Cultivation of enteroids from fresh and cryopreserved bovine duodenal tissues.

This study aimed to develop a method for intestinal tissue cryopreservation and resuscitation for enteroid cultivation. Two different types of tissues, fresh duodenal tissues (n = 3, from Angus steers) and duodenal tissues cryopreserved in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO; n = 3, from Holstein calves), were collected to develop enteroids. Crypts were isolated using 2 mM EDTA/phosphate-buffered saline from both fresh and cryopreserved tissues and embedded in basement membrane extract. Embedded crypts were seeded in a 24-well plate and cultured in IntestiCult Organoid Growth Medium (Mouse) with inhibitors cocktail and Primocin. The upper opening of crypts became sealed, and crypts formed sphere structures (i.e., enteroids) within 24 h. Primary (passage 0) enteroids showed budding crypt domains from d 3 of cultivation at the earliest. After 7 d of cultivation, enteroids were passaged in a new 24-well plate. Fragments from passaged d 7 enteroids also formed sphere structures within 24 h after seeding and showed budding crypt domains from d 3 of cultivation at the earliest. The area of enteroids was measured in each animal during d 1 to 7 in passage 0 and 1, and the area of enteroids derived from both tissues increased during d 1 to 7 in passage 0 and 1. The area increased from d 1 to 7 of cultivation, and the area of passage 1 was greater than that of passage 0. F-actin staining using phalloidin revealed that brush border microvilli were distributed on the luminal side of the enteroids. In conclusion, a cryopreserved solution consisting of FBS and DMSO is useful for cryopreservation and resuscitation of bovine intestine for enteroid cultivation. This method allows researchers to investigate intestinal function and health in the laboratory using enteroids derived from fresh and cryopreserved tissues collected from cattle.

Action slips in food choices: A measure of habits and goal-directed control.

We report a new, simple instrumental action-slip task, which sets goal-directed action against putative S-R associations. On each training trial, participants were presented with one of two stimuli (blue or green coloured screen). One stimulus (S1) signalled that one joystick response (R1-left or right push) would earn one of two rewards (O1-jellybeans or Pringles points). A second stimulus (S2) signalled a different instrumental relationship (S2:R2-O2). On each test trial, participants were told which outcome could be earnt (O1/O2) on that trial. They were required to withhold responding until the screen changed colour to S1 or S2. On congruent test trials, the stimulus presented (e.g., S1) was associated with the same response (R1) as the outcome available on that trial (O1). On incongruent test trials, in contrast, the outcome (e.g., O1) preceded a stimulus that was associated with a different response (e.g., S2). Hence, in order to obtain the outcome (O1) on incongruent trials, participants were required to suppress any tendency they might have to make the response associated with the stimulus (R2 in response to S2). In two experiments, participants made more errors on incongruent than congruent trials. This result suggests that, on incongruent trials, the stimulus drove responding (e.g., S2 increased R2 responding) in a manner that was inconsistent with goal-directed action (e.g., R1 responding to obtain O1)-an action slip. The results are discussed in terms of popular dual-process theories of instrumental action and a single-process alternative.

Addition of dietary methionine but not dietary taurine or methyl donors/receivers to a grain-free diet increases postprandial homocysteine concentrations in adult dogs.

Grain-based ingredients are replaced in part by pulse ingredients in grain-free pet foods. Pulse ingredients are lower in methionine and cysteine, amino acid (AA) precursors to taurine synthesis in dogs. Although recent work has investigated plasma and whole blood taurine concentrations when feeding grain-free diets, supplementation of a grain-free diet with various nutrients involved in the biosynthesis of taurine has not been evaluated. This study aimed to investigate the effects of supplementing a complete grain-free dry dog food with either methionine (MET), taurine (TAU), or methyl donors (choline) and methyl receivers (creatine and carnitine; CCC) on postprandial AA concentrations. Eight healthy Beagle dogs were fed one of the three treatments or the control grain-free diet (CON) for 7 d in a 4 × 4 Latin square design. On day 7, cephalic catheters were placed and one fasted sample (0 min) and a series of nine post-meal blood samples were collected at 15, 30, 60, 90, 120, 180, 240, 300, and 360 min. Data were analyzed as repeated measures using the PROC GLIMMIX function in SAS (Version 9.4). Dogs fed MET had greater plasma and whole blood methionine concentrations from 30 to 360 min after a meal (P < 0.0001) and greater plasma homocysteine concentrations from 60 to 360 min after a meal (P < 0.0001) compared with dogs fed CON, TAU, and CCC. Dogs fed TAU had greater plasma taurine concentrations over time compared with dogs fed CON (P = 0.02) but were not different than dogs fed MET and CCC (P > 0.05). In addition, most AAs remained significantly elevated at 6 h post-meal compared with fasted samples across all treatments. Supplementation of creatine, carnitine, and choline in grain-free diets may play a role in sparing the methionine requirement without increasing homocysteine concentrations. Supplementing these nutrients could also aid in the treatment of disease that causes metabolic or oxidative stress, including cardiac disease in dogs, but future research is required.

Oversupplying metabolizable protein in late gestation for beef cattle: effects on prepartum BW, ruminal fermentation, nitrogen balance, and skeletal muscle catabolism.

The objective of the study was to determine the effect of oversupplying MP during late gestation on maternal BW, ruminal fermentation, nitrogen balance, and skeletal muscle catabolism. Crossbred Hereford heifers (n = 24) were assigned to a control treatment designed to meet MP requirements (CON) or a treatment providing 133% of the MP requirement (HMP). Heifers were individually fed their treatment from day -55 ± 3 relative to parturition and DMI was summarized by week. BW was measured on day -55 ± 3, -41 ± 3, -27 ± 3, and -8 ± 3. Ruminal digesta samples were collected on day -34 ± 5 and -15 ± 4 for short-chain fatty acid and ammonia-N (NH3-N) concentration. Plasma was collected the day prior to ruminal digesta samples and analyzed for plasma urea-N. Nitrogen balance was measured over a 6-d period starting on day -34 ± 4 and -15 ± 4. Following completion of the N balance periods, muscle biopsies were collected from the longissimus dorsi and analyzed for abundance of proteins relating to skeletal muscle catabolism. Data were analyzed as a randomized complete block (date of parturition) design with repeated measures using the MIXED procedure of SAS. Heifers fed HMP increased conceptus-corrected BW by a greater magnitude than CON at day -8 relative to -55 and -41 (treatment × day, P < 0.01). DMI increased (P < 0.01) by 18% on week -2 compared to -8, but then decreased (P < 0.01) by 8.0% for week -1. N-intake, apparent N digestion, N excretion, and N retention (g/d) were all greater (P < 0.01) for HMP heifers than CON but did not differ when expressed as a proportion of N intake. Ruminal NH3-N decreased (treatment × day, P < 0.01) as parturition approached for HMP (10.1 to 8.6 mg/dL); whereas, NH3-N was not affected for CON (1.0 to 1.3 mg/dL). Consequently, plasma urea-N was greater (P < 0.01) for HMP heifers (15.0 vs. 7.5 mg/dL). Heifers fed HMP had improved (P < 0.01) DM, OM, and NDF digestibility relative to CON heifers. The abundance of calpastatin was greater (P = 0.03) and calpain tended to be greater (P = 0.085) for CON cows compared to HMP. Feeding greater quantities of MP during late gestation may improve ruminal fermentation, N balance, and improve BW gain prepartum.

Oversupplying metabolizable protein in late gestation for beef cattle: effects on postpartum ruminal fermentation, blood metabolites, skeletal muscle catabolism, colostrum composition, milk yield and composition, and calf growth performance.

The objective of the study was to determine whether oversupplying MP prepartum affects postpartum cow BW, colostrum composition, milk production and composition, protein catabolism in the dam, and calf growth. Crossbred Hereford heifers were individually fed a control treatment designed to meet MP requirements (CON; n = 10) or 133% of the MP requirement (HMP; n = 11) from day -55 ± 4 until parturition. All cows were provided a common postpartum diet. Cow BW was measured on days 7 ± 1, 14 ± 2, 28 ± 3, 57 ± 4, 82 ± 5, and 111 ± 3 relative to parturition. DMI and ruminal pH were measured daily and summarized by week until day 33. Milk yield was estimated based on a 12-h two-quarter milk yield on days 7 ± 1, 12 ± 1, 28 ± 3, 33 ± 3, 70 ± 3, and 112 ± 3. Urine samples were collected from cows over a 6-d period starting on days 7 ± 1 and 28 ± 3 and the composited samples were analyzed for 3-methylhistidine (3-MH) and creatinine. Muscle samples were collected from cows on day 13 ± 1 while calf muscle samples were collected on days 2 and 111 ± 3 of age. Muscle samples from cows were analyzed for markers of protein catabolism, and calf muscle samples were analyzed for genes regulating cell growth and differentiation. Data were analyzed as a randomized complete block design using the MIXED procedure of SAS accounting for repeated measures when necessary. Postpartum BW did not differ (P ≥ 0.30) by treatment, day, or the interaction of treatment and day (T × D), but rump fat decreased (P = 0.011) as lactation progressed. DMI decreased during weeks 2 and 3 compared to 1 and 4, whereas ruminal pH was less during weeks 2, 3, and 4 relative to week 1. Colostrum fat concentration was less (P = 0.003) for HMP than CON; but, milk production was not affected by treatment. Milk yield was greatest from days 7 to 33 and decreased thereafter (P < 0.01). Urinary 3-MH and the 3-MH:creatinine ratio did not differ by treatment, day, or the T × D (P ≥ 0.22) interaction, nor was there a difference (P ≥ 0.13) in the abundance of catabolic proteins. Calf growth was not affected by treatment, but HMP calves had greater expression (T × D, P = 0.05) of PPARG while PKM expression increased for CON calves (T × D, P = 0.04) at day 111 compared to their expression at day 2. Overfeeding MP during late gestation does not improve postpartum indicators of N balance or maternal muscle turnover but may alter colostrum composition and calf gene expression at weaning.

Effect of ruminal acidosis and short-term low feed intake on indicators of gastrointestinal barrier function in Holstein steers.

The objective of this study was to determine effect of ruminal acidosis (RA) and low feed intake [LFI] on the regional barrier function of the gastrointestinal tract. Twenty-one Holstein steers were fed for ad libitum intake for 5 d (control [CON]), fed at 25% of ad libitum intake for 5 d (LFI), or provided 2 d of ad libitum intake followed by 1-d of feed restriction (25% of ad libitum intake), 1 d where 30% of ad libitum dry matter intake (DMI) was provided as pelleted barley followed by the full allocation (RA) and fed for ad libitum intake the following day. Tissues and digesta from the rumen, omasum, duodenum, jejunum, ileum, cecum, proximal, and distal colon were collected. Permeability was assessed using the mucosal-to-serosal flux of inulin (JMS-inulin) and mannitol (JMS-mannitol). Digesta pH was 0.81, 0.63, and 0.42 pH units less for RA than CON in the rumen, cecum, and proximal colon; while, LFI had pH that was 0.47 and 0.36 pH units greater in the rumen and proximal colon compared to CON. Total ruminal short-chain fatty acid (SCFA) concentration were less for LFI (92 mM; P = 0.010) and RA (87 mM; P = 0.007) than CON (172 mM) steers. In the proximal colon, the proportion of butyrate (P = 0.025 and P = 0.022) and isobutyrate (P = 0.019 and P = 0.019) were greater, and acetate (P = 0.028 and P = 0.028) was less for LFI and RA, respectively, when compared to CON steers. Ruminal papillae length, width, perimeter, and surface area were 1.21 mm, 0.78 mm, 3.84 mm, and 11.15 mm2 less for LFI than CON; while, RA decreased papillae width by 0.52 mm relative to CON. The JMS-mannitol was less for LFI steers than CON in the proximal colon (P = 0.041) and in the distal colon (P = 0.015). Increased gene expression for claudin 1, occludin, tight-cell junction protein 1 and 2, and toll-like receptor 4 were detected for LFI relative to CON in the rumen, jejunum, and proximal colon. For RA steers, expression of toll-like receptor 4 in the rumen, and occludin and tight-cell junction protein 1 were greater in the jejunum than CON. An acute RA challenge decreased pH in the rumen and large intestine but did not increase tissue permeability due to increases in the expression of genes related to barrier function within 1 d of the challenge. Steers exposed to LFI for 5 d had reduced ruminal SCFA concentrations, smaller ruminal papillae dimensions, and increased tissue permeability in the proximal and distal colon despite increases for genes related to barrier function and immune function.

Maternal nutrient restriction in mid-to-late gestation influences fetal mRNA expression in muscle tissues in beef cattle.

BACKGROUND: Manipulating maternal nutrition during specific periods of gestation can result in re-programming of fetal and post-natal development. In this experiment we investigated how a feed restriction of 85% compared with 140% of total metabolizable energy requirements, fed to cows during mid-to-late gestation, influences phenotypic development of fetuses and mRNA expression of growth (Insulin-Like Growth Factor family and Insulin Receptor (INSR)), myogenic (Myogenic Differentiation 1 (MYOD1), Myogenin (MYOG), Myocyte Enhancer Factor 2A (MEF2A), Serum Response Factor (SRF)) and adipogenic (Peroxisome Proliferator Activated Receptor Gamma (PPARG)) genes in fetal longissimus dorsi (LD) and semitendinosus (ST) muscle. DNA methylation of imprinted genes, Insulin Like Growth Factor 2 (IGF2) and Insulin Like Growth Factor 2 Receptor (IGF2R), and micro RNA (miRNA) expression, were also examined as potential consequences of poor maternal nutrition, but also potential regulators of altered gene expression patterns. RESULTS: While the nutrient restriction impacted dam body weight, no differences were observed in phenotypic fetal measurements (weight, crown-rump length, or thorax circumference). Interestingly, LD and ST muscles responded differently to the differential pre-natal nutrient levels. While LD muscle of restricted fetal calves had greater mRNA abundances for Insulin Like Growth Factor 1 and its receptor (IGF1 and IGF1R), IGF2R, INSR, MYOD1, MYOG, and PPARG, no significant differences were observed for gene expression in ST muscle. Similarly, feed restriction had a greater impact on the methylation level of IGF2 Differentially Methylated Region 2 (DMR2) in LD muscle as compared to ST muscle between treatment groups. A negative correlation existed between IGF2 mRNA expression and IGF2 DMR2 methylation level in both LD and ST muscles. Differential expression of miRNAs 1 and 133a were also detected in LD muscle. CONCLUSIONS: Our data suggests that a nutrient restriction of 85% as compared to 140% of total metabolizable energy requirements during the 2nd half of gestation can alter the expression of growth, myogenic and adipogenic genes in fetal muscle without apparent differences in fetal phenotype. It also appears that the impact of feed restriction varies between muscles suggesting a priority for nutrient partitioning depending on muscle function and/or fiber composition. Differences in the methylation level in IGF2, a well-known imprinted gene, as well as differences in miRNA expression, may be functional mechanisms that precede the differences in gene expression observed, and could lead to trans-generational epigenetic programming.

Long-range communications between DNA sites by the dimeric restriction endonuclease SgrAI.

The SgrAI endonuclease displays its maximal activity on DNA with two copies of its recognition sequence, cleaving both sites concertedly. While most restriction enzymes that act concurrently at two sites are tetramers, SgrAI is a dimer in solution. Its reaction at two cognate sites involves the association of two DNA-bound dimers. SgrAI can also bridge cognate and secondary sites, the latter being certain sequences that differ from the cognate by one base-pair. The mechanisms for cognate-cognate and cognate-secondary communications were examined for sites in the following topological relationships: in cis, on plasmids with two sites in a single DNA molecule; on catenanes containing two interlinked rings of DNA with one site in each ring; and in trans, on oligoduplexes carrying either a single site or the DNA termini generated by SgrAI. Both cognate-cognate and cognate-secondary interactions occur through 3-D space and not by 1-D tracking along the DNA. Both sorts of communication arise more readily when the sites are tethered to each other, either in cis on the same molecule of DNA or by the interlinking of catenane rings, than when released from the tether. However, the dimer bound to an oligoduplex carrying either a cognate or a secondary site could be activated to cleave that duplex by interacting with a second dimer bound to the recognition site, provided both duplexes are at least 30 base-pairs long: the second dimer could alternatively be bound to the two duplexes that correspond to the products of DNA cleavage by SgrAI.

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI.

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.