Deep convolutional neural networks for the detection of diarrhea and respiratory disease in preweaning dairy calves using data from automated milk feeders.

The objective of the current study was to develop a predictive model for calf disease detection in the preweaning period using data from automated milk feeders (AMF). A deep convolutional neural network (CNN) architecture for the detection of respiratory disease and diarrhea in dairy calves was developed. German Holstein calves were fed milk replacer either ad libitum (up to 25 L/d; n = 32) or restrictively (6 L/d; n = 32) via AMF from 10 ± 3 d of life on. Concentrate, hay, and water were freely available. Calf health parameters were scored daily. The AMF measured milk replacer (MR) intake, number of rewarded visits, number of unrewarded visits, and drinking speed. A calf was considered sick if its fecal score was 3 or 4 and its respiratory score was 2 or 3. Only data from AMF up to 47 d of age were included in the analysis. This cut in the data was made to avoid data from the weaning period. Data were split in 80:20 ratios for training and testing data sets according to the Pareto principle. A minimum sensitivity of 80% was considered an appropriate requirement for the prediction models. Considering all calves in group housing, cross-validation of the test data set showed a sensitivity of 83% and a specificity of 79%, with a positive predictive value and a negative predictive value of 37 and 97%, respectively. The area under the curve of the receiver operating characteristic for the deep CNN model was 0.81 for all group-housed calves. The CNN model yielded sensitivity and specificity of 83 and 71%, respectively (for ad libitum-fed calves), and 82 and 87%, respectively (for restricted-fed calves), with good area under the curve-receiver operating characteristic (0.77 to 0.87), indicating that the CNN models can predict calf disease in both groups with different MR allowances. The permutation feature importance was measured by the decrease in model accuracy, and features (behaviors) were summarized in descending order of their relative importance to the CNN model. Drinking speed and MR intake were the main factors to predict calf disease in calves fed ad libitum. The number of unrewarded visits to the milk feeder and MR intake were the main factors to predict calf disease in restricted-fed calves. Despite the relatively small sample size, the results provide strong evidence that daily feeding behavior data from AMF can be used to identify calves at risk for disease. In conclusion, despite a very good testing performance of the CNN model, the relatively low daily prevalence of calf disease in the present study resulted in a high proportion of false-positive alarms.

Branched-chain amino acids: Abundance of their transporters and metabolizing enzymes in adipose tissue, skeletal muscle, and liver of dairy cows at high or normal body condition.

Branched-chain amino acids (BCAA) are major components of milk protein and important precursors for nonessential AA. Thus, the BCAA transport and break-down play a key role in the metabolic adaptation to the high nutrient demands in lactation. However, in monogastrics, increased BCAA levels have been linked with obesity and certain metabolic disorders such as impaired insulin sensitivity. Our objective was to study the effect of over-conditioning at calving on plasma BCAA levels as well as the tissue abundance of the most relevant BCAA transporters and degrading enzymes in dairy cows during late pregnancy and early lactation. Thirty-eight Holstein cows were allocated 15 wk antepartum to either a normal- (NBCS) or over-conditioned (HBCS) group, receiving 6.8 or 7.2 MJ of NEL/kg of DM, respectively, during late lactation to reach the targeted differences in body condition score (BCS) and back fat thickness (BFT; NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until dry-off. During the dry period and next lactation, cows were fed the same diets, whereby differences in BCS and BFT were maintained: prepartum means were 3.16 ± 0.06 and 1.03 ± 0.07 cm (NBCS) vs. 3.77 ± 0.08 and 1.89 ± 0.11 cm (HBCS), postpartum means were 2.89 ± 0.06 and 0.81 ± 0.05 cm (NBCS) vs. 3.30 ± 0.06 and 1.38 ± 0.08 cm (HBCS). Blood and biopsies from liver, semitendinosus muscle, and subcutaneous adipose tissue (scAT) were sampled at d 49 antepartum, 3, 21, and 84 postpartum. Free BCAA were analyzed and the mRNA abundance of solute carrier family 1 member 5 (SLC1A5), SLC7A5, and SLC38A2 as well as branched-chain aminotransferase 2 (BCAT2), branched-chain α-keto acid dehydrogenase E1α (BCKDHA), and branched-chain α-keto acid dehydrogenase E1ß (BCKDHB) as well as the protein abundance of BCKDHA were assessed. Concentrations of all BCAA changed with time, most markedly in HBCS cows, with a nadir around calving. Apart from Ile, neither individual nor total BCAA differed between groups. The HBCS group had greater BCKDHA mRNA as well as higher prepartum BCKDHA protein abundance in scAT than NBCS cows, pointing to a greater oxidative capacity for the irreversible degradation of BCAA transamination products in scAT of over-conditioned cows. Prepartum hepatic BCKDHA protein abundance was lower in HBCS than in NBCS cows. In both groups, SLC1A5, SLC7A5, and BCAT2 mRNA were most abundant in scAT, whereas SLC38A2 was higher in scAT and muscle compared with liver, and BCKDHA and BCKDHB mRNA were greatest in liver and muscle, respectively. Our results indicate that scAT may be a major site of BCAA uptake and initial catabolism, with the former, however, being independent of BCS and time relative to calving in dairy cows.

Metabolome profiling in skeletal muscle to characterize metabolic alterations in over-conditioned cows during the periparturient period.

The transition from late gestation to early lactation is associated with extensive changes in metabolic, endocrine, and immune functions in dairy cows. Skeletal muscle plays an important role in maintaining the homeorhetic adaptation to the metabolic needs of lactation. The objective of this study was to characterize the skeletal muscle metabolome in the context of the metabolic changes that occur during the transition period in dairy cows with high (HBCS) versus normal body condition (NBCS). Fifteen weeks antepartum, 38 pregnant multiparous Holstein cows were assigned to 1 of 2 groups, which were fed differently to reach the targeted BCS and back fat thickness (BFT) until dry-off at -49 d before calving (HBCS: >3.75 and >1.4 cm; NBCS: <3.5 and <1.2 cm). During the dry period and the subsequent lactation, both groups were fed identical diets. The differences in both BCS and BFT were maintained throughout the study. The metabolome was characterized in skeletal muscle samples (semitendinosus muscle) collected on d -49, 3, 21, and 84 relative to calving using a targeted metabolomics approach (AbsoluteIDQ p180 kit; Biocrates Life Sciences AG, Innsbruck, Austria), which allowed for the quantification of up to 188 metabolites from 6 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and hexoses). On d -49, the concentrations of citrulline and hydroxytetradecadienyl-l-carnitine in muscle were higher in HBCS cows than in NBCS cows, but those of carnosine were lower. Over-conditioning did not affect the muscle concentrations of any of the metabolites on d 3. On d 21, the concentrations of phenylethylamine and linoleylcarnitine in muscle were lower in HBCS cows than in NBCS cows, and the opposite was true for lysophosphatidylcholine acyl C20:4. On d 84, the significantly changed metabolites were mainly long-chain (>C32) acyl-alkyl phosphatidylcholine and di-acyl phosphatidylcholine, along with 3 long-chain (>C16) sphingomyelin that were all lower in HBCS cows than in NBCS cows. These data contribute to a better understanding of the metabolic adaptation in skeletal muscle of dairy cows during the transition period, although the physiological significance and underlying molecular mechanisms responsible for the regulation of citrulline, hydroxytetradecadienyl-l-carnitine, carnosine, and phenylethylamine associated with over-conditioning are still elusive and warrant further investigation. The changes observed in muscle lysophosphatidylcholine and phosphatidylcholine concentrations may point to an alteration in phosphatidylcholine metabolism, probably resulting in an increase in membrane stiffness, which may lead to abnormalities in insulin signaling in the muscle of over-conditioned cows.

Effects of ad libitum milk replacer feeding and butyrate supplementation on the epithelial growth and development of the gastrointestinal tract in Holstein calves.

Intensive milk feeding and butyrate supplementation in calves stimulate body growth and affect gastrointestinal development. The aim of the present study was to investigate the synergistic effects of ad libitum milk replacer (MR) feeding and butyrate supplementation of MR on rumen and small intestinal growth and on gene expression in the small intestine related to growth and energy metabolism at weaning. Male Holstein calves (n = 32) received colostrum from birth to d 3 of age and MR either ad libitum (Adl) or restrictively (Res; 6 L of MR/d; 12.5% solids) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate from d 4 until wk 8 of age. From wk 9 to 10, all calves were weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. At d 80, calves were slaughtered, volatile fatty acids were measured in rumen fluid, and rumen and small intestine samples were taken for histomorphometric measurements. The expression of mRNA associated with the local insulin-like growth factor (IGF) system and glucose metabolism as well as lactase and maltase activities were measured in the intestinal mucosa. The small intestine was 3 m longer in Adl than in Res. In the atrium ruminis, papilla width was greater in Res than in Adl. Villus circumference, cut surface, and height in the duodenum, proximal jejunum, and ileum were greater in Adl than in Res and in the proximal, mid, and distal jejunum and ileum were greater in calves treated with butyrate. Crypt depth in the duodenum and proximal jejunum was greater in Adl than in Res and in the ileum was smaller in calves treated with butyrate. The villus height:crypt depth ratio was greatest in AdlB+ calves. In the proximal and mid jejunum, IGF1 mRNA abundance was lower in calves treated with butyrate. In the proximal jejunum, INSR mRNA abundance was greater in Res than in Adl. The abundance of PCK2 mRNA was greater in Res than in Adl in the duodenum and was greatest in ResB- in the mid jejunum. Lactase activity tended to be greater in Res than in Adl and after butyrate treatment in the proximal jejunum. The results indicated an elevated growth of the small intestinal mucosa at weaning due to intensive milk feeding and butyrate supplementation, and the local IGF system was involved in intestinal growth regulation. Rumen development was not affected by butyrate supplementation of MR and was slightly delayed due to ad libitum MR feeding.

Mammalian target of rapamycin signaling and ubiquitin-proteasome-related gene expression in skeletal muscle of dairy cows with high or normal body condition score around calving.

The objective of the current study was to investigate the effects of overconditioning around calving on gene expression of key components of the mammalian target of rapamycin (mTOR) pathway and ubiquitin-proteasome system (UPS) in skeletal muscle as well as the AA profiles in both serum and muscle of periparturient cows. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to either a high body condition group (HBCS; n = 19) or a normal body condition group (NBCS; n = 19) and were fed different diets until dry-off (d -49 relative to calving) to amplify the difference. The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg). At dry-off, the NBCS cows (parity: 2.42 ± 1.84; body weight: 665 ± 64 kg) had a body condition score (BCS) <3.5 and backfat thickness (BFT) <1.2 cm, whereas the HBCS cows (parity: 3.37 ± 1.67; body weight: 720 ± 57 kg) had a BCS >3.75 and BFT >1.4 cm. During the dry period and the subsequent lactation, both groups were fed identical diets but maintained the BCS and BFT differences. Blood samples and skeletal muscle biopsies (semitendinosus) were repeatedly (d -49, +3, +21, and +84 relative to calving) collected for assessing the concentrations of free AA and the mRNA abundance of various components of mTOR and UPS. The differences in BCS and BFT were maintained throughout the study. The circulating concentrations of most AA with the exception of Gly, Gln, Met, and Phe increased in early lactation in both groups. The serum concentrations of Ala (d +21 and +84) and Orn (d +84) were lower in HBCS cows than in NBCS cows, but those of Gly, His, Leu, Val, Lys, Met, and Orn on d -49 and Ile on d +21 were greater in HBCS cows than in NBCS cows. The serum concentrations of 3-methylhistidine, creatinine, and 3-methylhistidine:creatinine ratio increased after calving (d +3) but did not differ between the groups. The muscle concentrations of all AA (except for Cys) remained unchanged over time and did not differ between groups. The muscle concentrations of Cys were greater on d -49 but tended to be lower on d +21 in HBCS cows than in NBCS cows. On d +21, mTOR and eukaryotic translation initiation factor 4E binding protein 1 mRNA abundance was greater in HBCS cows than in NBCS cows, whereas ribosomal protein S6 kinase 1 was not different between the groups. The mRNA abundance of ubiquitin-activating enzyme 1 (d +21), ubiquitin-conjugating enzyme 1 (d +21), atrogin-1 (d +21), and ring finger protein-1 (d +3) enzymes was greater in HBCS cows than in NBCS cows, whereas ubiquitin-conjugating enzyme 2 was not different between the groups. The increased mRNA abundance of key components of mTOR signaling and of muscle-specific ligases of HBCS cows may indicate a simultaneous activation of anabolic and catabolic processes and thus increased muscle protein turnover, likely as a part of the adaptive response to prevent excessive loss of skeletal muscle mass during early lactation.

Biogenic amines: Concentrations in serum and skeletal muscle from late pregnancy until early lactation in dairy cows with high versus normal body condition score.

Biogenic amines (BA) are a class of nitrogenous compounds that are involved in a wide variety of physiological processes, but their role in transition cows is poorly understood. Our objectives were to describe the longitudinal changes of BA in serum and in skeletal muscle during the transition period and to characterize temporal responses of BA in relation to body condition score (BCS) of periparturient dairy cows. Fifteen weeks before calving, 36 multiparous Holstein cows were assigned to 2 groups (n = 18 per group) that were fed differently to reach either high [HBCS; net energy for lactation (NEL) = 7.2 MJ/kg of dry matter (DM)] or normal BCS (NBCS; NEL = 6.8 MJ/kg of DM) at dry-off. The targeted BCS and back fat thickness (BFT) at dry-off (HBCS, >3.75 and >1.4 cm; NBCS, <3.5 and <1.2 cm) were reached. Thereafter, both groups were fed identical diets. Blood samples and muscle (semitendinosus) biopsies were collected at d -49, +3, +21, and +84 relative to parturition. In serum and skeletal muscle, BA concentrations were measured using a targeted metabolomics assay. The data were analyzed as a repeated measure using the MIXED procedure of SAS. The serum concentrations of most BA (i.e., creatinine, taurine, carnosine putrescine, spermine, α-aminoadipic acid, acetylornithine, kynurenine, serotonin, hydroxyproline, asymmetric dimethylarginine, and symmetric dimethylarginine) fluctuated during the transition period, while others (i.e., spermidine, phenylethylamine) did not change with time. The muscle concentrations of BA remained unchanged over time. Creatinine had the highest concentrations in the serum, while carnosine had the highest concentration among the muscle BA. The serum concentrations of creatinine (d +21), putrescine (d +84), α-aminoadipic acid (d +3), and hydroxyproline (d +21) were or tended to be higher for HBCS compared with NBCS postpartum. The serum concentrations of symmetric dimethylarginine (d -49) and acetylornithine (d +84) were or tended to be lower for HBCS compared with NBCS, respectively. The serum kynurenine/tryptophan ratio was greater with HBCS than with NBCS (d +84). Compared with NBCS, HBCS was associated with lower muscle concentrations of carnosine, but those of hydroxyproline were higher (d -49). In both serum and muscle, the asymmetric dimethylarginine concentrations were greater with HBCS than with NBCS (d -49). No correlation was found between serum and skeletal muscle BA. This study indicates that overconditioning of dairy cows may influence serum and muscle BA concentrations in the periparturient period.

Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves.

Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.

Effects of ad libitum milk replacer feeding and butyrate supplementation on behavior, immune status, and health of Holstein calves in the postnatal period.

Animal welfare in dairy calf husbandry depends on calf rearing and is probably improved by intensive milk feeding programs. In addition, butyrate supplementation in milk replacer (MR) stimulates postnatal growth and may affect the immune system in calves. We have investigated the combined effects of ad libitum MR feeding and butyrate supplementation on feeding behavior, health, and the immune responses in calves. Holstein calves (n = 64) were examined from birth until wk 11 of age. Calves received MR either ad libitum (Adl) or restrictively (Res) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate supplementation starting on d 4. From wk 9 to 10, all calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Calves were housed in straw-bedded group pens with automatic MR feeders, where feed intake and feeding behavior were documented. Blood was drawn on d 1 before the first colostrum intake; on d 2, 4, and 7; and weekly thereafter until the end of the study to measure plasma concentrations of total protein, albumin, the immunoglobulins IgG1, IgG2, and IgM, and the acute phase proteins fibrinogen, serum amyloid A, and haptoglobin. Liver samples were taken on d 50 and 80 to determine gene expression related to acute phase proteins. Body temperature was measured daily for the first 3 wk, and clinical traits were scored daily. Ad libitum MR feeding resulted in greater MR intake, greater MR intake per meal, slower sucking rate, and greater body weight, but in a lower number of unrewarded visits and lower concentrate intake when compared with Res. Butyrate reduced the sucking rate but increased MR intake per meal. Immunoglobulins in the blood plasma increased after colostrum intake in all calves, with only minor differences among groups throughout the study. Plasma fibrinogen and serum amyloid A increased in the first week of life in all calves, and fibrinogen was greater in Res than in Adl on d 21, 49, and 63. Hepatic gene expression of fibrinogen on d 80 was greater in Adl than in Res. Gene expression of SAA2 was greater on d 50 in Adl than in Res and on d 80 was greater in ResB+ than in ResB-. Body temperature was greater in Adl than in Res during the first 2 wk, but neither MR feeding nor butyrate affected the health status. An improved animal welfare in Adl calves is supported by fewer signs of hunger, but intensive milk feeding and butyrate did not affect the health and immune status of the calves in a consistent manner.

Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.

The enhanced growth performance of calves fed a higher plane of nutrition pre-weaning is well documented, and the effect of butyrate on the development of the gastrointestinal tract in calves has been evaluated. The aim of this study was to examine the synergistic effects of ad libitum milk replacer (MR) feeding and butyrate supplementation on growth performance and energy metabolism in calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (Res) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate supplementation. Colostrum and transition milk were fed in predefined amounts (Res or Adl) for the first 3 d postpartum. Ad libitum and restrictive MR feeding with or without butyrate was performed from d 4 until wk 8 of age. From wk 9 to 10, all calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate (CON), hay, and water were freely available. Intakes of MR and CON were measured daily. Calves were weighed at birth and weekly thereafter. Blood was drawn on d 1 before the first colostrum intake; on d 2, 4, and 7; and weekly thereafter until the end of the study to measure plasma concentrations of metabolites and hormones. Liver samples were taken at d 50 and at the end of the study to determine gene expression related to glucose metabolism. Milk, MR, and total nutrient intake were greater, but CON intake was lower in Adl than in Res calves, resulting in a greater body weight, but partially lower gain to feed ratio in Adl than in Res. Plasma concentrations of glucose and insulin were higher during the ad libitum milk-feeding period, whereas plasma ß-hydroxybutyrate was lower in Adl than in Res. Plasma concentrations of nonesterified fatty acids, lactate, total bilirubin, and cortisol were lower, but triglyceride and cholesterol concentrations were higher in Adl than in Res at specific time points. Feed intake, growth performance, and metabolic and endocrine changes were insignificantly affected by butyrate, and hepatic gene expression of enzymes related to endogenous glucose production was barely influenced by ad libitum MR feeding and butyrate supplementation. Intensive MR feeding indicated greater stimulation of growth and anabolic metabolism, but butyrate supplementation did not further improve postnatal growth or anabolic processes either in intensive or restrictive MR-fed calves.

The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation.

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been suggested that inflammation associated with the development of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, we investigated the hypothesis that feeding grape seed and grape marc meal extract (GSGME) as a plant extract rich in flavonoids attenuates inflammation and ER stress in the liver of dairy cows. Two groups of cows received either a total mixed ration as a control diet or the same total mixed ration supplemented with 1% of GSGME over the period from wk 3 prepartum to wk 9 postpartum. Dry matter intake during wk 3 to 9 postpartum was not different between the 2 groups. However, the cows fed the diet supplemented with GSGME had an increased milk yield and an increased daily milk protein yield. Cows supplemented with GSGME moreover had a significantly reduced mRNA abundancy of fibroblast growth factor (FGF) 21, a stress hormone induced by various stress conditions, in the liver in wk 1 and 3 postpartum. In contrast, mRNA abundances of a total of 3 genes involved in inflammation and 14 genes involved in ER stress response, as well as concentrations of triacylglycerols and cholesterol, in liver samples of wk 1 and 3 postpartum did not differ between the 2 groups. Overall, this study shows that supplementation of GSGME did not influence inflammation or ER stress in the liver but increased milk yield, an effect that could be due to effects on ruminal metabolism.

Blood and adipose tissue steroid metabolomics and mRNA expression of steroidogenic enzymes in periparturient dairy cows differing in body condition.

In high-yielding dairy cows, the rapidly increasing milk production after parturition can result in a negative nutrient balance, since feed intake is insufficient to cover the needs for lactation. Mobilizing body reserves, mainly adipose tissue (AT), might affect steroid metabolism. We hypothesized, that cows differing in the extent of periparturient lipomobilization, will have divergent steroid profiles measured in serum and subcutaneous (sc)AT by a targeted metabolomics approach and steroidogenic enzyme profiles in scAT and liver. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to a high (HBCS) or normal body condition (NBCS) group fed differently until week 7 antepartum to either increase (HBCS BCS: 3.8 ± 0.1 and BFT: 2.0 ± 0.1 cm; mean ± SEM) or maintain BCS (NBCS BCS: 3.0 ± 0.1 and BFT: 0.9 ± 0.1 cm). Blood samples, liver, and scAT biopsies were collected at week -7, 1, 3, and 12 relative to parturition. Greater serum concentrations of progesterone, androsterone, and aldosterone in HBCS compared to NBCS cows after parturition, might be attributed to the increased mobilization of AT. Greater glucocorticoid concentrations in scAT after parturition in NBCS cows might either influence local lipogenesis by differentiation of preadipocytes into mature adipocytes and/or inflammatory response.

Comparison of performance and metabolism from late pregnancy to early lactation in dairy cows with elevated v. normal body condition at dry-off.

Excessive mobilization of body reserves during the transition from pregnancy to lactation imposes a risk for metabolic diseases on dairy cows. We aimed to establish an experimental model for high v. normal mobilization and herein characterized performance, metabolic and endocrine changes from 7 weeks antepartum (a.p.) to 12 weeks postpartum (p.p.). Fifteen weeks a.p., 38 pregnant multiparous Holstein cows were allocated to two groups that were fed differently to reach either high or normal body condition scores (HBCS: 7.2 NEL MJ/kg dry matter (DM); NBCS: 6.8 NEL MJ/kg DM) at dry-off. Allocation was also based on differences in body condition score (BCS) in the previous and the ongoing lactation that was further promoted by feeding to reach the targeted BCS and back fat thickness (BFT) at dry-off (HBCS: >3.75 and >1.4 cm; NBCS: <3.5 and <1.2 cm). Thereafter, both groups were fed identical diets. Blood samples were drawn weekly from 7 weeks a.p. to 12 weeks p.p. to assess the serum concentrations of metabolites and hormones. The HBCS cows had greater BCS, BFT and BW than the NBCS cows throughout the study and lost more than twice as much BFT during the first 7 weeks p.p. compared with NCBS. Milk yield and composition were not different between groups, except that lactose concentrations were greater in NBSC than in HBCS. Feed intake was also greater in NBCS, and NBCS also reached a positive energy balance earlier than HBCS. The greater reduction in body mass in HBCS was accompanied by greater concentrations of non-esterified fatty acids, and ß-hydroxybutyrate in serum after calving than in NBCS, indicating increased lipomobilization and ketogenesis. The mean concentrations of insulin across all time-points were greater in HBCS than in NBCS. In both groups, insulin and IGF-1 concentrations were lower p.p than in a.p. Greater free thyroxine (fT4) concentrations and a lower free 3-3'-5-triiodothyronine (fT3)/fT4 ratio were observed in HBCS than in NBCS a.p., whereas p.p. fT3/fT4 ratio followed a reverse pattern. The variables indicative for oxidative status had characteristic time courses; group differences were limited to greater plasma ferric reducing ability values in NBSC. The results demonstrate that the combination of pre-selection according to BCS and differential feeding before dry-off to promote the difference was successful in obtaining cows that differ in the intensity of mobilizing body reserves. The HBCS cows were metabolically challenged due to intense mobilization of body fat, associated with reduced early lactation dry matter intake and compromised antioxidative capacity.

Different milk diets have substantial effects on the jejunal mucosal immune system of pre-weaning calves, as demonstrated by whole transcriptome sequencing.

There is increasing evidence that nutrition during early mammalian life has a strong influence on health and performance in later life. However, there are conflicting data concerning the appropriate milk diet. This discrepancy particularly applies to ruminants, a group of mammals that switch from monogastric status to rumination during weaning. Little is known regarding how the whole genome expression pattern in the juvenile ruminant gut is affected by alternative milk diets. Thus, we performed a next-generation-sequencing-based holistic whole transcriptome analysis of the jejunum in male pre-weaned German Holstein calves fed diets with restricted or unlimited access to milk during the first 8 weeks of life. Both groups were provided hay and concentrate ad libitum. The analysis of jejunal mucosa samples collected 80 days after birth and four weeks after the end of the feeding regimes revealed 275 differentially expressed loci. While the differentially expressed loci comprised 67 genes encoding proteins relevant to metabolism or metabolic adaptation, the most distinct difference between the two groups was the consistently lower activation of the immune system in calves that experienced restricted milk access compared to calves fed milk ad libitum. In conclusion, different early life milk diets had significant prolonged effects on the intestinal immune system.

Proliferation of human colonic mucosa as an intermediate biomarker of carcinogenesis: effects of butyrate, deoxycholate, calcium, ammonia, and pH.

A high-fat/high-protein diet has been reported to promote colon cancer by increasing luminal bile acid and ammonia concentrations, whereas butyrate, calcium, and low colonic pH may have protective effects. In this study, bromodeoxyuridine labeling of colonic epithelium was investigated after incubating biopsies from the ascending colon of 70 patients with HCl (20 mM, pH 6.0), butyric acid (H-BUT, 20 mM, pH 6.0), sodium butyrate (Na-BUT, 10 mM, pH 8.0), CaCl2 (10 mM), calcium butyrate (Ca-BUT, 10 mM), ammonium butyrate (NH4-BUT, 10 mM), deoxycholic acid (DCA, 5 microM), and a combination of DCA and Na-BUT (DCA/Na-BUT, 5 microM/10 mM). Compared to NaCl, H-BUT and Na-BUT increased the whole crypt-labeling index significantly, whereas HCl and CaCl2 had no effect. Reduced labeling, however, occurred with Ca-BUT in comparison to equimolar Na-BUT. No differences in the labeling indexes were found for NH4-BUT compared to Na-BUT, but increased labeling with expansion of the proliferative zone to the upper 40% of the crypt was seen with DCA compared to NaCl. DCA-induced hyperproliferation was abolished by coincubation with DCA/Na-BUT. These data suggest that butyrate, calcium, and DCA have complex influences on mucosal proliferation. Since luminal concentrations of these compounds are influenced by dietary interventions, the findings of this study may be of particular interest with regard to colon cancer development and prevention.

Effects of resistant starch on the colon in healthy volunteers: possible implications for cancer prevention.

Recent evidence suggests that resistant starch (RS) is the single most important substrate for bacterial carbohydrate fermentation in the human colon. During two 4-wk periods. 12 healthy volunteers consumed a controlled basal diet enriched with either amylomaize starch (55.2 +/- 3.5 g RS/d; high-RS diet) or available cornstarch (7.7 +/- 0.3 g RS/d; low-RS diet). Approximately 90% of the RS consumed disappeared during intestinal passage; increased fermentation was verified by elevated breath-hydrogen excretion. During the high-RS diet, fecal wet and dry weight increased 49% and 56%, respectively (P < or = 0.005), whereas stool water content did not change significantly. Fecal concentrations and daily excretion of short-chain fatty acids were not different in the two study periods. During the high-RS diet, bacterial beta-glucosidase activity decreased by 26% (P < or = 0.05). Fecal concentrations of total and secondary bile acids were significantly lower during the high-RS than during the low-RS period [a decrease of 30% (P < or = 0.05) and 32% (P < or = 0.01), respectively, in total and secondary bile acids] whereas concentrations of primary bile acids were unaffected by RS consumption. During the high-RS diet, fecal concentrations of total neutral sterols decreased by 30% (P < or = 0.005) and fecal concentrations of 4-cholesten-3-one decreased by 36% (P < or = 0.05). These data suggest that RS has potentially important effects on bacterial metabolism in the human colon that may be relevant for cancer prevention.

Effects of sodium selenite on deoxycholic acid-induced hyperproliferation of human colonic mucosa in short-term culture.

It has been shown that in vitro incubation of human colonic biopsies with the secondary bile acid deoxycholic acid (DCA) leads to the hyperproliferation of colonic crypt cells with an expansion of the proliferative zone, which is regarded as a biomarker of increased cancer risk. Sodium selenite (SSE), on the other hand, has been implicated as a protective agent in experimental studies, but toxic effects were reported as well, depending on the dose of SSE. To elucidate the effects of SSE on human colonic mucosa, biopsies from endoscopically normal sigmoid colon tissue of 30 subjects were incubated with 5 microM DCA or a combination of 5 microM DCA and SSE in concentrations of 5, 10, 20, 50, 80, and 100 microM, respectively. Equimolar NaCl incubations served as a control. Proliferating cells were labeled by bromodeoxyuridine immunohistochemistry, and the labeling index (LI) was computed. In the experiments using 5, 10, and 20 microM SSE, the whole crypt LI was significantly lower after DCA + SSE incubation (0.136, 0.118, and 0.110, respectively) compared to that after incubation with DCA alone (0.172, 0.157, and 0.165, respectively; P < 0.01). The corresponding LIs during DCA + SSE incubation were comparable to the LIs obtained after NaCl incubation (average LI = 0.14). Contrary to this finding, severe cell damage was observed in the biopsies that were incubated with the higher SSE concentrations of 50 microM and above. The antiproliferative effects of SSE may indicate a possible protective effect in the prevention of human colon cancer development. However, the observed toxic effects of higher SSE concentrations strongly suggest the need for additional studies before general recommendations for the use of SSE in colon cancer prevention can be made.

Comparisons of the anti-proliferative effects of butyrate and aspirin on human colonic mucosa in vitro.

The short-chain fatty acid butyrate is regarded as a regulative agent in haemostasis of mucosal cell turnover. Inhibition of prostaglandin E2 synthesis is particularly involved in this regulation process. In the present study, proliferation was stimulated in colonic biopsies of 12 healthy subjects (age 51.3 years, range 25-81) by incubation with deoxycholic acid (5 micromol/l DCA). The anti-proliferative and cyclo-oxygenase-inhibiting properties of butyrate (10 mmol/l BUT) and of aspirin (555 micromol/l ASA) were investigated. Colonic cell proliferation was determined by bromodeoxyuridine immunohistochemistry. PGE2 release into the incubation medium was measured by radioimmunoassay. Incubation with DCA/ASA, DCA/BUT and DCA/ASA/BUT revealed a significant reduction in crypt cell proliferation as measured by the labelling index of the whole crypt in comparison to incubation with DCA alone (DCA/ASA: 0.14, P < 0.01; DCA/BUT: 0.15, P < 0.05; DCA/ASA/BUT: 0.15, P < 0.05, versus DCA: 0.18). The labelling index for the upper 40% of the crypt was only lower after incubation with DCA/ASA (0.023) compared to DCA (0.028) (P < 0.05). PGE2 release from biopsy specimens was only significantly decreased in the incubation media where ASA was added (DCA/ASA: 29.0 pg/mg mucosa/h, P < 0.005; DCA/ASA/BUT: 31.4 pg/mg mucosa/h, P < 0.01 versus DCA: 56.9 pg/mg mucosa/h). Butyrate and aspirin showed no synergistic effects. The results indicate a normalization of DCA-induced hyperproliferation of colonic mucosa by butyrate, and, even more efficiently, by aspirin. The data support the hypothesis that butyrate and aspirin can act as chemopreventive agents in colon carcinogenesis.

Antiproliferative effect of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on colonic epithelium of patients with adenomatous polyps in vitro.

We have consistently shown that the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a superior cancer chemopreventive agent and less toxic than selenite or certain naturally-occurring selenoamino acids. To elucidate the effects of p-XSC on human colonic mucosa, biopsies from endoscopically normal sigmoid colon of 30 patients with adenomatous polyps were incubated with p-XSC at concentrations of 1, 2 and 5 micromol/l dissolved in dimethylsulphoxide (DMSO). Biopsies incubated with DMSO or pure culture medium served as a control. Proliferating cells were labelled by bromodeoxyuridine immunohistochemistry and the labelling index (LI) was computed. Upper crypt labelling index (LI of crypt compartments 4+5) and Phih value, which are both discriminators of the expansion of the proliferative zone, were significantly lower after incubation with 1 and 5 micromol/l p-XSC, respectively (LI 4+5: 0.8 and 1.0; Phih value: 2.1 and 2.4), as compared with DMSO (LI 4+5: 3.6 and 4.5; Phih value: 7.0 and 8.3) or culture medium (LI 4+5: 3.3 and 4.5; Phih value: 7.2 and 8.1) (P<0.005 and P<0.05 by Friedman's block test). A trend towards lower levels of LI 4+5 (P=0.059) and Phih value (P=0.075) were seen after 2 micromol/l p-XSC incubation compared with DMSO. Since hyperproliferation of colonic crypt cells with expansion of the proliferative zone is regarded as a biomarker of increased cancer risk, the antiproliferative effects of p-XSC especially on upper crypt LI and Phih value may indicate a possible protective effect of this organoselenium compound in the prevention of human colon cancer development.

Missing anti-proliferative effect of fish oil on rectal epithelium in healthy volunteers consuming a high-fat diet: potential role of the n-3:n-6 fatty acid ratio.

Several studies have indicated dietary fish oil (FO) as a protective agent in colon carcinogenesis. Rectal cell proliferation as an intermediate biomarker of cancer risk was shown to be reduced by dietary FO in patients with adenomatous polyps and healthy subjects consuming a low-fat diet. Because the synthesis of prostaglandins (PG) which seem to be involved in this process is dependent on the ratio of n-3:n-6 fatty acids in the diet, the present study was designed to investigate whether this FO effect is also detectable in volunteers eating a high-fat diet (50% of energy) with a low n-3:n-6 ratio of 0.25. Twelve healthy volunteers received in addition to a controlled basal diet either FO (4.4 g n-3 fatty acids/day) or corn oil supplements (double-blind, crossover) for two 4-week periods. No significant differences between the two study periods were found for rectal cell proliferation as assessed by bromodeoxyuridine immunohistochemistry and ornithine decarboxylase activity, as well as for mucosal PGE2 release and mucosal membrane fatty acid composition. The results emphasize the importance of dietary n-3:n-6 ratio in determining the effects of FO on rectal cell proliferation.