Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins (INPs) of common environmental bacteria like Pseudomonas syringae and Pseudomonas borealis. However, individually, these 100 kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an INP gene into Escherichia coli led to efficient ice nucleation. Here, we demonstrate that a positively charged subdomain at the C-terminal end of the central ß-solenoid of the INP is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the INP multimers form fibres that are ~5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Ice nucleation proteins self-assemble into large fibres to trigger freezing at near 0 °C.

In nature, frost can form at a few degrees below 0 °C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins of common environmental bacteria like Pseudomonas syringae and P. borealis. However, individually, these 100-kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an ice nucleation protein gene into Escherichia coli led to efficient ice nucleation. Here we demonstrate that a positively-charged sub-domain at the C-terminal end of the central beta-solenoid of the ice nucleation protein is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the ice nucleation protein multimers form fibres that are ~ 5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

Diet Preference, Feed Efficiency and Expression of the Sodium-Dependent Glucose Transporter Isoform 1 and Sweet Taste Receptors in the Jejunum of Lambs Supplemented with Different Flavours.

This study investigated the effect of dietary flavour supplements on the preference, feed efficiency and expression of the sweet taste receptor family 1 members 2 and 3 (T1R2 + T1R3), and sodium-glucose linked transporter 1 (SGLT1) genes in the lambs' small intestines. Eight, five-month-old, Israeli crossbred Assaf lambs were offered 16 different non-nutritive commercial flavours in rolled barley and ground corn. Capsicum and sucram were the most preferred non-aroma flavours (p = 0.020), while milky (p < 0.001) was the most preferred powder-aroma flavour. For the metabolic and relative gene expression study, eight lambs were randomly assigned to either sucram, capsicum, a mix containing sucram and capsicum at 1:1 ratio or no flavour for control in a 4 × 2 cross-over design. The total collection of urine (females only), faeces and refusals was carried out, and T1R2, T1R3 and SGLT1 relative gene expression evaluated from the proximal jejunum biopsies. Flavour had no significant effect on the feed intake (p = 0.934), but capsicum increased the average daily weight gain per metabolic body weight (p = 0.049). The T1R3 gene was expressed highest in the mix treatment (1.7; p = 0.005). Collectively, our findings indicate that flavours can be used to motivate feed acceptance and improve the weight gain in lambs.

Supply and demand of creatine and glycogen in broiler chicken embryos.

Optimal embryonic development and growth of meat-type chickens (broilers) rely on incubation conditions (oxygen, heat, and humidity), on nutrients and on energy resources within the egg. Throughout incubation and according to the embryo's energy balance, the main energy storage molecules (creatine and glycogen) are continuously utilized and synthesized, mainly in the embryonic liver, breast muscle, and the extraembryonic yolk sac (YS) tissue. During the last phase of incubation, as the embryo nears hatching, dynamic changes in energy metabolism occur. These changes may affect embryonic survival, hatchlings' uniformity, quality and post hatch performance of broilers, hence, being of great importance to poultry production. Here, we followed the dynamics of creatine and glycogen from embryonic day (E) 11 until hatch and up to chick placement at the farm. We showed that creatine is stored mainly in the breast muscle while glycogen is stored mainly in the YS tissue. Analysis of creatine synthesis genes revealed their expression in the liver, kidney, YS tissue and in the breast muscle, suggesting a full synthesis capacity in these tissues. Expression analysis of genes involved in gluconeogenesis, glycogenesis, and glycogenolysis, revealed that glycogen metabolism is most active in the liver. Nevertheless, due to the relatively large size of the breast muscle and YS tissue, their contribution to glycogen metabolism in embryos is valuable. Towards hatch, post E19, creatine levels in all tissues increased while glycogen levels dramatically decreased and reached low levels at hatch and at chick placement. This proves the utmost importance of creatine in energy supply to late-term embryos and hatchlings.

Water-organizing motif continuity is critical for potent ice nucleation protein activity.

Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section's deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity.

Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the ß-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.

Intra-amniotic administration of l-glutamine promotes intestinal maturation and enteroendocrine stimulation in chick embryos.

Initial nutritional stimulation is a key driving force for small intestinal maturation. In chick embryos, administration of l-glutamine (Gln) into the amniotic fluid stimulates early development of the small intestinal epithelium by promoting enterocyte differentiation. In this study, we evaluated the effects of intra-amniotic administration of Gln on enterocyte morphology and function, and elucidated a potential enteroendocrine pathway through which Gln stimulates small intestinal maturation. Our results show that Gln stimulation at embryonic day 17 significantly increased enterocyte and microvilli dimensions by 10 and 20%, respectively, within 48 h. Post-hatch, enterocytes and microvilli were 20% longer in Gln-treated chicks. Correspondingly, Gln stimulation significantly upregulated mRNA expression of brush border nutrient transporters PepT-1 and SGLT-1 and tight junction proteins TJP-1 and TJP-2, before and after hatch (P < 0.05). Since GLP-2 signaling from intestinal L-cells is associated with enterocyte growth, functionality and integrity, we examined the effects of Gln stimulation on mRNA expression of key hormones and receptors within this enteroendocrine pathway and found significant increases in GLP-2R, IGF-1 and IGF-1R expression before and after hatch (P < 0.05). In conclusion, our findings link primary nutrient stimulation in the developing small intestine with enterocyte morphological and functional maturation and enteroendocrine signaling.

Nutritional stimulation by in-ovo feeding modulates cellular proliferation and differentiation in the small intestinal epithelium of chicks.

Nutritional stimulation of the developing small intestine of chick embryos can be conducted by in-ovo feeding (IOF). We hypothesized that IOF of glutamine and leucine can enhance small intestinal development by promoting proliferation and differentiation of multipotent small intestinal epithelial cells. Broiler embryos (n = 128) were subject to IOF of glutamine (IOF-Gln), leucine (IOF-Leu), NaCl (IOF-NaCl) or no injection (control) at embryonic d 17 (E 17). Multipotent, progenitor and differentiated cells were located and quantified in the small intestinal epithelium between E 17 and d 7 after hatch (D 7) in all treatment groups by immunofluorescence of SRY-box transcription factor 9 (Sox9) and proliferating cell nuclear antigen (PCNA), in-situ hybridization of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) and peptide transporter 1 (PepT1) and histochemical goblet cell staining. The effects of IOF treatments at E 19 (48 h post-IOF), in comparison to control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and multipotent cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significance, 36% increase in progenitor cell counts. All IOF treatments shifted Lgr5+ stem cell localizations to villus bottoms. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Post-hatch, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to control chicks (P < 0.05). We conclude IOF of glutamine stimulates small intestinal maturation and functionality during the peri-hatch period by promoting multipotent cell proliferation and differentiation, resulting in enhanced compartmentalization of multipotent and differentiated cell niches and expansions of the absorptive surface area.

Size-Resolved Community Structure of Bacteria and Fungi Transported by Dust in the Middle East.

The atmosphere plays an important role in transporting microorganisms on a global scale, yet the processes affecting the composition of the airborne microbiome, the aerobiome, are not fully outlined. Here we present the community compositions of bacteria and fungi obtained by DNA amplicon-sequencing of aerosol samples collected in a size-resolved manner during nine consecutive days in central Israel. The campaign captured dust events originating from the Sahara and the Arabian deserts, as well as days without dust ("clear days"). We found that the source of the aerosol was the main variable contributing to the composition of both fungal and bacterial communities. Significant differences were also observed between communities representing particles of different sizes. We show evidence for the significant transport of bacteria as cell-aggregates and/or via bacterial attachment to particles during dust events. Our findings further point to the mixing of local and transported bacterial communities, observed mostly in particles smaller than 0.6 µm in diameter, representing bacterial single cells. Fungal communities showed the highest dependence on the source of the aerosols, along with significant daily variability, and without significant mixing between sources, possibly due to their larger aerodynamic size and shorter atmospheric residence times. These results, obtained under highly varied atmospheric conditions, provide significant assurances to previously raised hypotheses and could set the course for future studies on aerobiome composition.

Intestinal brush border assembly during the peri-hatch period and its contribution to surface area expansion.

Microvilli generate the small intestinal brush border, the main site of nutrient digestion and absorption. Mucosal structuring of the small intestine of chicken during the perihatch period has been widely researched, yet the developmental dynamics of microvilli during this period have not been fully characterized. In this study, we examined the structural and molecular characteristics of microvilli assembly and maturation during the perihatch period. Small intestines of broiler embryos and chicks were sampled at prehatch ages 17 E and 19 E, at day of hatch (DOH) and at 1, 3, 7, and 10 d posthatch. Morphological evaluations and measurements were conducted by scanning electron microscopy (SEM) and light microscopy (LM) (n = 3/timepoint), and expression of microvilli structural genes Plastin 1, Ezrin, and Myo1a was examined by Real-Time qPCR (n = 6/timepoint). Results revealed dissimilar patterns of microvilli and villi development during the perihatch period. From 19 E to 1 d, microvilli lengths increased 4.3-fold while villi lengths increased 2.8-fold (P < 0.0001). From 3 to 7 d, villi lengths increased by 20% (P < 0.005), while microvilli lengths decreased by 41% (P = 0.001). At 10 d, microvilli lengths stabilized, while villi continued to elongate by 26% (P < 0.0001). Estimations of the microvilli amplification factor (MAF) and total enterocyte surface area (TESA) revealed similar trends, with peak values of 78.53 and 1961.67 µm2, respectively, at 3 d. Microvilli structural gene expression portrayed diverse patterns. Expression of Plastin 1, which bundles and binds actin cores to the terminal web, increased 8.7-fold between 17 E and DOH (P = 0.005), and gradually increased up to 7 d (P = 0.045). Ezrin and Myo1a, both actin core-cell membrane cross-linkers, portrayed different expression patterns throughout the perihatch period, as Ezrin expression was relatively stable, while Myo1a expression increased 15.8-fold between 17 E and 10 d (P < 0.0001). We conclude that microvilli assembly during the perihatch period is a rapid, coordinated process, which dramatically expands the digestive and absorptive surface area of the small intestine before the completion of villi maturation.

Incubation temperature affects yolk utilization through changes in expression of yolk sac tissue functional genes.

The yolk sac tissue (YST) is a multifunctional metabolic organ supporting chicken embryonic development. This study examined whether incubation temperatures (ITs) affect YST functions. For this purpose, 300 eggs were assigned to 3 groups and incubated at control IT of 37.8°C, at 1.5°C below, 36.3°C (cold IT), and at 1.5°C above, 39.3°C (hot IT). For each group, 6 embryos' whole body mass and residual yolk (RSY) weights were recorded during incubation, and YST was sampled for both histology and gene expression analysis. YST functionality during incubation was examined by regression analysis, comparing changes in expression patterns of genes involved in lipid uptake and metabolism (LRP2, ApoA1), oligopeptides uptake (PepT1), gluconeogenesis (FBP1), glycogenesis (GYS2), and thyroid hormones regulation (TTR, DIO1, DIO2). Results show that hot and cold ITs affected YST gene expression and yolk utilization. PepT1 expression decreased towards hatch, in both hot and cold ITs, while in the Control IT, it reached a plateau. ApoA1 and DIO2 expression showed a moderate linear fit compared to polynomial fit in the control. GYS2 expression had no change along incubation, while in the control IT, it showed a polynomial fit. Expression of LRP2, FBP1, and DIO1 genes was affected by either cold or hot IT's. TTR expression patterns were similar in all IT groups. The variations in gene expression patterns observed in the 3 ITs can explain the changes in yolk utilization, an important parameter for hatchling quality. While the control IT showed optimal utilization, with an RSY value of 11.12% at the day of hatch, the cold and hot IT groups exhibited lower utilization with an RSY value of 18.18 and 29.99%, respectively. These findings are the first to show that ITs change the expression of key YST genes, leading to variations in yolk utilization by the embryo.

Stochastic nucleation processes and substrate abundance explain time-dependent freezing in supercooled droplets.

Atmospheric immersion freezing (IF), a heterogeneous ice nucleation process where an ice nucleating particle (INP) is immersed in supercooled water, is a dominant ice formation pathway impacting the hydrological cycle and climate. Implementation of IF derived from field and laboratory data in cloud and climate models is difficult due to the high variability in spatio-temporal scales, INP composition, and morphological complexity. We demonstrate that IF can be consistently described by a stochastic nucleation process accounting for uncertainties in the INP surface area. This approach accounts for time-dependent freezing, a wide range of surface areas and challenges phenomenological descriptions typically used to interpret IF. The results have an immediate impact on the current description, interpretation, and experiments of IF and its implementation in models. The findings are in accord with nucleation theory, and thus should hold for any supercooled liquid material that nucleates in contact with a substrate.

On-chip analysis of atmospheric ice-nucleating particles in continuous flow.

Ice-nucleating particles (INPs) are of atmospheric importance because they catalyse the freezing of supercooled cloud droplets, strongly affecting the lifetime and radiative properties of clouds. There is a need to improve our knowledge of the global distribution of INPs, their seasonal cycles and long-term trends, but our capability to make these measurements is limited. Atmospheric INP concentrations are often determined using assays involving arrays of droplets on a cold stage, but such assays are frequently limited by the number of droplets that can be analysed per experiment, often involve manual processing (e.g. pipetting of droplets), and can be susceptible to contamination. Here, we present a microfluidic platform, the LOC-NIPI (Lab-on-a-Chip Nucleation by Immersed Particle Instrument), for the generation of water-in-oil droplets and their freezing in continuous flow as they pass over a cold plate for atmospheric INP analysis. LOC-NIPI allows the user to define the number of droplets analysed by simply running the platform for as long as required. The use of small (∼100 µm diameter) droplets minimises the probability of contamination in any one droplet and therefore allows supercooling all the way down to homogeneous freezing (around -36 °C), while a temperature probe in a proxy channel provides an accurate measure of temperature without the need for temperature modelling. The platform was validated using samples of pollen extract and Snomax®, with hundreds of droplets analysed per temperature step and thousands of droplets being measured per experiment. Homogeneous freezing of purified water was studied using >10 000 droplets with temperature increments of 0.1 °C. The results were reproducible, independent of flow rate in the ranges tested, and the data compared well to conventional instrumentation and literature data. The LOC-NIPI was further benchmarked in a field campaign in the Eastern Mediterranean against other well-characterised instrumentation. The continuous flow nature of the system provides a route, with future development, to the automated monitoring of atmospheric INP at field sites around the globe.

From broiler breeder hen feed to the egg and embryo: The molecular effects of guanidinoacetate supplementation on creatine transport and synthesis.

Supplementation of broiler breeder hens with beneficial additives bears great potential for affecting nutrient deposition into the fertile egg. Guanidinoacetate (GAA) is the endogenous precursor of creatine that is used as a feed additive for improving cellular energy metabolism in animal nutrition. In the present study, we have investigated whether GAA supplementation in broiler breeder feed affects creatine deposition into the hatching egg and molecular mechanisms of creatine transport and synthesis within hens and their progeny. For this, broiler breeder hens of 47 wk of age were supplemented with 0.15% GAA for 15 wk, and samples from their tissues, hatching eggs and progeny were compared with those of control, nonsupplemented hens. A significant increase in creatine content was found within the yolk and albumen of hatching eggs obtained from the GAA group, compared with the control group. The GAA group exhibited a significant increased creatine transporter gene expression compared with the control group in their small intestines and oviduct. In GAA group progeny, a significant decrease in creatine transporter expression at embryonic day 19 and day of hatch was found, compared with control group progeny. At the day of hatch, creatine synthesis genes (arginine glycine amidinotransferase and guanidinoacetate N-methyltransferase) exhibited significant decrease in expression in the GAA group progeny compared with control group progeny. These results indicate that GAA supplementation in broiler breeder feed increases its absorbance and deposition into hatching eggs, subsequently affecting GAA and creatine absorbance and synthesis within broiler progeny.

It's All About Timing: Early Feeding Promotes Intestinal Maturation by Shifting the Ratios of Specialized Epithelial Cells in Chicks.

The small intestine (SI) of chicks (Gallus gallus) matures rapidly during the initial post-hatch period and acquires digestive, absorptive, and secretive capabilities. The effects of the timing of first feeding on the quantities and distribution of specialized epithelial cells, which generate and maintain SI morphology and functionality, have not yet been examined. In this study, we identified specialized SI epithelial cell sub-types, including stem, progenitor, proliferating, and differentiated cells within crypts and villi of chicks during the first 10 days post-hatch, by in situ hybridization (ISH), immunofluorescence (IF), and histochemical staining. We then examined their quantities and ratios between day of hatch and d10 in chicks that were fed upon hatch [early feeding (EF)], compared to chicks that were fed 24 h post-hatch [delayed feeding (DF)]. Results showed that EF increased total cell quantities in the crypts and villi at days 1, 3, 7, and 10, compared to DF (p < 0.0001). At d3, EF, in comparison to DF, decreased crypt stem cell proportions (p < 0.0001), increased crypt proliferating (p < 0.01) and differentiated (p < 0.05) cell proportions, and increased villus enterocyte proportions (p < 0.01). By d10, EF increased both the quantities and proportions of villus enterocytes and goblet cells, compared to DF. We conclude that feeding upon hatch, compared to 24 h-delayed feeding, enhanced SI maturation and functionality by increasing the quantities and proportions of proliferating and differentiated cells, thus expanding the digestive, absorptive, and secretive cell populations throughout the initial post-hatch period.

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas.

Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.

Contrasting Behavior of Antifreeze Proteins: Ice Growth Inhibitors and Ice Nucleation Promoters.

Several types of natural molecules interact specifically with ice crystals. Small antifreeze proteins (AFPs) adsorb to particular facets of ice crystals, thus inhibiting their growth, whereas larger ice-nucleating proteins (INPs) can trigger the formation of new ice crystals at temperatures much higher than the homogeneous ice nucleation temperature of pure water. It has been proposed that both types of proteins interact similarly with ice and that, in principle, they may be able to exhibit both functions. Here we investigated two naturally occurring antifreeze proteins, one from fish, type-III AFP, and one from beetles, TmAFP. We show that in addition to ice growth inhibition, both can also trigger ice nucleation above the homogeneous freezing temperature, providing unambiguous experimental proof for their contrasting behavior. Our analysis suggests that the predominant difference between AFPs and INPs is their molecular size, which is a very good predictor of their ice nucleation temperature.

Detecting thresholds for bitter, umami, and sweet tastants in broiler chicken using a 2-choice test method.

The sense of taste has a key role in nutrient sensing and food intake in animals. A standardized and simple method for determination of tastant-detection thresholds is required for chemosensory research in poultry. We established a 24-h, 2-alternative, forced-choice solution-consumption method and applied it to measure detection thresholds for 3 G-protein-coupled receptor-mediated taste modalities-bitter, sweet, and umami-in chicken. Four parameters were used to determine a significant response: 1) tastant-solution consumption; 2) water (tasteless) consumption; 3) total consumption (tastant and water together); 4) ratio of tastant consumption to total consumption. Our results showed that assignment of the taste solutions and a water control to 2 bottles on random sides of the pen can be reliably used for broiler chicks, even though 47% of the chicks groups demonstrated a consistently preferred side. The detection thresholds for quinine (bitter), L-monosodium glutamate (MSG) (umami), and sucrose (sweet) were determined to be 0.3 mM, 300 mM, and 1 M, respectively. The threshold results for quinine were similar to those for humans and rodents, but the chicks were found to be less sensitive to sucrose and MSG. The described method is useful for studying detection thresholds for tastants that have the potential to affect feed and water consumption in chickens.