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PubMed1996

Effect of maturity on digestion kinetics of water-soluble and water-insoluble fractions of alfalfa and brome hay.

Alfalfa and bromegrass, each harvested at five different stages of maturity, were separated into water-insoluble and -soluble fractions. The NDF concentrations ranged from 19 to 43% for alfalfa and from 42 to 58% for brome. The rates of digestion, by mixed ruminal microflora, of the unfractionated forage and of the water-insoluble and -soluble fractions were measured in vitro using pressure sensors to monitor gas production. Both forages showed the expected decline in fiber digestibility with increasing maturity. A dual-pool logistic model gave pool sizes, specific rates, and a single lag time for both the faster- and slower-digesting fractions. The main difference between alfalfa and brome was in the soluble pool. This pool produced approximately 40% of the total gas in alfalfa, 25% in brome. The specific digestion rates of the brome soluble pool were approximately 50% higher than those for alfalfa. Net VFA production showed a somewhat higher acetate: propionate ratio for brome (3.2) compared with alfalfa (2.2), but there was little change with increasing maturity within a given forage. Gas production curves for the unfractionated forage showed a 0 to 10% positive deviation from curves created by adding data from separate digestion of the insoluble and soluble forage fractions. Gas measurements offer a promising approach to the study of the water-soluble extracts of forages and the interaction of the soluble- and insoluble-fractions during fermentation.

Acetates↗

PubMed2000

Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant, and formic acid fed to lambs.

The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages. Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively. Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs. Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage. Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant. Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19%. Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage. Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species. Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19%. Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18%. Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant.

Animal Feed↗

PubMed2000

Effect of replacing alfalfa silage with high moisture corn on nutrient utilization and milk production.

Twenty-four multiparous lactating Holstein cows were blocked by days in milk and assigned to treatment sequences in a replicated 4x4 Latin square with 21-d periods. The four diets, formulated from alfalfa silage plus a concentrate mix based on ground high moisture ear corn, contained [dry matter (DM) basis]: 1) 20% concentrate, 80% alfalfa silage (24% nonfiber carbohydrates; NFC), 2) 35% concentrate, 65% alfalfa silage (30% NFC), 3) 50% concentrate, 50% alfalfa silage (37% NFC), or 4) 65% concentrate, 35% alfalfa silage (43% NFC). Soybean meal and urea were added to make diets isonitrogenous with equal nonprotein N (43% of total N). Intake of DM and milk yield indicated that adaptation was complete within 7 d of changing the diets within the Latin square. There were linear increases in apparent digestibility of DM and organic matter, and a linear decrease in neutral detergent fiber (NDF) digestibility with increasing dietary NFC. Solutions of significant quadratic equations yielded estimated maxima for intake of DM, organic matter, digestible organic matter, and NDF at, respectively, 37, 38, 43, and 27% dietary NFC. There were linear increases in yields of milk, protein, lactose, and solids not fat with increasing dietary NFC. Feed efficiency (milk/DM intake) yielded a quadratic response with a minimum at 27% dietary NFC. Maxima for milk fat content, fat yield, and fat-corrected milk yield were estimated to occur at, respectively, 30, 34 and 38% dietary NFC. In this short-term trial, maximal DM intake and fat-corrected milk yield indicated that the optimum concentrate for cows fed high moisture ear corn plus alfalfa silage as the only forage was equivalent to 37 to 38% dietary NFC; however, yields of milk, protein and solids not fat were still increasing at 65% dietary concentrate (43% NFC).

Animal Nutritional Physiological Phenomena↗

PubMed2002

Influence of particle size on the effectiveness of the fiber in alfalfa silage.

The objective was to determine the influence of alfalfa silage particle length on milk yield, milk composition, and chewing activity. Sixteen multiparous lactating Holsteins were used in each of two separate feeding trials over a 2-yr period providing two repetitions. Each trial was based on 4 x 4 Latin squares with 21-d periods. All four diets (2-yr average, dry basis) contained a basal level of 10.7% neutral detergent fiber from corn silage and 2.3% neutral detergent fiber from chopped alfalfagrass hay. One of the diets served as a low forage, low fiber control and contained only the basal forages. The other three diets contained an additional 8.6% neutral detergent fiber from coarser alfalfa silage, finely rechopped alfalfa silage or an equal mixture ofthe two. An increase in the forage content above the basal amount using alfalfa silage increased 4% fat-corrected milk yield, milk fat yield and concentration, eating time, and total chewing time. Dry matter intake was not influenced by diet. Linear increases in rumination and total chewing times were observed as the mean particle size of the alfalfa silage increased from finer to coarser. There was no linear effect of alfalfa silage particle size on milk yield, 4% fat-corrected milk yield, dry matter intake, or milk composition.

Animals↗

PubMed1991

Immunoreactive fraction 1 leaf protein and dry matter content during wilting and ensiling of ryegrass and alfalfa.

In four experiments with ryegrass and alfalfa, cut herbage was wilted in the field and silage made in 1- or 200-L silos. Direct-cut (mean DM, 20.3%), low wilt (mean DM, 26.0%), medium wilt (mean DM, 36.2%) and high wilt (mean DM, 47.7%) herbages were used. Fraction 1, the most abundant leaf protein, was measured by crossed immunoelectrophoresis using rabbit anti-Fraction 1 serum. In two ryegrass and one alfalfa experiments in which weather conditions allowed rapid drying to high wilt herbage in 24 h, there was no significant loss of Fraction 1 protein. In the second alfalfa experiment, in which wilting was prolonged to 3 d by adverse weather, there was a 70% loss of Fraction 1. Ensiling proceeded normally in the four experiments, with rapid fall in pH and production of VFA, lactate, and NPN; the extent and rates of production were inversely related to DM content. In alfalfa and ryegrass, pH fell below the isoelectric point of Fraction 1 within 8 d. In each ryegrass experiment, a high proportion (58 to 100%) of Fraction 1 in medium and high wilt silages survived fermentation for 28 and 68 d, with lesser amounts in other silages. With alfalfa, however, almost all Fraction 1 protein was degraded at all DM concentrations during fermentation. Fiber-associated protein increased markedly with increases in DM during wilting, and these differences were present in the mature silage of both ryegrass and alfalfa. Digestibility studies with fistulated sheep showed that appreciable amounts of immunoreactive Fraction 1 protein in ryegrass silages were undegraded in the rumen.

Animals↗

PubMed1992

Dynamic model prediction of the value of reduced solubility of alfalfa silage protein for lactating dairy cows.

A net carbohydrate and protein system was used to develop model diets for lactating dairy cattle with various protein solubilities in the alfalfa silage component of the diet. The objective was to determine the level to which alfalfa silage could be used to replace supplemental protein sources as the silage protein solubility decreased and to estimate the value of silage treatments needed to reduce protein solubility. Four cow groups were considered: early lactation multiparous cows, primiparous cows, midlactation cows, and late lactation cows. Diets were balanced for metabolizable protein, metabolizable energy, and ammonia and peptides for rumen bacteria; limits on DMI and effective NDF were enforced. Lower protein solubility was predicted to increase the yield of bacteria per unit of alfalfa silage DM and the yield of metabolizable protein per unit of alfalfa silage CP. Because of reduced protein supplements, diet costs were decreased. The savings per unit of silage in these rations increased as alfalfa silage protein solubility decreased. For example, with a reduction in solubility from 61 to 51% of CP, the savings ranged from $2.96 to $3.26/tonne of silage across the four cow groups. The value of acid treatment of silage needed to effect these reductions exhibited diminishing returns as application rate increased and appeared to be most cost effective when used on high quality alfalfa fed to high producing cows with application rates less than 2 kg/tonne. Management practices that reduce silage temperatures were predicted to save $.50 to $1.50/tonne of silage when the diets were balanced to account for protein degradability.

Animals↗

PubMed1992

Epiphytic microflora on alfalfa and whole-plant corn.

Epiphytic microflora were identified and counted on four cuttings of alfalfa, each harvested at three stages of maturity, and on three whole-plant corn hybrids. Enterobacteriaceae were predominant on both crops. Yeasts and molds also were major epiphytic microorganisms on whole-plant corn. The group--including lactobacilli, pediococci, and leuconostocs, which are genera that produce lactic acid and thus are instrumental in silage preservation--constituted only a small proportion of the total population (less than .5%) on both crops. Lactate-fermenting clostridial spores were not detected on standing alfalfa, and occurrences of these spores on standing corn plants were due to soil contamination from rainfall prior to harvest. The numbers of epiphytic microorganisms, except for the lactobacilli, pediococci, and leuconostocs group, were higher on standing corn than on alfalfa. The epiphytic microflora on alfalfa increased with increasing temperature during the growing season. However, neither cutting number nor maturity affected the epiphytic microflora on standing alfalfa, and wilting following mowing had little effect on most populations. Higher temperatures during wilting increased yeast and mold counts but had no effect on other microbial counts. The chopping process tended to increase the epiphytic microflora populations compared with those on the standing crops, and the group containing lactobacilli, pediococci, and leuconostocs was most enhanced. Only yeast and mold counts on the chopped alfalfa increased with greater DM content and buffering capacity.

Animal Feed↗

PubMed1993

Observations on in situ degradation of forage cell components in alfalfa and Italian ryegrass.

The rate and extent of degradation of forage feed fractions contained in alfalfa and Italian ryegrass hays were determined. Nylon bags filled with 4 g of each forage were suspended in the rumen of two cannulated cows immediately before feeding and incubated for 10 different times (0, 2, 4, 8, 12, 24, 48, 72, 120, and 168 h). The alfalfa hay, which had lower NDF, showed a lower extent, but a higher rate, of NDF degradation than the Italian ryegrass (41.1 vs. 59.8% and 4.64 vs. 2.91%/h, respectively). Alfalfa cell walls were degraded more rapidly than Italian ryegrass even though their lignin content was higher. The hemicellulose fermentation of alfalfa showed a longer lag time (13 h) and an undegradable fraction nearly twice that for Italian ryegrass (63.3 vs. 37.1%). Cellulose from alfalfa was degraded at a higher rate than NDF or ADF, indicating that cellulose may be the primary site of hydrolysis of the cell wall in the rumen. Calculations based on in situ degradability indicate that alfalfa can have a higher inclusion than Italian ryegrass in diets for dairy cows because of lower NDF and greater availability of cell contents.

Animal Feed↗

PubMed1993

Effectiveness of neutral detergent fiber in whole cottonseed and dried distillers grains compared with alfalfa haylage.

Sixteen Holstein cows in midlactation were used in a 4 x 4 Latin square design to determine the effect of replacing alfalfa NDF with NDF from whole, linted cottonseed or dried distillers grains. Low and high fiber control diets (13 and 19% of dietary DM from alfalfa haylage NDF, respectively) were compared with diets designed to contain 13% of DM from alfalfa haylage NDF plus 6% of DM from either cottonseed NDF or distillers grains NDF. Dry matter intake, milk yield, and milk protein yield were lower from the high fiber control diet. Milk fat percentage was lower for the low fiber control diet. The cottonseed diet was equal to the high fiber control diet in stimulating rumination. Rumen acetate to propionate ratio was higher for the high fiber control and cottonseed diets. Replacing alfalfa with either of these high fiber by-product feeds increased feed intake and yields of milk fat and protein. The effectiveness of the NDF in distillers grains and cottonseed was not significantly different from that of alfalfa NDF for maintaining milk fat yield. Whole cottonseed and dried distillers grains appear to be good sources of effective fiber for maintaining milk fat test when they are substituted for alfalfa haylage fiber in lactating cow rations.

Animals↗

PubMed1995

Partial replacement of alfalfa fiber with fiber from ground corn cobs or wheat middlings.

This trial examined the effect of using corn cobs or wheat middlings to replace alfalfa partially as the dietary fiber source for lactating cows. Multiparous midlactation cows were used in three 4 x 4 Latin squares with 21-d periods. A low fiber, basal diet contained 26.8 g of total NDF, including 14.7 g of alfalfa NDF/100 g of dietary DM. Dietary fiber was increased by addition of more alfalfa, corn cobs, or wheat middlings to obtain 27.4, 28.9, and 27.9%, respectively, of total dietary NDF. Intake (24.9 kg of DM/d) and milk yield (31.6 kg/d) were not different among treatments. Nonforage fiber sources raised milk fat concentration above basal amounts (3.1% to 3.4% fat) and decreased ruminating time below that of the high alfalfa diet (423 to 390 min/d). Fat test was raised approximately one-half as much per unit of NDF from these nonforage feeds as it was per unit of NDF from alfalfa. When additional dietary fiber came from nonforage sources, milk protein concentration (3.3%) was greater than when alfalfa provided the added fiber (3.2%).

Animal Feed↗

PubMed1997

Replacement of alfalfa neutral detergent fiber with a combination of nonforage fiber sources.

Sixteen Holstein cows in midlactation were used in a 4 x 4 Latin square design to determine the effect of replacing alfalfa neutral detergent fiber (NDF), with NDF from a combination of whole linted cottonseed, dried distillers grains, and wheat middlings. The four diets were a basal control diet that was low in forage and fiber [(5.9 g of corn silage NDF and 6.1 g of alfalfa NDF/100 g of dry matter (DM)], a normal forage diet (low forage plus 10 g of additional alfalfa NDF/100 g of DM), and two low forage diets with either 5 or 10 g of NDF from the nonforage fiber sources added per 100 g of DM. Milk yield, milk protein yield, and milk protein percentage were higher, and milk fat percentage and fat yield were lower, for cows fed the low forage diets than for those fed the alfalfa control diet that was higher in fiber. Among the low forage diets, dry matter intake, milk fat percentage, and fat yield all increased linearly as NDF content increased. The ratio of acetate to propionate in the rumen and rumination times were greater for the normal forage control diet than for the high nonforage fiber diet. Added NDF from these nonforage fiber sources increased milk fat percentage and yield, but this increase was less than the NDF from alfalfa and less than predicted. In agreement with results of similar previous trials, milk protein yield and percentage were increased when alfalfa NDF was replaced with fiber from nonforage fiber sources.

Analysis of Variance↗

PubMed1997

Physical effectiveness of the neutral detergent fiber of whole linted cottonseed relative to that of alfalfa silage at two lengths of cut.

The effectiveness of the neutral detergent fiber (NDF) of whole linted cottonseed to stimulate chewing was examined relative to the effectiveness of the NDF of alfalfa silage at two lengths of cut. Twelve Holstein cows (125 d of lactation) were fed diets containing alfalfa silages differing in theoretical length of cut, with or without cottonseed substituted for alfalfa silage at 27% of alfalfa NDF. The experiment was a replicated (n = 3) 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. Length of cut had no effect on milk production or composition, and substitution of cottonseed increased milk production but did not affect milk composition. An interaction was observed between treatment effects for dry matter intake, total chews per day, and total chewing time (minutes per day). Physical effectiveness of cottonseed NDF was calculated by dividing the total chewing time per unit of cottonseed NDF by total chewing time per unit of alfalfa silage NDF. Physical effectiveness of cottonseed NDF was calculated to be 50% of long-cut and 127% of short-cut alfalfa silage NDF. The interaction observed between main effects for total chewing time per day indicated that the physical effectiveness of whole linted cottonseed was relative and depended on the characteristics of the forage that it replaced.

Animal Nutritional Physiological Phenomena↗

PubMed1997

Timothy grass or alfalfa silage for cows in midlactation: effect of supplementary barley.

Twenty-four multiparous Holstein cows averaging 104 d of lactation were used in a trial with a split-plot design to evaluate the nutritive value of two silages, timothy grass or alfalfa, both treated with formic acid and stored in plastic bag silos. Silages were offered for ad libitum intake either alone or with 17 or 34% (dry matter basis) dry-rolled barley. Both silages contained similar amounts of acid detergent fiber (ADF) (27.5 and 26.7% for timothy grass and alfalfa, respectively). After 110 d of storage, alfalfa silage contained higher amounts of organic acids and NH3 N but had lower soluble N. Total dry matter intake (DMI) and silage DMI were similar between cows fed both silages. Increased barley proportion decreased silage DMI (19.2 to 14.2 kg/d). Apparent total tract digestibility of dry matter was unaffected by treatment. The digestibility of neutral detergent fiber and ADF was higher for the timothy grass silage than for alfalfa silage and was unaffected by the barley percentage added to either silage. Milk yield was lower (23.9 to 22.6 kg/d) for cows fed the highest proportion of barley. The 4% fat-corrected milk yield was unaffected by treatment. Percentages of fat, protein, and total solids in milk were higher for cows fed diets with the higher barley content. Milk fat and protein yields were similar among treatments. Urea in blood was lower for cows fed timothy grass silage than for cows fed alfalfa silage (4.68 vs. 6.23 mg/100 ml). These results suggest that timothy grass silage and alfalfa silage, when stored at a similar ADF content, have comparable nutritive value for midlactation cows.

Animal Feed↗

PubMed1999

Effect of year and cutting on equations for estimating net energy of alfalfa forage.

The net energy of lactation (NEL) concentration of forages is important for formulating diets. The equations presently used to estimate NEL of alfalfa are based on limited data. Our objective was to determine whether a larger database would provide more relevant equations. One hundred eighty samples of alfalfa were taken over four cuttings and 2 yr, combined into 45 composites and analyzed for neutral detergent fiber, acid detergent fiber (ADF), and in vitro true digestibility. Finally, NEL values were estimated using neutral detergent fiber and in vitro true digestibility. Prediction equations were obtained by regressing NEL upon ADF concentration. Changes in NEL with age of alfalfa at harvest differed with year and cutting. Estimation of NEL would be more accurate with equations specific for cutting and year, but such an approach would be difficult to use in practice. When data were pooled across years, four cutting-specific equations were obtained; these had different intercepts and slopes and gave different NEL values for a given ADF content. Differences among estimates of NEL increased as ADF increased. Data were combined across years and cuttings to give an overall equation that was similar to published prediction equations. Compared with NEL values from the cutting-specific equations, the overall equation underestimated NEL for first cutting alfalfa and overestimated NEL of second cutting alfalfa. A lack of precision in estimating NEL could equate to 1.5 to 2.5 kg of milk/d. Cutting specific equations should be used to estimate the NEL of first- and second-cutting alfalfa; NEL of the third and fourth cuttings could be estimated using an overall equation.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Effect of replacing alfalfa silage with high moisture corn on ruminal protein synthesis estimated from excretion of total purine derivatives.

Twenty-four multiparous dairy cows (eight with ruminal cannulae) were blocked by days in milk and assigned to six balanced 4 x 4 Latin squares with 21-d periods. The four diets, formulated from alfalfa silage plus a concentrate mix based on ground high moisture ear corn, contained (dry matter basis): 1) 20% concentrate, 80% alfalfa silage (24% nonfiber carbohydrate; NFC), 2) 35% concentrate, 65% alfalfa silage (30% NFC), 3) 50% concentrate, 50% alfalfa silage (37% NFC), or 4) 65% concentrate, 35% alfalfa silage (43% NFC). Soybean meal and urea were added to make diets isonitrogenous with equal nonprotein nitrogen (NPN) (43% of total N). Total urine was collected with indwelling Folley catheters for 24 h during each period. There was no effect of diet on urinary creatinine excretion (average 29 mg/kg of BW/d). There were quadratic effects of diet on total urinary ecretion of allantoin, uric acid, and purine derivatives (allantoin plus uric acid), and on ruminal synthesis of microbial N estimated from purine derivatives; maxima occurred at about 35% dietary NFC. Urinary excretion also was estimated with spot urine samples from creatinine concentration and the mean daily creatinine excretion. Daily excretion of allantoin, uric acid, and purine derivatives estimated from spot urine sampling followed the same pattern as that observed with total collection; differences between measured and estimated urine volume were significant only for 35% dietary concentrate. Spot urine sampling appeared to yield satisfactory estimates of purine derivative excretion. Maximal urea N excretion was estimated to occur at about 31% dietary NFC. Milk allantoin secretion increased linearly with concentrate and accounted for 4 to 6% of the total purine derivative excretion. Microbial yield was maximal at 35% dietary NFC, suggesting that this was the optimal level for utilization of dietary NPN from alfalfa silage and other sources.

Allantoin↗

PubMed2001

Wrinkled alfalfa seeds harbor more aerobic bacteria and are more difficult to sanitize than smooth seeds.

At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.

Bacteria, Aerobic↗

PubMed2003

Inactivation of Escherichia coli O157:H7 and Salmonella by gamma irradiation of alfalfa seed intended for production of food sprouts.

Inonizing irradiation was determined to be a suitable method for the inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the production of food sprouts. The radiation D (dose resulting in a 90% reduction of viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on alfalfa seeds were higher than the D-values for their inactivation on meat or poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for vegetable-associated isolates were not significantly different. The D-values for nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55 +/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the relatively high D-values were not due to the low moisture content or the low water activity of the seed. The D-values for Salmonella on alfalfa seeds from two different sources did not differ significantly, even though there were significant differences in seed size and water activity. The increased moisture content of the seed after artificial inoculation did not significantly alter the D-value for the inactivation of Salmonella. The results of this study demonstrate that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing radiation, which will permit satisfactory commercial yields of sprouts from alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.

Dose-Response Relationship, Radiation↗

PubMed1992

Isolation and structural determination of a novel TRH-like tripeptide, pyroGlu-Tyr-Pro amide, from alfalfa.

The tripeptide pyroGlu-Tyr-Pro amide was isolated from an aqueous extract prepared from dried alfalfa pellets. The tripeptide was quantitated using a competitive radioimmunoassay in which 125I-labeled thyrotropin-releasing hormone (TRH), is displaced from antibody specific to TRH (pyroGlu-His-Pro amide). The pyroGlu-Tyr-Pro amide was purified by passing the filtered extract through QAE-Sephadex A25 at pH 5, followed by open bed chromatography on C18 silica using an H2O/methanol gradient, then preparative high performance liquid chromatography (HPLC) on microbondapak C18 using a 10 mM HCl/methanol gradient, followed by G-10-Sephadex chromatography, SP-C25-Sephadex chromatography, QAE-Sephadex chromatography at pH 10.1, analytical HPLC on a microbondapak C18 column eluted with 10 mm HCl/acetonitrile, and analytical HPLC reverse phase chromatography on an APEX phenyl column eluted with H2O/acetonitrile. The tripeptide was essentially homogeneous after the final chromatography step, as judged by correspondence of immunoreactivity with A280. The sequence of the alfalfa tripeptide was determined to be Glu-Tyr-Pro by gas phase sequencing, after hydrolysis of pyroglutamic acid by mild acid hydrolysis. The mass of the alfalfa tripeptide was 389.1, as determined by fast ion bombardment mass spectroscopy, and was found to be identical to the mass of synthetic pyroGlu-Tyr-Pro amide. The sequence of the alfalfa tripeptide was also verified using B/E-linked scanning. I conclude that the tripeptide isolated from alfalfa differs from human thyrotropin-releasing hormone only by the substitution of tyrosine for histidine at position 2. The role of pyroGlu-Tyr-Pro amide in alfalfa is not known, but the existence of a family of thyrotropin-related peptides occurring in both the animal and the plant kingdoms indicates that the thyrotropin related peptides have a wide phylogenetic distribution.

Chromatography, Gel↗