Alternative hormone-free reproduction management of a dairy sheep flock disrupts the farm's annual feeding system calendar and its associated strategies.

Hormone-free (HF) reproduction in dairy sheep is a way to meet current societal demands, but it requires being prepared for collateral impacts on related system components. The efficiency of HF practices (e.g., using the male effect for estrus induction and synchronization) is uncertain compared with hormonal treatment (HT). For example, these practices can lead to higher variability in the flock physiological stage patterns throughout the year, which has direct consequences for feeding regimens. The objective of this work was to simulate the impacts of HF reproduction management, including artificial insemination (AI), on the temporal distribution of productive performance and nutritional requirements of a conventional dairy sheep flock. Using the REPROsheep2.0 model, 6 scenarios were compared over one typical production season for the same flock (n = 597 Lacaune ewes) intensively reared in the Roquefort region of France. These scenarios depicted reproduction with HT and AI in mid-May (Early); HT and AI in July (Summer Late); HT and AI in November (Autumn Late); and their HF versions (HF-Early; HF-Summer Late, and HF-Autumn Late, respectively). In all HF scenarios, a reduction in the number of ewes lambing and consequently in the annual milk production of the farm was observed (-1 to -7%). This affected annual performance with a subsequent decrease of total annual nutritional requirements (-2 to -6%). The HF scenarios resulted in a staggering of lambing events with a 7- to 14-d shift in the appearance of milk production peaks and related nutritional requirements compared with the HT scenarios. Transitioning from conventional to HF reproduction management, while preserving AI, would increase farm workload, lengthen milking period operations, and necessitate a readjustment of feeding management strategies with regard to available feed resources. Depending on the production season, the observed delay in the distribution of nutritional requirements could be either an attractive or an unfavorable outcome for farmers. The delay may be concordant, for example, with the recently observed impacts of climate change on seasonal forage availability in Mediterranean regions (less spring herbage production and warmer temperatures) that are affecting farmers' decision-making about the most efficient use of forage and feed resources.

The use of 3-dimensional imaging of Holstein cows to estimate body weight and monitor the composition of body weight change throughout lactation.

Three-dimensional (3D) imaging offers new possibilities in animal phenotyping. Here, we investigated how this technology can be used to study the morphological changes that occur in dairy cows over the course of a single lactation. First, we estimated the individual body weight (BW) of dairy cows using traits measured with 3D images. To improve the quality of prediction, we monitored body growth (via 3D imaging), gut fill (via individual dry matter intake), and body reserves (via body condition score) throughout lactation. A group of 16 Holstein cows-8 in their first lactation, 4 in their second lactation, and 4 in their third or higher lactation-was scanned in 3D once a month for an entire lactation. Values of morphological traits (e.g., chest depth or hip width) increased continuously with parity, but cows in their first lactation experienced the largest increase during the monitoring period. Values of partial volume, estimated from point of shoulder to pin bone, predicted BW with an error of 25.4 kg (R2 = 0.92), which was reduced to 14.3 kg when the individual effect of cows was added to the estimation model. The model was further improved by the addition of partial surface area (from point of shoulder to pin bone), hip width, chest depth, diagonal length, and heart girth, which increased the R2 of BW prediction to 0.94 and decreased root mean square error to 22.1 kg. The different slopes for individual cows were partly explained by body condition score and morphological traits, indicating that they may have reflected differences in body density among animals. Changes in BW over the course of lactation were mostly due to changes in growth, which accounted for around two-thirds of BW gain regardless of parity. Body reserves and gut fill had smaller but still notable effects on body composition, with a higher gain in body reserves and gut fill for cows in their first lactation compared with multiparous cows. This work demonstrated the potential for rapid and low-cost 3D imaging to facilitate the monitoring of several traits of high interest in dairy livestock farming.

Measuring liveweight changes in lactating dairy ewes with an automated walk-over-weighing system.

Monitoring liveweight (LW) is an important part of sound management practices at the individual and flock level (e.g., controlling for nutritional status based on body condition, reproduction, and health-related issues), but it is time consuming and stressful. To our knowledge, no literature has reported on the evaluation of automated weighing systems in dairy sheep as an alternative to conventional static scales. The objective of this research was to evaluate the practical feasibility of using an automated walk-over-weighing (WoW) prototype to measure daily LW changes in dairy ewes without human intervention. We used adult Lacaune dairy ewes in 2 complementary trials conducted indoors. Trial 1 aimed at evaluating the repeatability, precision, and accuracy of LW measures recorded using WoW scales compared with a static scale (the gold standard). Forty-two adult ewes (LW ± standard deviation = 71.3 ± 10.4 kg) were randomly drafted from the main flock and used in a 1-day session. The trial included 3 passages. In each passage, ewes were weighed first on a static scale; once a static position was achieved and LW recorded, they continued the circuit and immediately traversed the WoW scale for an automated LW record. Trial 2 aimed to demonstrate the feasibility of using the WoW device under real-world conditions in a dairy sheep-farming system. The WoW scale was installed in the exit race of the milking parlor and evaluated over 7 wk with adult ewes in mid lactation (n = 93; LW 78.5 ± 8.1 kg). Once the ewes were acclimated to the WoW system, 1 group of ewes (n = 48) continued to receive the same feeding regimen (controls), and the other group (n = 45) underwent a nutritional challenge [challenged; 2 wk of undernutrition and then back to control regimen (refeeding) for 1 wk]. We evaluated the ability of the WoW to detect small changes in LW. We collected LW data (2 weighings per ewe per day) from the WoW after each of the 2 milking sessions (morning and evening). We also obtained LW values by weighing the ewes using a static scale once a week. The automated WoW system showed substantial agreement with the gold standard when assessed using Lin's concordance correlation coefficient and Bland and Altman's method, largely due to high repeatability. The WoW system was adequate for detecting small daily variations in LW during undernutrition and refeeding periods. Misbehaviors resulted in spurious WoW values in trial 2, requiring us to use filtration methods to exclude outlier weights and allow meaningful assessment of small LW changes. The WoW system evaluated here is an alternative to the static scales conventionally used on dairy sheep farms. If sound filtration of raw data is applied, WoW could contribute to the close (daily) monitoring of individual LW without operator intervention (i.e., voluntary weighing) and taking animal welfare into account (i.e., no stress related to the weighing session on static scales).

Iron excess upregulates SPNS2 mRNA levels but reduces sphingosine-1-phosphate export in human osteoblastic MG-63 cells.

We aimed to study the mechanisms involved in bone-related iron impairment by using the osteoblast-like MG-63 cell line. Our results indicate that iron impact the S1P/S1PR signalizing axis and suggest that iron can affect the S1P process and favor the occurrence of osteoporosis during chronic iron overload. INTRODUCTION: Systemic iron excess favors the development of osteoporosis, especially during genetic hemochromatosis. The cellular mechanisms involved are still unclear despite numerous data supporting a direct effect of iron on bone biology. Therefore, the aim of this study was to characterize mechanisms involved in the iron-related osteoblast impairment. METHODS: We studied, by using the MG-63 cell lines, the effect of iron excess on SPNS2 gene expression which was previously identified by us as potentially iron-regulated. Cell-type specificity was investigated with hepatoma HepG2 and enterocyte-like Caco-2 cell lines as well as in iron-overloaded mouse liver. The SPNS2-associated function was also investigated in MG-63 cells by fluxomic strategy which led us to determinate the S1P efflux in iron excess condition. RESULTS: We showed in MG-63 cells that iron exposure strongly increased the mRNA level of the SPNS2 gene. This was not observed in HepG2, in Caco-2 cells, and in mouse livers. Fluxomic study performed concomitantly on MG-63 cells revealed an unexpected decrease in the cellular capacity to export S1P. Iron excess did not modulate SPHK1, SPHK2, SGPL1, or SGPP1 gene expression, but decreased COL1A1 and S1PR1 mRNA levels, suggesting a functional implication of low extracellular S1P concentration on the S1P/S1PR signalizing axis. CONCLUSIONS: Our results indicate that iron impacts the S1P/S1PR signalizing axis in the MG-63 cell line and suggest that iron can affect the bone-associated S1P pathway and favor the occurrence of osteoporosis during chronic iron overload.

Somatic cell count-based selection reduces susceptibility to energy shortage during early lactation in a sheep model.

During the transition from late gestation to early lactation ruminants experience a negative energy balance (NEB), which is considered to increase susceptibility to mammary infections. Our previous study in 2 divergent lines of sheep selected for high and low somatic cell score (SCS) suggested an association between the response to NEB and genetic susceptibility to mastitis. Forty-eight early-lactation primiparous dairy ewes from the 2 SCS genetic lines were allocated to 2 homogeneous subgroups-an NEB group, which was energy restricted and received 60% of the energy requirements for 15 d, and a control-fed group-to obtain 4 balanced groups of 12 ewes: high-SCS positive energy balance, low-SCS positive energy balance, high-SCS NEB, and low-SCS NEB. High-SCS ewes showed greater weight loss and increased plasmatic concentrations of ß-hydroxybutyrate and nonesterified fatty acids than low-SCS ewes when confronted with an induced NEB. The aim of this study was to further characterize this interaction by combining transcriptomic and phenotypic data with a generalized partial least squares discriminant analysis using mixOmics package framework. A preliminary analysis using 3 blocks of phenotypes (fatty acids, weight and production, blood metabolites) revealed a high correlation between fat-to-protein ratio, ß-hydroxybutyrate, and nonesterified fatty acids concentrations with milk long-chain fatty acid yields. These phenotypes allowed good discrimination of the energy-restricted high-SCS ewes and confirmed a high level of adipose tissue mobilization in this group. A second analysis, which included RNA-seq data, revealed high correlations between the long-chain fatty acid yields in milk and PDK4, CPT1A, SLC25A20, KLF10, and KLF11 expression, highlighting the relationship between mobilization of body reserves and enhanced fatty acids utilization for energy production in blood cells. Finally, analysis of milk composition measured in 1,025 ewes from the 2 genetic lines over 10 yr confirmed significant higher fat-to-protein ratio in high-SCS ewes in early lactation. Altogether, our results strongly confirmed a genetic link between susceptibility to mastitis and metabolic adaptation to energy shortage. Improving genetic resistance to mastitis using SCS should be accompanied by a favorable effect on the response to metabolic stress, especially in highly stressful early lactation. Moreover, this study suggests that the fat-to-protein ratio could be used as a low-cost tool for monitoring energy balance and ketosis during this critical phase of lactation.

Synthesis and photophysical properties of extended π conjugated naphthalimides.

A series of π conjugated naphthalimide derivatives having an imide group as an acceptor conjugated with a methoxy arylethynyl or a methoxyphenyl triazole as a donor were prepared by Sonogashira coupling or "click" chemistry. Their photophysical properties were investigated by steady state and time resolved fluorescence spectroscopy and modelled by TD-DFT calculations. Compound Naphth-AlkyneOMe has a high fluorescence quantum yield and displays efficient photoinduced charge transfer in solution as well as in the powder state. Compound Naphth-TriazoleOMe exhibits a very high Stokes shift and its fluorescence quantum yield is low, which can be rationalized by theoretical calculations.

Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis.

Dairy ruminants experiencing a severe postpartum negative energy balance (NEB) are considered to be more susceptible to mastitis. Although the genetic variability of mastitis resistance is well established, the biological basis of the link between energy metabolism and resistance is mostly unknown. The aim of this study was to characterize the effect of NEB on metabolism and immune response according to the genetic background for mastitis resistance or susceptibility. Forty-eight ewes from high and low somatic cell score (SCS) genetic lines were allocated to 2 homogeneous subgroups 2 wk after lambing: one group (NEB) received an energy-restricted diet to cover 60% of their energy requirements, and the other group received a control (positive energy balance: PEB) diet. Both diets met the protein requirements. After 10 d on either the NEB or PEB diet, all ewes were injected with a Pam3CSK4/MDP solution in one half-udder to induce an inflammatory response. The ewes were monitored for milk production, somatic cell count (SCC), body weight (BW), body condition score (BCS), and blood metabolites. Differential milk cell counts were determined by flow cytometry. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), and triiodothyronine were determined. Energy restriction resulted in an increased fat:protein ratio in milk and decreased milk yield, BW, and BCS. The NEB ewes had significantly higher NEFA and BHB and lower plasma glucose concentrations than PEB ewes, reflecting a mobilization of body reserves and ketone body synthesis. High-SCS ewes had a higher SCS than low-SCS throughout the experiment, except after the inflammatory challenge, which resulted in similar SCS in all 4 groups. A noteworthy interaction between genetic background and diet was evidenced on metabolic parameters and BW. Indeed, high-SCS ewes subjected to NEB showed greater decrease in BW and increased NEFA and BHB concentrations compared with low-SCS ewes. Thus, NEB in early lactation led to extensive mobilization of body reserves and intense ketone body synthesis in mastitis-susceptible sheep. These results reinforce the hypothesis of a genetic association between mastitis susceptibility and energy metabolism and open the way to further studies on the biological basis for this association.

Solvent tuned single molecule dual emission in protic solvents: effect of polarity and H-bonding.

Phen-PENMe2 has recently been proposed as a promising new molecule displaying solvent-tuned dual emission, highlighting an original and newly-described charge transfer model. The study of the photophysical behaviour of this molecule was extended to include protic solvents. The effects of polarity and hydrogen bonding lead to an even more evident dual emission associated with a large multi-emission band in some solvents like methanol, highlighting Phen-PENMe2 as a promising candidate for white light emission.

Solvent-tuned dual emission: a structural and electronic interplay highlighting a novel planar ICT (OPICT).

Displaying a dual emission, a Phen-PENMe2 compound can be foreseen as a new model for fundamental studies. It is based on an excited state cumulene-type structure, involving orthogonal π orbital (OPICT). In contrast to the "Twisted Intramolecular Charge Transfer" (TICT) emission, the OPICT emissive state is planar. This new compound is also a potential candidate for local ratiometric probes of medium polarity (mixture of solvents and biological systems) and white emission.

Feed restriction affects milk performances and decreases milk lipolysis in dairy ewes.

Spontaneous lipolysis results in the breakdown of milk fat by the lipoprotein lipase (EC: 3.1.1.34), an enzyme present in milk. Free fatty acids (FFAs) and by-products released in milk during lipolysis can alter both the organoleptic value of milk (off-flavors release) and technological properties of dairy products (decrease in creaming capabilities). Current climate change is having significant impacts on the feeding of grazing animals, with negative consequences on the availability and quality of grass. We and others have demonstrated that dietary restriction increases milk lipolysis in the cow species. However, no data about the impact of feed restriction on milk lipolysis is available in the ewe species. Thus, this paper aims to investigate the effect of feed restriction on milk characteristics with regard to lipolysis values in dairy ewes. Two groups of 24 multiparous Lacaune ewes in mid-lactation received a "non-restricted" control diet (100% of ad libitum DM intake) or a "restricted" (RESTR) diet (65% of ad libitum DM intake) according to a 2 × 2 crossover design. Milk gross composition together with lipolysis analyses were performed. Blood samples were also screened for metabolites or hormone concentrations. The RESTR treatment induced a decrease in milk production (- 21% compared with control treatment) and a modification of the metabolism of dairy ewes characterized by an increase in plasma non-esterified fatty acids (NEFAs), which represents the balance between adipose tissue mobilization and the use of NEFA by other tissues (+153%), cholesterol (+17%) and ß-hydroxybutyrate (+4 %) levels. As a result, a decrease in BW of dairy ewes was observed (-7%). Feed restriction also resulted in a decrease in milk lipolysis estimated by the milk FFA measured by the copper-soap method (-63 and -62%, respectively, for morning and evening milking) or by the reference Bureau of Dairy Industry method (-51 and -57%, respectively, for morning and evening milking). The decrease in milk spontaneous lipolysis under feed restriction was not associated with a decrease in lipoprotein lipase activity in ewes. These results will be completed with proteomic and lipidomic studies in milk samples to better understand mechanisms initiated in the ewe species specifically with regard to lipolysis in milk.

Opportunities and limits of commercial farm data to study the genetic determinism of feed efficiency throughout lactation in dairy sheep.

The collective economic and environmental interest of the whole dairy sheep sector is to reduce feed costs and the negative impact of milk production on the environment. Thus, this study focused on the characterisation and genetic selection potential of feed efficiency in the Lacaune breed. Estimates for feed efficiency in dairy ewes are limited, mainly due to a lack of individual feed intake measurements in the sheepfold or in the pasture. We estimated the genetic parameters for two approximated (not entirely based on individual data) feed efficiency traits (lactation feed conversion ratio (LFCR) and residual energy intake (REI)) and daily milk yield (DMY) at different stages of lactation and throughout lactation. The accuracy of the efficiency traits was first evaluated on samples from Lacaune dairy ewes that were monitored individually, especially for their feed intake. Then, feed efficiency estimation methods were applied on eight commercial farms corresponding to 4 680 Lacaune dairy ewes over two milk lactations (30 854 records). Animals were collectively (for a large part of feed intake) or individually (for milk performance and dynamics of body fat reserves) monitored at different lactation stages. The heritabilities of LFCR and REI were estimated over lactations at 0.10 ± 0.01 and 0.11 ± 0.01, respectively. High genetic correlations were observed between the two efficiency traits and milk production traits, with a genetic correlation between LFCR and DMY of 0.74 ± 0.04 and between REI and DMY of -0.79 ± 0.04. A strong influence of environmental factors such as farm, year of milk production and lactation stage affected the genetic link between REI and milk production traits. Efficiency values observed in early lactation when animals were bred in the sheepfold were less genetically correlated with values obtained later in lactation when animals were grass-fed. However, individual characterisation of feed efficiency remains difficult due to the collective feeding context in dairy ewe farms.

Hypocholesterolaemic and antioxidant properties of Olea europaea L. leaves from Chlef province, Algeria using in vitro, in vivo and in silico approaches.

Aqueous and ethanol extracts prepared from leaves of Olea europaea L. were evaluated for in vitro antioxidant and in vivo hypocholesterolemic effect. The result of administration of O. europaea leaf extracts on serum total cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) in hypercholesterolaemic mice was evaluated. In addition, rutin and luteolin, reported to occur naturally in O. europaea leaves, were docked against HMG-CoA reductase, the rate-limiting enzyme in cholesterol metabolism. Mice treated with both extracts showed reduced total cholesterol (246.6 and 163.4 mg/dl, for mice groups treated with respective extracts) and LDL (150.16 and 81.28 mg/dl, for mice groups treated with respective extracts) levels as compared to the hypercholesterolaemic group (total cholesterol 253.00 mg/dl and LDL 160.00 mg/dl). Mice treated with aqueous extract (200 mg/kg body weight) showed significantly reduced triglyceride and VLDL levels as compared to the group treated with atorvastatine. HDL level of mice administered with O. europaea aqueous extract was comparable to the atorvastatine-treated group. The ethanol extract of O. europeae leaves was a potent antioxidant (IC50 69.15 mg/ml, % inhibition 54.98, 82.63 mg ascorbic acid equivalent/g extract, 7.53 mol of Fe2+/g extract, and % inhibition 49.71, for the DPPH, ß-carotene bleaching, total antioxidant capacity, FRAP, and ferric thiocyanate assays, respectively). Docking studies revealed that rutin showed higher binding affinity with HMG-CoA reductase as compared to luteolin. Data gathered from this study support the development of a prophylactic biomedicine from O. europaea leaves for the management of hypercholesterolemia and atherosclerosis.

Ligands playing musical chairs with G-quadruplex DNA: a rapid and simple displacement assay for identifying selective G-quadruplex binders.

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex- over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplex- and duplex-DNA matrices. The original protocol was tested using various quadruplex- and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays.

Application of molecular technologies to monitor the microbial content of biosolids and composted biosolids.

Disposal of human biosolids is a source of concern for public health and the environment. Composting appears to be an interesting alternative to traditional disposal methods as it can decrease the load of human pathogenic microorganisms often present in biosolids and yield an end-product rich in nutrients for use as a soil supplement. Assessing the exact microbial content of biosolids, both for biosafety and operational reasons, has traditionally relied on the use of standard microbiological methods. Recent developments in molecular-based technologies now offer more rapid and specific monitoring of microorganisms in biosolids than culture-based methods. In this study, denaturing gradient gel electrophoresis (DGGE) was adapted to monitor the succession of bacteria in composted biosolids through different steps of compost production. Secondly, a TaqMan quantitative real time PCR (qPCR) approach was developed to detect and quantify the presence of Salmonella species, a model human pathogenic bacterium, susceptible to be found in biosolids. DGGE results indicated that the bacterial content of composted biosolids of different ages belongs to various taxa and significantly changes with age. qPCR results indicated that the quantity of Salmonella species found in composted biosolids ranging from 1 to 24 months significantly decreases with composting time.

Thiazole orange: a useful probe for fluorescence sensing of G-quadruplex-ligand interactions.

Fluorimetric titrations were performed to gain insight into parameters that govern the association of thiazole orange (TO) and G-quadruplex-DNA (G4-DNA). Use of loop-containing and loop-lacking quadruplexes evidenced the critical influence of the loops on the stoichiometry of the association and on the fluorescence exaltation of TO. We subsequently tried to benefit from this sensitivity to evaluate the influence of G4-DNA cationic environment on ligand binding via a recently reported G4-FID assay.

A unique, environmentally sustainable and cost-effective programme to re-vegetate military training lands utilising composted wastewater biosolids at a large Canadian military training centre.

The Greater Moncton Sewerage Commission has developed, in concert with National Defence Canada, an environmentally sustainable and cost effective biosolids management and land reclamation programme at the Canadian Forces Base (CFB) Gagetown, New Brunswick, Canada (the second largest land based Military Training Facility in the British Commonwealth). The use of composted biosolids to revegetate military training lands is thought to be a unique application for the beneficial use of biosolids. Results and practical experience gained from this approach to successfully re-vegetate initial sections of extremely large and vast tracts of these lands are described. The paper also overviews the Commission's modern 115000 m3 x d(-1) advanced, chemically assisted primary wastewater treatment facility and associated alkaline (lime) sludge stabilisation process. Planning strategies, security aspects, special and unique challenges in operating adjacent to an active military training facility, costs, spreading techniques, monitoring, next steps and conclusions are also presented.

Stable and unstable surface evolution during the drying of a polymer solution drop.

Drying of a sessile drop of a complex liquid can lead to intriguing complex shapes. We report here a study dealing with a model system, made of a hydrosoluble polymer that is glassy when pure. Under solvent evaporation, polymers accumulate near the vapor/drop interface and may form a glassy skin, which bends as the volume of liquid it encloses decreases. The conditions for the occurrence of this buckling instability have been investigated; the experimental results are well explained by a model that compares the characteristic times for drying and for the formation of a glassy skin. Depending on the experimental conditions, different types of shape distortion take place; secondary instabilities that break the axisymmetry are also observed.

Rapid settling of a colloidal gel.

We study the rapid collapse of gels formed from strongly aggregating colloidal suspensions. This gravity-driven collapse is associated with the apparition of fractures in the bulk of the gels that provide an easy route to the gel-supernatant interface for the solvent and are the cause for the strong increase of the settling velocity. We propose a model that connects the apparition of a fracture in the gel to the settling velocity of the interface. This description takes into account the microscopic structure of the gel and is consistent with the experimental results.

Morphologies resulting from the directional propagation of fractures.

When growing in a stress gradient, cracks have a directional growth. We investigate here this type of instability in the case of a colloidal gel deposited on a substrate and left to dry. The use of various materials reveals the existence of two distinct types of dynamics. When the crack nucleation is easy a well known situation is reached: an array of periodic fractures forms, which grow parallel to each other and move quasistatically with the stressed region. In contrast, in materials where the crack nucleation is difficult, a subcritical process is observed with the retarded formation of isolated cracks which move faster and which display an arch shaped trajectory. This type of process appears to be generic in all cases where there is delayed nucleation. This is confirmed by experiments on the directional propagation of cracks in thermally stressed glass.

Cadmium content of wheat grain from a long-term field experiment with sewage sludge.

Grain Cd concentrations were determined in the wheat (Triticum aestivum L.) cultivars Soissons, Brigadier, and Hereward grown in 1994,1996, and 1999, respectively, in soils of a long-term field experiment to which sewage sludges contaminated with Zn, Cu, Ni, or Cr had previously been added. Soil pore water soluble Cd and free Cd2+ increased linearly with increasing total soil Cd (R2=0.82 and 0.84, respectively; P<0.001). Similarly, soil pore water free Cd2+ increased linearly with increasing soil pore water soluble Cd (R2=0.98; P<0.001). There was no evidence of a plateau in soil pore water Cd concentrations with increasing soil Cd concentrations. Grain Cd concentrations were significantly correlated with total soil Cd (P<0.001), soil pore water Cd (P<0.001), and free Cd2+ (P<0.001). A slight curvilinear relationship between grain Cd and soil Cd was apparent, but there was no plateau, even at the maximum soil Cd concentration of about 2.7 mg kg(-1). The relationship between soil pore water Cd and grain Cd was linear for all three cultivars. The slopes were in the order 1994 > 1996 > 1999, with more Cd being taken up into the grain by Soissons grown in 1994, and least by Hereward grown in 1999. For Soissons, Cd concentration in the grain greater than the EU limit (0.24 mg kg(-1) dry wt.) occurred at soil Cd less than the current UK limit of 3 mg kg(-1) for soils receiving sewage sludge. In contrast, for Brigadier and Hereward, grain Cd concentrations were near to and less than the EU limit, respectively, at soil Cd concentrations of 3 mg kg(-1).