Buffer-soluble Protein Research Articles

Nutrient and Rumen Fermentation Studies of Indian Pasture Legumes for Sustainable Animal Feed Utilisation in Semiarid Areas

This study evaluated 5 annual and 11 perennial Indian pasture legumes species for their nutritive value, dry matter and mineral contents and in vitro fermentation parameters. Legume species differed significantly (p < 0.05) in various nutritional aspects such as organic matter, crude protein (CP), ether extract, fibres and protein fractions. Perennial Clitoria ternateaa had higher (p < 0.05) buffer soluble protein (477), while neutral detergent soluble protein was highest in annually grown Lablab purpureus (420 g/kg CP). Atylosia scarabaeoides (AS) had higher levels of nonstructural carbohydrates (NSCs) (392 g/kg dry matter (DM)) than structural carbohydrates (SC) (367 g/kg DM). Its rapidly degradable fraction (51.7 g/kg (total carbohydrate) tCHO) was lower (p < 0.05) than other fractions of carbohydrates. Total digestible nutrients, digestible energy and metabolisable energy varied, with Desmodium virgatus (DV) having higher values and Stylosanthas seabrana (SSe) having the lowest. Predicted dry matter intake, digestible dry matter and relative feed value also showed significant differences (p < 0.05). Annual grasses such as Dolichos biflorus, Macroptilium atropurpureum, Rhynchosia minima (RM) were found to be better balanced with micro minerals. In vitro dry matter degradability, partition factor, short-chain fatty acids and microbial protein production of legumes varied significantly (p < 0.05). Gas and CH4 production (mL/g and mL/g (digestible DM) DDM) also varied, with Clitoria ternatea-blue having the highest gas production and C. ternatea -white (CT-w) and AS having lower CH4 production. Methane in total gas was low for DV, RM and CT-w (8.99%, 9.72% and 9.51%). Loss of DE and ME as CH4 varied (p < 0.05) among the legumes. Each legume offers unique benefits, potentially allowing for tailored combinations of annual and perennial legumes to optimize rumen feed efficiency.

Degradability of Vicia ervilia Grain Using In Situ and CNCPS Methods, and Model-Based Analysis of Its Ruminal Degradation

Using nylon bag techniques, Cornell net carbohydrates and protein systems (CNCPS), and scanning electron microscopy, the authors examined the digestibility and structure of Vicia ervilia (ervil, bitter vetch) after steam flaking, roasting, and microwave processing. During the in situ technique, the samples were incubated at 0, 2, 4, 6, 8, 12, 16, 24, 36, and 48 h. For the description of the ruminal DM (dry matter) and CP (crude protein) degradation kinetics of treated and untreated Vicia ervilia, different models were selected as the best fit for the dry matter (DM) and crude protein (CP) degradation parameters of steam flaked samples. The results showed that both the steam flaking and microwave treatment samples contained high levels of non-protein nitrogen and buffer soluble protein, respectively. In comparison with steam flaking and microwave treatment, roasting decreased and increased the buffer soluble protein and neutral detergent insoluble protein, respectively. The control treatments showed the highest levels of neutral detergent soluble protein and the lowest levels of acid detergent soluble protein. Moreover, steam flaking and roasting decreased and increased the amount of acid detergent insoluble protein, respectively. When using dry heat (microwave and roasting), the acid detergent soluble and insoluble protein fractions were increased. As a result of this experiment, the nitrogen fractions were altered using heat processing. Hence, protein fermented feed and ruminal fermentation conditions can be expressed using these results.

Effect of lactobacilli inoculation on protein and carbohydrate fractions, ensiling characteristics and bacterial community of alfalfa silage.

Alfalfa (Medicago sativa L.) silage is one of the major forages with high protein for ruminants. The objective of this study was to investigate the effects of lactobacilli inoculants on protein and carbohydrate fractions, ensiling characteristics and bacterial community of alfalfa silage. Wilted alfalfa (35% dry matter) was inoculated without (control) or with Lactobacillus coryniformis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus pentosus and ensiled for 7, 15, and 60 days. Silage inoculated with L. pentosus was superior to L. coryniformis, L. casei, L. plantarum in improving the fermentation quality of alfalfa silage, as indicated by the lowest ammonia nitrogen content and silage pH during ensiling. There was minor difference in water soluble carbohydrates content among all silages, but L. pentosus inoculants was more efficient at using xylose to produce lactic acid, with lower xylose content and higher lactic acid content than the other inoculants. Compared with the control, L. pentosus inoculants did not affect true protein content of silage, but increased the proportions of buffer soluble protein and acid detergent soluble protein. The L. pentosus inoculants reduced the bacterial diversity In alfalfa silage with lower Shannon, Chao1, and Ace indices, and promoted relative abundance of lactobacillus and decreased the relative abundance of Pediococcus compared with the control. As well as L. pentosus inoculants up-regulated amino acid, carbohydrate, energy, terpenoids, and polypeptides metabolism, and promoted lactic acid fermentation process. In summary, the fermentation quality and nutrient preservation of alfalfa silage were efficiently improved by inoculated with L. pentosus.

Potassium and Phosphorus Fertilizer Impacts on Alfalfa Taproot Carbon and Nitrogen Reserve Accumulation and Use During Fall Acclimation and Initial Growth in Spring.

Phosphorus (P) and potassium (K) impact alfalfa (Medicago sativa L.) performance, but how these nutrients alter taproot physiology during fall acclimation and subsequent growth in spring is unclear. Our objectives were to: (1) determine seasonal patterns for taproot P and K concentrations during fall acclimation and during initial shoot growth in spring; (2) determine how P and K nutrition impacts accumulation of taproot C and N reserves during fall and their subsequent use when shoot growth resumes in spring; and (3) assess how addition of P and K fertilizer impacts survival and shoot growth in spring. Two P (0 and 75 kg ha−1) and two K (0 and 400 kg ha−1) treatments were applied and taproots were sampled between September and December, and again from March to May over 2 years. Concentrations of taproot sugar, starch, buffer-soluble protein, amino-N, and RNA pools were determined. While P and K fertilizer application increased taproot P and K concentrations two- to three-fold, concentrations of P and K in taproots over time did not change markedly during cold acclimation in fall, however, taproot P declined in spring as plant growth resumed. Compared to the 0K-0P treatment, taproots of plants fertilized with 400K-75P had higher starch, protein, amino-N, and RNA, but reduced sugar concentrations in fall. Concentrations of all these pools, except starch, declined during the initial 2 weeks of sampling beginning in late March as shoot growth resumed in spring. Herbage yield in May was highest for the 400K-75P treatment and least for the 0K-0P treatment, differences that were associated with variation in mass shoot−1 and not shoots m−2. High yield of the 400K-75P plants in May was consistently associated with greater concentrations and use of amino-N, soluble protein, and RNA pools in taproots, and not with accumulation and use of starch and sugar pools. Understanding factors leading to the accumulation of taproot N reserves and RNA during cold acclimation in fall and their use during the initial growth in spring should enhance efforts to improve alfalfa growth and herbage yield in spring.

Replacing Alfalfa with Paper Mulberry in Total Mixed Ration Silages: Effects on Ensiling Characteristics, Protein Degradation, and In Vitro Digestibility.

Simple SummaryThe usage of alfalfa (Medicago sativa L.) as a dietary protein source for ruminants in China is limited by forage quality and planting scale. Paper mulberry (Broussonetia papyrifera L., RY) has emerged as a new and representative high-protein woody forage resource for ruminants. However, information is less available regarding how substituting RY for alfalfa affect the fermentation and protein quality in total mixed ration (TMR) silages. This study evaluated ensiling characteristics, protein quality, and in vitro digestibility in TMR silages by mixing RY with alfalfa at different rations. The TMR were made with alfalfa and RY mixtures (36.0%), maize meal (35.0%), oat grass (10.0%), soybean meal (7.5%), brewers’ grain (5.0%), wheat bran (5.0%), premix (1.0%), and salt (0.5%) on a dry matter basis, respectively. The alfalfa and RY mixtures were made in the following ratios of dry matter: 36:0 (RY0), 27:9 (RY9), 18:18 (RY18), 9:27 (RY27), and 0:36 (RY36). The results showed that RY substitution had no adverse effect on fermentation quality and nutritional composition, but inhibited true protein degradation, while decreasing in vitro dry matter and crude protein digestibility. Therefore, RY and alfalfa mixtures at a ratio of 18:18 is suitable for silage-based TMR.To develop an alternative high-protein forage resource to alleviate ruminant feed shortages, we investigated the effects of replacing alfalfa (Medicago sativa L.) with different ratios of paper mulberry (Broussonetia papyrifera L., RY) on fermentation quality, protein degradation, and in vitro digestibility of total mixed ration (TMR) silage. The TMR were made with alfalfa and RY mixtures (36.0%), maize meal (35.0%), oat grass (10.0%), soybean meal (7.5%), brewers’ grain (5.0%), wheat bran (5.0%), premix (1.0%), and salt (0.5%) on a dry matter basis, respectively. The alfalfa and RY mixtures were made in the following ratios of dry matter: 36:0 (RY0), 27:9 (RY9), 18:18 (RY18), 9:27 (RY27), and 0:36 (RY36). After ensiling for 7, 14, 28, and 56 days, fermentation quality, protein degradation, and microbial counts were examined, and chemical composition and in vitro digestibility were analyzed after 56 days of ensiling. All TMR silages, irrespective of the substitution level of RY, were well preserved with low pH and ammonia nitrogen content, high lactic acid content, and undetectable butyric acid. After ensiling, the condensed tannin content for RY18 silages was higher than the control, but non-protein nitrogen, peptide nitrogen, and free amino acid nitrogen contents was lower, while the fraction B1 (buffer-soluble protein) was not different among all the silages. Dry matter and crude protein digestibility for RY27 and RY36 silages was lower than the control, but there was no difference between control and RY18 silages. This study suggested that ensiling RY with alfalfa inhibited true protein degradation, but decreased in vitro dry matter and crude protein digestibility of TMR silages, and that 18:18 is the optimal ratio.

Expression of Arabidopsis SHN1 in Indian Mulberry (Morus indica L.) Increases Leaf Surface Wax Content and Reduces Post-harvest Water Loss.

Mulberry (Morus species) leaf is the sole food for monophagous silkworms, Bombyx mori L. Abiotic stresses such as drought, salinity, and high temperature, significantly decrease mulberry productivity and post-harvest water loss from leaves influence silkworm growth and cocoon yield. Leaf surface properties regulate direct water loss through the cuticular layer. Leaf surface waxes, contribute for cuticular resistance and protect mesophyll cells from desiccation. In this study we attempted to overexpress AtSHN1, a transcription factor associated with epicuticular wax biosynthesis to increase leaf surface wax load in mulberry. Agrobacterium mediated in vitro transformation was carried out using hypocotyl and cotyledonary explants of Indian mulberry (cv. M5). Mulberry transgenic plants expressing AtSHN1 displayed dark green shiny appearance with increased leaf surface wax content. Scanning electron microscopy (SEM) and gas chromatograph–mass spectrometry (GC-MS) analysis showed change in pattern of surface wax deposition and significant change in wax composition in AtSHN1 overexpressors. Increased wax content altered leaf surface properties as there was significant difference in water droplet contact angle and diameter between transgenic and wild type plants. The transgenic plants showed significant improvement in leaf moisture retention capacity even 5 h after harvest and there was slow degradation of total buffer soluble protein in detached leaves compared to wild type. Silkworm bioassay did not indicate any undesirable effects on larval growth and cocoon yield. This study demonstrated that expression of AtSHN1, can increase surface wax load and reduce the post-harvest water loss in mulberry.

Nutritional evaluation of different varieties of sorghum stovers in sheep

Nutritive value of stovers from 4 sorghum varieties (Pant Chari 5, PKV 809, CSV 17 and CSV 20) that were selected from a previous breeding program for their relative nutritive value was assessed using the in vitro and in vivo methods. Samples for in vitro and in vivo studies were harvested separately. For the in vivo studies, 16 adult Jalauni sheep (19.1±0.35kg) were randomly divided into 4 groups (n=4) and offered chopped stover from CSV 20, PKV 809, Pant Chari 5 and CSV 17 varieties, respectively along with 200g of concentrate mixture. After a 30-day feeding, 5days of metabolism trial was conducted to determine dry matter intake (DMI), nutrients digestibility and nitrogen balance. For in vitro study, crude protein (CP) concentration of CSV 20 was lowest (P<0.05) compared with the other varieties. The NDF and hemicellulose contents were highest (P<0.001) in CSV 20. The CP content in chaffed stover used for the animal study increased marginally for all varieties except for Pant Chari 5 (45.3g/kg DM) which had the lowest CP content. Buffer soluble protein (PB1), PA and PC fractions of the stovers were higher (P=0.006) in CSV 17 compared with the other stovers. Although CSV 20 had the lowest (P<0.001) PA and PC fractions, it had similar (P>0.05) PB1, PB2 and PB3 fractions with one or more of the other stovers. In vitro dry matter digestibility was highest (P=0.002) for CSV 17 and lowest for Pant Chari 5. Stover estimated DMI values varied between 0.0156 to 0.0164 of BW of the sheep with lower (P<0.001) DMI noted for CSV 20 versus the other varieties. Greater (P<0.05) cumulative gas production and rate of gas production (k) were noted for CSV 20 and Pant Chari 5 compared with others. Similar trend was also noted in the in vitro methane production, PF and SCFA concentration of the stovers. No difference was noted in the intake and digestibility of DM of the stover varieties. Sheep fed CSV 17 had significantly higher (P<0.05) nitrogen intake and fecal nitrogen (11.2 and 4.18g/d, respectively), and a tendency (P=0.07) for higher urinary nitrogen and nitrogen balance. Overall, results showed that sheep had higher stover intake and better nutrients digestibility, more nitrogen balance and higher nutritive value (digestible CP and metabolizable energy) from CSV 17 and PKV 809 sorghum stover based diets.

The effect of ensiling in round bales on the content of nitrogen fractions in lucerne and red clover protein

The aim of the study was to determine changes in nitrogen fractions during the ensiling of lucerne and red clover herbage in bales. Protein nitrogen and non-protein nitrogen compounds in herbage and silage were distinguished. Furthermore, buffer soluble nitrogen compounds (BSN) were determined and divided into buffer soluble protein and non-protein nitrogen (BSPN and NPBSN, respectively). In addition, peptide nitrogen and amino acid nitrogen were distinguished within the NPBSN fraction, while nitrogen compounds were divided into those of low solubility (NDIN) and completely insoluble (ADIN). The research findings confirmed the effect of a legume plant species on changes in the content of nitrogen fractions during the ensiling in round bales. The ensiling of lucerne herbage grossly decreased the protein nitrogen fraction while raising the total soluble fraction of nitrogen. Nitrogen in the form of soluble proteins, NDIN and ADIN was the least affected. The study demonstrated a different response of the two legume plant species to ensiling with respect to all fractions. A high proportion of protein nitrogen in round bale wilted red clover silage was accompanied by a large contribution of insoluble nitrogen fractions. A large share of the protein nitrogen fraction in red clover silage does not necessarily mean a higher nutritional value of the protein because it may contain a large amount of protein compounds that are non-hydrolysable by bacterial and intestinal enzymes.

Assessment of genetic diversity among some elite cultivars of ginger (Zingiber officinale Rosc.) using isozyme and protein markers

Protein and isozyme markers were used to characterize 20 elite cultivars of ginger. Four different isozymes (acid phosphatase, esterase, peroxidase and polyphenolic oxidase) were analyzed to assess the genomic relationship among 20 varieties of ginger. Buffer soluble protein was isolated and resolved in SDS-PAGE. A total of 108 types of proteins were resolved, out of which 33 were unique. A total of 85 bands were detected for 4 isozymes, among them only 3 were found to be unique. The den- drogram constructed based on pooled data (protein and isozyme) divided the varieties into two major clusters each containing 10 members, respectively. The varieties Singj- hara and Waynad local were found to be the most closely related while Singjhara and Sleeva local were distantly apart. The present study gave an indication of usefulness of the isozyme and protein markers for genetic discrimi- nation between different varieties of ginger. The result of the present investigation will help in future breeding program.

Nitrogen fractions and amino acid content in alfalfa and red clover immediately after cutting and after wilting in the field

The aim of this study was to determine the composition of nitrogen fractions in alfalfa and red clover, which differ in proteolytic activity, and to evaluate the effect of wilting on changes in nitrogen fractions in alfalfa and red clover herbage. Total nitrogen was divided into protein and non-protein nitrogen, and the amino acid profile of protein was analyzed. Buffer-soluble nitrogen (BSN), including buffer-soluble protein nitrogen (BSPN) and non-protein buffer-soluble nitrogen (NPBSN), was determined. The NPBSN fraction was further subdivided into peptide nitrogen, amino acid nitrogen, neutral detergent-insoluble nitrogen (NDIN) and acid detergent-insoluble nitrogen (ADIN). Wilting in the field to 40% dry matter content (the swath was tedded once) did not reduce the total nitrogen content of alfalfa and red clover herbage, although it affected the concentration of the BSNfraction, in particular NPBSN, and free amino acid nitrogen. During alfalfa wilting, the soluble protein content decreased and the concentrations of non-protein nitrogen compounds increased, mostly due to an increase in free amino acid nitrogen. A reverse trend was observed during red clover wilting – the concentrations of non-protein nitrogen compounds decreased and soluble protein content increased. A decrease was also noted in peptide nitrogen, NDIN and ADIN. Wilting of alfalfa and red clover had no adverse effect on the amino acid profile of protein. The concentrations of essential amino acids that limit milk protein synthesis, i.e. Lys, Met, His and Arg, did not decrease. Legume wilting in the field contributes to an increase in the concentrations of soluble nitrogen in the plant material intended for ensiling.

조사료 자원의 단백질 분획 및 Buffer 추출이 In Vitro 발효 성상, 분해율 및 Gas 생성량에 미치는 효과

Buffer solubility and protein fractionation were evaluated from the hays (timothy, alfalfa and klein) and straws (tall fescue and rice), and trial was conducted to examine the effect of buffer extraction on fermentation characteristics, degradability and gas ( and ) production. Buffer soluble protein (SP) content and A fraction in total protein were highest in alfalfa hay as 61% and 41.77%, respectively while lowest in rice straw (42.8% and 19.78%, respectively). No difference was observed in B1 fraction among forages but B2 fraction was slightly increased in klein hay (12.34%) and tall fescue straw (10.05%) compared with other forages (6.34~8.85%). B3 fraction of tall fescue was highest as 38.49% without difference among other forages while C fraction was highest in rice straw. pH in incubation solution was higher in all forages after extraction than before extraction at 3h (P) proportion was also increased(P from hays were mostly higher (P production from all the forages uo to 24h incubation and its production was greater (P) production was also greater (P VFA concentration, degradability and gas ( and ) production. Thus, measurement of buffer solubility and protein fractionation of forages might be useful to improve TMR availability in the ruminants.

Modeling degradation ratios and nutrient availability of anthocyanidin-accumulating Lc-alfalfa populations in dairy cows

Dairy cattle eating fresh or ensiled alfalfa as the main portion of their diet often have low protein efficiency because of the rapid initial rate of ruminal protein degradation of fresh and ensiled alfalfa. Ruminal protein degradation of alfalfa might be reduced by introducing a gene that stimulates the accumulation of mono- or polymeric anthocyanidins in alfalfa. The objectives of this study were to fractionate protein and carbohydrates by in situ and chemical approaches, to evaluate in situ ruminal degradation characteristics and synchronization ratios, to determine protein availability to dairy cattle using the 2007 digestible intestinal protein/rumen-degraded protein balance (DVE/OEB) protein system, and to determine net energy for lactation using the Dutch net energy for lactation (VEM) system for 3 newly developed transgenic winter hardy anthocyanidin-accumulating T1 Lc-alfalfa populations. These T1 Lc-alfalfa populations, called T1BeavLc1, T1RambLc3, and T1RangLc4, had an average anthocyanidin accumulation of 163.4 μg/g of DM, whereas AC Grazeland (selected for a low initial rate of degradation) did not accumulate anthocyanidin. The basic chemical composition of the original samples, soluble and potentially degradable fractions, and degradation characteristics of crude protein and carbohydrates were similar in T1 Lc-alfalfa and AC Grazeland. The undegradable in situ crude protein and neutral detergent fiber fraction had 1.3% lower CP and 4.8% lower CHO, respectively, in T1 Lc-alfalfa compared with the amounts in AC Grazeland. The T1 Lc-alfalfa had a 0.34 MJ/kg of DM higher calculated net energy for lactation and 1.9% of CP higher buffer soluble protein compared with that in AC Grazeland. By the protein evaluation model, it was predicted that T1 Lc-alfalfa tended to have 11.9, 6.9, and 8.4 g/kg of DM higher rumen degradable protein, OEB, and intestinal available protein, respectively, compared with the amounts in AC Grazeland. The hourly OEB included an initial and substantial peak (oversupply) of protein relative to energy, which was highest in T1RangLc4 and lowest in T1RambLc3. The hourly OEB between 4 and 24h was similar and more balanced for all 4 alfalfa populations. In conclusion, T1 Lc-alfalfa accumulated anthocyanidin, tended to have higher predicted intestinal protein availability, and had higher predicted net energy of lactation availability for dairy cattle than did AC Grazeland.

Screening of selenium containing proteins in the Tris-buffer soluble fraction of African catfish (Clarias gariepinus) fillets by laser ablation-ICP-MS after SDS-PAGE and electroblotting onto membranes

The anti-carcinogenic properties of selenium against certain types of cancer when present in organic forms justify the increasing interest in development of selenium fortified food. In this particular study, African catfish (Clarias gariepinus) were fed with a Se-enriched diet in order to enhance the selenium concentration in the fish fillet up to 0.85 ± 0.06 mg Se kg−1 (0.26 ± 0.02 mg Se kg−1 in control). Selenium distribution in proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was studied for better understanding of the metabolism of this element in the organism. Soluble proteins (Tris buffer) were investigated by conventional application of a glass homogenizer in comparison to application of ultrasound probe sonication, and the latter was convincing due to higher protein extraction efficiency and the ease and speed of application. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was successfully applied for screening of selenium in proteins in fillets of African catfish separated by one-dimensional SDS-PAGE and after electroblotting onto nitrocellulose membranes, with a total Se amount below 0.4 ng loaded into a well. Selenium was detected in more than 11 protein spots. In order to improve the protein separation of one-dimensional SDS-PAGE, pre-fractionation of the Tris buffer soluble protein extract by size exclusion was carried out. Tryptic digestion of the Se-containing bands was performed for protein identification by nano-HPLC coupled to electrospray-MS/MS. A database search revealed several proteins which are located in muscle tissue. However, the very low Se concentration circumvents the detection of selenium containing amino acids in the tryptic peptides by electrospray-MS/MS.

Mechanical Maceration Divergently Shifts Protein Degradability in Condensed‐Tannin vs. o‐Quinone Containing Conserved Forages

Conditioning and conservation methods may interact with polyphenols to alter forage crude protein (CP) solubility and degradability. In this study, forages with ∼200 g CP kg−1 dry matter were roll conditioned or macerated, conserved as hay or silage, and then analyzed for CP fractions. Shifting from roll conditioning to maceration of polyphenol‐free alfalfa (Medicago sativa L.) reduced buffer soluble protein (SP) with little effect on protease rumen‐undegradable protein (RUP) and intestinal available protein (IAP, RUP minus acid‐detergent insoluble CP). In birdsfoot trefoil (Lotus corniculatus L.), condensed tannin (CT) and maceration independently reduced SP and increased RUP to yield up to 62% more IAP in hay and 145% more IAP in silage than alfalfa. Based on results with trefoil, roll‐conditioned forage with 70 to 120 g CT kg−1 CP or macerated forage with more modest CT levels should meet a 350 g RUP kg−1 CP target to support 35 kg d−1 milk yield by cattle. In roll‐conditioned red clover (Trifolium pratense L.), o‐quinones formed by polyphenol oxidase reduced SP and increased RUP to yield 50% more IAP in hay and 88% more IAP in silage than alfalfa. Surprisingly, maceration reduced SP, RUP, and IAP in clover. Following maceration, RUP and IAP in conserved clover and trefoil responded similarly to CT, suggesting that maceration disabled o‐quinone protection of protein substrates. Although red clover has high RUP, low reported milk yields indicate o‐quinones might depress IAP.

Can herbage nitrogen fractionation in Lolium perenne be improved by herbage management?

The high degradability of grass protein is an important factor in the low nitrogen (N) utilization of grazing bovines in intensive European grassland systems. We tested the hypothesis that protein degradability as measured by the Cornell Net Carbohydrate and Protein System (CNCPS) protein fractionation scheme, can be manipulated by herbage management tools, with the aim to reduce N loss to the environment. A field experiment comprising the factorial combinations of three fertilizer N application rates (0, 90 and 390 kg N ha −1 year −1), three regrowth periods (2–3, 4–5, and 6–7 weeks), two perennial ryegrass ( Lolium perenne L.) cultivars [Aberdart (high sugar content) and Respect (low sugar content)] and two cutting heights (approximately 8 and 12 cm) was conducted at Teagasc, Johnstown Castle Research Centre, Wexford, Ireland. The plots were sampled during four seasons [September/October 2002 (late season), April 2003 (early season), May/June 2003 (mid season) and September 2003 (late season)] and protein fractions were determined in both sheath and lamina material. The protein was highly soluble and on average 19% and 28% of total N was in the form of non-protein N, 16% and 19% in the form of buffer-soluble protein, 52% and 40% in the form of buffer-insoluble protein, and 12% and 13% in the form of potentially available cell wall N for lamina and sheath material, respectively. In both materials only 0.9% of total N was present as unavailable cell wall N. In general the herbage management tools investigated did not have much effect on protein fractionation. The effects of regrowth period, cultivar and cutting height were small and inconsistent. High N application rates significantly increased protein degradability, especially during late season. This is relevant, as it has been shown that enhanced protein degradation increases the potential N loss through urine excretion at a time when urine-N excreted onto pasture is prone to leaching. However, the effect was most evident for sheath material, which forms only a small proportion of the animals' intake. It was concluded that there appears to be little scope for manipulating the herbage-N fractionation through herbage management. The consequences for modelling herbage quality could be positive as there does not seem to be a need to model the individual N fractions; in most cases the N fractions can be expressed as a fixed proportion of total N instead.

Chemical composition, ensiling characteristics and ruminal degradability of forage soybean cultivars

A study was conducted to determine the effects of forage soybean cultivar on chemical composition, ensiling characteristics, and ruminal degradability of silage. Two cultivars of forage soybean (Kodiak and Mammouth) were field-grown, harvested at the R6 stage, and ensiled in mini-silos (n = 3) for 0, 2, 4, 8, 16 and 45 d. Two ruminally fistulated cows were used to determine in situ ruminal nutrient degradabilities of the 45-d silages. Both cultivars went through slow fermentation as indicated by a gradual decline in pH up to day 45 post-ensiling. Lactic acid concentration increased throughout ensiling and was higher for Mammouth than Kodiak except at day 45 post-ensiling. Analysis of the 45-d silages showed that Mammouth contained higher neutral detergent fiber (NDF, 490 vs. 444 g kg-1), acid detergent fiber (371 vs. 353 g kg-1) and acid detergent lignin (81 vs. 64 g kg-1) than Kodiak. However, crude protein (CP) concentration was higher for Kodiak than Mammouth. Mammouth silage had lower buffer soluble protein and higher neutral and acid detergent insoluble protein concentrations than Kodiak silage. Results of the in situ study indicated that Kodiak silage had greater ruminal dry matter (606 vs. 549 g kg-1), CP (828 vs. 752 g kg -1) and NDF (272 vs. 227 g kg-1) degradabilities than Mammouth. It was concluded that chemical composition and ruminal nutrient degradabilities of forage soybean silage were significantly influenced by cultivar. Key words: Soybean [Glycine max (L.) Merill.], silage, ensiling; forage quality, nutrient degradability

Protein degradation kinetics of untreated and treated soybean meal using SDS-PAGE

This study was completed to determine dry matter (DM) and crude protein (CP) degradation characteristics of untreated, autoclaved (127°C for 10 min), ethanol (700 ml/l for 1 h) and xylose (20 g/kg DM) treated soybean meal (SBM) using nylon bags and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. Nylon bags of untreated and treated SBM were suspended in the rumen of three Holstein steers from 0 to 48 h, and data was fitted to a non-linear degradation model to calculate effective rumen degradation (ERD). Autoclave, ethanol and xylose treatments decreased (P<0.05) ERD of CP. The ERD of untreated, autoclaved, alcohol and xylose treated SBM CP at rumen outflow rate of 0.05/h were 0.683, 0.480, 0.466 and 0.299, respectively. From slab gel analysis and densitometric scanning, SBM proteins were composed of two major components; β-conglycinin and glycinin, accounting for approximately 30 and 40% of buffer soluble meal protein, respectively. The approximate molecular weights of the β-conglycinin ά, α and β subunits were 90.5, 71.5 and 55.2 kDa and the glycinin acidic and basic subunits were 37.6 and 19.8 kDa, respectively. Electrophoretic patterns of untreated, autoclaved, ethanol and xylose treated SBM protein residues revealed that β-conglycinin α and ά subunits were degraded completely within 2, 12, 12 and 6 h of incubation in the rumen, whereas the β subunit of β-conglycinin were degraded after 8, 12, 24 and 12 h of incubation, respectively. Acidic subunits of glycinin were degraded after 12 and 48 h of incubation, but basic subunits of glycinin were not degraded completely until 48 h of incubation in untreated and treated SBM, respectively. The in vitro digestibility (mimic of post-ruminal digestion) of undegradable CP from untreated and treated SBM at 0, 8, 12 and 24 h of incubation increased as incubation time increased. Autoclaving and ethanol treatment increased, but xylose treatment decreased (P<0.05), in vitro digestibility of CP. Processing of SBM by autoclave and with ethanol had greater potential to increase rumen undegradable protein, with positive effects on in vitro CP digestibility, than xylose treatment. SDS-PAGE indicated that the basic subunit of glycinin make an appreciable contribution to metabolizable protein when untreated SBM (whereas β-conglycinin and glycinin when autoclaved, xylose or ethanol treated SBM) is fed to ruminants.

Measurement of soluble protein degradation in the rumen

Twenty-five feeds were screened for buffer soluble N (BSN) and its constituent protein N, peptide N, amino acid N and ammonia N. In vitro degradation rates of buffer soluble protein were determined in 11 of these feeds. To estimate rumen in vivo degradation rates, buffer soluble protein from peas or cold pressed rapeseed cake was also given as a pulse dose, together with a liquid marker (CoEDTA), to lactating rumen-fistulated dairy cows. Buffer soluble N ranged from 36 to 802 g/kg N. There were also large differences in BSN composition. Buffer soluble protein N ranged from 0 to 874 g/kg BSN with peas and lupine seeds containing the highest amounts. There were differences ( P<0.05) in the buffer soluble protein in vitro degradation rates among the feeds. Degradation rate was highest for casein (1.0 h −1) and lowest for linseed cake (0.18 h −1). Buffer soluble protein from soybean meal, peas and lupine were degraded at intermediate rates, 0.46, 0.39, and 0.34 h −1, respectively. In the in vivo experiment, soluble proteins were rapidly degraded and ruminal protein concentrations had reached baseline values within 1 h post dosing. Results were, however, compromised by very slow mixing in the rumen. The increase in ammonia N concentration when cows received soluble protein from pea, in contrast to rapeseed cake, supports the in vitro results, where peas were degraded at a higher rate (0.39 h −1 versus 0.19 h −1). Soluble proteins from different feeds were degraded at substantially different rates. Feeds high in soluble proteins, like peas and lupines, should be studied in vitro and not in sacco, since the latter method cannot measure the degradation rates of soluble proteins.

Biochemical changes during seed development in Pinus taeda L.

Amino acids, polyamines, 3-indoleacetic acid (IAA), abscisic acid (ABA), buffer-soluble protein and starch contents and dry matter accumulation were analyzed in megagametophytes containing developing embryos during seed development in Pinus taeda. The highest total amino acids and polyamine contents occurred at the cotyledonary stage, followed by a significant decrease in the mature seed. Free polyamines exhibited higher levels than conjugated ones, with putrescine being the predominant type until the cotyledonary stage, and spermidine at the mature seed stage. IAA content increased continually from the globular stage reaching the maximum at the cotyledonary stage, followed by a decrease in the mature seed. The highest ABA level occurred at the globular stage, followed by a continuous reduction until stabilization at the pre-cotyledonary stage. Buffer-soluble protein and starch contents, and dry matter increased progressively during development, reaching their maximum values at the mature stage.

Inter-relationship between electrophoretic characteristics of pseudocereal and cereal proteins and their microscopic structure for possible substitution based on nutritional evaluation

Amaranth, soybean and maize were screened for proteins and their nutritional value. Isopropanol-soluble protein and buffer-soluble protein fractions were extracted from seeds and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The comparison of the identity and differences between investigated plants was carried out by the obtained SDS-PAGE electrophoretic patterns, and their microstructure was determined by scanning electron microscopy. Electrophoretic patterns of extracted proteins have shown that the main protein subunits were concentrated between 10 and 50 kDa. Variations were found in major fractions and minor bands as well as in the fine structure. The microstructure of pseudocereal and cereal protein fractions was inter-related with the results obtained by their electrophoretic separation. Pseudocereal amaranth can be used as a nutritive substitute of cereal maize in functional foods.