Effect of zinc source (zinc sulfate or zinc hydroxychloride) on relative abundance of fecal Treponema spp. in lactating dairy cows.

Previous research revealed a potential effect of dietary trace mineral source on both ruminal and fecal microbiota. However, the effect of Zn source, specifically, has not previously been considered. Based on reported postruminal solubility, we hypothesized that Zn hydroxychloride would decrease Treponema spp. fecal excretion relative to cows fed Zn sulfate. To test this hypothesis, lactating Holstein cows (n = 24; 685 ± 9 kg of body weight; 159 ± 8 d in milk; parity 3 ± 0.2) were randomly assigned to 1 of 2 dietary treatments: control (75 mg/kg Zn from ZnSO4) or Zn hydroxychloride (HYD; 75 mg/kg IntelliBond Z; Micronutrients USA LLC). Single fecal grab samples were collected on d 1 before dietary treatments and on d 27 after dietary treatments were applied. Fecal microbial DNA was extracted and sequenced to establish taxonomy using a universal primer for the 16S rRNA gene. Supplementation of HYD decreased the relative abundance of Treponema 2 by 3-fold (14.7% vs. 4.9%). Poor sequencing resolution at the species level limited inference of Treponema spp. toward management or gut health implications of HYD supplementation. However, the inclusion of pathogenic species among Treponema spp. indicates a potential implication of HYD feeding to reduce environmental exposure of the dairy cow to Treponema spp.

Effect of chromium on bioenergetics and leukocyte dynamics following immunoactivation in lactating Holstein cows.

Activated immune cells are insulin sensitive and utilize copious amounts of glucose. Because chromium (Cr) increases insulin sensitivity and may be immunomodulatory, our objective was to evaluate the effect of supplemental Cr (KemTrace Cr propionate, 20 g/d; Kemin Industries Inc., Des Moines, IA) on immune system glucose utilization and immune system dynamics following an intravenous endotoxin challenge in lactating Holstein cows. Twenty cows (320 ± 18 d in milk) were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF and Cr supplemented (PF-Cr; 5 mL of saline; n = 5), (3) lipopolysaccharide (LPS)-euglycemic clamp and control supplemented (LPS-CON; 0.375 µg/kg of body weight LPS; n = 5), and (4) LPS-euglycemic clamp and Cr supplemented (LPS-Cr; 0.375 µg/kg of body weight LPS; n = 5). The experiment was conducted serially in 3 periods (P). During P1 (3 d), cows received their respective dietary treatments and baseline values were obtained. At the initiation of P2 (2 d), either a 12-h LPS-euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During P3 (3 d), cows consumed feed ad libitum and continued to receive their respective dietary treatment. During P2, LPS administration decreased dry matter intake (DMI; 40%) similarly among diets, and by experimental design the pattern and magnitude of reduced DMI were similar in the PF cohorts. During P3, LPS-Cr cows tended to have decreased DMI (6%) relative to LPS-CON cows. Relative to controls, milk yield from LPS-challenged cows decreased (58%) during P2 and LPS-Cr cows produced less (16%) milk than LPS-CON cows. During P3, milk yield progressively increased similarly in LPS-administered cows, but overall milk yield remained decreased (24%) compared with PF controls. There were no dietary treatment differences in milk yield during P3. Circulating insulin increased 9- and 15-fold in LPS-administered cows at 6 and 12 h postbolus, respectively, compared with PF controls. Compared with LPS-CON cows, circulating insulin in LPS-Cr cows was decreased (48%) at 6 h postbolus. Relative to PF cows, circulating LPS binding protein and serum amyloid A from LPS-administered cows increased 2- and 5-fold, respectively. Compared with PF cows, blood neutrophil counts in LPS-infused cows initially decreased, then gradually increased 163%. Between 18 and 48 h postbolus, the number of neutrophils was increased (12%) in LPS-Cr versus LPS-CON cows. The 12-h total glucose deficit was 220 and 1,777 g for the PF and LPS treatments, respectively, but glucose utilization following immune activation was not influenced by Cr. In summary, supplemental Cr reduced the insulin response and increased circulating neutrophils following an LPS challenge but did not appear to alter the immune system's glucose requirement following acute and intense activation.

Meta-analysis to predict the effects of metabolizable amino acids on dairy cattle performance.

Meta-analytic methods were used to determine statistical relationships between metabolizable AA supplies and milk protein yield, milk protein percentage, and milk yield in lactating dairy cows. Sixty-three research publications (258 treatment means) were identified through a search of published literature using 3 search engines and met the criteria for inclusion in this meta-analysis. The Cornell Net Carbohydrate and Protein System (CNCPS) version 6.5 was used to determine dietary nutrient parameters including metabolizable AA. Two approaches were used to analyze the data. First, mixed models were fitted to determine whether explanatory variables predicted responses. Each mixed model contained a global intercept, a random intercept for each experiment, and data were weighted by the inverse of the SEM squared. The second analysis approach used classical effect size meta-analytical evaluation of responses to treatment weighted by the inverse of the treatment variance and with a random effect of treatment nested within experiment. Regardless of the analytical approach, CNCPS-predicted metabolizable Met (g/d) was associated with milk protein percentage and yield. Milk yield was positively associated with CNCPS-predicted metabolizable His, Leu, Trp, Thr, and nonessential AA (g/d). Milk true protein yield was also associated with CNCPS-predicted metabolizable Leu (g/d). Predicted metabolizable Lysine (g/d) did not increase responses in production outcomes. However, mean metabolizable Lys supply was less than typically recommended and the change with treatment was minimal (157 vs. 162 g; 6.36 vs. 6.38% metabolizable protein). Experiments based solely on Lys or Met interventions were excluded from the study database. It is possible that the inclusion of these experiments may have provided additional insight into the effect of these AA on responses. This meta-analysis supports other research indicating a positive effect of Met and His as co-limiting AA in dairy cows and suggests Leu, Trp, and Thr be given greater consideration in future research.

Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows.

Multiparous Holstein cows (n=61) were used to determine the effects of chromium propionate (Cr-Pro) supplementation during the periparturient period and early lactation on metabolism, performance, and the incidence of cytological endometritis (CE). After a 1-wk preliminary period, cows were assigned randomly to 1 of 2 treatments from 21 d before expected calving through 63 d postpartum: (1) control (n=31) and (2) Cr-Pro (n=30) administered by daily topdress at a rate of 8 mg/d of Cr. A tendency was detected for increased dry matter intake (DMI) during the prepartum period for cows fed Cr-Pro. Moreover, cows fed Cr-Pro tended to have lower plasma concentrations of nonesterified fatty acids during the prepartum period. However, effects of Cr-Pro supplementation on postpartum DMI and milk yield were not significant. Cows fed Cr-Pro tended to have higher urea N concentrations in milk. An interaction of treatment and day existed during the postpartum period, such that cows fed Cr-Pro had lower plasma glucose concentrations within the first day postpartum compared with controls. Plasma haptoglobin concentration was not affected by treatment during the postpartum period. Blood neutrophil glycogen concentrations were not affected by treatment when sampled at either 7 d postpartum or on one day between 40 and 60 d (48 d ± 0.44 standard error) postpartum. Evaluation of endometrial cytology by low volume lavage at 7 d postpartum (first lavage) and on one day between 40 and 60 d (second lavage) postpartum revealed that cows fed Cr-Pro tended to have a higher percentage of neutrophils at first lavage and decreased incidence of CE as assessed at second lavage. In conclusion, supplementation with Cr-Pro resulted in trends for increased DMI and lower plasma nonesterified fatty acids prepartum. Postpartum production and energy metabolism were not affected by treatment; however, Cr-Pro supplementation tended to affect the postpartum influx of neutrophils into the uterus and decreased the incidence of CE, suggesting positive effects of Cr-Pro supplementation on uterine health.

Effects of supplemental chromium propionate and rumen-protected amino acids on nutrient metabolism, neutrophil activation, and adipocyte size in dairy cows during peak lactation.

The objective of this study was to evaluate effects of chromium propionate (CrPr), rumen-protected lysine and methionine (RPLM), or both on metabolism, neutrophil function, and adipocyte size in lactating dairy cows (38 ± 15 d in milk). Forty-eight individually fed Holstein cows (21 primiparous, 27 multiparous) were stratified by calving date in 12 blocks and randomly assigned to 1 of 4 treatments within block. Treatments were control, CrPr (8 mg/d of Cr, KemTRACE brand chromium propionate 0.04%, Kemin Industries Inc., Des Moines, IA), RPLM (10 g/d lysine and 5 g/d methionine intestinally available, from LysiPEARL and MetiPEARL, Kemin Industries Inc.), or CrPr plus RPLM. Treatments were fed for 35 d; blood plasma samples were collected ond 21 and 35 of treatment, and blood neutrophils were isolated from 24 cows for analysis of tumor necrosis factor α (TNFα) and interleukin 1ß (IL-1ß) transcript abundance in the basal state and after 12h of lipopolysaccharide (LPS) activation. Tailhead subcutaneous adipose tissue samples were collected ond 35 for measurement of adipocyte size. Plasma glucose, nonesterified fatty acids, and glucagon concentrations were unaffected by treatments, whereas plasma insulin concentration was increased by RPLM. Basal TNFα transcript abundance in neutrophils was not affected by treatment, but basal IL-1ß transcript abundance was decreased by RPLM and tended to be increased by CrPr. After LPS activation, CrPr increased neutrophil TNFα transcript abundance. In addition, RPLM×parity interactions were detected for both TNFα and IL-1ß abundance after LPS activation, reflecting enhanced responses in primiparous cows and attenuated responses in multiparous cows supplemented with RPLM. Adipocyte size was not affected by treatment. Supplemental CrPr and RPLM had minimal effects on metabolism when fed for 35 d near peak lactation but may modulate innate immune function in lactating dairy cows.

Effects of supplemental chromium propionate and rumen-protected amino acids on productivity, diet digestibility, and energy balance of peak-lactation dairy cattle.

Chromium (Cr) feeding in early lactation increased milk production in some studies, but responses to dietary Cr during peak lactation have not been evaluated. Furthermore, interactions of essential amino acids (AA) and Cr have not been explored. Our objective was to evaluate responses to CrPr (KemTRACE chromium propionate 0.04%, Kemin Industries Inc., Des Moines, IA) and rumen-protected Lys (LysiPEARL, Kemin Industries Inc.) and Met (MetiPEARL, Kemin Industries Inc.) and their interaction in peak-lactation cows. Forty-eight individually fed Holstein cows (21 primiparous, 27 multiparous, 38 ± 15 d in milk) were stratified by calving date in 12 blocks and randomly assigned to 1 of 4 treatments within block. Treatments were control, CrPr (8 mg/d of Cr), RPLM (10 g/d of Lys and 5 g/d of Met, intestinally available), or CrPr plus RPLM. Treatments were premixed with ground corn and top-dressed at 200 g/d for 35 d. Diets consisted of corn silage, alfalfa hay, and concentrates, providing approximately 17% crude protein, 31% neutral detergent fiber, and 40% nonfiber carbohydrates. Dry matter intake (DMI) significantly increased with the inclusion of CrPr (22.2 vs. 20.8 ± 0.67 kg/d), and energy-corrected milk (ECM) yield tended to increase. In addition, CrPr increased milk protein yield and tended to increase DMI in primiparous cows but not in multiparous cows. A CrPr×week interaction was detected for milk lactose content, which was increased by CrPr during wk 1 only (4.99 vs. 4.88 ± 0.036%). As a proportion of plasma AA, lysine increased and methionine tended to increase in response to RPLM, but the inclusion of RPLM decreased N efficiency (milk protein N:N intake). Digestible energy intake, gross energy digestibility, and energy balance were not affected by treatments. We observed no treatment effects on feed efficiency or changes in body weight or body condition score. In summary, feeding CrPr increased DMI and tended to increase ECM in cows fed for 5 wk near peak lactation, with primiparous cows showing greater responses in DMI and milk protein yield than multiparous cows.

The digestive fate of Escherichia coli glutamate dehydrogenase deoxyribonucleic acid from transgenic corn in diets fed to weanling pigs.

Corn containing genetically engineered plasmid DNA encoding an Escherichia coli glutamate dehydrogenase (gdhA) was fed to 19-d-old weanling swine to trace the digestive fate of the transgenic DNA. Eight pens of 8 pigs were fed a commercial (nongdhA) starter for 2 wk. One pig was randomly selected from each pen for 0-h control samples. The remaining 56 pigs were transitioned onto a corn-soybean meal diet and fed a diet containing 58% gdhA corn for approximately 1 wk; immediately thereafter, liver, 10th rib muscle, white blood cells, and plasma from the hepatic portal vein and ingesta from the stomach, distal ileum, and large intestine were collected. The DNA was extracted and the concentration determined via spectrophotometry. Polymerase chain reaction and gel electrophoresis were performed with primers designed to amplify 490 bp that included the plasmid's ligation site between the maize ubiquitin and the gdhA genes. The gdhA corn-derived DNA and diet served as positive assay controls, and conventional corn DNA and distilled water acted as negative assay controls. Detection limits were 0.99 fg of target DNA confounded with 500 ng of conventional corn DNA per each 20 &L reaction. Transgenic DNA was detected in 71.43% of the stomach and 1.79% of the ileal ingesta samples from treatment animals but was not detected in the large intestine, white blood cells, plasma, liver, or muscle samples. Transgenic DNA was not detected in any sample from 0-h control animals. Stomach and ileal ingesta samples were further analyzed using real-time PCR. With an estimated limit of detection of 1.049 ag/microL, 89.29% of the stomach ingesta samples were positive (average 1.56 fg target DNA). The proportion of transgenic DNA to total DNA differed between diet and stomach ingesta samples (P < 0.001). Despite the greater sensitivity of real-time PCR, target DNA was detected in only 1.79% of ileal ingesta. These data suggest that the gdhA transgene began degradation in the stomach and was nondetectable in the large intestine.

Fecal prevalence and diversity of Salmonella species in lactating dairy cattle in four states.

Salmonella is one of the most serious foodborne pathogenic bacteria in the United States, causing an estimated 1.3 million human illnesses each year. Dairy cows can be reservoirs of foodborne pathogenic bacteria, including Salmonella spp.; it is estimated that from 27 to 31% of dairy herds across the United States are colonized by Salmonella. The present study was designed to examine the occurrence of Salmonella spp. on dairies and to examine the serotypic diversity of Salmonella isolates on sampled dairies from across the United States. Fecal samples (n = 60 per dairy) were collected from 4 dairies in each of 4 states for a total of 960 fecal samples representing a total population of 13,200 dairy cattle. In the present study, 93 of 960 samples (9.96%) collected were culture-positive for Salmonella enterica. At least one Salmonella fecal-shedding cow was found in 9 of the 16 herds (56%) and the within-herd prevalence varied in our study from 0% in 7 herds to a maximum of 37% in 2 herds, with a mean prevalence among Salmonella-positive herds of 17%. Seventeen different serotypes were isolated, representing 7 different Salmonella serogroups. There were 2 or more different serogroups and serotypes present on 7 of the 9 Salmonella-positive farms. Serotypes Montevideo and Muenster were the most frequent and widespread. From our data, it appears that subclinical colonization with Salmonella enterica is relatively common on dairy farms and is represented by diverse serotypes on US dairy farms.

Effects of substrate, passage rate, and pH in continuous culture on flows of conjugated linoleic acid and trans C18:1.

A dual-flow continuous culture system consisting of 4 fermenters was used in a 4 x4 Latin square design. The objective of the research was to evaluate the effects of solid dilution rate (SDR), pH, and concentration of linoleic acid (LA) in the feed mixture on the production of conjugated linoleic acid (CLA) and trans-C18:1. The 4 treatments were 1) control = pH 6.5, 1% LA, 4%/h SDR; 2) high solid dilution rate (HSDR) = pH 6.5, 1% LA, 8%/h SDR; 3) high linoleic acid (HLA) = pH 6.5, 3% LA, 4%/h SDR; and 4) low pH (LPH) = pH 5.8, 1% LA, 4%/h SDR. Inoculum was collected 6 h after feeding from a cow fed 40% alfalfa hay and 60% grain. Liquid dilution rate was held at 0.12/h. All treatments except HLA contained 2% tallow. The LA was dissolved in buffer and continuously infused into the fermenters. The CLA flows were 16.5, 20.4, 23.2, and 25.2 mg/d for control, HSDR, HLA, and LPH, respectively. Compared with control, LPH increased flows of CLA, cis-C18:1, and C18:2, and decreased flow of C18:0. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibilities were not affected by pH. The HSDR tended to increase CLA flow compared to control, possibly because a shorter solid retention time led to incomplete biohydrogenation (BH). The NDF and ADF digestibilities and bacterial numbers were reduced by HSDR. With more LA available as a substrate for CLA, HLA resulted in a higher flow of CLA than control. The HLA resulted in the highest acid detergent fiber and fatty acid digestibilities, bacterial numbers, and BH. Increasing solids passage rate, reducing pH, and increasing dietary LA appears to increase in vitro CLA production.

Nutritional value of a corn containing a glutamate dehydrogenase gene for growing pigs.

Eight female PIC Line 42 pigs (initial BW = 47.5 +/- 1.8 kg) were used in a two-period switchback design (n = 4 per treatment per period) to evaluate the nutritional difference between a genetically modified corn and a similar nontransgenic corn. The genetically altered corn (gdhA+) contained a glutamate dehydrogenase gene isolated from Escherichia coli. The non-transgenic corn was the same variety lacking the transgenic cassette, grown at the same two locations. Pigs were surgically fitted with steered ileocecal valve cannulas for collection of ileal digesta. Diets were made up of primarily one of the two corn sources. Dietary AA profiles were adjusted using crystalline AA to match Illinois Ideal Protein Ratios. Pigs were limit-fed at 8% of metabolic body weight (BW0.75) in two equal feedings at 0600 and 1800 daily throughout the experiment. The study consisted of two 15-d periods. Each period consisted of a 7-d acclimation period, a 3-d total collection of feces and urine, two 12-h ileal collections, and a 3-d adjustment period between ileal collections to ensure adequate hydration. Crude protein, leucine, methionine, alanine, aspartic acid, glutamic acid, and tyrosine concentrations were greater (P < 0.05) in the gdhA+ corn than in the nontransgenic variety. The presence of the gene did not alter (P > 0.17) BW gain. Similarly, DM digestibility, fecal N excretion (grams per day), apparent total-tract N digestibility, N balance, net protein utilization, and N retained as percentages of absorbed were not affected (P > or = 0.32) by the gene modification. Apparent ileal AA digestibility values did not differ (P > 0.31) between the two dietary treatments. Results of this study suggest corn that contains the E coli. gene for glutamate dehydrogenase was nutritionally equivalent to the unaltered variety.

Domperidone can ameliorate deleterious reproductive effects and reduced weight gain associated with fescue toxicosis in heifers.

The aim of this study was to evaluate the effect of a dopamine antagonist, domperidone, in nonpregnant, reproductively cycling heifers consuming endophyte-infected (EI) fescue diets. Thirty crossbred heifers (Angus x Holstein or Hereford x Holstein) were assigned to one of three treatment groups (n = 10); endophyte-free (EF) fescue diet, EI fescue diet, or endophyte-infected diet and treated with domperidone (EID). Heifers fed EI diets had decreased weight gains compared with heifers fed EF or EID (P < 0.05) during a 21-d treatment period. Ovarian structures were monitored via transrectal ultrasound to determine follicle size and day of ovulation. Blood plasma samples were collected daily and analyzed for progesterone concentration to determine luteal function. Heifers ingesting EI diets had estrous cycles of shorter duration and lower mid-cycle progesterone concentrations than heifers in the EF or EID treatments (P < 0.05). Ovaries from a subset of heifers in each group (n = 3 per group) were harvested and in vitro secretion of progesterone from luteal tissue extracts was determined. No differences in progesterone concentrations were detected among luteal tissue incubates (P > 0.05). These results suggest that domperidone supplementation of heifers consuming EI fescue may ameliorate certain symptoms of fescue toxicosis.

Effectiveness of short-term feeding strategies for altering conjugated linoleic acid content of beef.

A steer finishing trial was performed to determine the effect of short-term dietary regimens on conjugated linoleic acid (CLA) content of muscle tissues. The experimental design was an incomplete 3 x 2 factorial, with three levels of soybean oil (SBO; 0, 4, and 8% of diet DM) and two levels of forage (20 vs. 40% of diet DM). Forty Angus x Hereford steers averaging 504 +/- 29.0 kg were allotted randomly to one of four treatments for the last 6 wk of the finishing period. Treatments were: 80:20 concentrate:forage control diet (C); 80:20 concentrate:forage + 4% SBO (C4); 60:40 concentrate:forage + 4% SBO (F4); and 60:40 concentrate:forage + 8% SBO (F8). After 42 d on the experimental diets, steers were sacrificed and samples were collected from the chuck, loin, and round muscle groups. Fatty acid (FA; mg/100 mg of FA) composition was determined by gas-liquid chromatography. Data were statistically analyzed with mixed models procedures. The performance and carcass quality model included the effects of SBO and forage. The model for FA composition included the effects of SBO, forage, muscle group, and interactions. Orthogonal contrasts were used to determine linear effects of SBO. There were no differences in growth performance among treatments (P > 0.05). Increasing dietary SBO linearly decreased dressing percent (P = 0.04), and tended to linearly decrease marbling score (P = 0.12) and quality grade (P = 0.08). The only CLA isomer detected in tissue samples was cis-9,trans-11. Addition of SBO to diets linearly increased linoleic acid (18:2n-6; P = 0.04) and tended to linearly increase linolenic acid (18:3n-3; P = 0.10) in muscle tissues. The CLA in lean tissues was decreased (P = 0.005) with SBO-containing diets. These findings suggest that increased PUFA may limit ruminal production of CLA and trans-vaccenic acid (VA) and/or may depress stearoyl-CoA desaturase expression or activity in lean tissues, which in turn limits CLA formation and accretion in tissues. Increasing dietary forage tended to increase 18:0, 18:2n-6, CLA, and 18:3n-3 (P < 0.15), suggesting that increased forage may mitigate toxic effects of PUFA on ruminal biohydrogenation, thereby increasing the pool of CLA and VA available for CLA formation and accretion in tissues. Short-term feeding of elevated SBO and forage levels can alter FA profiles in muscle tissues.

Effects of urea infusion and ruminal degradable protein concentration on microbial growth, digestibility, and fermentation in continuous culture.

The effects of urea and rumen-degradable protein (RDP) on microbial growth, digestibility, and fermentation were examined using dual-flow continuous culture. The experimental design was a 4 x 4 Latin square with a 2 x 2 factorial arrangement of treatments. Factors were urea infusion (0.4 g/L of artificial saliva) and RDP concentration, and the treatments were as follows: 1) low RDP (8% of dietary dry matter) without urea (LDNU), 2) high RDP (11% of dietary dry matter) without urea (HDNU), 3) low RDP (8% of dietary dry matter) with urea (LDU), and 4) high RDP (11% of dietary dry matter) with urea (HDU). The LDNU (i.e., negative control) and HDNU treatments were formulated to be nitrogen limiting. Results indicated that infusion of urea increased all digestibility measurements (P < 0.05), which in turn increased (P < 0.05) volatile fatty acid, NH3 nitrogen, trichloroacetic acid-soluble nitrogen, and soluble protein concentrations. Increasing dietary RDP improved dry matter and organic matter digestibility (P < 0.05) but did not alter acid detergent fiber or nonfiber carbohydrate digestibilities (P > 0.05). Isobutyrate concentration decreased (P = 0.05) with increased RDP. Increased dietary RDP increased crude protein degradation and soluble protein concentration (P < 0.05), but NH3 nitrogen, trichloroacetic acid-soluble nitrogen, and peptide nitrogen were unaffected by changing RDP levels. Microbial growth efficiency was 19.9, 24.9, 28.0, and 32.2 g N/g organic matter truly digested for LDNU, HDNU, LDU, and HDU, respectively, and was significantly improved both by urea infusion (P = 0.002) and increased RDP concentration (P = 0.021). The interactions of urea and RDP (P < 0.05) were explained by the high digestibility of neutral detergent fiber, nonstructural carbohydrate, and especially hemicellulose, with the HDNU treatment. The results of this study indicated that hemicellulose-degrading bacteria were able to effectively compete with nonstructural carbohydrate-degrading bacteria for available peptide and amino acid nitrogen. Further, the extent of protein degradation was dependent on the availability of NH3 nitrogen in the system.

Proteolytic activities of the starch-fermenting ruminal bacterium, Streptococcus bovis.

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160-200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies.

Diversity of extracellular proteolytic activities among Prevotella species from the rumen.

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B(1)4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH(4)Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B(1)4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100-200 kDa) and two metalloproteases (90-100 kDa). P. brevis GA33 generated a diffuse clearing zone (95-160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B(1)4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult.

Degradation of protein and utilization of the hydrolytic products by a predominant ruminal bacterium, Prevotella ruminicola B1(4).

Degradation and utilization of protein by Prevotella ruminicola B1(4), a proteolytic bacterium that is prominent in the rumen, was examined. In preliminary experiments, proteinaceous N sources produced faster growth rates than did NH4Cl, based on changes in optical density over time. However, ammonium chloride produced a greater maximum cell density than did proteinaceous N sources. Of the proteinaceous N sources, an enzymatic hydrolysate of soybean protein with a relative peptide size of 3 AA residues produced a greater growth rate and maximum cell density compared with the other proteinaceous N sources. Further experiments revealed that P. ruminicola B1(4) grew faster and to a greater final dry weight with soybean protein than with casein. Degradation of both proteins was low as was indicated by the slow disappearance of soluble protein, low concentrations of free AA and peptides, and the decrease in ammonia concentrations over time. Patterns of degradation did differ between the two proteins, however. Accumulation of peptides and free AA from soybean protein peaked 2 h earlier than those from casein, and concentrations of free AA and peptides from soybean protein were lower on average than those from casein. Prevotella ruminicola B1(4) preferentially utilized Asp, Ile, Leu, Lys, and Arg from soybean protein compared with casein. The relative size of peptides that accumulated from both proteins, as determined by the ratio of ninhydrin reaction after HCl hydrolysis to ninhydrin reaction before HCl hydrolysis, suggested that part of the proteolytic activity of P. ruminicola B1(4) is a dipeptidase. Our findings suggest that P. ruminicola may have a greater impact on peptide degradation than on protein degradation in the rumen.

Effect of form of nitrogen on growth of ruminal microbes in continuous culture.

A study was conducted to determine the effect of various forms of N on the growth of ruminal microbes in a continuous culture system with solids and liquid dilution rates comparable to those of a high-producing dairy cow. Nitrogen forms were isolated soy protein, soy peptides, individual amino acids (AA) blended to profile soy protein, and urea, which were fed alone and in combinations so that the total N provided was 1.6% of the diet DM. The 100% soy protein treatment resulted in reduced digestion of N and nonstructural carbohydrate compared with other N forms, and outflow of bacterial N/24 h was less than when peptides were fed. This suggested that proteolysis rather than peptide uptake was the rate-limiting step in N utilization in this study. Non-urea N forms increased ADF digestion, total VFA production and the molar percentages of isobutyrate, isovalerate, and valerate compared to urea, which reflected the contribution of carbon skeletons of AA. When combinations of N forms were used, each form contributed an equal quantity of N, 50% of the total treatment, which was .8% of the diet DM. Combinations of N forms did not enhance, and in most cases reduced, ADF and NDF digestion when compared with individual N forms, and no combinations increased microbial growth over that of the individual forms. These results confirm that N forms other than ammonia are needed not only for maximum microbial growth, and they further demonstrate a need for non-protein N for the fiber digestion. In addition, results of this study suggest a requirement for a minimum level of peptide or AA N, which was met only when individual N forms were fed.

Amelioration of cardiotoxic effects of alcohol by vitamin E.

Injection of mice with a single ip dose of 2g/kg of ethanol leads to time dependent increases of lactic dehydrogenase plasma isoenzymes indicative of myocardial damage. Electron microscopic analysis of the myocardium shows changes in mitochondrial structure, endoplasmic reticulum and myofibrils. Pretreatment of the animals with 86 units of alpha tocopherol partially prevented the changes in isoenzyme patterns and reduced the electron microscopic evidence of myocardial damage. The study supports previous findings that some of the toxic effects of alcohol might be mediated through free radical mechanisms leading to lipid peroxidation and that the ameliorating effect of alpha tocopherol could relate to its function as antioxidant and free radical scavenger.