A Novel Strategy to Identify Endolysins with Lytic Activity against Methicillin-Resistant Staphylococcus aureus.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the dairy industry has become a fundamental concern. Endolysins are bacteriophage-derived peptidoglycan hydrolases that induce the rapid lysis of host bacteria. Herein, we evaluated the lytic activity of endolysin candidates against S. aureus and MRSA. To identify endolysins, we used a bioinformatical strategy with the following steps: (1) retrieval of genetic information, (2) annotation, (3) selection of MRSA, (4) selection of endolysin candidates, and (5) evaluation of protein solubility. We then characterized the endolysin candidates under various conditions. Approximately 67% of S. aureus was detected as MRSA, and 114 putative endolysins were found. These 114 putative endolysins were divided into three groups based on their combinations of conserved domains. Considering protein solubility, we selected putative endolysins 117 and 177. Putative endolysin 117 was the only successfully overexpressed endolysin, and it was renamed LyJH1892. LyJH1892 showed potent lytic activity against both methicillin-susceptible S. aureus and MRSA and showed broad lytic activity against coagulase-negative staphylococci. In conclusion, this study demonstrates a rapid strategy for the development of endolysin against MRSA. This strategy could also be used to combat other antibiotic-resistant bacteria.

Dietary lysophospholipids supplementation inhibited the activity of lipolytic bacteria in forage with high oil diet: an in vitro study.

OBJECTIVE: The objective of this study was to evaluate the effects of lysophospholipids (LPL) supplementation on rumen fermentation, degradability, and microbial diversity in forage with high oil diet in an in vitro system. METHODS: Four experimental treatments were used: i) annual ryegrass (CON), ii) 93% annual ryegrass +7% corn oil on a dry matter (DM) basis (OiL), iii) OiL with a low level (0.08% of dietary DM) of LPL (LLPL), and iv) OiL with a high level (0.16% of dietary DM) of LPL (HLPL). An in vitro fermentation experiment was performed using strained rumen fluid for 48 h incubations. In vitro DM degradability (IVDMD), in vitro neutral detergent fiber degradability, pH, ammonia nitrogen (NH3-N), volatile fatty acid (VFA), and microbial diversity were estimated. RESULTS: There was no significant change in IVDMD, pH, NH3-N, and total VFA production among treatments. The LPL supplementation significantly increased the proportion of butyrate and valerate (Linear effect [Lin], p = 0.004 and <0.001, respectively). The LPL supplementation tended to increase the total bacteria in a linear manner (p = 0.089). There were significant decreases in the relative proportions of cellulolytic (Fibrobacter succinogenes and Ruminococcus albus) and lipolytic (Anaerovibrio lipolytica and Butyrivibrio proteoclasticus) bacteria with increasing levels of LPL supplementation (Lin, p = 0.028, 0.006, 0.003, and 0.003, respectively). CONCLUSION: The LPL supplementation had antimicrobial effects on several cellulolytic and lipolytic bacteria, with no significant difference in nutrient degradability (DM and neutral detergent fiber) and general bacterial counts, suggesting that LPL supplementation might increase the enzymatic activity of rumen bacteria. Therefore, LPL supplementation may be more effective as an antimicrobial agent rather than as an emulsifier in the rumen.

Replacement of corn with rice grains did not alter growth performance and rumen fermentation in growing Hanwoo steers.

OBJECTIVE: This study was realized to evaluate the nutritional value of rice grains as a replacement for corn grains in the diet of growing Hanwoo steers. METHODS: Two experimental diets were prepared: i) Corn total mixed ration (TMR) consisting of 20% corn grains and ii) Rice TMR consisting of 20% rice grains, in a dry matter (DM) basis. These treatments were used for in vitro rumen fermentation and in vivo growth trials. In the rumen fermentation experiment, the in vitro DM digestibility (IVDMD), in vitro crude protein digestibility (IVCPD), in vitro neutral detergent fiber digestibility, pH, ammonia nitrogen, and volatile fatty acids (VFA) were estimated at 48 h, and the gas production was measured at 3, 6, 12, 24, and 48 h. Twenty four growing Hanwoo steers (9 months old; body weight [BW]: 259±13 kg) were randomly divided into two treatment groups and the BW, dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR) were measured. RESULTS: The in vitro experiment showed that the IVDMD, IVCPD, and VFA production of the Rice TMR were higher than those of the Corn TMR (p<0.05). The growth trial showed no differences (p>0.05) in the final BW, ADG, DMI, and FCR between the two TMRs. CONCLUSION: The use of rice grains instead of corn grains did not exhibit any negative effects on the rumen fermentation or growth performance, thereby rice grains with a DM of less than 20% could be used as a starch source in the diet of growing steers.

Changes in the ruminal fermentation and bacterial community structure by a sudden change to a high-concentrate diet in Korean domestic ruminants.

OBJECTIVE: To investigate changes in rumen fermentation characteristics and bacterial community by a sudden change to a high concentrate diet (HC) in Korean domestic ruminants. METHODS: Major Korean domestic ruminants (each of four Hanwoo cows; 545.5±33.6 kg, Holstein cows; 516.3±42.7 kg, and Korean native goats; 19.1±1.4 kg) were used in this experiment. They were housed individually and were fed ad libitum with a same TMR (800 g/kg timothy hay and 200 g/kg concentrate mix) twice daily. After two-week feeding, only the concentrate mix was offered for one week in order to induce rapid rumen acidosis. The rumen fluid was collected from each animals twice (on week 2 and week 3) at 2 h after morning feeding using an oral stomach tube. Each collected rumen fluid was analyzed for pH, volatile fatty acid (VFA), and NH3-N. In addition, differences in microbial community among ruminant species and between normal and an acidosis condition were assessed using two culture-independent 16S polymerase chain reaction (PCR)-based techniques (terminal restriction fragment length polymorphism and quantitative real-time PCR). RESULTS: The HC decreased ruminal pH and altered relative concentrations of ruminal VFA (p<0.01). Total VFA concentration increased in Holstein cows only (p<0.01). Terminal restriction fragment length polymorphism and real-time quantitative PCR analysis using culture-independent 16S PCR-based techniques, revealed rumen bacterial diversity differed by species but not by HC (p<0.01); bacterial diversity was higher in Korean native goats than that in Holstein cows. HC changed the relative populations of rumen bacterial species. Specifically, the abundance of Fibrobacter succinogenes was decreased while Lactobacillus spp. and Megasphaera elsdenii were increased (p<0.01). CONCLUSION: The HC altered the relative populations, but not diversity, of the ruminal bacterial community, which differed by ruminant species.

Effects of reducing inclusion rate of roughages by changing roughage sources and concentrate types on intake, growth, rumen fermentation characteristics, and blood parameters of Hanwoo growing cattle (Bos Taurus coreanae).

OBJECTIVE: Reducing roughage feeding without negatively affecting rumen health is of interest in ruminant nutrition. We investigated the effects of roughage sources and concentrate types on growth performance, ruminal fermentation, and blood metabolite levels in growing cattle. METHODS: In this 24-week trial, 24 Hanwoo cattle (224±24.7 kg) were fed similar nitrous and energy levels of total mixed ration formulated using two kinds of roughage (timothy hay and ryegrass straw) and two types of concentrate mixes (high starch [HS] and high fiber [HF]). The treatments were arranged in a 2×2 factorial, consisting of 32% timothy-68% HS, 24% timothy-76% HF, 24% ryegrass-76% HS, and 17% ryegrass-83% HF. Daily feed intakes were measured. Every four weeks, blood were sampled, and body weight was measured before morning feeding. Every eight weeks, rumen fluid was collected using a stomach tube over five consecutive days. RESULTS: The mean dry matter intake (7.33 kg) and average daily gain (1,033 g) did not differ among treatments. However, significant interactions between roughage source and concentrate type were observed for the rumen and blood parameters (p<0.05). Total volatile fatty acid concentration was highest (p<0.05) in timothy-HF-fed calves. With ryegrass as the roughage source, decreasing the roughage inclusion rate increased the molar proportion of propionate and decreased the acetate-to-propionate ratio; the opposite was observed with timothy as the roughage source. Similarly, the effects of concentrate types on plasma total protein, alanine transaminase, Ca, inorganic P, total cholesterol, triglycerides, and creatinine concentrations differed with roughage source (p<0.05). CONCLUSION: Decreasing the dietary roughage inclusion rate by replacing forage neutral detergent fiber with that from non-roughage fiber source might be a feasible feeding practice in growing cattle. A combination of low-quality roughage with a high fiber concentrate might be economically beneficial.

Essential oil mixture on rumen fermentation and microbial community - an in vitro study.

OBJECTIVE: The objective of this study was to investigate the effects of essential oil mixture (EOM) supplementation on rumen fermentation characteristics and microbial changes in an in vitro. METHODS: Three experimental treatments were used: control (CON, no additive), EOM 0.1 (supplementation of 1 g EOM/kg of substrate), and EOM 0.2 (supplementation of 2 g EOM/kg of substrate). An in vitro fermentation experiment was carried out using strained rumen fluid for 12 and 24 h incubation periods. At each time point, in vitro dry matter digestibility (IVDMD), neutral detergent fiber digestibility (IVNDFD), pH, ammonia nitrogen (NH3-N), and volatile fatty acid (VFA) concentrations, and relative microbial diversity were estimated. RESULTS: After 24 h incubation, treatments involving EOM supplementation led to significantly higher IVDMD (treatments and quadratic effect; p = 0.019 and 0.008) and IVNDFD (linear effect; p = 0.068) than did the CON treatment. The EOM 0.2 supplementation group had the highest NH3-N concentration (treatments; p = 0.032). Both EOM supplementations did not affect total VFA concentration and the proportion of individual VFAs; however, total VFA tended to increase in EOM supplementation groups, after 12 h incubation (linear; p = 0.071). Relative protozoa abundance significantly increased following EOM supplementation (treatments, p<0.001). Selenomonas ruminantium and Ruminococcus albus (treatments; p<0.001 and p = 0.005), abundance was higher in the EOM 0.1 treatment group than in CON. The abundance of Butyrivibrio fibrisolvens, fungi and Ruminococcus flavefaciens (treatments; p< 0.001, p<0.001, and p = 0.005) was higher following EOM 0.2 treatment. CONCLUSION: The addition of newly developed EOM increased IVDMD, IVNDFD, and tended to increase total VFA indicating that it may be used as a feed additive to improve rumen fermentation by modulating rumen microbial communities. Further studies would be required to investigate the detailed metabolic mechanism underlying the effects of EOM supplementation.

The co-injection of antioxidants with foot-and-mouth disease vaccination altered growth performance and blood parameters of finishing Holstein steers.

OBJECTIVE: This study was conducted to evaluate whether the co-injection of antioxidants together with foot-and-mouth disease (FMD) vaccination has the potential to attenuate the negative effects caused by vaccination in Holstein finishing steers. METHODS: A total of 36 finishing Holstein steers (body weight [BW]: 608±45.6 kg, 17 months old) were randomly allocated to one of three treatments: i) control (CON, only FMD vaccination without any co-injection), ii) co-injection of commercial non-steroidal anti-inflammatory drugs (NSAID) with FMD vaccination at a ratio of 10:1 (NSAID vol/FMD vaccine vol) as a positive control (PCON), iii) co-injection of commercial mixture of vitamin E and selenium with FMD vaccination (VITESEL) (1 mL of FMD vaccine+1 mL of antioxidants per 90 kg of BW). Changes in growth performance and blood parameters because of treatments were determined. RESULTS: No significant difference in BW, average daily gain, and dry matter intake of the steers was observed among the treatments. The FMD vaccination significantly increased white blood cells (WBC), neutrophils, platelets, and mean platelet volume (p<0.01) in blood analysis. The count of lymphocyte tended to increase after vaccination (p = 0.08). In blood analysis, steers in VITESEL tended to have higher numbers of WBC, neutrophils, and platelets compared to that of other treatments (p = 0.09, 0.06, and 0.09, respectively). Eosinophils in VITESEL were higher than those in PCON (p<0.01). Among blood metabolites, blood urea nitrogen and aspartate transaminase were significantly increased, but cholesterol, alanine transferase, inorganic phosphorus, Mg, and albumin were decreased after FMD vaccination (p<0.01). CONCLUSION: The use of antioxidants in FMD vaccination did not attenuate growth disturbance because of FMD vaccination. The metabolic changes induced by vaccination were not controlled by the administration of antioxidants. The protective function of antioxidants was effective mainly on the cell counts of leukocytes.

The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse.

OBJECTIVE: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme 1α (IRE1α)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates IRE1α signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. METHODS: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. RESULTS: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. CONCLUSION: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

Molecular cloning, purification, expression, and characterization of ß-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen.

OBJECTIVE: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. METHODS: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. RESULTS: The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 µmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s-1) and 6.88 (mL/mg/s), respectively. CONCLUSION: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs.

OBJECTIVE: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. METHODS: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. RESULTS: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. CONCLUSION: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Development of a novel endolysin, PanLys.1, for the specific inhibition of Peptostreptococcus anaerobius.

OBJECTIVE: The objective of this study was to develop a novel endolysin (PanLys.1) for the specific killing of the ruminal hyper-ammonia-producing bacterium Peptostreptococcus anaerobius (P. anaerobius). METHODS: Whole genome sequences of P. anaerobius strains and related bacteriophages were collected from the National Center for Biotechnology Information database, and the candidate gene for PanLys.1 was isolated based on amino acid sequences and conserved domain database (CDD) analysis. The gene was overexpressed using a pET system in Escherichia coli BL21 (DE3). The lytic activity of PanLys.1 was evaluated under various conditions (dosage, pH, temperature, NaCl, and metal ions) to determine the optimal lytic activity conditions. Finally, the killing activity of PanLys.1 against P. anaerobius was confirmed using an in vitro rumen fermentation system. RESULTS: CDD analysis showed that PanLys.1 has a modular design with a catalytic domain, amidase-2, at the N-terminal, and a cell wall binding domain, from the CW-7 superfamily, at the C-terminal. The lytic activity of PanLys.1 against P. anaerobius was the highest at pH 8.0 (p<0.05) and was maintained at 37°C to 45°C, and 0 to 250 mM NaCl. The activity of PanLys.1 significantly decreased (p<0.05) after Mn2+ or Zn2+ treatment. The relative abundance of P. anaerobius did not decrease after administration PanLys.1 under in vitro rumen conditions. CONCLUSION: The application of PanLys.1 to modulate P. anaerobius in the rumen might not be feasible because its lytic activity was not observed in in vitro rumen system.

Negative association between high temperature-humidity index and milk performance and quality in Korean dairy system: big data analysis.

The aim of this study was to investigate the effects of heat stress on milk traits in South Korea using comprehensive data (dairy production and climate). The dataset for this study comprised 1,498,232 test-day records for milk yield, fat- and protein-corrected milk, fat yield, protein yield, milk urea nitrogen (MUN), and somatic cell score (SCS) from 215,276 Holstein cows (primiparous: n = 122,087; multiparous: n = 93,189) in 2,419 South Korean dairy herds. Data were collected from July 2017 to April 2020 through the Dairy Cattle Improvement Program, and merged with meteorological data from 600 automatic weather stations through the Korea Meteorological Administration. The segmented regression model was used to estimate the effects of the temperature-humidity index (THI) on milk traits and elucidate the break point (BP) of the THI. To acquire the least-squares mean of milk traits, the generalized linear model was applied using fixed effects (region, calving year, calving month, parity, days in milk, and THI). For all parameters, the BP of THI was observed; in particular, milk production parameters dramatically decreased after a specific BP of THI (p < 0.05). In contrast, MUN and SCS drastically increased when THI exceeded BP in all cows (p < 0.05) and primiparous cows (p < 0.05), respectively. Dairy cows in South Korea exhibited negative effects on milk traits (decrease in milk performance, increase in MUN, and SCS) when the THI exceeded 70; therefore, detailed feeding management is required to prevent heat stress in dairy cows.

Effects of the Use of Rice Grain on Growth Performances, Blood Metabolites, Rumen Fermentation, and Rumen Microbial Community in Fattening Hanwoo Steers.

This study aimed to assess the influence of rice grain in the total mixed ration (TMR) on the growth performance, blood metabolites, rumen fermentation, and rumen microbial community of fattening Hanwoo steers. Two experimental diets were prepared: (i) a TMR containing 33% dry matter (DM) corn grains (Corn TMR) and (ii) a TMR containing 33% DM rice grains (Rice TMR). Twenty-two Hanwoo steers (body weight [BW], 498 ± 32 kg; months, 17 ± 0.5) were distributed into two treatment groups in a completely randomized block design according to BW. The Rice TMR group had a higher final BW and DM intake (DMI) compared to those in the Corn TMR group (p < 0.01). However, no difference was observed in the average daily gain (ADG) and feed conversion ratio (FCR) between the two treatments. For the rumen fermentation parameters, the molar portion of butyrate in the Rice TMR was higher than in the Corn TMR (p < 0.01). Streptococcus bovis tended to be higher in the Rice TMR (p = 0.09). The results of this study suggest that using rice grain as the primary starch source in TMRs may be an alternative option for fattening Hanwoo steers.

Obese dogs exhibit different fecal microbiome and specific microbial networks compared with normal weight dogs.

Canine obesity is a major health concern that predisposes dogs to various disorders. The fecal microbiome has been attracting attention because of their impact on energy efficiency and metabolic disorders of host. However, little is known about specific microbial interactions, and how these may be affected by obesity in dogs. The objective of this study was to investigate the differences in fecal microbiome and specific microbial networks between obese and normal dogs. A total of 20 beagle dogs (males = 12, body weight [BW]: 10.5 ± 1.08 kg; females = 8, BW: 11.3 ± 1.71 kg; all 2-year-old) were fed to meet the maintenance energy requirements for 18 weeks. Then, 12 beagle dogs were selected based on body condition score (BCS) and divided into two groups: high BCS group (HBCS; BCS range: 7-9, males = 4, females = 2) and normal BCS group (NBCS; BCS range: 4-6, males = 4, females = 2). In the final week of the experiment, fecal samples were collected directly from the rectum, before breakfast, for analyzing the fecal microbiome using 16S rRNA gene amplicon sequencing. The HBCS group had a significantly higher final BW than the NBCS group (P < 0.01). The relative abundances of Faecalibacterium, Phascolarctobacterium, Megamonas, Bacteroides, Mucispirillum, and an unclassified genus within Ruminococcaceae were significantly higher in the HBCS group than those in the NBCS group (P < 0.05). Furthermore, some Kyoto Encyclopedia of Genes and Genomes (KEGG) modules related to amino acid biosynthesis and B vitamins biosynthesis were enriched in the HBCS group (P < 0.10), whereas those related to carbohydrate metabolism were enriched in the NBCS group (P < 0.10). Microbial network analysis revealed distinct co-occurrence and mutually exclusive interactions between the HBCS and NBCS groups. In conclusion, several genera related to short-chain fatty acid production were enriched in the HBCS group. The enriched KEGG modules in the HBCS group enhanced energy efficiency through cross-feeding between auxotrophs and prototrophs. However, further studies are needed to investigate how specific networks can be interpreted in the context of fermentation characteristics in the lower gut and obesity in dogs.

Effects of Dietary Fat Level of Concentrate Mix on Growth Performance, Rumen Characteristics, Digestibility, Blood Metabolites, and Methane Emission in Growing Hanwoo Steers.

This study investigated the effect of different dietary fat levels in concentrate mixes on the growth performance, rumen characteristics, digestibility, blood metabolites, and methane emissions in growing Hanwoo steers. Thirty steers (386 ± 24.6 kg of body weight [BW]; 12 months old), blocked by BW, were randomly assigned to three dietary treatments with varying fat concentrations in concentrate mix (48, 74, and 99 g of ether extract per kg dry matte [DM]). The fat intake of the low-fat treatment represented 4.15% of the total dry matter intake (DMI), while the medium- and high-fat treatments accounted for 5.77% and 7.23% of total DMI, respectively. Concentrate mix DMI decreased with increasing fat level (p < 0.01). The growth rate and digestibility did not significantly differ based on the fat level (p > 0.05). As the fat level increased, propionate in the total ruminal volatile fatty acids increased, and butyrate and acetate-to-propionate decreased (p < 0.01). Cholesterol in blood serum increased significantly with increasing dietary fat levels (p < 0.01). Methane emissions exhibited a linear decrease with increasing fat level (p < 0.05). In conclusion, elevating fat content in the concentrates up to 100 g/kg DM reduced methane emissions without compromising the growth performance of growing Hanwoo steers.

Oral administration of Pinus koraiensis cone essential oil reduces rumen methane emission by altering the rumen microbial composition and functions in Korean native goat (Capra hircus coreanae).

This study aimed to investigate Pinus koraiensis cone essential oil (PEO) as a methane (CH4) inhibitor and determine its impact on the taxonomic and functional characteristics of the rumen microbiota in goats. A total of 10 growing Korean native goats (Capra hircus coreanae, 29.9 ± 1.58 kg, male) were assigned to different dietary treatments: control (CON; basal diet without additive) and PEO (basal diet +1 g/d of PEO) by a 2 × 2 crossover design. Methane measurements were conducted every 4 consecutive days for 17-20 days using a laser CH4 detector. Samples of rumen fluid and feces were collected during each experimental period to evaluate the biological effects and dry matter (DM) digestibility after PEO oral administration. The rumen microbiota was analyzed via 16S rRNA gene amplicon sequencing. The PEO oral administration resulted in reduced CH4 emission (eructation CH4/body weight0.75, p = 0.079) without affecting DM intake; however, it lowered the total volatile fatty acids (p = 0.041), molar proportion of propionate (p = 0.075), and ammonia nitrogen (p = 0.087) in the rumen. Blood metabolites (i.e., albumin, alanine transaminase/serum glutamic pyruvate transaminase, creatinine, and triglyceride) were significantly affected (p < 0.05) by PEO oral administration. The absolute fungal abundance (p = 0.009) was reduced by PEO oral administration, whereas ciliate protozoa, total bacteria, and methanogen abundance were not affected. The composition of rumen prokaryotic microbiota was altered by PEO oral administration with lower evenness (p = 0.054) observed for the PEO group than the CON group. Moreover, PICRUSt2 analysis revealed that the metabolic pathways of prokaryotic bacteria, such as pyruvate metabolism, were enriched in the PEO group. We also identified the Rikenellaceae RC9 gut group as the taxa potentially contributing to the enriched KEGG modules for histidine biosynthesis and pyruvate oxidation in the rumen of the PEO group using the FishTaco analysis. The entire co-occurrence networks showed that more nodes and edges were detected in the PEO group. Overall, these findings provide an understanding of how PEO oral administration affects CH4 emission and rumen prokaryotic microbiota composition and function. This study may help develop potential manipulation strategies to find new essential oils to mitigate enteric CH4 emissions from ruminants.

Effects of Red Ginseng Byproducts on Rumen Fermentation, Growth Performance, Blood Metabolites, and mRNA Expression of Heat Shock Proteins in Heat-Stressed Fattening Hanwoo Steers.

The objective of this study was to evaluate the effects of dietary supplementation with red ginseng byproduct (RGB) on rumen fermentation, growth performance, blood metabolites, and mRNA expression of heat shock proteins (HSP) in fattening Hanwoo steers under heat stress. Two experimental total mixed rations (TMR) were prepared: (1) a TMR meeting the requirement of fattening beef having an average daily gain (ADG) 0.8 kg/day (CON) and (2) a TMR that included 2% RGB on a dry matter (DM) basis (GINSENG). In vitro rumen fermentation and in vivo growth experiments were conducted using two experimental diets. A total of 22 Hanwoo steers were distributed to two treatments (CON vs. GINSENG) in a completely randomized block design according to body weight (BW). The experiment was conducted during the summer season for five weeks. The final BW, ADG, DM intake, and feed conversion ratio did not differ between treatments in the growth trial. In the mRNA expression results, only HSP 90 showed an increasing tendency in the GINSENG group. The use of 2%DM RGB did not improve the growth performance or alleviate heat stress in fattening Hanwoo steers during the summer season.

Red seaweed extracts reduce methane production by altering rumen fermentation and microbial composition in vitro.

A series of in vitro batch culture incubations were carried out to investigate changes in rumen fermentation characteristics, methane (CH4) production, and microbial composition in response to supplementation with five different red seaweed species (Amphiroa anceps, AANC; Asparagopsis taxiformis, ATAX; Chondracanthus tenellus, CTEN; Grateloupia elliptica, GELL; and Gracilaria parvispora, GPAR). Prior to the incubations, the total flavonoid and polyphenol content of the red seaweed extracts was quantified. The incubated substrate consisted of timothy hay and corn grain [60:40 dry matter (DM) basis]. Treatments were substrate mixtures without seaweed extract (CON) or substrate mixtures supplemented with 0.25 mg/mL of red seaweed extract. Samples were incubated for 6, 12, 24, 36, and 48 h. Each sample was incubated in triplicates in three separate runs. In vitro DM degradability, fermentation parameters (i.e., pH, volatile fatty acids, and ammonia nitrogen), total gas production, and CH4 production were analyzed for all time points. Microbial composition was analyzed using 16S rRNA amplicon sequencing after 24 h of incubation. The highest CH4 reduction (mL/g DM, mL/g digested DM, and % of total gas production) was observed in ATAX (51.3, 50.1, and 51.5%, respectively, compared to CON; P < 0.001) after 12 h of incubation. The other red seaweed extracts reduced the CH4 production (mL/g DM; P < 0.001) in the range of 4.6-35.0% compared to CON after 24 h of incubation. After 24 h of incubation, supplementation with red seaweed extracts tended to increase the molar proportion of propionate (P = 0.057) and decreased the acetate to propionate ratio (P = 0.033) compared to the CON. Abundances of the genus Methanobrevibacter and total methanogens were reduced (P = 0.050 and P = 0.016) by red seaweed extract supplementation. The linear discriminant analysis effect size (P < 0.05, LDA ≥ 2.0) showed that UG Succinivibrionaceae, Anaeroplasma, and UG Ruminococcaceae, which are associated with higher propionate production, starch degradation, and amylase activity were relatively more abundant in red seaweed extracts than in the CON. Our results suggest that supplementation with red seaweed extracts altered the microbiota, leading to the acceleration of propionate production and reduction in CH4 production.

Specific inhibition of Streptococcus bovis by endolysin LyJH307 supplementation shifts the rumen microbiota and metabolic pathways related to carbohydrate metabolism.

BACKGROUND: Endolysins, the bacteriophage-originated peptidoglycan hydrolases, are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance. The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S. bovis and to explore changes in rumen fermentation and microbiota in an in vitro system. Two treatments were used: 1) control, corn grain without LyJH307; and 2) LyJH307, corn grain with LyJH307 (4 U/mL). An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers (450 ± 30 kg) and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time. In vitro dry matter digestibility, pH, volatile fatty acids, and lactate concentration were estimated at 12 h, and the gas production was measured at 6, 9, and 12 h. The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing. RESULTS: LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S. bovis (approximately 70% compared to control, P = 0.0289) and increased ruminal pH (P = 0.0335) at the 12 h incubation. The acetate proportion (P = 0.0362) was significantly increased after LyJH307 addition, whereas propionate (P = 0.0379) was decreased. LyJH307 supplementation increased D-lactate (P = 0.0340) without any change in L-lactate concentration (P > 0.10). There were no significant differences in Shannon's index, Simpson's index, Chao1 estimates, and evenness (P > 0.10). Based on Bray-Curtis dissimilarity matrices, the LyJH307 affected the overall shift in microbiota (P = 0.097). LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium, WCHB1-41, unclassified genus Selenomonadaceae, Paraprevotella, vadinBE97, Ruminococcus gauvreauii group, Lactobacillus, Anaerorhabdus furcosa group, Victivallaceae, Desulfuromonadaceae, and Sediminispirochaeta. The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism. CONCLUSIONS: LyJH307 caused a reduction of S. bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism. This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.

High-level dietary crude protein decreased backfat thickness and increased carcass yield score in finishing Hanwoo beef cattle (Bos taurus coreanae).

Recently, a high level of dietary crude protein (CP) has become of interest as a possible practice to improve the carcass quality of beef cattle, and its level has been increasing in the field. However, there is little scientific evidence that supports this. This study was conducted to test whether a high dietary CP level would improve growth performance, body metabolism, and carcass traits in Hanwoo beef cattle. A total of 32 Hanwoo finishing beef cattle (18 multiparous cows, six heifers, and eight steers) participated in a 12-weeks feeding trial. Two kinds of total mixed rations were prepared to contain two different CP; 156 g/kg for the control (CON) and 173 g/kg of CP for the treatment (HCP), while maintaining a similar level of metabolizable energy. The experiment was ended when more than half of the steers reached the target body weight (730 kg). Blood was collected at the end of the experiment. After harvesting, the carcass trait was evaluated at the slaughterhouse according to Korean standards. The carcass yield score and grade were also calculated based on revised criteria. Overall, dry matter intake, average daily gain, blood metabolites concentration, and the carcass traits, except for backfat thickness and the yield score, did not differ between the treatments. The HCP had lower backfat thickness than those of CON. There was no difference in the carcass yield grade, but the yield score was higher in the HCP treatment. According to the newly revised carcass grading criteria, both yield score and grade were higher in HCP than in CON. Increasing CP supply decreased the carcass's backfat thickness without altering growth performance and body metabolism, resulting in improved yield score and grade. Therefore, feeding a high CP diet may be beneficial in the farm income, although it may also increase feed cost and nitrogen excretion to the environment.