No size-dependent net particle retention in the hindgut of horses.

Sieve analyses of hindgut contents of horses as well as observations in horses where plastic markers had been applied to a caecal cannula suggested that there may be a discrimination by particle size in the passage or retention of digesta. Here, we performed a similar experiment with five caecum-cannulated horses (562 ± 31 kg) fed a constant amount (6.81 kg dry matter/day) of grass hay. Passage markers representing the liquid (Co-EDTA) as well as the particulate digesta phase (Yb-undefined; Cr mordanted fibre 1-2 mm; Ce-mordanted fibre 8 mm) were given as a pulse-dose into the cannula to measure their mean retention times (MRT). The MRTs were compared by repeated-measurements analysis of variance. The MRT in the hindgut was 22.2 ± 2.4 h for Co, 25.0 ± 3.4 h for Yb, 26.2 ± 1.6 h for Cr and 26.3 ± 1.5 h for Ce. Whereas differences between the particle marker MRTs were not significant (padj. > 0.05), significant differences were observed between the solute marker Co and each of the particle markers Cr and Ce (padj. < 0.009). The results confirm the well-known significant, albeit small, difference in MRT in horses between the fluid and the particle digesta phase, and corroborate another recent study that used a combination of whole, marked hay and individual marker analysis in different particle size fractions of the faeces, which also did not detect a selective retention of any particle size class.

The effects of processing barley and maize on metabolic and digestive responses in horses.

The competition for customers increases the search for new grain processing methods for equine feed, but the effect on starch digestibility and metabolic responses varies. Therefore, to evaluate the effect of the processing methods, toasting and micronizing, on starch digestion and the effect on metabolic responses, the mobile bag technique (MBT) and plasma glucose and insulin concentrations in the blood were used to estimate nutrient disappearance and metabolic responses pre-cecally. Further, cecal pH, ammonium nitrogen (N), and short-chain fatty acid (SCFA) concentrations were used to estimate the metabolic response in the cecum. Four cecally cannulated horses (body weight [BW] 565 ± 35 kg) were used in a 4 × 4 Latin square design with four periods of 8 d of diet adaptation and 2 d of data collection. Diets were formulated using hay and processed grains: micronized barley (MB), toasted barley (TB), micronized maize (MM), and toasted maize (TM) and were balanced to provide 1 g starch/kg BW in the morning meal. On day 9 in each period, blood and cecal fluid samples were taken before the morning meal and hourly thereafter for 8 h. On day 10 in each period, 15 bags of either MB, TB, MM, or TM (1 × 1 × 12 cm; 15 µm pore size; 1 g feed) were placed in the stomach, respectively. The dry matter disappearance was highest for the MM at all time points compared with the other feedstuffs (P < 0.001). Maize and micronizing had the highest starch disappearance (P = 0.048) compared with barley and toasting. No treatment effect was measured for any of the glucose and insulin parameters. No feed effect was measured for the insulin parameters. Plasma glucose peaked later (P = 0.045) for maize than for barley, and TB had a larger area under the curve for glucose than MB, MM, and TM (P = 0.015). The concentration of total SCFA increased after feeding (P < 0.001), with a higher concentration for barley than for maize (P = 0.044). No treatment or feed effects were measured for ammonium N or pH, but both were affected by time (P < 0.001). In conclusion, toasting was not as efficient as micronizing to improve pre-cecal starch digestibility; therefore, the preferred processing method for both barley and maize is micronizing. Further, the amount of starch escaping enzymatical digestion in the small intestine was higher than expected.

Body condition score, morphometric measurements and estimation of body weight in mature Icelandic horses in Denmark.

BACKGROUND: Obesity is related to the development of several diseases like insulin resistance and laminitis in horses. The prevalence of obesity among mature Icelandic horses in Denmark has not been investigated previously. This study aimed to find the prevalence of obesity, to compare body condition score (BCS) based on owner perception with that of an experienced person and to correlate the BCS to body weight (BW) and morphometric measures in a group of mature Icelandic horses in Denmark. A total of 254 Icelandic horses (≥4 years; 140 geldings, 105 mares, 9 stallions) from 46 different farms were included. All horses were assigned a BCS on a scale from 1 to 9 (1 is poor, 5 is moderate and 9 is extremely fat) by their owner and by an experienced person. Two weight tapes were used to assess BW. Girth circumference (GC), neck circumference (NC) and height at withers (HW) were measured, and the GC:HW and NC:HW ratios were calculated. RESULTS: Categorising the horses into four groups, 5.9 % were underweight (BCS 3-4), 70.1 % were optimal (BCS 5-6), 13.8 % were overweight (BCS 7) and 10.2 % were obese (BCS 8-9). The GC:HW and NC:HW ratios increased with increasing BCS, as did the BW estimated with the weight tapes. A GC:HW ratio >1.21 might indicate overweight or obesity in Icelandic horses. Horse owners underestimated the BCS of their horses compared to an experienced person. CONCLUSIONS: The results from this study show that 24.0 % of mature Icelandic horses in Denmark are overweight or obese, and that owners tend to underestimate the BCS of their Icelandic horses. The GC:HW ratio might indicate overweight or obesity, however, the ratio for Icelandic horses is different than reported for horses and ponies of other breeds.

Diet-Dependent Modular Dynamic Interactions of the Equine Cecal Microbiota.

Knowledge on dynamic interactions in microbiota is pivotal for understanding the role of bacteria in the gut. We herein present comprehensive dynamic models of the horse cecal microbiota, which include short-chained fatty acids, carbohydrate metabolic networks, and taxonomy. Dynamic models were derived from time-series data in a crossover experiment in which four cecum-cannulated horses were fed a starch-rich diet of hay supplemented with barley (starch intake 2 g kg-1 body weight per day) and a fiber-rich diet of only hay. Cecal contents were sampled via the cannula each h for 24 h for both diets. We observed marked differences in the microbial dynamic interaction patterns for Fibrobacter succinogenes, Lachnospiraceae, Streptococcus, Treponema, Anaerostipes, and Anaerovibrio between the two diet groups. Fluctuations and microbiota interactions were the most pronounced for the starch rich diet, with Streptococcus spp. and Anaerovibrio spp. showing the largest fluctuations. Shotgun metagenome sequencing revealed that diet differences may be explained by modular switches in metabolic cross-feeding between microbial consortia in which fermentation is linked to sugar alcohols and amino sugars for the starch-rich diet and monosaccharides for the fiber-rich diet. In conclusion, diet may not only affect the composition of the cecal microbiota, but also dynamic interactions and metabolic cross-feeding.

The oral [(13)C]bicarbonate technique for measurement of short-term energy expenditure of sled dogs and their physiological response to diets with different fat:carbohydrate ratios.

The oral [(13)C]bicarbonate technique (o(13)CBT) was assessed for the determination of short-term energy expenditure (EE) under field conditions. A total of eight Alaskan huskies were fed two experimental diets in a cross-over experiment including two periods of 3 weeks. Effects of diets on EE, apparent total tract digestibility (ATTD) and on plasma hormones, blood lactate and glucose were furthermore investigated. The percentages of metabolisable energy derived from protein (P), fat (F) and carbohydrates (C) were 26:58:16 in the PFC diet and 24:75:1 in the PF diet. Measurements of EE were performed in the post-absorptive state during rest. Blood samples were collected during rest and exercise and ATTD was determined after days with rest and with exercise. EE was higher (P < 0·01) in period 2 than in period 1 (68 v. 48 kJ/kg body weight(0·75) per h). The ATTD of organic matter, crude protein and crude fat was higher (P < 0·01) in the PF diet compared with the PFC diet, and lower (P < 0·01) for total carbohydrates. Exercise did not affect ATTD. Higher (P < 0·01) insulin-like growth factor 1 and leptin concentrations were measured when fed the PF diet compared with the PFC diet. Concentrations of insulin decreased (P < 0·01), whereas cortisol and ghrelin increased (P < 0·05), after exercise. There was no effect of diet on blood lactate and glucose, but higher (P < 0·001) lactate concentrations were measured in period 1 than in period 2. The results suggest that the o(13)CBT can be used in the field to estimate short-term EE in dogs during resting conditions. Higher ATTD and energy density of the PF diet may be beneficial when energy requirements are high.

Evaluation of the oral (13)C-bicarbonate technique for measurements of energy expenditure in dogs before and after body weight reduction.

BACKGROUND: Overweight and obesity are the most common nutritional disorders in dogs and may lead to various secondary diseases and decreased lifespan. In obesity research, measurement of energy expenditure (EE) and determination of the energy requirements are essential. The objective with this study was to validate and evaluate the suitability of the oral (13)C-bicarbonate technique (o(13)CBT) for measuring EE in dog obesity studies. A further objective was to investigate the impact of body weight (BW) reduction and changes in body composition on the EE when measured under conditions corresponding to the basal metabolic rate (BMR). RESULTS: The EE in five privately owned, overweight dogs was measured simultaneously with the o(13)CBT and indirect calorimetry (IC) for comparison of the results. Two measurements per dog were performed under the same standardised conditions (i.e. fasted and resting state) at the start, and after completing a 12-week BW reduction program. Additionally, measurements of body composition by Dual-energy X-ray absorptiometry (DEXA) were conducted at the beginning and at the end of the BW reduction program. There were no differences in EE results obtained by the o(13)CBT and IC. Overweight and the BW reduction did not affect the estimates for the respiratory quotient (RQ) or the recovery factor for the (13)C-tracer (RF), both needed when using the o(13)CBT. The dogs lost 16% (SD ± 2.0) of their initial BW in reduced fat mass (P < 0.001), whereas fat free mass (FFM) remained unchanged. There was no effect of the BW reduction on the determined EE expressed in kJ/kg BW/d, or in kJ/kg BW(0.75)/d. However, EE was lower (P < 0.001) after the BW reduction program when expressed in relation to FFM (kJ/kg FFM/d). CONCLUSIONS: Results from the present study show that the o(13)CBT can be a used in obesity research to determine EE in fasted dogs and under resting conditions. Furthermore, the results suggest that the BMR does not change with reduced BW in overweight dogs as long as the FFM remains unchanged. This indicates that the BMR to maintain one gram of fat is equal to maintaining one gram of FFM in overweight dogs.

Growth conditions regulate the requirements for Caulobacter chromosome segregation.

Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small-molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated.

Location and architecture of the Caulobacter crescentus chemoreceptor array.

A new method for recording both fluorescence and cryo-EM images of small bacterial cells was developed and used to identify chemoreceptor arrays in cryotomograms of intact Caulobacter crescentus cells. We show that in wild-type cells preserved in a near-native state, the chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of nanometers away from the flagellar motor that they control. The arrays were always found on the convex side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of receptor dimers at each vertex of the lattice accounts well for the density and agrees with other constraints. Based on this model for the arrangement of receptors, there are between one and two thousand receptors per array.

Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography.

While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy-resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near-native state, producing three-dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as 'inner curvature', 'cytoplasmic', 'polar' and 'ring-like'. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as-yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected.

Analysis of the terminus region of the Caulobacter crescentus chromosome and identification of the dif site.

The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.

Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome.

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells.

Proteorhodopsin is a family of over 50 proteins that provide phototrophic capability to marine bacteria by acting as light-powered proton pumps. The potential importance of proteorhodopsin to global ocean ecosystems and the possible applications of proteorhodopsin in optical data storage and optical signal processing have spurred diverse research in this new family of proteins. We show that proteorhodopsin expressed in Escherichia coli is functional and properly inserted in the membrane. At high expression levels, it appears to self-associate. We present a method for determining spectral properties of proteorhodopsin in intact E. coli cells that matches results obtained with detergent-solubilized, purified proteins. Using this method, we observe distinctly different spectra for protonated and deprotonated forms of 21 natural proteorhodopsin proteins in intact E. coli cells. Upon protonation, the wavelength maxima red shifts between 13 and 53 nm. We find that pKa values between 7.1 and 8.5 describe the pH-dependent spectral shift for all of the 21 natural variants of proteorhodopsin. The wavelength maxima of the deprotonated forms of the 21 natural proteorhodopsins cluster in two sequence-related groups: blue proteorhodopsins (B-PR) and green proteorhodopsins (G-PR). The site-directed substitution Leu105Gln in Bac31A8 proteorhodopsin shifts this G-PR's wavelength maximum to a wavelength maximum the same as that of the B-PR Hot75m1 proteorhodopsin. The site-directed substitution Gln107Leu in Hot75m1 proteorhodopsin shifts this B-PR's wavelength maximum to a wavelength maximum as that of Bac31A8 proteorhodopsin.

Cell-cycle-regulated expression and subcellular localization of the Caulobacter crescentus SMC chromosome structural protein.

Structural maintenance of chromosomes proteins (SMCs) bind to DNA and function to ensure proper chromosome organization in both eukaryotes and bacteria. Caulobacter crescentus possesses a single SMC homolog that plays a role in organizing and segregating daughter chromosomes. Approximately 1,500 to 2,000 SMC molecules are present per cell during active growth, corresponding to one SMC complex per 6,000 to 8,000 bp of chromosomal DNA. Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle. Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. Stalked cells, which are actively engaged in DNA replication, have three or four SMC foci per cell. The SMC foci appear randomly distributed in the cell. Many predivisional cells have bright polar SMC foci, which are lost upon cell division. Thus, chromosome compaction likely involves dynamic aggregates of SMC bound to DNA. The aggregation pattern changes as a function of the cell cycle both during and upon completion of chromosome replication.

Dynamic localization of proteins and DNA during a bacterial cell cycle.

A cellular differentiation programme that culminates in an asymmetric cell division is an integral part of the cell cycle in the bacterium Caulobacter crescentus. Recent work has uncovered mechanisms that ensure the execution of many events at different times during the cell cycle and at specific places in the cell. Surprisingly, in this one-micron bacterial cell, the dynamic spatial disposition of regulatory proteins, structural proteins and specific regions of the chromosome are important components of both cell-cycle progression and the generation of daughter cells with different cell fates.