Measurement of cow comfort during milking on different cluster removal settings through the use of leg-mounted accelerometers.

Increasing levels of data are routinely collected on modern dairy farms. These include multiple variables measured by milking machine sensors and software and cow-attached sensor data, used predominantly for fertility and health monitoring. Following milking efficiency principles, including milking gently, quickly, and completely, there is utility in investigating how various milking machine settings affect gentleness of milking through a proxy measurement of cow comfort during milking. The use of leg-mounted accelerometers was investigated as a noninvasive labor-efficient means of estimating cow comfort on different automatic cluster remover (ACR) milk flow-rate switch-point settings. Accelerometer step count measurements during milking were collected from 37 cows divided into 2 groups allocated to either an ACR milk flow-rate switch-point setting of 0.2 kg/min or 0.8 kg/min for a 2-wk period and then crossed over to the other setting. Significantly more rear leg stepping occurred during daily milking (combined step count during a.m. and p.m. milkings) where the ACR activated at 0.2 kg/min (11.7 steps) compared with 0.8 kg/min (10.1 steps). Shorter milking interval between a.m. and p.m. milkings resulted in lower udder fill and reduced milk flow-rate. Under these lower udder fill conditions, rear leg movement, as an indicator of cow comfort, reduced when milk flow-rate switch-point for cluster removal increased from 0.2 kg/min (5.75 steps) to 0.8 kg/min (4.96 steps). There was no significant difference between stepping rates on both cluster removal settings during a.m. milkings. Similarly, no significant differences were noted in assessed postmilking teat condition, which was conducted after a.m. milking. The 0.2 kg/min setting extended total daily milking time by 70 s, resulting in lower mean flow-rates while producing similar milk yield. Higher vacuum levels at the teat-end were also recorded on this milking setting. This provides further incentive to consider cluster removal settings above 0.2 kg/min.

Identifying strategies to enhance the milking and operator efficiency of herringbone and rotary parlor systems in Ireland.

International trends of increasing dairy herd sizes coupled with scarcity of labor has necessitated the enhancement of labor efficiency for dairy production systems. This study quantified the effects of infrastructure, automation, and management practices on the milking and operator efficiency of herringbone and rotary parlors used on pasture-based farms in Ireland. Data from 592 milkings across 26 farms (16 herringbones and 10 rotaries) was used. The metrics of cows milked per hour (cows/h), cows milked per operator per hour (cows/h per operator) and liters of milk harvested per hour (L/h) described milking efficiency. The metrics of total process time per cow (TPT, s/cow), milk process time per cow (MPT, s/cow), work routine time (WRT, s/cow), cluster time (CT, s/cluster), and attachment time per cow (ATC, s/cow) described operator efficiency. Automations investigated were backing gates, cluster flush, plant wash, cluster removers (ACRs), feeders, entry gates, rapid-exit, and teat spray. Additional operator presence at milking was also investigated. Herringbone and rotary parlors were assigned to quartiles from their cows/h per operator values to examine infrastructure, automations, and management practices variations. Fourth quartile herringbones based on cows/h per operator values (Q4) averaged 93 cows/h per operator using average system sizes of 24 clusters with 5 parlor automations. Q4 rotaries averaged 164 cows/h per operator using average system sizes of 47 clusters and an average CT of 13 s/cluster. Cows/h per operator values for Q4 herringbone and rotary parlors were 82% and 54% higher, respectively, than values observed on Q1 parlors, indicating the considerable potential to improve efficiency. To determine if infrastructure, automations, or additional operators at milking significantly affected operator efficiencies, general linear mixed models were developed. For parlor infrastructure, additional clusters had greater significance on operator efficiencies (MPT) for herringbones (-1.3 s/cow) as opposed to rotaries (-0.2 s/cow). Hence, increases in system size was likely to result in improved efficiencies for herringbones but less so for rotaries. For automations, ACRs significantly reduced herringbone TPT, CT, and WRT values by 13.3 s/cow, 18.9 s/cluster, and 32.6 s/cow, respectively, whereas rapid-exit significantly lowered CT by 18.6 s/cluster. We found no significant effect on rotary TPT, MPT, CT, or WRT values from the use of automatic teat sprayers. An additional operator at milking was found to significantly reduce herringbone TPT but not MPT or CT. For rotaries, a second operator had no significant effect on TPT, MPT, CT, or WRT values. We documented strong negative correlations between operator efficiencies (TPT, MPT) and milking efficiency (cows/h) for both herringbone (-0.91, -0.84) and rotaries (-0.98, -0.89). Strong negative correlations between the herringbone automation count and TPT (-0.80), MPT (-0.72), and CT (-0.75) suggested highly automated parlors were likely to achieve greater operator efficiencies than less automated parlors. The strong negative correlation (-0.81) between rotary milking efficiency (cows/h) and CT suggested lower CT values (i.e., rotation speed) resulted in increased milking efficiency. In conclusion, our study quantified the effects of parlor infrastructure, automation, and management practices on the milking and operator efficiency of herringbone and rotary parlors.

Survey of farm, parlour and milking management, parlour technologies, SCC control strategies and farmer demographics on Irish dairy farms.

BACKGROUND: This cross-sectional study describes a survey designed to fill knowledge gaps regarding farm management practices, parlour management practices and implemented technologies, milking management practices, somatic cell count (SCC) control strategies, farmer demographics and attitudes around SCC management on a sample of Irish dairy farms. RESULTS: We categorized 376 complete responses by herd size quartile and calving pattern. The average respondent herd was 131 cows with most (82.2%) operating a seasonal calving system. The median monthly bulk tank somatic cell count for seasonal calving systems was 137,000 cells/ml (range 20,000 - 1,269,000 cells/ml), 170,000 cells/ml for split-calving systems (range 46,000 - 644,000 cells/ml) and 186,000 cells/ml for 'other' herds (range 20,000 - 664,000 cells/ml). The most common parlour types were swing-over herringbones (59.1%) and herringbones with recording jars (22.2%). The average number of units across herringbone parlours was 15, 49 in rotary parlours and two boxes on automatic milking system (AMS) farms. The most common parlour technologies were in-parlour feeding systems (84.5%), automatic washers on the bulk tank (72.8%), automatic cluster removers (57.9%), and entrance or exit gates controlled from the parlour pit (52.2%). Veterinary professionals, farming colleagues and processor milk quality advisors were the most commonly utilised sources of advice for SCC management (by 76.9%, 50.0% and 39.2% of respondents respectively). CONCLUSIONS: In this study, we successfully utilised a national survey to quantify farm management practices, parlour management practices and technology adoption levels, milking management practices, SCC control strategies and farmer demographics on 376 dairy farms in the Republic of Ireland. Rotary and AMS parlours had the most parlour technologies of any parlour type. Technology add-ons were generally less prevalent on farms with smaller herds. Despite finding areas for improvement with regard to frequency of liner changes, glove-wearing practices and engagement with bacteriology of milk samples, we also found evidence of high levels of documentation of mastitis treatments and high use of post-milking teat disinfection. We discovered that Irish dairy farmers are relatively content in their careers but face pressures regarding changes to the legislation around prudent antimicrobial use in their herds.

The effects of seasonality, management, infrastructure, and automation on the milking efficiency of herringbone and rotary milking parlors in Ireland.

The objective of this study was to document the milking efficiency of a sample of Irish dairy farms and to understand the effects of (1) seasonality, (2) management practices, (3) parlor infrastructure, and (4) parlor automations on milking efficiency metrics. A novel methodology based on empirical data from video cameras, infrastructure surveys, and milk yield data allowed for the accurate computation of milking efficiency metrics and quantification of the effects of seasonality, number of operators, and parlor automations on milking efficiency across 2 parlor types. The data for this study were collected over 2 periods: period 1 (July 28, 2020, to October 23, 2020, peak-late production) and period 2 (April 12, 2021, to May 19, 2021, early-peak production) from a sample of 16 herringbone and 10 rotary commercial Irish dairy farms. Milking efficiency was evaluated on each farm using 3 key performance indicators: (1) cows milked per hour (cows/h), (2) cows milked per operator per hour (cows/h per operator), and (3) liters of milk harvested per hour (L/h). Milking efficiency key performance indicators were calculated using "total process time," defined as the time between the first cow entering the holding yard and the end of the cleaning process. Average herd sizes for herringbone and rotary farms were 180 and 425 cows, respectively. Average system sizes for herringbone and rotary farms were 20 and 50 clusters, respectively. For herringbone farms, the average milking efficiency was 94 cows/h, 73 cows/h per operator, and 1,012 L/h, whereas rotary farms achieved an average milking efficiency of 170 cows/h, 132 cows/h per operator, and 1,534 L/h. Parlor size was strongly correlated with milking efficiency (cows/h) for herringbone parlors (0.91) but was only moderately correlated for rotary parlors (0.50). Hence, we documented the effect of parlor size on milking efficiency is relative to parlor type. Cluster utilization values on herringbone farms were 5 cows/cluster per h, 4 cows/cluster per operator per h, and 51 L/cluster per h, which were 67%, 33%, and 65% greater than rotary farms, respectively. We found for both herringbone and rotary farms hourly cow throughput (cows/h, cows/h per operator) were greatest during period 1 and that the volume of milk harvested per hour (L/h) was greatest for period 2. Thus, we documented an inverse seasonal relationship between hourly rates of cows milked and milk harvested. We observed that for herringbone farms, milking efficiency (cows/h, L/h) had a strong positive correlation (0.75, 0.74) with the levels of automation use. However, the minimal variation in automations used among rotary farms made it difficult to evaluate their effect on milking efficiency. Similarly, we found that the effect of automations on milking efficiency was dependent on parlor type. On average, a second operator at milking for both herringbone (H) and rotary (R) farms increased values for cows/h (+19%, H; +34%, R) and L/h (+21%, H; +12%, R) but lowered values for cows/h per operator (-35%, H; -12%, R). The holistic methodology applied in this study allowed us to add novel data to the literature by quantifying the effects of seasonality, the number of operators present at milking, and parlor automation use on milking efficiency across 2 parlor types.

Effect of environmental enrichment and group size on the water use and waste in grower-finisher pigs.

The grower-finisher stage accounts for 64% of the total on-farm herd water use. Part of this is consumed by the pigs, but a part is also wasted. Drinking water usage and wastage is affected by different factors. We investigated how different group sizes and different levels of enrichment affect water usage (ingested plus wasted), water wastage, behavior and performance in grower-finisher pigs. Pigs (n = 672), 11 weeks of age (77 ± 2 days) were used for the experiment. The effect of group size: SMALL (12 pigs), MEDIUM (24 pigs), and LARGE (48 pigs) was assessed across two levels of enrichment (LOW-wooden post, hanging rubber toy, HIGH-Same as LOW + fresh grass). There was no effect of group size on water use or wastage. Pigs with HIGH enrichment (10.4 ± 0.4 L/pig/day) used less water than LOW enrichment (11.0 ± 0.4 L/pig/day; p < 0.001). The water wastage/drinker/hour was lower in pens with HIGH enrichment than LOW (p = 0.003). The drinking bout number (p = 0.037) and total occupancy/hour (p = 0.048) was also higher for pens with LOW than HIGH enrichment. Aggressive and harmful behaviour were performed less in LARGE groups and pens with HIGH enrichment. Thus, HIGH enrichment allowance reduced water usage and wastage so may have benefits for the environment, as well as animal welfare.

Effect of different cleaning procedures on water use and bacterial levels in weaner pig pens.

Pork is one of the most globally eaten meats and the pig production chain contributes significantly to the water footprint of livestock production. However, very little knowledge is available about the on-farm factors that influence freshwater use in the pig production chain. An experiment was conducted to quantify the effect of three different washing treatments on freshwater use, bacterial levels [(total bacterial counts; TBC), Enterobacteriaceae and Staphylococcus] and cleaning time in washing of pens for weaning pigs. Three weaner rooms were selected with each room having 10 pens and a capacity to hold up to 14 pigs each. Pigs were weaned and kept in the pens for 7 weeks. Finally, the pens were cleaned before the next batch of pigs moved in. The washing treatments used were power washing and disinfection (WASH); presoaking followed by power washing and disinfection (SOAK), and presoaking followed by detergent, power washing and disinfection (SOAK + DETER). A water meter was used to collect water use data and swab samples were taken to determine the bacterial levels. The results showed that there was no overall effect of washing treatments on water use. However, there was an effect of treatment on the washing time (p<0.01) with SOAK and SOAK+DETER reducing the washing time per pen by 2.3 minutes (14%) and 4.2 minutes (27%) compared to WASH. Nonetheless, there was an effect of sampling time (before or after washing) (p<0.001) on the levels of TBC and Staphylococcus, but no effect was seen on Enterobacteriaceae levels. Thus, the washing treatments used in this study had no effect on the water use of the pork production chain. Although there was no difference in both water use and bacterial load, from a producer perspective, presoaking and detergent use can save time and labour costs, so this would be the preferred option.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

IRE1α cleaves select microRNAs during ER stress to derepress translation of proapoptotic Caspase-2.

The endoplasmic reticulum (ER) is the primary organelle for folding and maturation of secretory and transmembrane proteins. Inability to meet protein-folding demand leads to "ER stress," and activates IRE1α, an ER transmembrane kinase-endoribonuclease (RNase). IRE1α promotes adaptation through splicing Xbp1 mRNA or apoptosis through incompletely understood mechanisms. Here, we found that sustained IRE1α RNase activation caused rapid decay of select microRNAs (miRs -17, -34a, -96, and -125b) that normally repress translation of Caspase-2 mRNA, and thus sharply elevates protein levels of this initiator protease of the mitochondrial apoptotic pathway. In cell-free systems, recombinant IRE1α endonucleolytically cleaved microRNA precursors at sites distinct from DICER. Thus, IRE1α regulates translation of a proapoptotic protein through terminating microRNA biogenesis, and noncoding RNAs are part of the ER stress response.

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1ß (IL-1ß) secretion. Txnip gene deletion reduces pancreatic ß cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1ß secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.

Application of low-temperature CP-Sil 88 column for the isomeric analysis of toxic 2,378-substituted PCDD/Fs in incinerator flyash and sewage sludge using a triple quadrupole GC-MS/MS.

The seventeen 2378-substituted polychlorinated dibenzo-p-dioxins and dibenzo-p-furans (PCDD/Fs) congeners have been separated and analyzed in sewage sludge and incinerator flyash samples using a CP-Sil 88 column (50 m × 0.25 mm I.D., 0.25 µm film thickness) operating at a maximum oven temperature of 240°C. The column was used on a Varian 450-GC with a Varian 320-MS Triple Quadrupole. Calibration standards were used to determine the transition chemistries of the 2378-substituted PCDD/F congeners in the gas chromatography/mass spectrometry (GC-MS/MS) system. The five-point calibration curve for each of the congeners showed very good linearity with R(2) values greater than 0.999. The recovery of labelled compounds ranged from 50% to 120%. Analytical results from a reference flyash (BCR-490) and a reference sewage sludge (BCR-677) compared very well with the certified values, giving percentage deviations in I-TEQ (international toxic equivalents) of 4.93% and 0.53%, respectively. Results from 'real' flyash samples underscored the level of progress made in the abatement of dioxin emissions from incinerators; the old incinerator flyash contained much higher PCDD/F concentrations than the modern one. In addition, the concentrations profiles of PCDD/Fs in the 'real' sewage sludge from two UK wastewater treatment plants (WWTPs) showed that one contained a total PCDD/Fs content of 314 ng I-TEQ kg(-1), while the other gave a total of 53 ng I-TEQ kg(-1). Over an 18-month period of operation, no significant loss of analytical performance was observed from the low-temperature column.

A 3' terminal stem-loop structure in Nodamura virus RNA2 forms an essential cis-acting signal for RNA replication.

Nodamura virus (NoV; family Nodaviridae) contains a bipartite positive-strand RNA genome that replicates via negative-strand intermediates. The specific structural and sequence determinants for initiation of nodavirus RNA replication have not yet been identified. For the related nodavirus Flock House virus (FHV) undefined sequences within the 3'-terminal 50 nucleotides (nt) of FHV RNA2 are essential for its replication. We previously showed that a conserved stem-loop structure (3'SL) is predicted to form near the 3' end of the RNA2 segments of seven nodaviruses, including NoV. We hypothesized that the 3'SL structure from NoV RNA2 is an essential cis-acting element for RNA replication. To determine whether the structure can actually form within RNA2, we analyzed the secondary structure of NoV RNA2 in vitro transcripts using nuclease mapping. The resulting nuclease maps were 86% consistent with the predicted 3'SL structure, suggesting that it can form in solution. We used a well-defined reverse genetic system for launch of NoV replication in yeast cells to test the function of the 3'SL in the viral life cycle. Deletion of the nucleotides that comprise the 3'SL from a NoV2-GFP chimeric replicon resulted in a severe defect in RNA2 replication. A minimal replicon containing the 5'-terminal 17 nt and the 3'-terminal 54 nt of RNA2 (including the predicted 3'SL) retained the ability to replicate in yeast, suggesting that this region is able to direct replication of a heterologous mRNA. These data suggest that the 3'SL plays an essential role in replication of NoV RNA2. The conservation of the predicted 3'SL suggests that this common motif may play a role in RNA replication for the other members of the Nodaviridae.

IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates.

During endoplasmic reticulum (ER) stress, homeostatic signaling through the unfolded protein response (UPR) augments ER protein-folding capacity. If homeostasis is not restored, the UPR triggers apoptosis. We found that the ER transmembrane kinase/endoribonuclease (RNase) IRE1alpha is a key component of this apoptotic switch. ER stress induces IRE1alpha kinase autophosphorylation, activating the RNase to splice XBP1 mRNA and produce the homeostatic transcription factor XBP1s. Under ER stress--or forced autophosphorylation--IRE1alpha's RNase also causes endonucleolytic decay of many ER-localized mRNAs, including those encoding chaperones, as early events culminating in apoptosis. Using chemical genetics, we show that kinase inhibitors bypass autophosphorylation to activate the RNase by an alternate mode that enforces XBP1 splicing and averts mRNA decay and apoptosis. Alternate RNase activation by kinase-inhibited IRE1alpha can be reconstituted in vitro. We propose that divergent cell fates during ER stress hinge on a balance between IRE1alpha RNase outputs that can be tilted with kinase inhibitors to favor survival.

Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress.

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.

Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization.

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.

A kinase inhibitor activates the IRE1alpha RNase to confer cytoprotection against ER stress.

Unfolded proteins in the endoplasmic reticulum (ER) cause trans-autophosphorylation of the bifunctional transmembrane kinase IRE1alpha, inducing its RNase activity to splice XBP1 mRNA, in turn triggering a transcriptional program in the unfolded protein response (UPR). As we previously showed with the yeast IRE1 kinase ortholog, a single missense mutation in the ATP-binding pocket of murine IRE1alpha kinase sensitizes it to the ATP-competitive inhibitor 1NM-PP1, and subordinates RNase activity to the drug. This highly unusual mechanism of kinase signaling requiring kinase domain ligand occupancy-even through an inhibitor-to activate a nearby RNase has therefore been completely conserved through evolution. We also demonstrate that engagement of the drug-sensitized IRE1alpha kinase through this maneuver affords murine cells cytoprotection under ER stress. Thus kinase inhibitors of IRE1alpha are useful for altering the apoptotic outcome to ER stress, and could possibly be developed into drugs to treat ER stress-related diseases.

The use of direct toxicity assessment in the assessment and control of complex effluents in the UK: a demonstration programme.

This paper outlines the background, objectives, methodology, findings, outputs and recommendations from the Direct Toxicity Assessment (DTA) Demonstration Programme. This was a trial of a suite of bioassay methods and a seven-step protocol designed to deliver water quality improvements in catchments with well-defined water quality problems, where ecotoxicity from effluents was a contributing factor. The trial was run as a collaborative venture between the environmental regulators and water and manufacturing industries in the UK and was conducted at three project sites: -a reach of the river Aire near the city of Bradford in Yorkshire; -a reach of the River Esk near the town of Langholm on the border between Scotland and England; and -the lower Tees estuary on the north-east coast of England. The outcomes of each project are summarised in this paper. The learning points delivered by the programme were used to make recommendations to the regulators on how best to use bioassays for the assessment and control of complex effluents in the UK. Guidance was provided on how to carry out the bioassays and on how to use the data generated for regulatory decision-making. The programme also demonstrated how the regulators and the regulated can successfully work together to tackle environmental issues and deliver effective and workable solutions.