The in vitro and in vivo anti-inflammatory and anti-oxidative effects of an amino acid blend supplemented feed on pigs experimentally challenged with Salmonella Typhimurium.

Background: The in vitro and in vivo anti-inflammatory and anti-oxidative effects of an amino acid (AA) blend (tryptophan, threonine, and methionine) in pigs. Objective: This study aimed to evaluate the in vitro anti-inflammatory and anti-oxidative effects of an AA blend on intestinal porcine epithelial cells (IPEC-J2) and the in vivo anti-inflammatory and anti-oxidative effects in pigs experimentally challenged with Salmonella Typhimurium. Methods: IPEC-J2 were pretreated with an AA blend for 25 h and then treated with lipopolysaccharide (LPS), deoxynivalenol (DON), or H2O2 for in vitro evaluation. A controlled standard diet supplemented with 0.3% of the AA blend was orally fed to the treated group pigs for 14 days, beginning at 21 days of age. At the end of the feeding period, pigs were orally inoculated with Salmonella Typhimurium. Results: Pre-treatment with the AA blend reduced LPS/DON-induced interleukin (IL)-8 mRNA as a measurement of the anti-inflammatory effect and H2O2-induced reactive oxygen species (ROS) as a measurement of the anti-oxidative effect on IPEC-J2. Feeding with an AA blend resulted in a reduction of proinflammatory (tumor necrosis factor-α, IL-6, and IL-8) cytokine levels, while treated pigs experienced an increase in anti-inflammatory IL-10 cytokine in their sera. The addition of an AA blend-supplemented pig feed resulted in significantly lower Salmonella-induced cecal lesion scores compared to untreated pigs. Discussion: Supplementation of feed with an AA blend reduced intestinal inflammation and pathology in pigs and may be applied for the control of Salmonella Typhimurium infection, as demonstrated in this study.

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.

In Vivo Recovery of Bacteriophages and Their Effects on Clostridium perfringens-Infected Broiler Chickens.

The objectives of this study were to recover bacteriophages (BPs) from the intestinal digesta of BP-fed broilers and to evaluate the antibacterial effects of encapsulated or powdered BPs in broiler chickens challenged with Clostridium perfringens. Day-old broiler chicks (n = 320/experiment) were randomly assigned to 32 pens (n = 10 broilers/pen) and allocated to one of four dietary groups: (1) unchallenged group (NEG); (2) C. perfringens-challenged group (POS); (3) POS group fed a diet supplemented with powdered BPs; and (4) POS group fed a diet supplemented with encapsulated BPs. On days 21, 22, and 23 post-hatch, all chickens except NEG were orally inoculated twice a day with 2 mL C. perfringens (1.0 × 108 cfu/mL). Varying BP levels were detected in gut digesta at all ages and were numerically or significantly higher in the encapsulated BP group than in the powdered BP group. Dietary powder or encapsulated BPs reversed the C. perfringens-mediated increase in crypt depth. In addition, villus height to crypt depth ratio was elevated in the NEG and BP-treated/challenged groups compared with that in the POS group. C. perfringens counts in the cecum were significantly lower in the BP-fed chickens than in the POS group. The encapsulated BP-supplemented diet-fed chickens had the highest serum IgA levels. Collectively, our results suggest that dietary BP remains viable in intestinal digesta upon ingestion and can inhibit cecal C. perfringens counts.

Effects of Dietary Supplementation of Acetate and L-Tryptophan Conjugated Bypass Amino Acid on Productivity of Pre- and Post-Partum Dairy Cows and Their Offspring.

In this study, we investigated the effect of dietary supplementation with acetate and L-tryptophan-conjugated bypass amino acid (ACT) during late pregnancy on the production performance of cows pre- and postpartum and their offspring. Eight multiparous Holstein cows (calving date ±15 d, 2nd parity; n = 4) were supplied with diets without ACT supplementation (Control) or with 15 g/day ACT supplementation (ACT). The results showed that ACT improved the feed intake (FI) in dry cows. No differences in blood hematological parameters were found between the two groups of prepartum cows. The serum glutamic-oxaloacetic transaminase activity increased and the triglyceride concentration decreased in the ACT-treated group compared to the control group. In the postpartum cows, milk compositions were not affected by ACT supplementation. Saturated fatty acid (SFA) content in the colostrum was significantly lower in the ACT-treated group than in the control group. Serum glucose (GLC) level was significantly higher in the ACT-treated group than in the control group. Monocyte and GLC levels were lower in calves of groups where their dams had received ACT. Overall, we found higher FI in the dry cows, lower colostrum SFA levels, and heavier calf birth weight (5.5 kg) when the dams were supplemented with ACT, suggesting a positive nutrient compensation by ACT supplementation to dry cows.

Isolation, characterization, and genomic analysis of the novel T4-like bacteriophage ΦCJ20.

Pathogenic Escherichia coli infections have been consistently reported annually. The basic characteristics and genome of the newly isolated ΦCJ20 from swine feces was analyzed. To determine basic characteristics, dotting assays and double-layer agar assays were conducted. Bacteriophage particles were analyzed via transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to determine the sizes of major structural proteins. The complete genome of the phage was analyzed. Bacteriophage particles were identified as Myoviridae, with a head measuring 110.57 ± 1.89 nm and a contractile tail measuring 107.97 ± 3.20 nm and were found to infect E. coli. Major structural proteins of ΦCJ20 showed two well-pronounced bands of approximately 53.6 and 70.9 kDa. The genome size of ΦCJ20 was 169,884 bp, and 118 of 307 open reading frames were annotated. This study provides a baseline for the development of E. coli infection treatment strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00906-y.

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.

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.

"Dietary supplementation of L-tryptophan" increases muscle development, adipose tissue catabolism and fatty acid transportation in the muscles of Hanwoo steers.

This study investigated the effects of dietary rumen-protected L-tryptophan (TRP) supplementation (43.4 mg of L-tryptophan kg-1 body weigt [BW]) for 65 days in Hanwoo steers on muscle development related to gene expressions and adipose tissue catabolism and fatty acid transportation in longissimus dorsi muscles. Eight Hanwoo steers (initial BW = 424.6 kg [SD 42.3]; 477 days old [SD 4.8]) were randomly allocated to two groups (n = 4) of control and treatment and were supplied with total mixed ration (TMR). The treatment group was fed with 15 g of rumen-protected TRP (0.1% of TMR as-fed basis equal to 43.4 mg of TRP kg-1 BW) once a day at 0800 h as top-dressed to TMR. Blood samples were collected 3 times, at 0, 5, and 10 weeks of the experiment, for assessment of hematological and biochemical parameters. For gene study, the longissimus dorsi muscle samples (12 to 13 ribs, approximately 2 g) were collected from each individual by biopsy at end of the study (10 weeks). Growth performance parameters including final BW, average daily gain, and gain to feed ratio, were not different (p > 0.05) between the two groups. Hematological parameters including granulocyte, lymphocyte, monocyte, platelet, red blood cell, hematocrit, and white blood cell showed no difference (p > 0.05) between the two groups except for hemoglobin (p = 0.025), which was higher in the treatment than in the control group. Serum biochemical parameters including total protein, albumin, globulin, blood urea nitrogen, creatinine phosphokinase, glucose, nonesterified fatty acids, and triglyceride also showed no differences between the two groups (p > 0.05). Gene expression related to muscle development (Myogenic factor 6 [MYF6], myogenine [MyoG]), adipose tissue catabolism (lipoprotein lipase [LPL]), and fatty acid transformation indicator (fatty acid binding protein 4 [FABP4]) were increased in the treatment group compared to the control group (p < 0.05). Collectively, supplementation of TRP (65 days in this study) promotes muscle development and increases the ability of the animals to catabolize and transport fat in muscles due to an increase in expressions of MYF6, MyoG, FABP4, and LPL gene.

Phenylalanine and valine differentially stimulate milk protein synthetic and energy-mediated pathway in immortalized bovine mammary epithelial cells.

Studies on promoting milk protein yield by supplementation of amino acids have been globally conducted. Nevertheless, there is a lack of knowledge of what pathways affected by individual amino acid in mammary epithelial cells that produce milk in practice. Phenylalanine (PHE) and valine (VAL) are essential amino acids for dairy cows, however, researches on mammary cell levels are still lacking. Thus, the aim of this study was conducted to evaluate the effects of PHE and VAL on milk protein synthesis-related and energy-mediated cellular signaling in vitro using immortalized bovine mammary epithelial (MAC-T) cells. To investigate the effects of PHE and VAL, the following concentrations were added to treatment medium: 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. The addition of PHE or VAL did not adversely affect cell viability compared to control group. The concentrations of cultured medium reached its maximum at 0.9 mM PHE and 0.6 mM VAL (p < 0.05). Therefore, aforementioned 2 treatments were analyzed for proteomics. Glucose transporter 1 and mammalian target of rapamycin mRNA expression levels were up-regulated by PHE (166% and 138%, respectively) (p < 0.05). Meanwhile, sodium-dependent neutral amino acids transporter type 2 (ASCT2) and ß-casein were up-regulated by VAL (173% in ASCT2, 238% in and 218% in ß-casein) (p < 0.05). A total of 134, 142, and 133 proteins were detected in control group, PHE treated group, and VAL treated group, respectively. Among significantly fold-changed proteins, proteins involved in translation initiation or energy metabolism were detected, however, expressed differentially between PHE and VAL. Thus, pathway analysis showed different stimulatory effects on energy metabolism and transcriptional pathways. Collectively, these results showed different stimulatory effects of PHE and VAL on protein synthesis-related and energy-mediated cellular signaling in MAC-T cells.

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.

Effect of Dietary Rumen-Protected L-Tryptophan Supplementation on Growth Performance, Blood Hematological and Biochemical Profiles, and Gene Expression in Korean Native Steers under Cold Environment.

We assessed the growth performance, physiological traits, and gene expressions in steers fed with dietary rumen-protected L-tryptophan (RPT) under a cold environment. Eight Korean native steers were assigned to two dietary groups, no RPT (Control) and RPT (0.1% RPT supplementation on a dry matter basis) for six weeks. Maximum and minimum temperatures throughout the experiment were 6.7 °C and -7.0 °C, respectively. Supplementation of 0.1% RPT to a total mixed ration did not increase body weight but had positive effects of elevating average daily gain (ADG) and reducing the feed conversion ratio (FCR) on days 27 and 48. The metabolic parameter showed a higher glucose level (on day 27) in the 0.1% RPT group compared to the control group. Real-time PCR analysis showed no significant differences in the expression of muscle (MYF6, MyoD, and Desmin) metabolism genes between the two groups, whereas the expression of fat (PPARγ, C/EBPα, and FABP4) metabolism genes was lower in the 0.1% RPT group than in the control group. Thus, we demonstrate that long-term (six weeks) dietary supplementation of 0.1% RPT was beneficial owing to enhanced growth performance by increasing the ADG and glucose level, decreasing FCR, and maintaining homeostasis in immune responses in beef steers in a cold environment.