Entourage effect for phenolic compounds on production and metabolism of mammary epithelial cells.

Primary culture of mammary epithelial cells (MEC) was exposed to ethyl-acetate, chloroform and hexane extracts of Pistacia lentiscus (lentisk). The hexane extract contained mainly ethyl gallate whereas the chloroform extract contained mainly ethyl-gallate with smaller amount of gallic acid, and the ethyl-acetate extract contained mainly rutin, gallic acid and myricetin. Ethyl acetate extract increased secretion of protein and fat and improved mitochondrial activity. The enhancing effect on protein production was attributed to myricetin, one of the polyphenols in the ethyl-acetate extract whereas gallic acid did not affect protein production or secretion. Interestingly, exposure to the isolated polyphenols did not improve mitochondrial productivity and activity as effectively as exposure to the complete plant extract. The results indicated that polyphenols improve production of milk constituents by MEC, through different modes of action for different polyphenols suggesting an additive or even synergistic effect on production traits of mammary cells.

Pistacia lentiscus extract enhances mammary epithelial cells' productivity by modulating their oxidative status.

We assessed the potential of phenolic compounds from Pistacia lentiscus (lentisk) to enhance production of milk constituents in bovine mammary epithelial cells (MEC). MEC were exposed to 0 (control), 1 or 10 ppm of polyphenols from lentisk ethanolic extract (PLEE) for 24 h. PLEE were absorbed by the MEC plasma membrane, but also penetrated the cell to accumulate in and around the nucleus. PLEE increased triglyceride content in the cell and its secretion to the medium, and significantly increased intracellular lipid droplet diameter. Compared to control, PLEE increased dose-dependently the lactose synthesis, secretion of whey proteins, and contents of casein. To evaluate mitochondrial activity under pro-oxidant load, MEC were preincubated with PLEE and exposed for 2 h to H2O2. Exposure to H2O2 increased the proportion of cells with impaired mitochondrial membrane potential twofold in controls, but not in PLEE-pre-treated cells. Accordingly, proton leakage was markedly decreased by PLEE, and coupling efficiency between the respiratory chain and ATP production was significantly enhanced. Thus, lentisk polyphenols divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control; alternatively, PLEE enables MEC to maintain energy and oxidative status under extreme metabolic rate required for milk production and secretion, and reduces the limitation on energy required to support production.

The effect of willow fodder feeding on immune cell populations in the blood and milk of late-lactating dairy goats.

In a previous study, we showed that access to willow fodder decreased somatic cell counts (SCC) in the milk of local Mamber goats grazing in brushland at the end of lactation. To test whether the consumption of willow affects the cells of the immune system, Alpine crossbred dairy goats grazing in the same environment were either offered free access to freshly cut willow fodder (W, n = 24) or not (C, n = 24) for 2 weeks. The willow fodder contained 7.5 g/kg DM of salicin. The other major secondary compounds were catechin, myricitrin, hyperin and chlorogenic acid (2.2, 2.6, 1.0 and 0.75 g/kg DM, respectively). Udder health status was determined before the experiment, and each of the two groups included five (W) or six (C) goats defined as infected, as established by microbial cfu in milk, and 19 (W) or 18 (C) non-infected goats. Goats ingested, on average, 600 g of DM from willow (25% of food intake), resulting in minor changes in dietary quality compared to the controls, as established by faecal near-IR spectrometry. Throughout the 2 weeks of experiment, differences between groups in dietary CP contents were minor and affected neither by infection nor by access to willow; the dietary percentage of neutral detergent fibre (NDF) decreased in C and increased in W; dietary acid detergent fibre (ADF) increased; and the dietary tannin contents decreased for both treatments. However, milking performance and milk quality attributes in both W and C goats were similar. Initial SCC and milk neutrophil (cluster of differentiation (CD)18+ and porcine granulocyte (PG)68) cell counts were higher in infected than in non-infected goats; counts decreased significantly in W but not in C uninfected goats. The percentage of CD8+ T-cells increased in all C goats, while in the W group, a significant increase was found only for infected goats. The consumption of willow mitigated an increase in CD8+ in blood and triggered an increase in CD8+ in milk, suggesting an immune-regulatory effect independent of udder status. To our knowledge, this is the first report of a direct nutraceutical effect of fodder ingestion on the immune status of goats.

Impact of animal density on cattle nutrition in dry Mediterranean rangelands: a faecal near-IR spectroscopy-aided study.

In the context of determining the sustainable carrying capacity of dry-Mediterranean herbaceous rangelands, we examined the effect of animal density on cattle nutrition, which is fundamental to animal performance and welfare. The effects on dietary components of low (0.56 cows/ha; L) and high (1.11 cows/ha; H) animal densities were monitored for three consecutive years in grazing beef cows. In the dry season (summer and early autumn), cows had free access to N-rich poultry litter (PL) given as a dietary supplement. In each season, near-IR spectroscopy (NIRS) was used to predict the chemical composition of herbage samples (ash, NDF, CP, in vitro dry matter digestibility (IVDMD) and metabolizable energy (ME) content from IVDMD). Near-IR spectroscopy was applied also to faecal samples to determine the chemical composition of the diet selected by the animal, as well as the contents of ash, NDF and CP in the faeces themselves. A faecal-NIRS equation was applied to estimate the dietary proportion of PL. Seasonal categories were green, dry without PL supplementation and dry with it. We found no effects of animal density on nutrition during the green season but effects were apparent when cows consumed dry pasture. Ash content predicted by faecal NIRS was higher in the diet than in plant samples clipped from pasture, which infers that cows ingested soil. Dietary and faecal ash contents were higher (P<0.05) at the H, implying greater soil intake in these animals. During the dry period, dietary contents of ME were higher in L than in H (P<0.05). Poultry litter supplementation was associated with a marked increase (P<0.01) in dietary and faecal CP contents. Poultry litter represented 0.45 and 0.59 of the diet in treatments L and H, respectively (P<0.05). Consequently, treatment H had higher faecal protein (P<0.05). A tendency of higher dietary protein (P=0.08) and lower dietary NDF (P=0.10) in treatment H was probably related to greater PL ingestion. Given that high and sustained rates of poultry litter consumption are detrimental to animal health, the above results cast doubts on the long-term sustainability of the higher of the animal densities tested. Although it may be sustainable vis-à-vis the vegetation, treatment H may have exceeded the boundaries of what is acceptable for cow health. Chemical information revealed with NIRS can be used to evaluate whether animal densities are compatible with animal health and welfare standards and can play a role in determining the carrying capacity of Mediterranean rangelands.

Faecal near-IR spectroscopy to determine the nutritional value of diets consumed by beef cattle in east Mediterranean rangelands.

Rapid assessment of the nutritional quality of diets ingested by grazing animals is pivotal for successful cow-calf management in east Mediterranean rangelands, which receive unpredictable rainfall and are subject to hot-spells. Clipped vegetation samples are seldom representative of diets consumed, as cows locate and graze selectively. In contrast, faeces are easily sampled and their near-IR spectra contain information about nutrients and their utilization. However, a pre-requisite for successful faecal near-infrared reflectance spectroscopy (FNIRS) is that the calibration database encompass the spectral variability of samples to be analyzed. Using confined beef cows in Northern and Southern Israel, we calibrated prediction equations based on individual pairs of known dietary attributes and the NIR spectra of associated faeces (n=125). Diets were composed of fresh-cut green fodder of monocots (wheat and barley), dicots (safflower and garden pea) and natural pasture collected at various phenological states over 2 consecutive years, and, optionally, supplements of barley grain and dried poultry litter. A total of 48 additional pairs of faeces and diets sourced from cows fed six complete mixed rations covering a wide range of energy and CP concentrations. Precision (linearity of calibration, R2cal, and of cross-validation, R2cv) and accuracy (standard error of cross-validation, SEcv) were criteria for calibration quality. The calibrations for dietary ash, CP, NDF and in vitro dry matter digestibility yielded R2cal values >0.87, R2cv of 0.81 to 0.89 and SEcv values of 16, 13, 39 and 31 g/kg dry matter, respectively. Equations for nutrient intake were of low quality, with the exception of CP. Evaluation of FNIRS predictions was carried out with grazing animals supplemented or not with poultry litter, and implementation of the method in one herd over 2 years is presented. The potential usefulness of equations was also established by calculating the Mahalanobis (H) distance to the spectral centroid of a calibration population of 796 faecal samples collected throughout 2 years in four herds. Seasonal trends in pasture quality and responses to management practices were identified adequately and H<3.0 for 98% of faecal samples collected. We conclude that the development of FNIRS equations with confined animals is not only unexpensive and ethically acceptable, but their predictions are also sufficiently accurate to monitor dietary composition (but not intake) of beef cattle in east Mediterranean rangelands.

Self-medication with tannin-rich browse in goats infected with gastro-intestinal nematodes.

Primates self-medicate to alleviate symptoms caused by gastro-intestinal nematodes (GIN) by consuming plants that contain secondary compounds. Would goats display the same dietary acumen? Circumstantial evidence suggests they could: goats in Mediterranean rangelands containing a shrub - Pistacia lentiscus - with known anthelmintic properties consume significant amounts of the shrub, particularly in the fall when the probability of being infected with GIN is greatest, even though its tannins impair protein metabolism and deter herbivory. In order to test rigorously the self-medication hypothesis in goats, we conducted a controlled study using 21 GIN-infected and 23 non-infected goats exposed to browse foliage from P. lentiscus, another browse species - Phillyrea latifolia, or hay during the build-up of infection. GIN-infected goats showed clear symptoms of infection, which was alleviated by P. lentiscus foliage but ingesting P. lentiscus had a detrimental effect on protein metabolism in the absence of disease. When given a choice between P. lentiscus and hay, infected goats of the Mamber breed showed higher preference for P. lentiscus than non-infected counterparts, in particular if they had been exposed to Phillyrea latifolia before. This was not found in Damascus goats. Damascus goats, which exhibit higher propensity to consume P. lentiscus may use it as a drug prophylactically, whereas Mamber goats, which are more reluctant to ingest it, select P. lentiscus foliage therapeutically. These results hint at subtle trade-offs between the roles of P. lentiscus as a food, a toxin and a medicine. This is the first evidence of self-medication in goats under controlled conditions. Endorsing the concept of self-medication could greatly modify the current paradigm of veterinary parasitology whereby man decides when and how to treat GIN-infected animals, and result in transferring this decision to the animals themselves.

Polyphenols from Pistacia lentiscus and Phillyrea latifolia impair the exsheathment of gastro-intestinal nematode larvae.

The infection of grazing ruminants with gastro-intestinal nematodes (GINs) is a severe problem in the Middle East. However, goats that graze the south-western slopes of the Carmel Heights in Israel have very low faecal egg counts, despite high grazing density. We hypothesized that polyphenols from Pistacia lentiscus L. and/or Phillyrea latifolia L. - both prevalent woody species of the region that are consumed by goats - have anthelmintic bioactivity. We tested this hypothesis by using the larval exsheathment inhibition assay (LEIA). Extracts were prepared from leaves of either plant species using 70% ethanol (E70), 100% ethanol (E100), or boiling water (W). Larvae were incubated in a phosphate-buffered saline solution with or without plant extract (1200µg/ml) and then exposed to an exsheathment solution expected to elicit 100% exsheathment after one hour. All extraction methods of P. lentiscus were highly effective at inhibiting larval exsheathment, but higher potency was found for the E70 than for E100 extraction method, while W was intermediate. Only the E70 extract of P. latifolia was highly effective relative to the control. The E70 extract of P. lentiscus had more than 7 times the potency of the E70 extract of P. latifolia. Irrespective of solvent and tannin-equivalent used, P. lentiscus contained more than double the quantity of total polyphenols than P. latifolia. The polyphenols of P. lentiscus consisted mainly of galloyl derivatives (63.6%), flavonol glucosides (28.6%), and catechin (7.8%). In P. latifolia, oleuropein and its derivative tyrosol accounted for 49.3 and 23.1% of phenolics, respectively, the remainder being flavones (luteolin and quercetin) and their glucoside derivatives. Results of the LEIA test suggest that extracts of tannin-rich plants interfere with the very early stage of host invasion and that high concentration of galloylated derivatives may explain anthelmintic activity.

Consumption of Pistacia lentiscus foliage alleviates coccidiosis in young goats.

Coccidiosis near weaning is a major cause of diarrhea, ill-thrift, and impaired performance in small ruminants. A recent survey showed that in villages of the Samaria Hills, Israel, shepherds treat young, weaned goat kids afflicted with diarrhea by cutting and feeding them the foliage of Pistacia lentiscus L. (lentisk) or by tethering them close to lentisk bushes which they browse. The aim of the present study was to assess whether lentisk leaves do indeed have anti-coccidial value, and, if positive, to ascertain the role of tannins in this effect. We monitored for 24 (Experiment 1) and 30 (Experiment 2) days the effect of lentisk feeding on the development of naturally occurring coccidiosis in weaned kids artificially infected with parasitic nematodes. In Experiment 1, kids were infected with nematodes and fed lentisk foliage (PIS) or cereal hay (HAY). Coccidiosis developed at the early stage of the nematode infection, when dietary treatments were initiated. Kids in the PIS group had a lower (P<0.02) concentration of oocysts per gram feces (opg). In Experiment 2, aimed at verifying if tannins are the active component in lentisk foliage, coccidiosis occurred at the peak of the nematode infection, before experimental diets were initiated. Dietary treatments were: cereal hay (HAY), or lentisk foliage consumed without (PIS) or with (PISPEG) a 20-g daily supplement of polyethylene glycol (PEG; MW 4000), a molecule that impairs tannin-bonding with proteins. Goats fed the PIS diet had lower fecal opg counts than counterparts of the HAY (P<0.001) and PISPEG (P<0.002) treatments. Fecal opg counts for the HAY and PISPEG treatments did not differ, suggesting that the anti-coccidial moiety in lentisk was indeed tannins. Our results strongly suggest that: (i) in agreement with the ethno-veterinary anecdotal evidence, exposure of young, weaned goat kids to lentisk foliage alleviates coccidiosis; and (ii) this positive effect is associated with tannins. As coccidiosis is a major affliction of kids, providing them with tannin-rich browse near weaning could be an environmentally friendly way of improving their welfare and health status, in particular under bio-organic farm management.

Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse.

The Damascus and Mamber breeds of goats thrive in Middle Eastern Mediterranean regions where the tannin-rich (20% of polyethylene glycol-binding tannins) brush species Pistacia lentiscus L. (lentisk) is ubiquitous. In light of the increasing recognition of the anthelmintic activity of plant tannins, we examined the effect of offering lentisk foliage for 24 days on fecal egg excretion in 5.5-month-old Damascus and Mamber kid goats (n=28) following infection with 10,000 L3 larvae of mixed gastro-intestinal nematodes (GIN). Lentisk foliage was consumed with or without a daily supplement of 20 g polyethylene glycol (PEG; MW 4000). Lentisk tannins showed a strong protein-depletive effect that was totally reversed by the addition of PEG. At the peak of infection, kids of the two breeds lost weight unless they were fed with lentisk without PEG. Fecal egg counts (FEC) were lowest - and did not differ from 0 - in kids fed lentisk without PEG, highest in the controls fed hay as roughage, and intermediate in kids fed lentisk and PEG (241, 1293, and 705 eggs per gram, respectively, SEM 180; P<0.001); therefore, the anthelmintic activity of lentisk was only partly attributable to tannins. The suppressive effect of lentisk on FEC ceased when feeding was discontinued, suggesting that female parasites were not killed but their fertility was reversibly impaired. Damascus kids showed lower FEC than their Mamber counterparts, inferring that the effect of foraging on tannin-rich species is only additive to genetic differences between goat breeds in their sensitivity to GIN infection. On the basis of our results we would expect yearlong lentisk grazing to result in no or very low GIN infection, and Damascus goats to have some advantage over Mamber goats where chemical control of GIN is unfeasible. There appears to be a trade-off between the benefits of lentisk tannin as drug and its side-effects (protein depletion) when given at high level; how goats balance this trade-off requires further elucidation.

A fecal near-infrared reflectance spectroscopy-aided methodology to determine goat dietary composition in a Mediterranean shrubland.

An ecologically sound approach to the problem of brush encroachment onto Israeli rangeland might be their utilization by goats, but better knowledge of the feeding selectivity and ability of goats to thrive in encroached areas is required to devise viable production systems. Direct observation of bites could provide precise and accurate estimates of diet selection, but construction of a sufficiently large database would require too much time. The present study describes the first attempt to construct fecal near-infrared reflectance spectroscopy (NIRS) calibrations of the botanical and nutritional composition of the diet, and of the total intake of free-ranging goats, based on reference values determined with bite-count procedures. Calibration of fecal NIRS was based on 43 observations encompassing 3 goat breeds and 4 periods (spring, summer, and fall of 2004, and spring of 2005). Each observation comprised 242 min of continuous recording of the species and bite-type category selected by a single animal, on each of 2 consecutive days. The mass and chemical quality of each species and bite-type category-a total of more than 200,000 bites-were determined by using the simulated bite technique. Associated feces were scanned in the 1,100- to 2,500-nm range with a reflectance monochromator. Fecal NIRS calibrations had reasonable precision for dietary percentages of the 3 main botanical components: herbaceous vegetation (as one category; R(2) = 0.85), Phillyrea latifolia (R(2) = 0.89), and tannin-rich Pistacia lentiscus (R(2) = 0.77), with SE of cross-validation (SECV) of 7.8, 6.3, and 5.6% of DM, respectively. The R(2) values for dietary percentages of CP, NDF, IVDMD, and polyethylene glycol-binding tannins were 0.93, 0.88, 0.91, and 0.74, respectively, with SECV values of 0.9, 2.1, 4.3, and 0.9% of DM, respectively. The R(2) values for intakes of herbaceous vegetation, P. latifolia, and P. lentiscus were 0.80, 0.75, and 0.65, with SECV values of 71, 64, and 46 g of DM/d, respectively. The R(2) values for the daily nutrient intakes were below 0.60. Fecal NIRS data can be used to expand the databases of botanical and nutritional dietary composition when observed and resident animals graze simultaneously, but intakes should be calculated from fecal NIRS-predicted dietary DM composition and an independent evaluation of DMI.