Effect of seven days heat stress on feed and water intake, milk characteristics, blood parameters, physiological indicators, and gene expression in Holstein dairy cows.

This study examined the effects of 7 days of heat stress on eight early lactating Holstein cows in climate-controlled chambers. The early lactating Holstein cows (42 ± 2 days in milk, 29.27 ± 0.38 kg/day milk yield, 1.21 ± 0.05 parity) were subjected to two 14-day periods, each consisting of 7 days of adaptation and 7 days of heat stress. Conditions were set to 22 °C and 50% humidity during adaptation, followed by heat stress periods with low-temperature, low-humidity (LTLH, 71 THI) and high-temperature, high-humidity (HTHH, 86 THI) treatments. Data from the last 7 days were analyzed using a mixed procedure in SAS. In the study, the HTHH group displayed marked physiological and biochemical changes on 14 days of heat stress exposure compared to the LTLH group. Firstly, the HTHH group's dry matter intake decreased by approximately 12% while their water intake increased by about 23%. Secondly, both milk yield and milk protein production in the HTHH group decreased by 10% and 20%, respectively. Thirdly, there was a reduction in white blood cells, hemoglobin, mean corpuscular hemoglobin, and platelets in the HTHH group, with concurrent increases in glucose, non-esterified fatty acids, and albumin concentrations. Additionally, the HTHH group exhibited elevated plasma concentrations of cortisol and haptoglobin. Moreover, the gene expression of heat shock protein 70 and heat shock protein 90 was significantly upregulated in the HTHH group's peripheral blood mononuclear cells. Lastly, key physiological indicators such as rectal temperature, heart rate, and skin temperature showed substantial elevations in the HTHH group. Considering the enormous negative effects observed in the analyzed blood metabolites, milk yield and compositions, and heat shock protein gene expression, early lactating Holstein cows were found to be more vulnerable to HTHH than LTLH over a 7 days exposure to heat stress.

Effects of Rumen-Protected L-Tryptophan Supplementation on Productivity, Physiological Indicators, Blood Profiles, and Heat Shock Protein Gene Expression in Lactating Holstein Cows under Heat Stress Conditions.

In this study, we examined the effects of rumen-protected L-tryptophan supplementation on the productivity and physiological metabolic indicators in lactating Holstein cows under heat stress conditions. The study involved eight early lactating Holstein cows (days in milk = 40 ± 9 days; milk yield 30 ± 1.5 kg/day; parity 1.09 ± 0.05, p < 0.05), four cows per experiment, with environmentally controlled chambers. In each experiment, two distinct heat stress conditions were created: a low-temperature and low-humidity (LTLH) condition at 25 °C with 35-50% humidity and a high-temperature and high-humidity (HTHH) condition at 31 °C with 80-95% humidity. During the adaptation phase, the cows were subjected to LTLH and HTHH conditions for 3 days. This was followed by a 4-day heat stress phase and then by a 7-day phase of heat stress, which were complemented by supplementation with rumen-protected L-tryptophan (ACT). The findings revealed that supplementation with ACT increased dry matter intake as well as milk yield and protein and decreased water intake, heart rate, and rectal temperature in the HTHH group (p < 0.05). For plateletcrit (PCT, p = 0.0600), the eosinophil percentage (EOS, p = 0.0880) showed a tendency to be lower, while the monocyte (MONO) and large unstained cells (LUC) amounts were increased in both groups (p < 0.05). Albumin and glucose levels were lower in the HTHH group (p < 0.05). The gene expressions of heat shock proteins 70 and 90 in the peripheral blood mononuclear cells were higher in the ACT group (HTHH, p < 0.05). These results suggest that ACT supplementation improved productivity, physiological indicators, blood characteristics, and gene expression in the peripheral blood mononuclear cells of early lactating Holstein cows under heat-stress conditions. In particular, ACT supplementation objectively relieved stress in these animals, suggesting that L-tryptophan has potential as a viable solution for combating heat-stress-induced effects on the cattle in dairy farming.

Effects of different energy levels and two levels of temperature-humidity indices on growth, blood metabolites, and stress biomarkers in Korean native calves.

This study investigated the effects of dietary energy levels on growth, blood metabolites, and stress biomarkers in Korean native calves subjected to heat stress (HS). Twenty-four calves (BW: 221.5 ± 24.9 kg; age: 162 ± 4.8 d) were randomly housed in climate-controlled chambers using 3 × 2 factorial design. There were three treatment groups including low energy (LE = 2.53), medium energy (ME = 2.63), and high energy levels (HE = 2.72 Mcal/kg of DM) and two stress levels (threshold: THI = 70-73; severe: THI = 89-91). The calves were adapted to 22 °C for 7 days, then to the target THI level for 14 days. Energy intake, average daily gain, and gain to feed ratio were determined to decline (p < 0.05) under severe HS compared with threshold. Under severe HS, rectal temperature was increased 0.67 °C compared with threshold. Severe HS increased glycine, ammonia, and 3-methylhistidine concentrations compared with threshold (p < 0.05). Gluconeogenic AAs in the blood were increased among the various energy levels regardless of HS. In PBMCs the expression of HSP70 gene was increased in the LE group (p < 0.05), and the HSP90 gene expression was increased in LE and ME groups (p < 0.05) under severe HS. However, the expression of genes HSP70 and HSP90 in HE group did not differ under severe HS (p > 0.05). It has been suggested that HE intake may have a beneficial effect on PBMCs by mitigating ATP depletion. No differences in growth performance were found when increasing energy intake with high protein (CP 17.5%) under HS. However, the increase in energy levels resulted in increased gluconeogenic AAs but decreased urea and 3-methylhistidine in blood. In conclusion, increased energy levels are thought to improve HS adaptability by inhibiting muscle degradation and glucose production using gluconeogenic AAs in Korea native calves under HS condition.

A Clinical Decision Support System for Sleep Staging Tasks With Explanations From Artificial Intelligence: User-Centered Design and Evaluation Study.

BACKGROUND: Despite the unprecedented performance of deep learning algorithms in clinical domains, full reviews of algorithmic predictions by human experts remain mandatory. Under these circumstances, artificial intelligence (AI) models are primarily designed as clinical decision support systems (CDSSs). However, from the perspective of clinical practitioners, the lack of clinical interpretability and user-centered interfaces hinders the adoption of these AI systems in practice. OBJECTIVE: This study aims to develop an AI-based CDSS for assisting polysomnographic technicians in reviewing AI-predicted sleep staging results. This study proposed and evaluated a CDSS that provides clinically sound explanations for AI predictions in a user-centered manner. METHODS: Our study is based on a user-centered design framework for developing explanations in a CDSS that identifies why explanations are needed, what information should be contained in explanations, and how explanations can be provided in the CDSS. We conducted user interviews, user observation sessions, and an iterative design process to identify three key aspects for designing explanations in the CDSS. After constructing the CDSS, the tool was evaluated to investigate how the CDSS explanations helped technicians. We measured the accuracy of sleep staging and interrater reliability with macro-F1 and Cohen κ scores to assess quantitative improvements after our tool was adopted. We assessed qualitative improvements through participant interviews that established how participants perceived and used the tool. RESULTS: The user study revealed that technicians desire explanations that are relevant to key electroencephalogram (EEG) patterns for sleep staging when assessing the correctness of AI predictions. Here, technicians wanted explanations that could be used to evaluate whether the AI models properly locate and use these patterns during prediction. On the basis of this, information that is closely related to sleep EEG patterns was formulated for the AI models. In the iterative design phase, we developed a different visualization strategy for each pattern based on how technicians interpreted the EEG recordings with these patterns during their workflows. Our evaluation study on 9 polysomnographic technicians quantitatively and qualitatively investigated the helpfulness of the tool. For technicians with <5 years of work experience, their quantitative sleep staging performance improved significantly from 56.75 to 60.59 with a P value of .05. Qualitatively, participants reported that the information provided effectively supported them, and they could develop notable adoption strategies for the tool. CONCLUSIONS: Our findings indicate that formulating clinical explanations for automated predictions using the information in the AI with a user-centered design process is an effective strategy for developing a CDSS for sleep staging.

Identification of Potential Biomarkers and Metabolic Pathways of Different Levels of Heat Stress in Beef Calves.

Heat stress (HS) damages the global beef industry by reducing growth performance causing high economic losses each year. However, understanding the physiological mechanisms of HS in Hanwoo calves remains elusive. The objective of this study was to identify the potential biomarkers and metabolic pathways involving different levels of heat stress in Hanwoo calves. Data were collected from sixteen Hanwoo bull calves (169.6 ± 4.6 days old, BW of 136.9 ± 6.2 kg), which were maintained at four designated ranges of HS according to the temperature−humidity index (THI) including: threshold (22 to 24 °C, 60%; THI = 70 to 73), mild (26 to 28 °C, 60%; THI = 74 to 76), moderate (29 to 31 °C, 80%; THI = 81 to 83), and severe (32 to 34 °C, 80%; THI = 89 to 91) using climate-controlled chambers. Blood was collected once every three days to analyze metabolomics. Metabolic changes in the serum of calves were measured using GC-TOF-MS, and the obtained data were calculated by multivariate statistical analysis. Five metabolic parameters were upregulated and seven metabolic parameters were downregulated in the high THI level compared with the threshold (p < 0.05). Among the parameters, carbohydrates (ribose, myo-inositol, galactose, and lactose), organic compounds (acetic acid, urea, and butenedioic acid), fatty acid (oleic acid), and amino acids (asparagine and lysine) were remarkably influenced by HS. These novel findings support further in-depth research to elucidate the blood-based changes in metabolic pathways in heat-stressed Hanwoo beef calves at different levels of THI. In conclusion, these results indicate that metabolic parameters may act as biomarkers to explain the HS effects in Hanwoo calves.

Dietary supplementation of acetate-conjugated tryptophan alters feed intake, milk yield and composition, blood profile, physiological variables, and heat shock protein gene expression in heat-stressed dairy cows.

The purpose of this study was to investigate the effects of dietary supplementation of rumen-protected tryptophan (RPT) at four levels on milk yield, milk composition, blood profile, physiological variables, and heat shock protein gene expression in dairy cows under conditions of moderate-severe heat stress (MSHS, THI = 80~89). Sixteen early-lactating dairy cows (body weight = 719 ± 66.4 kg, days in milk = 74.3 ± 7.1, milk yield = 33.55 ± 3.74 kg, means ± SEM) were randomly assigned in a factorial arrangement to one of the four treatments: control group (n = 4, no RPT supplementation), 15 g/d RPT (n = 4), 30 g/d RPT (n = 4), or 60 g/d RPT group per cow (n = 4) supplemented to the TMR. A higher dry matter intake (DMI) and milk yield were found in the 30 g RPT group compared with the other groups, and the 3.5% fat-corrected milk yield, energy-corrected milk yield, milk fat, protein, ß-casein, mono-unsaturated fatty acid, and poly-unsaturated fatty acid contents, and serum glucose content were observed in the 30 g RPT group (p < 0.05). The milk lactose concentration was significantly higher in the 30 g RPT group compared with the control and 60 g RPT groups (p < 0.05). The plasma cortisol level was lower, while the serotonin and melatonin concentrations were higher in the 30 g group compared with the other groups (p < 0.05). Heat shock protein (HSP) 70 expression was downregulated in the control and 15 g RPT groups, whereas the expression of HSP90 and HSPB1 remained unchanged among the groups. In particular, the 30 g RPT group was considered to have an improved DMI, milk yield, and lactose concentration, as well as anti-heat stress effects due to the simulation of serotonin and melatonin during MSHS.

Responses of beef calves to long-term heat stress exposure by evaluating growth performance, physiological, blood and behavioral parameters.

The objective of this study was to explore the responses of beef calves to long-term heat stress (HS) exposure at various levels in comparison with the animals under thermoneutral conditions by evaluating growth performance, physiological, blood, and behavioural parameters. Data were collected from sixteen beef calves (BW: 136.9 ± 6.23 kg; age: 169.6 ± 4.60 d) kept at four stress levels of designated temperature humidity index (THI): threshold (22-24 °C, 60%; THI = 70 to 73), mild (26-28 °C, 60%; THI = 74 to 76), moderate (29-31 °C, 80%; THI = 81 to 83), and severe (32-34 °C, 80%; THI = 89 to 91) stress levels in climatic controlled chambers. Feed and water intake were recorded daily, and body weight was measured once a week. Blood was sampled every three days to analyse metabolite parameters. Dry matter intake (DMI) (p = 0.069, tendency) and blood glucose levels (p = 0.028) were decreased after sudden exposure to HS conditions (severe THI levels). Also, blood cortisol (p = 0.002), glutamic-oxaloacetic transaminase (GOT) (p = 0.009), blood urea nitrogen (BUN) (p = 0.004) and standing time (p = 0.009) were increased in moderate and severe THI levels compared with threshold after exposure to HS conditions. However, in the severe THI group, blood cortisol (p < 0.05), glucose (p < 0.05), GOT (p < 0.05) and BUN (p < 0.05) levels were returned to normal range after 6-13 days of continuous HS exposure. In conclusion, DMI, blood cortisol, GOT, glucose, BUN, and standing time were closely associated with long-term HS condition in beef calves. In addition, calves exposed to HS modulated their physiological responses that resulted in the regulation of the pertinent blood metabolites in the blood to maintain homeostasis during the long-term HS.

Supplementing with L-Tryptophan Increases Medium Protein and Alters Expression of Genes and Proteins Involved in Milk Protein Synthesis and Energy Metabolism in Bovine Mammary Cells.

The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. ß-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated (p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in ß-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.

Supplementing conjugated and non-conjugated L-methionine and acetate alters expression patterns of CSN2, proteins and metabolites related to protein synthesis in bovine mammary cells.

The experiments reported in this research paper aimed to determine the effect of supplementing different forms of L-methionine (L-Met) and acetate on protein synthesis in immortalized bovine mammary epithelial cell line (MAC-T cells). Treatments were Control, L-Met, conjugated L-Met and acetate (CMA), and non-conjugated L-Met and Acetate (NMA). Protein synthesis mechanism was determined by omics method. NMA group had the highest protein content in the media and CSN2 mRNA expression levels (P < 0.05). The number of upregulated and downregulated proteins observed were 39 and 77 in L-Met group, 62 and 80 in CMA group and 50 and 81 in NMA group from 448 proteins, respectively (P < 0.05). L-Met, NMA and CMA treatments stimulated pathways related to protein and energy metabolism (P < 0.05). Metabolomic analysis also revealed that L-Met, CMA and NMA treatments resulted in increases of several metabolites (P < 0.05). In conclusion, NMA treatment increased protein concentration and expression level of CSN2 mRNA in MAC-T cells compared to control as well as L-Met and CMA treatments through increased expression of milk protein synthesis-related genes and production of the proteins and metabolites involved in energy and protein synthesis pathways.

Dietary supplementation with combined extracts from garlic (Allium sativum), brown seaweed (Undaria pinnatifida), and pinecone (Pinus koraiensis) improves milk production in Holstein cows under heat stress conditions.

OBJECTIVE: This study was conducted to examine the effects of a mixture of pinecone oil, garlic, and brown seaweed extracts (PGBE) on milk production traits as well as physiological and ethological parameters in Holstein cows during the summer season (24 May to 03 July 2015, Korea). METHODS: Among the extract combinations tested, we found that the level of 2,2'-azino-bis (3-ethylberzothiazoline-6-sulphonic acid) cation radical scavenging activity of the 0.16% PBGE complex at ratio of 1:1:1 (vol/vol) was comparable to that of the control (ascorbic acid; 1 mg/mL). Additionally, the PBGE complex reduced lipopolysaccharide-induced COX-2 expression in bovine mammary epithelial cells. Based on these findings, 40 lactating Holstein cows were used to measure the effects of PBGE complex at ratio of 1:1:1 (vol/vol) on milk production, immune response, metabolites, and behavior patterns by dividing the cows into two groups fed diets containing PGBE complex (n = 20; 0.016%/kg feed dry matter basis) or not containing PGBE complex (control, n = 20) for 40 d. RESULTS: Results showed that PGBE complex did not influence milk composition, eating and ear surface temperature patterns, immune response, or metabolic parameters but promoted average milk yield throughout the experimental period. Additionally, a tendency of higher total antioxidant capacity and glutathione in the PGBE group was observed compared to the those in the control. When the temperature-humidity index (THI) exceeded 72 (average THI = 73.8), PGBE complex-fed cows experiencing heat stress showed increased milk yield and a tendency of increased rumination compared to the control. CONCLUSION: We suggest that incorporation of a combined mixture of 0.016% PGBE (1:1:1 ratio, vol/vol) to diet has the potential to improve milk yield and health status of cows under mild to moderate heat stress, denoting that it might be useful as an alternative anti-stressor in the diet of dairy cows under hot conditions.

Effect of water scarcity during thermal-humidity exposure on the mineral footprint of sheep.

OBJECTIVE: Combination of two stressors on alteration of mineral footprints in animals needs due attention to meet maximum production and welfare, particularly in grazing sheep. This study tested whether ewes (Ovis aries) exposed to water deprivation and thermal-humidity stressors had altered mineral footprints in their wool, serum, urine, and feces. METHODS: Nine ewes (age = 3 years; mean body weight = 41±3.5 kg) were divided among a control group with free access to water, and treatment groups with water deprivation lasting either 2 h (2hWD) or 3 h (3hWD) after feeding. Using a 3×3 Latin square design, animals were assigned to treatment groups for three sampling periods of 21 days each (n = 9). Blood was collected by jugular venipuncture. Wool was collected at the end of periods 2 and 3. Metabolic crates designed with metal grated floors were used for urine and feces collection. We measured sodium (Na), magnesium (Mg), phosphorus (P), chloride (Cl), calcium (Ca), manganese (Mn), copper (Cu), iron (Fe), and zinc (Zn). RESULTS: The wool mineral levels did not differ between the treatment groups, although K was marginally lower (p = 0.10) in the 2hWD group. The serum and urine mineral levels did not differ between the treatments (p>0.05). Fecal K was significantly lower in the 2hWD group than in the other groups (p≤0.05). CONCLUSION: In conclusion, water deprivation and thermal-humidity exposure altered the excretion of K, but not of other minerals, in the wool, urine, feces, or serum of ewes. Thus, no additional mineral supplementation is needed for water deprived ewes during thermalhumidity exposure.

Administration of encapsulated L-tryptophan improves duodenal starch digestion and increases gastrointestinal hormones secretions in beef cattle.

OBJECTIVE: This study investigated the effects of oral administration of rumen-protected L-tryptophan (RPL-T) on duodenal starch digestion and gastrointestinal hormones (GIH) secretions using Hanwoo beef steers as the animal models. METHODS: Four steers (423±24 kg) fitted with ruminal and duodenal cannulas were employed in a crossover design replicated twice. Treatments were control (basal diet) and RPL-T (basal diet+191.1 mg/kg body weight [BW]) group. Blood and duodenal samples were collected to measure serum GIH levels and pancreatic α-amylase activity at day 0, 1, 3, and 5 (-30, 30, 90, 150, and 210 min) of the study. Samples from each segment of the gastrointestinal tract were collected via ruminal and duodenal cannulas and were used to determine soluble protein and the starch digestion rate at days 6 (-30, 180, 360, and 540 min) and 8 (-30, 90, 270, and 450 min) of the experiment. RESULTS: No significant difference in ruminal pH, NH3-N, and total volatile fatty acid including the levels of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and the acetate-to-propionate ratio was observed between groups (p>0.05). Crude protein uptake was higher and feces starch content was lower in RPL-T group than the control group (p<0.05). The D-glucose contents of feces in RPL-T group decreased at day 5 compared to those in the control group (p<0.05), however, no change was found at day 0, 1, or 3 compared to the control group (p>0.05). Serum cholecystokinin (CCK), melatonin, duodenal pancreatic α-amylase activity, and starch digestion were significantly higher in RPL-T group than the control group (p<0.05). CONCLUSION: Taken together, oral administration of RPL-T at the rate of 191.1 mg/kg BW consistently increased CCK concentration, pancreatic α-amylase activity in duodenal fluids, and starch digestion rate in the small intestine and thus found to be beneficial.

Effect of fermented spent instant coffee grounds on milk productivity and blood profiles of lactating dairy cows.

OBJECTIVE: This study was conducted to evaluate the fermentation characteristics under low mesophilic temperature of spent instant coffee ground (SICG) and to estimate the effect of fermented SICG (FSICG) as alternative feed ingredient on milk productivity of dairy cows. METHODS: In the fermentation trial, fermentation of SICG was performed to investigate changes in characteristics using the microbial mixture (Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis = 1:1:1) for 21 days at 20°C under anaerobic conditions. Molasses was added at 5% of dry mass. In the animal trial, eighteen Holstein Friesian cows were used to evaluate the nutritive value of the FSICG which was fermented for 14 days under the same condition as the fermentation trial. RESULTS: In the fermentation trial, the dry matter (DM) and organic matter content linearly decreased with fermentation time (p<0.001 and p = 0.008, respectively). The acid detergent insoluble nitrogen content linearly decreased with fermentation time (p = 0.037). The microorganism counts linearly increased for Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis across fermentation time (p<0.001). In the animal trial, the DM intake of the control and FSICG treatment were not significantly different, as were milk yield, 4% fat corrected milk, fat-protein corrected milk, and feed to milk conversion content. Fat, protein, lactose, non-fat solids, milk urea nitrogen, and somatic cell counts were also not significantly different in milk composition between treatments. CONCLUSION: FSICG should be considered a sufficient substitute for cottonseed as a feed component, and 5% DM of a dietary FSICG level was appropriate for dairy cow diets.

Effects of dietary phytoncides extracted from Korean pine (Pinus koraiensis) cone on performance, egg quality, gut microflora, and immune response in laying hens.

This study was conducted to evaluate the effects of dietary phytoncides extracted from discarded Korean pine cones (Pinus koraiensis) on the performance, egg quality, immune response and gut microflora in laying hens. A total of 400 Hy-Line brown laying hens (50-week old) were allotted into four dietary treatments including a control diet or a diet supplemented with phytoncides at 0.002%, 0.004% and 0.008%. During the 6 weeks of experimental feeding, 0.008% of dietary phytoncides improved egg production, feed conversion ratio (p < 0.05), but not feed intake, egg weight or feed efficiency. Although dietary phytoncides had no effect on egg quality, decreases in Haugh units depending on storage periods were improved by 0.008% of dietary phytoncides (p < 0.05). To investigate the roles of dietary phytoncides on the alteration of the immune response during inflammation, lipopolysaccharide (LPS) or saline was intraperitoneally injected into 10 hens per diet group on the end date of the experimental feeding period. Serum immunoglobulins and splenic cytokine expression at mRNA levels were then measured at 4 hr postinjection. Although the levels of IgA were decreased by LPS injection in all dietary groups, dietary phytoncides at 0.008% showed a higher level of IgA by LPS (p < 0.05). Interestingly, although LPS injection resulted in an enhanced expression of proinflammatory cytokines such as IL-1ß and IL-6, dietary phytoncides at 0.008% showed less increased levels of them (p < 0.05). Gut microflora was examined from 10 hens per diet group at the end of the experimental period. While the number of Lactobacillus spp. was increased (p < 0.05), Escherichia coli counts in the cecal contents were decreased by 0.008% of dietary phytoncides. Taken together, these results demonstrate that dietary supplementation of 0.008% phytoncides improved the egg production, immune responses during inflammation and gut microflora in laying hens.

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine ß-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine ß-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the ß-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine ß-casein gene.

Chemerin is a novel regulator of lactogenesis in bovine mammary epithelial cells.

Chemerin is a chemoattractant cytokine (chemokine) produced by adipocytes and hepatocytes; it regulates insulin sensitivity and adipocyte differentiation. The objective of this study was to investigate the effect of chemerin on the expression of genes related to lactogenesis and the regulators of chemerin signaling in a bovine mammary epithelial cell line (MAC-T). Two types of chemerin receptors, chemokine like-receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2), were detected in cultured MAC-T cells, whereas chemerin was not detected. G protein-coupled receptor 1 (GPR1), another receptor of chemerin, was undetectable in MAC-T cells. Chemerin upregulated transcript expression of CMKLR1, CCRL2, and genes associated with fatty acid synthesis, glucose uptake, insulin signaling, and casein synthesis in MAC-T cells. Lactogenic hormones (insulin, growth hormone, and prolactin) downregulated the expression of CMKLR1 in MAC-T cells. Adiponectin suppressed CMKLR1 expression. TNF-α suppressed CMKLR1, but induced CCRL2 expression. These data suggest chemerin is a novel regulator of lactogenesis via its own receptor in bovine mammary epithelial cells.

Heat-shock protein beta 1 regulates androgen-mediated bovine myogenesis.

To elucidate the functional significance of heat-shock protein beta 1 (HSPB1) in androgen-mediated myogenesis of bovine cells, we conducted 'loss and gain of function of HSPB1' assays by siRNA inhibition and gene overexpression. siRNA inhibition of HSPB1 expression reduced the expression of desmin (a myogenic marker) and repressed the formation of myotubes in cells induced for myogenic differentiation. In contrast, overexpression of HSPB1 enhanced the expression of desmin and accelerated formation of myotubes. The loss and gain of HSPB1 function was closely associated with the expression level of androgen receptor (AR). Our findings suggest that HSPB1 mediates androgen signaling by binding directly to AR and then enhancing androgen-mediated myogenesis in myogenic cells.

Association of protein expression in isolated milk epithelial cells and cis-9, trans-11 conjugated linoleic acid proportions in milk from dairy cows.

BACKGROUND: The effects of the individual variation among dairy cows on the synthesis of cis-9, trans-11 conjugated linoleic acid (CLA) are still not well characterised. Therefore, the protein expression profiles of isolated milk epithelial cells (MECs) were detected by two-dimensional electrophoresis and their correlation with the various proportion of cis-9, trans-11 CLA were evaluated. RESULTS: Although animals were offered the same diet, the proportion of cis-9, trans-11 CLA in group High (1.02 ± 0.10%) was twice as high as that in group Low (0.59 ± 0.14%) (P < 0.05). MECs with the characteristics of native epithelial cells were successfully isolated from the milk and these cells had no obvious RNA degradation or were hardly contaminated with leucocytes or blood red cells. Moreover, the protein expression pattern of cathelicidin 5 in isolated MECs was positive, whereas annexin I (confirmed by real-time polymerase chain reaction), ZW10 interactor and κ-casein were negatively related to the proportion of cis-9, trans-11 CLA in the milk fat. CONCLUSION: The varied individual content of cis-9, trans-11 CLA in cows may be associated with annexin I. These findings may provide some theoretical basis for studies concerning the effects of the individual variation among dairy cows of the synthesis of cis-9, trans-11 CLA. © 2013 Society of Chemical Industry.

Effect of Encapsulating Nitrate in Sesame Gum on In vitro Rumen Fermentation Parameters.

Encapsulation is a method used to protect material from certain undesirable environments, for controlled release at a more favorable time and place. Animal productivity would be enhanced if feed additives are delivered to be utilized at their site of action, bypassing the rumen where they are likely to be degraded by microbial action. A novel method of encapsulation with sesame gum was used to coat nitrate, a known enteric methane mitigating agent, and tested for the effect on methane reduction and other in vitro fermentation parameters using rumen fluid from cannulated Hanwoo steers. Orchard grass was used as basal diet for fermentation. The treatments were matrix (1.1 g sesame gum+0.4 g sesame oil cake) only, encapsulated nitrate (matrix+nitrate [21 mM]), free nitrate (21 mM), and a control that contained no additive. Analyses of fermentation parameters were done at 0, 3, 6, 9, 12, 24, and 48 h time periods. In comparison to control, both free and encapsulated nitrate produced significantly reduced (p<0.01) methane (76% less) and also the total volatile fatty acids were reduced. A significantly higher (p<0.01) concentration of ammonia nitrogen was obtained with the encapsulated nitrate treatment (44%) compared to the free form (28%) and matrix only (20%) (p = 0.014). This might suggest slow release of encapsulated nitrate so that it is fully reduced to ammonia. Thus, this pioneering study found a significant reduction in methane production following the use of sesame gum encapsulated nitrate that shows the potential of a controlled release system in enhancing sustainability of ruminant production while reducing/eliminating the risk of nitrite toxicity.

Effects of Centella asiatica Extracts on Rumen In Vitro Fermentation Characteristics and Digestibility.

Two in vitro experiments were conducted to evaluate the effects of Centella asiatica extract (CAE) supplementation on the rumen's in vitro fermentation characteristics. In the first experiment, CAE with five concentrations (C: 0%; T1: 3.05%; T2: 6.1%; T3: 12.2%; and T4: 24.4% CAE in diet) was supplemented in the rumen fluid and incubated for 6, 24, and 48 h to determine the optimal dosage. The total gas and methane production increased in all incubation times, and the total volatile fatty acids increased at 6 and 48 h. Ammonia nitrogen, branched chain volatile fatty acids, acetate, and butyrate were increased by CAE supplementation. T1 was chosen as the optimal dosage based on the total volatile fatty acids, branched chain volatile fatty acids, and ammonia nitrogen production. The CAE with the identified optimal dosage (T1) was incubated to identify its effect on the rumen's in vitro degradability in the second experiment. The CAE supplementation did not influence the in vitro dry matter, crude protein, or neutral detergent fiber degradability. In conclusion, CAE has no CH4 abatement or digestion promotion effects. However, CAE could be utilized as a feed additive to increase the rumen's total volatile fatty acid production without an adverse effect on the in vitro dry matter, crude protein, or neutral detergent fiber degradability.