thus, were excreted in the faeces. Hence, feeding of dried browse leaves can contribute to lower methane emission.

Tannin phenotyping of the Vitaceae reveals a phylogenetic linkage of epigallocatechin in berries and leaves.

BACKGROUND AND AIMS: Condensed tannins, responsible for berry and wine astringency, may have been selected during grapevine domestication. This work examines the phylogenetic distribution of condensed tannins throughout the Vitaceae phylogenetic tree. METHODS: Green berries and mature leaves of representative true-to-type members of the Vitaceae were collected before 'véraison', freeze-dried and pulverized, and condensed tannins were measured following depolymerization by nucleophilic addition of 2-mercaptoethanol to the C4 of the flavan-3-ol units in an organic acidic medium. Reaction products were separated and quantified by ultrahigh pressure liquid chromatography/diode array detection/mass spectrometry. KEY RESULTS AND CONCLUSIONS: The original ability to incorporate epigallocatechin (EGC) into grapevine condensed tannins was lost independently in both the American and Eurasian/Asian branches of the Vitaceae, with exceptional cases of reversion to the ancestral EGC phenotype. This is particularly true in the genus Vitis, where we now find two radically distinct groups differing with respect to EGC content. While Vitis species from Asia are void of EGC, 50 % of the New World Vitis harbour EGC. Interestingly, the presence of EGC is tightly coupled with the degree of leaf margin serration. Noticeably, the rare Asian EGC-forming species are phylogenetically close to Vitis vinifera, the only remnant representative of Vitis in Eurasia. Both the wild ancestral V. vinifera subsp. sylvestris as well as the domesticated V. vinifera subsp. sativa can accumulate EGC and activate galloylation biosynthesis that compete for photoassimilates and reductive power.

Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures.

Infections with Cryptosporidium spp. constitute a substantial public health burden and are responsible for widespread production losses in cattle herds. Reducing disease and shedding of Cryptosporidium spp. oocysts is an important One Health goal. There are very few therapeutic options available to treat cryptosporidiosis. Interest in plant bioactive compounds to mitigate the spread of anthelmintic resistance in ruminants has led to investigation of these phytocompounds against other parasitic taxa. Condensed tannins (CTs) are plant secondary metabolites that have shown potential against nematodes in vitro and in vivo but their applicability to Cryptosporidium spp. is comparatively under-explored. Cryptosporidium parvum infected human ileocecal colorectal adenocarcinoma (HCT)-8 cell cultures were treated with escalating doses of highly purified and well-characterized CTs from five plant species, big trefoil (Lotus pedunculatus), black currant (Ribes nigrum), sainfoin (Onobrychis viciifolia), white clover (Trifolium repens) and grapeseed (Vitis vinifera) for 44 h. Quantitative-PCR (qPCR) analysis revealed that none of the CTs examined demonstrated inhibitory potential against the parasite. Substantial inhibition of C. parvum by paromomycin was observed in positive controls in all assays (76.94-90.72% inhibition), proving the validity of the assay. Despite the lack of inhibition, these results represent an important step towards identifying alternative treatment options against this parasite.

The D165H Polymorphism of QiMYB-like-1 Is Linked to Interactions between Tannin Accumulation, Herbivory and Biogeographical Determinants of Quercus ilex.

The accumulation in the leaves and young stems of phenolic compounds, such as hydrolyzable and condensed tannins, constitutes a defense mechanism of plants against herbivores. Among other stressing factors, chronic herbivory endangers Quercus ilex, a tree playing a central role in Mediterranean forests. This work addressed the connections between the chemical defenses of Q. ilex leaves and their susceptibility to herbivory, quantitative traits whose relationships are modulated by environmental and genetic factors that could be useful as molecular markers for the selection of plants with improved fitness. A search for natural variants detected the polymorphism D165H in the effector domain of QiMYB-like-1, a TT2-like transcription factor whose family includes members that control the late steps of condensed tannins biosynthesis in different plant species. QiMYB-like-1 D165H polymorphism was screened by PCR-RFLP in trees from six national parks in Spain where Q. ilex has a relevant presence, revealing that, unlike most regions that match the Hardy-Weinberg equilibrium, homozygous plants are over-represented in "Monfragüe" and "Cabañeros", among the best examples to represent the continental Mediterranean (cM) ecosystem. Accordingly, the averages of two stress-related quantitative traits measured in leaves, herbivory index and accumulation of condensed tannins, showed asymmetric distributions depending on the clustering of trees based on ecological and genetic factors. Thus, the impact of herbivory was greater in managed forests with a low density of trees from the cM region, among which QiMYB-like-1 D165 homozygotes stand out, whereas condensed tannins accumulation was higher in leaves of QiMYB-like-1 H165 homozygotes from low-density forests, mainly in the Pyrenean (Py) region. Besides, the correlation between the contents of condensed tannins and total tannins vanished after clustering by the same factors: the cM region singularity, forest tree density, and QiMYB-like-1 genotype, among which homozygous shared the lowest link. The biogeographical and genetic constraints that modulate the contribution of condensed tannins to chemical defenses also mediated their interactions with the herbivory index, which was found positively correlated with total phenolics or tannins, suggesting an induction signal by this biotic stress. In contrast, a negative correlation was observed with condensed tannins after tree clustering by genetics factors where associations between tannins were lost. Therefore, condensed tannins might protect Q. ilex from defoliation in parks belonging to the cM ecosystem and carrying genetic factor(s) linked to the QiMYB-like-1 D165H polymorphism.

Optimization of the Extraction of Proanthocyanidins from Grape Seeds Using Ultrasonication-Assisted Aqueous Ethanol and Evaluation of Anti-Steatosis Activity In Vitro.

Conventional extraction methods of proanthocyanidins (PAC) are based on toxic organic solvents, which can raise concerns about the use of extracts in supplemented food and nutraceuticals. Thus, a PAC extraction method was developed for grape seeds (GS) and grape seed powder using food-grade ethanol by optimizing the extraction conditions to generate the maximum yield of PAC. Extraction parameters, % ethanol, solvent: solid (s:s) ratio, sonication time, and temperature were optimized by the central composite design of the response surface method. The yields of PAC under different extraction conditions were quantified by the methylcellulose precipitable tannin assay. The final optimum conditions were 47% ethanol, 10:1 s:s ratio (v:w), 53 min sonication time, and 60 °C extraction temperature. High-performance liquid chromatography analysis revealed the presence of catechin, procyanidin B2, oligomeric and polymeric PAC in the grape seed-proanthocyanidin extracts (GS-PAC). GS-PAC significantly reduced reactive oxygen species and lipid accumulation in the palmitic-acid-induced mouse hepatocytes (AML12) model of steatosis. About 50% of the PAC of the GS was found to be retained in the by-product of wine fermentation. Therefore, the developed ethanol-based extraction method is suitable to produce PAC-rich functional ingredients from grape by-products to be used in supplemented food and nutraceuticals.

Behaviour of Extractives in Norway Spruce (Picea abies) Bark during Pile Storage.

The current practices regarding the procurement chain of forest industry sidestreams, such as conifer bark, do not always lead to optimal conditions for preserving individual chemical compounds. This study investigates the standard way of storing bark in large piles in an open area. We mainly focus on the degradation of the most essential hydrophilic and hydrophobic extractives and carbohydrates. First, two large 450 m3 piles of bark from Norway spruce (Picea abies) were formed, one of which was covered with snow. The degradation of the bark extractives was monitored for 24 weeks. Samples were taken from the middle, side and top of the pile. Each sample was extracted at 120 °C with both n-hexane and water, and the extracts produced were then analysed chromatographically using gas chromatography with flame ionisation or mass selective detection and high-performance liquid chromatography. The carbohydrates were next analysed using acidic hydrolysis and acidic methanolysis, followed by chromatographic separation of the monosaccharides formed and their derivatives. The results showed that the most intensive degradation occurred during the first 4 weeks of storage. The levels of hydrophilic extractives were also found to decrease drastically (69% in normal pile and 73% in snow-covered pile) during storage, whereas the decrease in hydrophobic extractives was relatively stable (15% in normal pile and 8% in snow-covered pile). The top of the piles exhibited the most significant decrease in the total level of extractives (73% in normal and snow-covered pile), whereas the bark in the middle of the pile retained the highest amount of extractives (decreased by 51% in normal pile and 47% in snow-covered pile) after 24-week storage.

Fatty acid profile, secondary compounds and antioxidant activities in the fresh forage, hay and silage of sainfoin (Onobrychis viciifolia) and sulla (Hedysarum coronarium).

BACKGROUND: Sainfoin (Onobrychis viciifolia) and sulla (Hedysarum coronarium) are forage legumes usually preserved to optimize their utilization as feedstuffs. However, the method of preservation modifies the chemical composition differently in both legume species. Secondary compounds (such as proanthocyanidins, fatty acids, carotenoids and tocopherols) present in forages affect the quality of animal products. Therefore, the effect of preservation on the contents of secondary compounds should be investigated. Accoridngly, samples of sainfoin and sulla were directly freeze-dried (fresh), dried at ambient temperature (hay) and vacuum-packaged for 82 days (silage). RESULTS: In both legumes, the total fatty acid and C18:3 n-3 contents decreased and C16:0 increased with preservation (P < 0.001), with a greater effect for the hays than for the silages. For both legumes, the lutein, neoxanthin and violaxanthin contents decreased to a greater extent in the silages than in the hays (P < 0.001). Both hays exhibited the lowest ß-carotene concentrations (P < 0.001). The α-tocopherol contents decreased in hays, but not in silages, compared to the fresh forages of both legumes (P < 0.001). The antioxidant activities were lower in the silages than in the hays and fresh forages (P < 0.001) and were very strongly related to the contents of polyphenols and proanthocyanidins (P < 0.001). CONCLUSION: Haymaking affected the fatty acid, carotenoid and α-tocopherol contents to a greater extent but had a lesser effect on the antioxidant activities than silage-making, which were very strongly related to their contents of polyphenols and proanthocyanidins. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Intake and performance of growing lambs supplemented with quebracho tannins.

Tannins can modulate rumen fermentation and increase the bypass protein; however, the use of different doses and sources of condensed tannins (CT) in animal diets has divergent results. The aim of this study was to evaluate the effect of different levels of dietary quebracho extract inclusion, as a CT source, in the performance and carcass characteristics of growing lambs. Forty-eight weaned lambs (Texel, Suffolk, and Hampshire Down crossbred) were homogenously blocked into four groups and supplemented at 0, 10, 30, and 60 g tannins/kg dry matter intake (DMI) (CONT, T1%, T3%, and T6%, respectively). The DMI, the roughage to concentrate ratio, the feed conversion rate (FCR), the average daily gain (ADG), and the body condition score (BCS) were evaluated. After the trial, animals were slaughtered, and the cold carcass weight, carcass yield, and subcutaneous fat thickness (SFT) were determined. Higher levels of CT in T3% and T6% reduced (P < 0.001) the total DMI and intake of concentrate without impacting (P = 0.071) the roughage intake (% body weight). ADG was the lowest for T6%, although the total weight gain and BCS were the lowest also for T3% (P < 0.001). FCR was worst for T6% (P < 0.001). Regarding carcass characteristics, T6% had the lowest weight of carcass (P = 0.003) and SFT (P = 0.021) compared to CONT. Carcass yields were similar among all the groups (P = 0.247). T1% did not differ from CONT to any parameter (P > 0.05). Supplementing quebracho CT at a low dose did not show beneficial effects; moreover, higher doses of CT led to detrimental effects in intake and animal performance.

Blend of secondary metabolites from mesquite to improve nutrient digestibility, microbial protein, efficient use of nitrogen, ruminal parameters, and blood metabolites in sheep.

Although chemical additives are able to improve the efficiency of ruminal fermentation, they can leave residues in the meat. However, a blend of secondary metabolites can improve ruminal fermentation without harming the population welfare. Five levels (0.0, 1.5, 3.0, 4.5, and 6.0 g/day) of a blend of secondary metabolites from mesquite extract in sheep feed to promote increases in the nutritional value, ruminal parameters, nitrogen (N) use efficiency, microbial protein (MP) synthesis, and blood metabolites. Ten intact male Santa Inês sheep with average body weight of 55 ± 9.81 kg were used in a 5 × 5 Latin square design, replicated twice. There was a quadratic response of the digestibility of dry matter (DM), organic matter (OM), crude protein (CP), and total digestible nutrients (TDN). Microbial protein concentrations, MP synthesis efficiency, propionic acid levels, and acetic/propionic acid ratio also showed a quadratic response. The blend promoted a quadratic effect on plasma glucose and lactate levels. On the other hand, it decreased the concentrations of ammoniacal nitrogen, plasma urea, and plasma cholesterol. It is recommended to supply a blend of secondary metabolites at 3.43 g/day.

The effect of varying levels of purified condensed tannins on performance, blood profile, meat quality and methane emission in male Bapedi sheep fed grass hay and pellet-based diet.

This study determined the effect of purified condensed tannin inclusion levels in a diet on production, haematological indices, blood biochemical components, meat quality and methane emission by yearling indigenous male Bapedi sheep on a grass hay and sheep pellet-based diet in a 28-day trial. The diets contained similar (P > 0.05) nutrients but with different (P < 0.05) purified condensed tannin supplementation levels. A complete randomized design was used. Twenty-four yearling male Bapedi sheep were assigned to four dietary treatments having different purified condensed tannin levels of 0 (GH80P20PCT0), 30 (GH80P20PCT30), 40 (GH80P20PCT40) and 50 (GH80P20PCT50) g/kg DM. A quadratic type of equation was also used to determine condensed tannin supplementation levels for optimal performance and methane emission reduction by sheep. Supplementing diets with purified condensed tannins did not affect (P > 0.05) diet intake, digestibility and live weight gain of male Bapedi sheep. Supplementing diets with purified condensed tannins did not affect (P > 0.05) blood components of male Bapedi sheep. Inclusion of condensed tannins in the diets did not affect (P > 0.05) Bapedi sheep meat pH and sensory attributes. However, supplementing diets with purified condensed tannins decreased (P < 0.05) methane emission by 51 to 60%. A 49.08 g supplementation level with purified condensed tannins per kg DM diet was calculated, with the use of quadratic equations, to result in the lowest methane emission by male Bapedi sheep. The meat of male Bapedi rams on diets containing 30, 40 or 50 g of purified condensed tannins per kg DM contained higher (P < 0.05) antioxidant activities than those from rams fed a diet without purified condensed tannins. These results indicate that purified condensed tannin supplementation levels of 0, 30, 40 or 50 g/kg DM diet had no adverse effects on growth performance, blood profiles and meat sensory attributes of male Bapedi sheep. However, supplementation levels of 30, 40 or 50 g of purified condensed tannins per kg DM diet reduced methane emission by 51 to 60%, and increased sheep meat antioxidant activity values. Supplementing diets with purified condensed tannins has the potential to reduce methane production and emission by sheep. However, long-term studies are recommended to ascertain the present findings.

Responses of Lotus corniculatus to environmental change 3: The sensitivity of phenolic accumulation to growth temperature and light intensity and effects on tissue digestibility.

MAIN CONCLUSION: Growth temperature and light intensity are major drivers of phenolic accumulation in Lotus corniculatus resulting in major changes in carbon partitioning which significantly affects tissue digestibility and forage quality. The response of plant growth, phenolic accumulation and tissue digestibility to light and temperature was determined in clonal plants of three genotypes of Lotus corniculatus (birdsfoot trefoil) cv Leo, with low, intermediate or high levels of proanthocyanidins (condensed tannins). Plants were grown from 10 °C to 30 °C, or at light intensities from 20 to 500 µm m-2 s-1. Plants grown at 25 °C had the highest growth rate and highest digestibility, whereas the maximum tannin concentration was found in plants grown at 15 °C. Approximately linear increases in leaf flavonol glycoside levels were found with increasing growth temperature in the low tannin genotype. Tannin hydroxylation increased with increasing growth temperature but decreased with increasing light intensity. The major leaf flavonols were kaempferol glycosides of which kaempferol-3-glucoside and kaempferol-3,7-dirhamnoside were the major components. Increases in both tannin and total flavonol concentrations in leaves were linearly related to light intensity and were preceded by a specific increase in the transcript level of a non-legume type chalcone isomerase. Changes in growth temperature and light intensity, therefore, result in major changes in the partitioning of carbon into phenolics, which significantly affects tissue digestibility and nutritional quality with a high correlation between tannin concentration and leaf digestibility.

Responses of Lotus corniculatus to environmental change. 4: Root carbohydrate levels at defoliation and regrowth climatic conditions are major drivers of phenolic content and forage quality.

MAIN CONCLUSION: Differential accumulation of root carbohydrates at defoliation have a higher impact than regrowth environmental conditions on the phenolic content and feed quality of the perennial forage legume Lotus corniculatus. The unpredictable nature of proanthocyanidin (condensed tannin) accumulation in regrowth vegetation of the perennial forage legume Lotus corniculatus represents a dilemma to the wider use of this species in agriculture, and a potential problem in the nutritional ecology of some terrestrial herbivores, as variable condensed tannin levels can result in either beneficial or detrimental effects on animal nutrition. However, the source of this variation has not been extensively explored. High levels of carbon allocation to roots during low-temperature preconditioning of clonal plants were found to significantly increase condensed tannin and flavonol levels in regrowth foliage, while low levels of carbon allocation to roots during periods of high-temperature preconditioning significantly decreased condensed tannin and flavonol levels. Phenolic accumulation and tissue digestibility were also differentially affected by regrowth of these defoliated plants at high CO2 concentrations and by drought. Lower rates of digestion generally paralleled increases in tannin levels in regrowth leaves under the different environmental conditions, with rates of digestion falling in high tannin plants, despite correspondingly higher levels of leaf carbohydrates. Differential accumulation of root carbohydrates between seasons and years may therefore explain some of the variability found in the nutritional quality of the forage of this species.

Trait plasticity and trade-offs shape intra-specific variation in competitive response in a foundation tree species.

The ability to tolerate neighboring plants (i.e. degree of competitive response) is a key determinant of plant success in high-competition environments. Plant genotypes adjust their functional trait expression under high levels of competition, which may help explain intra-specific variation in competitive response. However, the relationships between traits and competitive response are not well understood, especially in trees. In this study, we investigated among-genotype associations between tree trait plasticity and competitive response. We manipulated competition intensity in experimental stands of trembling aspen (Populus tremuloides) to address the covariance between competition-induced changes in functional trait expression and aspects of competitive ability at the genotype level. Genotypic variation in the direction and magnitude of functional trait responses, especially those of crown foliar mass, phytochemistry, and leaf physiology, was associated with genotypic variation in competitive response. Traits exhibited distinct plastic responses to competition, with varying degrees of genotypic variation and covariance with other trait responses. The combination of genotypic diversity and covariance among functional traits led to tree responses to competition that were coordinated among traits yet variable among genotypes. Such relationships between tree traits and competitive success have the potential to shape stand-level trait distributions over space and time.