Comparison of methods to determine methane emissions from dairy cows in farm conditions.

Nutritional and animal-selection strategies to mitigate enteric methane (CH4) depend on accurate, cost-effective methods to determine emissions from a large number of animals. The objective of the present study was to compare 2 spot-sampling methods to determine CH4 emissions from dairy cows, using gas quantification equipment installed in concentrate feeders or automatic milking stalls. In the first method (sniffer method), CH4 and carbon dioxide (CO2) concentrations were measured in close proximity to the muzzle of the animal, and average CH4 concentrations or CH4/CO2 ratio was calculated. In the second method (flux method), measurement of CH4 and CO2 concentration was combined with an active airflow inside the feed troughs for capture of emitted gas and measurements of CH4 and CO2 fluxes. A muzzle sensor was used allowing data to be filtered when the muzzle was not near the sampling inlet. In a laboratory study, a model cow head was built that emitted CO2 at a constant rate. It was found that CO2 concentrations using the sniffer method decreased up to 39% when the distance of the muzzle from the sampling inlet increased to 30cm, but no muzzle-position effects were observed for the flux method. The methods were compared in 2 on-farm studies conducted using 32 (experiment 1) or 59 (experiment 2) cows in a switch-back design of 5 (experiment 1) or 4 (experiment 2) periods for replicated comparisons between methods. Between-cow coefficient of variation (CV) in CH4 was smaller for the flux than the sniffer method (experiment 1, CV=11.0 vs. 17.5%, and experiment 2, 17.6 vs. 28.0%). Repeatability of the measurements from both methods were high (0.72-0.88), but the relationship between the sniffer and flux methods was weak (R(2)=0.09 in both experiments). With the flux method CH4 was found to be correlated to dry matter intake or body weight, but this was not the case with the sniffer method. The CH4/CO2 ratio was more highly correlated between the flux and sniffer methods (R(2)=0.30), and CV was similar (6.4-8.8%). In experiment 2, cow muzzle position was highly repeatable (0.82) and influenced sniffer and flux method results when not filtered for muzzle position. It was concluded that the flux method provides more reliable estimates of CH4 emissions than the sniffer method. The sniffer method appears to be affected by variable air-mixing conditions created by geometry of feed trough, muzzle movement, and muzzle position.

Integrating spot short-term measurements of carbon emissions and backward dietary energy partition calculations to estimate intake in lactating dairy cows fed ad libitum or restricted.

The objective of this study was to use spot short-term measurements of CH4 (QCH4) and CO2 (QCO2) integrated with backward dietary energy partition calculations to estimate dry matter intake (DMI) in lactating dairy cows. Twelve multiparous cows averaging 173±37d in milk and 4 primiparous cows averaging 179±27d in milk were blocked by days in milk, parity, and DMI (as a percentage of body weight) and, within each block, randomly assigned to 1 of 2 treatments: ad libitum intake (AL) or restricted intake (RI=90% DMI) according to a crossover design. Each experimental period lasted 22d with 14d for treatments adaptation and 8d for data and sample collection. Diets contained (dry matter basis): 40% corn silage, 12% grass-legume haylage, and 48% concentrate. Spot short-term gas measurements were taken in 5-min sampling periods from 15 cows (1 cow refused sampling) using a portable, automated, open-circuit gas quantification system (GreenFeed, C-Lock Inc., Rapid City, SD) with intervals of 12h between the 2daily samples. Sampling points were advanced 2h from a day to the next to yield 16 gas samples per cow over 8d to account for diurnal variation in QCH4 and QCO2. The following equations were used sequentially to estimate DMI: (1) heat production (MJ/d)=(4.96 + 16.07 ÷ respiratory quotient) × QCO2; respiratory quotient=0.95; (2) metabolizable energy intake (MJ/d)=(heat production + milk energy) ± tissue energy balance; (3) digestible energy (DE) intake (MJ/d)=metabolizable energy + CH4 energy + urinary energy; (4) gross energy (GE) intake (MJ/d)=DE + [(DE ÷ in vitro true dry matter digestibility) - DE]; and (5) DMI (kg/d)=GE intake estimated ÷ diet GE concentration. Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) and Fit Model procedure in JMP (α=0.05; SAS Institute Inc.). Cows significantly differed in DMI measured (23.8 vs. 22.4kg/d for AL and RI, respectively). Dry matter intake estimated using QCH4 and QCO2 coupled with dietary backward energy partition calculations (Equations 1 to 5 above) was highest in cows fed for AL (22.5 vs. 20.2kg/d). The resulting R(2) were 0.28 between DMI measured and DMI estimated by gaseous measurements, and 0.36 between DMI measured and DMI predicted by the National Research Council model (2001). Results showed that spot short-term measurements of QCH4 and QCO2 coupled with dietary backward estimations of energy partition underestimated DMI by 7.8%. However, the approach proposed herein was able to significantly discriminate differences in DMI between cows fed for AL or RI.

Differential rumination, intake, and enteric methane production of dairy cows in a pasture-based automatic milking system.

Proper performance monitoring of cows on pasture-based diets is crucial to inform nutritional recommendations that minimize undesirable effects of high ruminant CH4 emissions into the environment. The prediction of linkages between rumination patterns, methane emissions, and correlated production traits of cows in a pasture-based automatic milking system was tested. A previous 10-d baseline measurement of rumination activity by acoustic methodology of 156 Holstein-Friesian cows was used for frequency analysis of rumination time and identification of 2 treatment groups (n = 37 cows/group) represented by cows with consistently high (HR; 75th rumination percentile = 617.55 ± 81.37 min/d) or low (LR; 25th rumination percentile = 356.65 ± 72.67 min/d) rumination. The HR and LR cows were paired by nearest parity, days in milk, body weight (BW), and previous 10-d milk production, and within pairs randomly assigned to 1 of 2 experimental groups managed on a voluntary milking system with diets consisting of at least 75% pasture, plus concentrates. Animal traits, including rumination time, mass flux of CH4 (QCH4) and carbon dioxide (QCO2), milk production, and estimated dry matter intake according to individual QCO2 fluxes over a 22-d period were analyzed with repeated measure mixed models for a completely randomized design, structural equation modeling, and nonlinear regression. High rumination and methane was seen in older and heavier cows that had greater estimated dry matter intake and milk production. A consistent difference in rumination time and QCH4 across days was detected between HR and LR, even after adjustment for metabolic BW. Estimated dry matter intake had direct positive effects on rumination and QCH4, but no independent direct effect of rumination on QCH4 was detected. The LR cows produced more QCH4/milk, associated with lower milk, BW, concentrate intake, and greater activity at pasture. A typical dilution of maintenance effect on QCH4/milk was detected as a consequence of increasing milk yield and similar significant reduction of QCO2/milk. The results raise challenging questions regarding the rumination patterning of grazing dairy cows and alternatives to reduce ruminant methane emissions in grazing dairy cows.

Is differential use of Juniperus monosperma by small ruminants driven by terpenoid concentration?

Differential plant use by herbivores has been observed for several woody plant species and has frequently been attributed to plant secondary metabolites. We examined the relationship between terpenoid concentration and Juniperus monosperma herbivory by small ruminants. Two groups of animals (10 goats or 5 goats plus 4 sheep) browsed 16 paddocks (20 × 30 m) containing one-seed juniper for six days during two seasons. Juniper leaves were sampled from 311 saplings immediately after browsing. Saplings were categorized by size (short [<0.5 m], medium [0.5-1.0 m], or tall [>1.0 m]), and by browsing intensity (light [<33 %], moderate [33-66 %], or heavy [>66 %]). Juniper bark was collected from 12 saplings during spring. Total estimated terpenoid concentrations in leaves and bark were 18.3 ± 0.3 and 8.9 ± 0.8 mg/g, respectively, and the dominant terpene in both tissues was α-pinene (11.1 ± 0.2 and 7.6 ± 0.7 mg/g, respectively). Total terpenoid concentration of juniper leaves was greater in spring than summer (20.6 ± 0.5 vs. 16.7 ± 0.3 mg/g, respectively) and was lower in short saplings than medium or tall saplings (16.5 ± 0.6 vs. 19.8 ± 0.4 and 19.5 ± 0.4 mg/g, respectively). Total terpenoid concentration of leaves also differed among the three defoliation categories (21.2 ± 0.6, 18.7 ± 0.5, and 16.1 ± 0.4 mg/g for light, moderate, and heavy, respectively). The smallest subset of terpenoids able to discriminate between light and heavy browsing intensity categories included eight compounds ([E]-ß-farnesene, bornyl acetate, γ-eudesmol, endo-fenchyl acetate, γ-cadinene, α-pinene, cis-piperitol, and cis-p-menth-2-en-1-ol). Our results suggest terpenoid concentrations in one-seed juniper are related to season, sapling size, and browsing by small ruminants.

Grazing of cover crops in integrated crop-livestock systems.

Conventional agriculture is specializing rapidly into the management of few monoculture crops, threatening crop diversity and questioning the sustainability of extensive cropping systems. The grazing of cover crops in integrated crop-livestock systems could be a feasible biologically based technology to restore crop diversity and mitigate ecological issues in cropping systems. However, there is limited evidence on plausible synergies or trade-offs for the practice, and about how grazing plans could affect the herbage production and services from cover crops. This work assessed the effects of cattle grazing on the primary and secondary production of annual ryegrass (Lolium multiflorum) in an integrated ryegrass-soybean rotation system. Specifically, the prediction for synergistic effects of cattle grazing on the ryegrass herbage production, residual crop cover and animal performance were tested in a 2-year (2014 and 2015) study comprising a randomized complete block design of four grazing intensity treatments, replicated three times. A no-cattle grazing treatment (NG), used as control, or continuous grazing with Holstein heifers (~220kg live weight) at targeted sward heights of 5, 10, 15 and 20cm (hereafter referred as G5, G10, G15 and G20, respectively) was applied to ryegrass plots. The herbage production and residual herbage cover of ryegrass, and the average daily gain (ADG, kg/day) and live weight gain per hectare (LWG, kg/ha) of heifers were analyzed by ANOVA (P<0.05) and compared by the TukeyHSD test (P<0.05). Regression models were used to estimate relationships between herbage production, animal performance and sward height. The herbage production was 60% higher (P<0.01) for the grazing treatments compared to NG. The residual herbage for G15 and G20 was not different than that for NG and increased linearly as sward heights increased, reaching highest values for G15 and G20. Maximum ADG was 1.10kg/day for ryegrass grazed at a 20.6cm height, whereas maximum LWG was 427kg/ha for ryegrass grazed to a 16.1cm height. The results support the hypothesis for synergistic effects of using annual ryegrass as a dual forage and service cover crop. Moderate grazing intensity to sward height of 12-18cm with continuous stocking led to optimized forage production and utilization by dairy heifers.

Monitoring and assessment of ingestive chewing sounds for prediction of herbage intake rate in grazing cattle.

Accurate measurement of herbage intake rate is critical to advance knowledge of the ecology of grazing ruminants. This experiment tested the integration of behavioral and acoustic measurements of chewing and biting to estimate herbage dry matter intake (DMI) in dairy cows offered micro-swards of contrasting plant structure. Micro-swards constructed with plastic pots were offered to three lactating Holstein cows (608±24.9 kg of BW) in individual grazing sessions (n=48). Treatments were a factorial combination of two forage species (alfalfa and fescue) and two plant heights (tall=25±3.8 cm and short=12±1.9 cm) and were offered on a gradient of increasing herbage mass (10 to 30 pots) and number of bites (~10 to 40 bites). During each grazing session, sounds of biting and chewing were recorded with a wireless microphone placed on the cows' foreheads and a digital video camera to allow synchronized audio and video recordings. Dry matter intake rate was higher in tall alfalfa than in the other three treatments (32±1.6 v. 19±1.2 g/min). A high proportion of jaw movements in every grazing session (23 to 36%) were compound jaw movements (chew-bites) that appeared to be a key component of chewing and biting efficiency and of the ability of cows to regulate intake rate. Dry matter intake was accurately predicted based on easily observable behavioral and acoustic variables. Chewing sound energy measured as energy flux density (EFD) was linearly related to DMI, with 74% of EFD variation explained by DMI. Total chewing EFD, number of chew-bites and plant height (tall v. short) were the most important predictors of DMI. The best model explained 91% of the variation in DMI with a coefficient of variation of 17%. Ingestive sounds integrate valuable information to remotely monitor feeding behavior and predict DMI in grazing cows.

Lymphokine-independent, leukemia cell-mediated induction of tumor necrosis factor in human monocytes.

Unactivated human blood monocytes and monocytic THP-1 cells were found to respond to some leukemia cells by tumor necrosis factor (TNF) production. The TNF production by THP-1 cells in response to K562 cells was preceded by a rapid rise in [Ca2+]i, initiated within 1 h and terminated within 4 h as a refractory state took over. Neither the amount nor the duration of TNF production was enhanced by gamma-interferon. The P32/ISH cells did not induce a significant [Ca2+]i change of TNF production, while MOLT-4 cells failed to induce TNF despite their capacity to mobilize Ca2+ in THP-1 cells. The failure of P32/ISH or MOLT-4 to induce TNF was attributed primarily to a lack of stimulatory membrane molecules rather than to suppression by an inhibitory component, since liposomes carrying membrane components of K562 and MOLT-4 or P32/ISH in varying proportions elicited TNF production that precisely reflected the K562 proportion. The ability of K562 to induce TNF was selectively impaired by trypsin, whereas the ability to mobilize [Ca2+]i was more sensitive to glutaraldehyde, although once the latter activity was extinguished, the K562 cell could no longer induce TNF. These results suggest that some leukemia cells are equipped with two or more signaling membrane moieties which together stimulate monocytes for transient tumoricidal expression in the preimmune stage.

Heterogeneity of broad bean legumin.

Legumins from various cultivars were fractionated on a DEAE-Sephadex A-50 column and their subunit structures were investigated. The results obtained indicated the heterogeneity of legumin molecular species. The nature of the heterogeneity was common to all cultivars examined from the standpoint of the molecular sizes of the subunits. Four groups of molecular species with molecular weights of 320 000, 350 000, 380 000 and 400 000 were detected by gradient gel electrophoresis. The smallest molecular species was composed of only three kinds of subunit, with molecular weights of 20 500, 23 000 and 36 000, and the largest one was composed of five kinds of subunit with molecular weights of 19 000, 23 000, 36 000, 49 000 and 51 000. All molecular species were composed of intermediary subunits which consisted of acidic and basic subunits. The intermediary subunits with molecular weights of 61 700, 59 800 and 48 000 are composed of the acidic subunits with molecular weights of 51 000, 49 000 and 36 000 and the basic subunits with molecular weights of 19 000, 19 000 and 23 000 or 20 500, respectively. The possible molecular species constructed from the subunit groups classified according to their sizes were presented.

N-glycosylation does not affect assembly and targeting of proglycinin in yeast.

Glycinin, a simple protein, and beta-conglycinin, a glycoprotein, are the dominant storage proteins of soybean and are suggested to be derived from a common ancestor. To investigate why glycinin does not require glycosylation for its maturation, we attempted N-glycosylation of proglycinin A1aB1b using site-directed mutagenesis and yeast expression system. An N-glycosylation consensus sequence Asn-X-Ser/Thr was created at positions 103, 183, 196, 284 and 457 in the variable regions being strongly hydrophilic revealed from the alignment of amino acid sequences of various glycinin-type proteins. Among five mutant proglycinins (Q103N, H183N, G198T, S284N, N459T), Q103N was fully glycosylated, and H183N and N459T were partly (around 20% of the expressed proteins), whereas others were barely or not glycosylated. The glycosylated proglycinin was susceptible to endo-beta-N-acetylglucosamidase and N-glycanase cleavages. N-glycosylation did not cause inconveniences to processing of signal peptide, assembly into trimers and targeting into the vacuoles. Thermal and trypsin sensitivity analyses of the glycosylated proglycinin suggested that N-linked glycan prevents protein-protein interaction but does not stabilize the protein conformation. The reason why glycinin does not require N-glycosylation for its maturation is discussed.

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K.

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.

Crystal structure of soybean proglycinin A1aB1b homotrimer.

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.

Crystallization and preliminary X-ray crystallographic analysis of the soybrean proglycinin expressed in Escherichia coli.

Glycinin is one of the dominant storage proteins of soybean seeds. Soybean proglycinin expressed in Escherichia coli has been crystallized from Tris.HCl buffer (pH 7.6) by the dialysis equilibrium method. The crystals belong to the tetragonal system, space group P4(1) or P4(3), with unit cell dimensions of a = b = 115.2 A, and c = 147.1 A. The asymmetric unit contains three molecules of proglycinin, with crystal volume per protein mass (Vm) of 3.05 A3/Da and solvent content of 58.4% by volume. The crystals diffract X-rays to a resolution limit of at least 2.9 A and are resistant to X-ray radiation damage. They appear to be suitable for X-ray structure analysis.

Accumulation of soybean glycinin and its assembly with the glutelins in rice

Saline-soluble glycinins and insoluble glutelins are the major storage proteins in soybean (Glycine max) and rice (Oryza sativa), respectively. In spite of their differences in solubility properties, both proteins are members of the 11S globulin gene family based on their similarities in primary sequences and processing of the coded protein. Wild-type and methionine-modified glycinin coding sequences were expressed in transgenic rice plants under the control of the rice glutelin GluB-1 promoter. Glycinins were specifically synthesized in the endosperm tissue and co-localized with glutelins in type II protein bodies. They assembled into 7S and 11S species, similar to what was observed in developing soybean seeds. This pattern was quite different from that displayed by the rice glutelins in untransformed plants, in which processed subunits sedimenting at 2S were apparent. In glycinin-expressing transgenic plants, however, glutelins were observed sedimenting at 7S and 11S with lesser amounts in the 2S region. A portion of the glycinins was also found associated in the insoluble glutelin fraction. Renaturation experiments suggested that the hybrid glycinin-glutelin oligomers were formed through specific interactions. Overall, these results indicate that despite significant differences in the assembly of soybean glycinin and rice glutelin, both proteins can assemble with each other to form soluble hexameric oligomers or insoluble aggregates.

Conditions which favor the C1-fixation by mouse IgG1.

The capacity of mouse IgG1 to mediate activation of the classical complement pathway was reinvestigated with purified C1 and under various conditions. Monoclonal and polyclonal IgG1 antibodies to TNP, which failed to show a haemolytic activity on TNP-SRBC with low epitopic density at physiologic salt concentration, invariably restored their activity when reacted to TNP-SRBC with relatively higher epitopic density and at a reduced salt concentration. Unliganded, monomeric IgG1 too proved to possess an inherent C1-binding capacity, but the susceptibility to competing salt was extremely high. These results were seemingly in accordance with the reduced number of basic amino acids in the plausible C1-binding region of the molecule.

Co-operation between the pair of C gamma 2 domains in Clq-binding by rabbit IgG.

The single site binding constants of rabbit IgG and its plasmin-derived fragments F(acb)2, Facb and F(ab)2 for human C1q were measured by the sedimentation velocity method. The intact IgG and F(acb)2 having the paired C gamma 2 domains gave an identical association constant at 20 degrees C (Ka) of 3.02 X 10(4) M-1 in the presence of a physiological concn of salt and on the basis of six sites per C1q. The C1q-binding affinity was found to be decreased to 1.04 X 10(4) M-1 in the reduced, monomerized fragment Facb. Under the same conditions F(ab)2, which is completely unable to activate the classical complement cascade, gave an apparent C1q-affinity of 0.36 X 10(4) M-1. The results, together with previous observations, led us to the conclusion that the C1q-binding site of rabbit IgG is constituted associatively by the pair of C gamma 2 domains, each of which providing a limited, complementary part of the binding free energy between IgG and C1q.

Co-operative interaction of subcomponents of the first component of complement with IgG: a functional defect of dimeric Facb from rabbit IgG.

By following dissociation kinetics of radiolabelled C1q from rabbit IgG antibody-sensitized sheep red blood cells (SRBC) before and after its incorporation in the C1 complex, it was demonstrated that the binding stability is markedly enhanced by the presence of the C1r2-C1s2 subunit of C1 which by itself exhibits no significant binding capacity to immune complexes. The dissociation of C1q was decreased by up to 95%, the extent of decrease being pronounced as the cell surface IgG antibody density increased. However, such a stabilizing effect of C1r2-C1s2 was largely abolished when SRBC sensitized with the dimeric fragment F(acb)2 lacking C gamma 3 was used as the C1 binder, whereas the dissociation rate of uncomplexed C1q from F(acb)2-sensitized cells was similar to that from whole IgG-sensitized cells. It was also shown that, although the C1r2-C1s2 subunit is dissociated selectively from C1 bound to either IgG- or F(acb)2-sensitized cells in the presence of EDTA, it is held on much longer by the former cells than the latter cells. These results were taken to indicate that, although the C1 fixation by immune complexes of IgG is undertaken primarily by the interaction between C1q and the C gamma 2 domain, it is also strengthened by the secondary interaction between the C1r2-C1s2 subunit of C1 and the C gamma 3 domain or a structure which is dependent on the pair of C gamma 3 domains.

Preparation and biologic characterization of fragments containing dimeric and monomeric C gamma 2 domain of rabbit IgG.

A number of fragments derived from acid-treated rabbit IgG by digestion with plasmin have been separated by high-resolution gel filtration. Fragments isolated included a dimer and monomer Facb, named F(acb)2 and Facb, respectively and a heterodimer composed of Facb and Fab subunits, named F(acb)(ab). A C gamma 2 fragment was obtained by papain digestion of Facb. A heterodimer composed of Facb and Fab', named F(acb)(ab'), was also prepared by oxidizing a reduced mixture of these fragments. Fragments thus obtained are classified into two groups--those carrying paired C gamma 2 domains, i.e. F(acb)2, and the disulfide-linked dimeric C gamma 2 fragment; and those having a single C gamma 2 domain, i.e. reduced, alkylated Facb and C gamma 2 fragment, F(acb)(ab) and F(acb)(ab'). These fragments exhibited marked differences in their capacity to activate complement in assay systems of hemolysis and complement consumption by immune complexes or aggregates on polystyrene latex. Fragments of the former group could activate complement but with a definitely reduced efficiency (50%) compared to intact IgG, whereas fragments of the latter group were practically inactive. Although it was not determined whether the C1-binding capacity itself is changed by monomerization of the C gamma 2 domain, the results suggested that the intact paired C gamma 2 module is required at least for the activation process of complement.

Differential response to stocking rates and feeding by two genotypes of Holstein-Friesian cows in a pasture-based automatic milking system.

The throughput of automatic milking systems (AMS) is likely affected by differential traffic behavior and subsequent effects on the milking frequency and milk production of cows. This study investigated the effect of increasing stocking rate and partial mixed ration (PMR) on the milk production, dry matter intake (DMI), feed conversion efficiency (FCE) and use of AMS by two genotypes of Holstein-Friesian cows in mid-lactation. The study lasted 8 weeks and consisted in a factorial arrangement of two genotypes of dairy cattle, United States Holstein (USH) or New Zealand Friesian (NZF), and two pasture-based feeding treatments, a low stocking rate system (2 cows/ha) fed temperate pasture and concentrate, or a high stocking rate system (HSR; 3 cows/ha) fed same pasture and concentrate plus PMR. A total of 28 cows, 14 USH and 14 NZF, were used for comparisons, with 12 cows, six USH and six NZF, also used for tracking of animal movements. Data were analyzed by repeated measure mixed models for a completely randomized design. No differences (P>0.05) in pre- or post-grazing herbage mass, DMI and FCE were detected in response to increases in stocking rate and PMR feeding in HSR. However, there was a significant (P<0.05) grazing treatment×genotype×week interaction on milk production, explained by differential responses of genotypes to changes in herbage mass over time (P<0.001). A reduction (P<0.01) in hours spent on pasture was detected in response to PMR supplementation in HSR; this reduction was greater (P=0.01) for USH than NZF cows (6 v. 2 h, respectively). Regardless of the grazing treatment, USH cows had greater (P=0.02) milking frequency (2.51 v. 2.26±0.08 milkings/day) and greater (P<0.01) milk yield (27.3 v. 16.0±1.2 kg/day), energy-corrected milk (24.8 v. 16.5±1.0 kg/day), DMI (22.1 v. 16.6±0.8 kg/day) and FCE (1.25 v. 1.01±0.06 kg/kg) than NZF cows. There was also a different distribution of milkings/h between genotypes (P<0.001), with patterns of milkings/h shifting (P<0.001) as a consequence of PMR feeding in HSR. Results confirmed the improved FCE of grazing dairy cows with greater milk production and suggested the potential use of PMR feeding as a tactical decision to managing HSR and milkings/day in AMS farms.

Signal sequence of preproglycinin affects production of the expressed protein in Escherichia coli.

The effect of the signal peptide portion on the bacterial production of preproglycinin, a precursor of soybean storage protein, was examined. Nucleotide sequences corresponding to the signal peptide and the mature N-terminal region were deleted stepwise from the cDNA encoding the glycinin A1aB1b subunit precursor, and the deleted cDNAs were placed under the control of trc promoter in an expression vector pKK233-2. When the amounts of the protein products in Escherichia coli from each expression plasmid were determined, no accumulation of preproglycinin was observed from the plasmids with the full length or the five amino acids of the signal sequence. However, significant accumulation of the preproglycinin homologue proteins was noted from the plasmids retaining less than three amino acids of the signal sequence depending on the extent of deletion. N-terminal amino acid sequences of the products coincided with those predicted from the deleted cDNAs. The preproglycinin homologue proteins expressed from the mutant plasmids assembled into trimers of about 8S.

Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin(2-7).

The potent anti-hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2-7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean beta-conglycinin alpha' subunit. The native alpha' subunit as well as the modified, RPLKPW-containing alpha' subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion-exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW-containing alpha' subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW-containing alpha' subunit given orally at a dose of 10 mg/kg exerted an anti-hypertensive effect in spontaneously hypertensive rats, unlike the native alpha' subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site-directed mutagenesis could practically function in vivo even at a low dose.