Bos indicus cattle possess greater basal concentrations of HSP27, alpha B-crystallin, and HSP70 in skeletal muscle in vivo compared with cattle.

The objectives of the present study were to evaluate the basal concentrations of heat shock proteins (HSP) between and cattle and to determine if HSP basal concentrations change as an animal matures. A total of 40 cattle were used in a 2 × 2 factorial design to evaluate the effects of genotype and age (heifers and mature cows) on basal concentrations of heat shock protein 27 (HSP27), α B-crystallin (Cryab), and heat shock protein 70 (HSP70). Each experimental group of 10 animals was sampled on a separate day over a period of 4 wk during July 2014. A muscle sample was collected from the longissimus thoracis (LT) and concentrations of HSP were quantified using ELISA. There were no significant differences in HSP concentration for the interaction between age and genotype or for age alone. cattle had greater ( < 0.05) basal concentrations of HSP27, Cryab, and HSP70 in the LT than cattle. The results of this study show that basal in vivo HSP concentrations differ between and cattle. However, further studies are needed to investigate the relationship between HSP concentrations and meat tenderness with respect to genotypes to see if HSP concentrations account for at least some variability in tenderness differences.

Analysis of rumen microbial populations in lactating dairy cattle fed diets varying in carbohydrate profiles and Saccharomyces cerevisiae fermentation product.

The rumen microbial ecosystem is a critical factor that links diets to bovine physiology and productivity; however, information about dietary effects on microbial populations has generally been limited to small numbers of samples and qualitative assessment. To assess whether consistent shifts in microbial populations occur in response to common dietary manipulations in dairy cattle, samples of rumen contents were collected from 2 studies for analysis by quantitative real-time PCR (qPCR). In one study, lactating Holstein cows (n=8) were fed diets in which a nonforage fiber source replaced an increasing proportion of forages and concentrates in a 4×4 Latin square design, and samples of ruminal digesta were collected at 9-h intervals over 3 d at the end of each period. In the second study, lactating Holstein cows (n=15) were fed diets with or without the inclusion of a Saccharomyces cerevisiae fermentation product (SCFP) in a crossover design. In this study, rumen liquid and solid samples were collected during total rumen evacuations before and after feeding in a 42-h period. In total, 146 samples of ruminal digesta were used for microbial DNA isolation and analysis by qPCR. Validated primer sets were used to quantify total bacterial and anaerobic fungal populations as well as 12 well-studied bacterial taxa. The relative abundance of the target populations was similar to those previously reported. No significant treatment effects were observed for any target population. A significant interaction of treatment and dry matter intake was observed, however, for the abundance of Eubacterium ruminantium. Increasing dry matter intake was associated with a quadratic decrease in E. ruminantium populations in control animals but with a quadratic increase in E.ruminantium populations in cows fed SCFP. Analysis of sample time effects revealed that Fibrobacter succinogenes and fungal populations were more abundant postfeeding, whereas Ruminococcus albus tended to be more abundant prefeeding. Seven of the target taxa were more abundant in either the liquid or solid fractions of ruminal digesta. By accounting for the total mass of liquid and solid fractions in the rumen and the relative abundance of total bacteria in each fraction, it was estimated that 92% of total bacteria were found in the solid digesta fraction.

Short communication: Supplementing lysine and methionine in a lactation diet containing a high concentration of wet corn gluten feed did not alter milk protein yield.

Primiparous (n=33) and multiparous (n=63) lactating Holstein cows (186±51 d in milk) were used to evaluate the effects of supplementing metabolizable amino acids using lysine in a matrix of Ca salts of fatty acids (Megamine-L, Arm & Hammer Animal Nutrition, Princeton, NJ) and the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (MetaSmart, Adisseo Inc., Antony, France) in diets containing >26% wet corn gluten feed (dry matter basis). Cows were blocked by production level, parity, and pregnancy status, then randomly assigned to 1 of 8 pens and allowed a 7-d adaption period before receiving dietary treatments for 28 d. Pens were assigned randomly to either of 2 diets formulated to differ by metabolizable amino acid supply. Dry matter intake and production were monitored daily and milk components analyzed 3d/wk. Data were analyzed using mixed models with repeated measures. The original design of the study consisted of a control diet predicted to be deficient in lysine and methionine; however, after ingredient nutrients were analyzed and modeled with animal requirements at dry matter intake [26.6±0.35 kg/d (mean ± SEM)] and milk production levels achieved during the study (40.1±0.46 kg/d), only marginal deficiencies were predicted for the control (-8.1g/d for lysine; -1g/d for methionine) according to the National Research Council method, whereas the Cornell Net Carbohydrate and Protein System 5.0 and 6.1 models indicated positive balances for these amino acids (25.9 and 21.8 g/d for lysine, 14.7 and 18.9 g/d for methionine, respectively). Supplementing 30 g/d of metabolizable lysine in a Ca soap matrix and 2.4 g/d of metabolizable methionine as 2-hydroxy-4-(methylthio) butanoic acid led to positive predicted lysine and methionine balances by all 3 models, and predicted metabolizable lysine-to-methionine ratios ranging from 2.9 to 3.1. No treatment effects were observed for dry matter intake, milk yield, milk component concentrations or yields, or energy-corrected milk yield. Despite the negative lysine balance and low lysine-to-methionine ratio predicted by the National Research Council model, results provided no evidence of a lysine deficiency in the control diet.

Invited review: strategies for promoting productivity and health of dairy cattle by feeding nonforage fiber sources.

High-fiber byproducts are generated by several industries, and the supplies of some of these nonforage fiber sources (NFFS) are increasing. Although NFFS generally have limited utility in nonruminant diets, dairy cattle nutritionists can use these products to partially replace both forages and concentrates in lactation diets. Research has shown that production responses vary, but under certain conditions, NFFS-based diets can maintain or improve performance of dairy cattle. Traditional dietary formulation strategies are not ideal when formulating diets to contain large concentrations of NFFS. When feeding high levels of NFFS (≥15% inclusion rates, dry matter basis), less physically effective fiber is required; however, determining if this requirement has been met can be challenging, mainly because of the lack of a broadly applicable method for quantifying effective fiber in the field. Nutritionists must also be conscious of the nutrient variation that exists among many NFFS. Strategies to reduce risks associated with this variability include purchasing feed from a sole supplier who demonstrates product consistency and combining multiple NFFS at lower inclusion rates. A targeted approach whereby nonforage fiber primarily replaces some forage fiber for higher-producing cows but partially replaces some starch for lower-producing cows can optimize nutrient utilization without sacrificing animal health. In summary, the judicious use of NFFS represents an opportunity to improve the productivity and health of cattle in all stages of lactation while potentially controlling feed costs.

Effects of monensin on metabolic parameters, feeding behavior, and productivity of transition dairy cows.

The effects of monensin on transition cow metabolism may be dependent on modulation of feeding behavior, rumen pH, and expression of key metabolic genes. Multiparous Holstein cows were used to determine the effects of monensin (400mg/cow daily) on these variables. Cows were randomly assigned, based on calving date, to control or monensin treatments (n = 16 per treatment) 21 d before their expected calving date, and cows remained on treatments through 21 d postpartum. Feeding behavior and water intake data were collected daily. Liver biopsies were conducted after assessing BCS and BW on d -21, -7, 1, 7, and 21 relative to calving for analysis of triglyceride (TG) content as well as mRNA abundance of cytosolic phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase 1a, and apolipoprotein B. Blood samples were collected 21, 7, and 4 d before expected calving and 1 (day of calving), 4, 7, 14, and 21 d postpartum for nonesterified fatty acid, ß-hydroxybutyrate, glucose, insulin, and haptoglobin analyses. Ruminal pH was collected every 5 min on d 1 through 6 postpartum via a wireless indwelling probe. On d 7 postpartum, a caffeine clearance test was performed to assess liver function. Data were analyzed using mixed models with repeated measures over time. Monensin decreased mean plasma ß-hydroxybutyrate (734 vs. 616 ± 41 µM) and peak concentrations (1,076 vs. 777 ± 70 µM on d 4 postpartum). Monensin also decreased time between meals prepartum (143 vs. 126 ± 5.0 min) and postpartum (88.8 vs. 81.4 ± 2.9 min), which was likely related to a smaller ruminal pH standard deviation in the first day after cows changed to a lactation ration (0.31 vs. 0.26 ± 0.015). Monensin also increased liver mRNA abundance of carnitine palmitoyltransferase 1a (0.10 vs. 0.15 ± 0.002 arbitrary units), which corresponded to a slower rate of liver TG accumulation from d -7 to +7 (412 vs. 128 ± 83 mg of TG/g of protein over this time period). No significant effects of monensin supplementation were observed on milk production, liver cytosolic phosphoenolpyruvate carboxykinase, apolipoprotein B, plasma nonesterified fatty acid, glucose, insulin, or haptoglobin. No effects on disease incidence were detected, but sample size was small for detecting such effects. Overall, results confirm that the effects of monensin on transition cows extend beyond altered propionate flux.

Effects of feeding increasing levels of wet corn gluten feed on production and ruminal fermentation in lactating dairy cows.

An experiment was conducted to evaluate the effects of increasing dietary inclusion rates of wet corn gluten feed (WCGF; Sweet Bran; Cargill Inc., Blair, NE) on milk production and rumen parameters. Four primiparous and 4 multiparous ruminally cannulated Holstein cows averaging 90±13 d in milk (mean ± SD) were randomly assigned to 1 of 4 sequences in a replicated 4 × 4 Latin square experiment with 28-d periods. Treatments were diets containing 0, 11, 23, and 34% WCGF on a dry matter basis; alfalfa hay, corn silage, corn grain, soybean meal, expeller soybean meal, and mineral supplements were varied to maintain similar nutrient concentrations across diets. Performance and measures of ruminal fermentation were monitored. Linear and quadratic effects of increasing WCGF inclusion rate were assessed using mixed-model analysis. Increasing dietary WCGF linearly increased dry matter intake (26.7, 25.9, 29.3, and 29.7 kg/d for 0, 11, 23, and 34% WCGF, respectively) and milk production (36.8, 37.0, 40.1, and 38.9 kg/d). Concentrations of milk components did not differ among treatments; however, protein and lactose yields increased linearly and fat yield tended to increase linearly when more WCGF was fed. This led to greater production of energy-corrected milk (38.2, 38.8, 41.7, and 40.4 kg/d) and solids-corrected milk (35.2, 35.7, 38.5, and 37.2 kg/d), but efficiency of production linearly decreased. Increased WCGF in the diet tended to linearly decrease ruminal pH (6.18, 6.12, 6.14, and 5.91), possibly because mean particle size was below typical recommendations for all diets, and diets with greater proportions of WCGF had a smaller mean particle size. Ruminal acetate concentration decreased linearly and propionate increased linearly as WCGF inclusion rate increased. Treatments had a quadratic effect on ammonia concentration, with greater concentrations for the 0 and 34% WCGF diets. In situ digestibility of soybean hulls showed a significant diet-by-time interaction, and increasing dietary levels of WCGF linearly decreased in situ neutral detergent fiber disappearance at 24h. Change in body condition score increased linearly with increasing WCGF inclusion rate. Results indicate that adding WCGF to dairy rations can increase energy-corrected milk yield, and this increase appears to be driven, at least in part, by an increase in dry matter intake.

Effects of a molasses-coated cottonseed product on diet digestibility, performance, and milk fatty acid profile of lactating dairy cattle.

An experiment was conducted to evaluate the effects of a molasses-coated cottonseed product on nutrient digestibility and milk fatty acid (FA) composition of lactating dairy cattle. The effect of a direct-fed microbial (DFM) product was also examined. Twelve Holstein cows (693+/-85kg of body weight, 127+/-39 d in milk, 2.08+/-0.29 lactations; mean +/- SD) were randomly assigned to sequence in a replicated 4 x 4 Latin square design balanced for carryover effects. Cows were fed 1 of 4 treatments during each of the four 14-d periods: a control diet including 11.4% (dry matter basis) reginned cottonseed (CON), a diet with 14.4% molasses-coated cottonseed to match the cottonseed inclusion rate of the control diet (TC), the control diet with the addition of a liquid form of the cotton coating used to produce molasses-coated cottonseed (LC), and the LC diet with the addition of a DFM (LC+DFM). Diets were formulated for equal concentrations of neutral detergent fiber, crude protein, ether extract, and macrominerals. Treatments had no effect on dry matter intake, apparent total-tract nutrient digestibility, or milk production. The molasses coat, in either form, tended to decrease concentrations of odd-chain FA (2.25 and 2.31 vs. 2.35 g/100g of FA for TC, LC, and CON, respectively) and unsaturated FA (31.4 and 31.1 vs. 32.1 g/100g of FA) in milk. This could be indicative of a mild shift in ruminal fermentation away from propionate-producing bacteria toward fiber-digesting bacteria responsible for biohydrogenation of FA. The form of the molasses coating had few effects, but LC significantly decreased concentrations of total trans-C18:1 (2.04 vs. 2.30+/-0.13 g/100g of FA) and polyunsaturated FA (4.81 vs. 5.01+/-0.17) compared with TC, implying that the liquid form slightly enhanced ruminal FA biohydrogenation. Furthermore, adding the DFM to the LC diet tended to increase the proportion of long-chain FA (FA >C16) and significantly increased the proportions of trans-C18:1 (2.22 vs. 2.04+/-0.13 g/100g of FA) and unsaturated FA (32.4 vs. 31.1+/-0.7 g/100g of FA), suggesting an inhibitory effect on ruminal biohydrogenation. Results suggest that coating cottonseed with a hardened molasses product does not significantly depress nutrient digestibility and may provide a convenient method of incorporating these ingredients into dairy rations.

Effects of alfalfa hay inclusion rate on productivity of lactating dairy cattle fed wet corn gluten feed-based diets.

An experiment was conducted to evaluate the effects of varying the alfalfa inclusion rate in diets containing 31% (dry matter basis) wet corn gluten feed (Sweet Bran, Cargill Inc.). Eighty primiparous and multiparous Holstein cows averaging 178 +/- 90 d in milk (mean +/- SD) were randomly assigned to 1 of 4 sequences in a 4 x 4 Latin square design with 28-d periods. Treatments were diets containing 0, 7, 14, or 21% alfalfa on a dry matter basis, with corn silage, corn grain, soybean meal, expeller soybean meal, and mineral supplements varying across diets to maintain uniform nutrient densities. Diets were formulated for similar crude protein, neutral detergent fiber, and nonfiber carbohydrate concentrations. Feed intake, milk production, body weight, and body condition score were monitored, and linear and quadratic effects of increasing the alfalfa inclusion rate were assessed using mixed model analysis. As the alfalfa inclusion rate increased, dry matter intake tended to increase linearly (26.7, 27.3, 27.4, and 27.5 kg/d for 0, 7, 14, and 21% alfalfa, respectively), and solids-corrected milk (29.9, 30.2, 30.8, and 30.5 kg/d) and energy-corrected milk production (32.9, 33.3, 33.8, and 33.6 kg/d) tended to increase linearly. Body weight gain decreased linearly (22.9, 18.0, 11.2, and 9.5 kg/28 d) with increasing alfalfa inclusion rate. Although increasing the inclusion rate of alfalfa increased the proportion of large particles in the diets, treatments had no effect on milk fat yield or concentration. Feeding more alfalfa (up to 21% of dry matter) tended to increase milk yield while decreasing body weight gain, suggesting that metabolizable energy utilization shifted from body weight gain to milk production in these treatments. However, adding alfalfa to the diet had only minor effects on productivity.

Evaluation of nitrogen utilization and the effects of monensin in dairy cows fed brown midrib corn silage.

Twenty midlactation Holstein cows (4 ruminally fistulated) averaging 101 +/- 34 d in milk and weighing 674 +/- 77 kg were used to compare rations with brown midrib corn silage (bm3) to rations with dual-purpose control silage (DP) on N utilization and milk production. The effect of monensin in these rations was also examined. Animals were assigned to one of five 4 x 4 Latin squares with treatments arranged in a 2 x 2 factorial. Cows were fed 1 of 4 treatments during each of the four 28-d periods. Treatments were 1) 0 mg/d monensin and bm3 corn silage, 2) 0 mg/d monensin and DP corn silage, 3) 300 mg/d monensin and bm3 corn silage, and 4) 300 mg/d monensin and DP corn silage. In vitro 30-h neutral detergent fiber (NDF) digestibility was greater for bm3 corn silage (61.0 vs. 49.1 +/- 0.62). Dry matter intake (DMI) tended to be greater for cows consuming bm3 corn silage (21.3 vs. 20.2 kg/d). Neither hybrid nor monensin affected milk production, fat, or protein (37.7 kg, 3.60%, or 3.04%). Monensin tended to increase rumen pH (5.89 vs. 5.79 +/- 0.07) compared with the control treatment. In addition, bm3 corn silage resulted in a significant decrease in rumen pH (5.72 vs. 5.98 +/- 0.07). Supplementing monensin had no effect on molar proportions of acetate, propionate, or butyrate. In contrast, an increase was observed in branched-chain volatile fatty acids. No treatment interactions were observed for rumen pH or molar proportion of propionate but monensin decreased the molar proportion of acetate and increased the molar proportion of butyrate when cattle consumed bm3 silage. Dry matter, N, and acid detergent fiber digestibility were lower for the bm3 ration, whereas NDF digestibility was not different between treatments. There was no effect of hybrid on microbial protein synthesis (1,140 g/d) as estimated by urinary concentration of purine derivatives. Cows consuming bm3 excreted more fecal N than cows consuming DP (38.2 vs. 34.4% N intake); however, based on spot sampling, estimated urinary and manure N were not different between treatments (35.8 and 71.9% N intake). Monensin had no effect on DMI, digestibility of any nutrients, or N metabolism, and there were no hybrid by monensin interactions. Rations including bm3 corn silage tended to increase DMI but did not affect production. The reduction in the digestibility of some nutrients when cows consumed bm3 may have been caused by increased DMI and possible increased digestion in the lower gut. This increase in DMI appeared to also have negatively affected N digestibility but not NDF digestibility. This resulted in a greater amount of N excreted in feces but did not affect total mass of manure N.

An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning.

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach.

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A >> T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.

Mutation detection using a novel plant endonuclease.

We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease-causing mutations in DNA fragments as long as 1120 bp in length.