Legume grains enhance ileal losses of specific endogenous serine-protease proteins in weaned pigs

Feeding legume grains to pigs usually increases losses of endogenous proteins at the terminal ileum. However, the identity of such proteins is largely unknown. This study was undertaken to determine the ileal flow and identity of soluble proteins present in large concentrations in ileal digesta of young pigs fed soybean meal (SBM), peas (P), faba beans (FB), or blue lupin (L) in expt. 1, and white (WPC) or black (BPC) chickpeas in expt. 2. Protein in the control diet (C) was provided by casein. Ileal digesta proteins were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, Coomassie blue staining, densitometry and N-terminal amino acid sequencing. Three protein bands at molecular masses of 25, 27, and 30 kDa had a higher ileal flow (P < 0.05) in the pigs fed the legume-based diets compared to those fed the control diet in expt. 2. This was true for the 25- and 30-kDa proteins (P < 0.05) and the 27-kDa protein (P < 0.10) in pigs fed the legume-containing diets in expt. 1. These proteins shared N-terminal amino acid sequences with enzymes of the serine protease family including pig trypsin (25 kDa) and blood coagulation factor IX or chymotrypsin (27 and 30 kDa).     

Comparison of the isotope dilution method for determination of the ileal endogenous amino acid losses with labelled diet and labelled pigs

The aims of the present study were first to compare the amino acid dilution method performed using labelled animals with that using labelled diets, and second to determine real digestibilities and total ileal endogenous losses of N and amino acids. Two diets containing pea cultivars (Solara and Amino) and a protein-free diet were compared in a 3 x 3 Latin-square experiment. The three pigs were each prepared with an ileo-rectal anastomosis and were continuously infused with [1-13C]leucine. For each cultivar, 15N-labelled and unlabelled diets were formulated. The real digestibility and endogenous losses of leucine were higher when obtained by labelling the pig than by labelling the foodstuff. This was due either to the inadequate estimation of the endogenous protein enrichment in the first case or to the importance of dietary N recycling in the second case. However, in both cases the ileal endogenous losses of N and amino acids were higher than the basal losses determined with the protein-free diet. There were significant differences between the two pea cultivars in terms of phenylalanine and leucine when measured with labelled diets. It is suggested that, although ileal endogenous losses may be underestimated, using labelled feedstuffs is of great interest due to the direct estimation of the individual amounts of amino acids.     

Identification of organochlorine pesticides in crude extracts by mass spectrometry

A method is described for identifying a chlorinated pesticide in crude extracts (i) at 1 ppm by low resolution mass spectrometry with the aid of a recently developed temperature controlled probe and (ii) at 0.01 ppm by high resolution techniques using a photoplate record to determine the mass deficient ions containing chlorine.This analytical method is feasible wherever ions of the same elemental composition as those derived from an unknown are not otherwise present in the crude extract mass spectrum.Issued as NRCC No. 11254.Presented at the CIS-ACS Meeting, Toronto, Canada, May 24–29, 1970.     

Identification of organochlorine pesticides in crude extracts by mass spectrometry

Bulletin of Environmental Contamination and Toxicology - A method is described for identifying a chlorinated pesticide in crude extracts (i) at 1 ppm by low resolution mass spectrometry with the...     

Identification of residual blood proteins in ticks by mass spectrometry proteomics

Mass spectrometry-based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including alpha- and beta-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks.     

The effect of dietary peptide concentration on endogenous ileal amino acid loss in the growing pig

The aim of the present study was to determine whether dietary peptide concentration had an effect on endogenous ileal amino acid flow in the growing pig. Eight 33 kg live weight entire male pigs had post-valve T-caecum (PVTC) cannulas surgically implanted for the collection of ileal digesta. The pigs were fed twice daily at 100 g/kg metabolic body weight per d and were given diets containing enzyme-hydrolysed casein (EHC) at 0, 50, 100 and 200 g/kg in a Latin-square design. A basal casein-based diet was fed to the pigs for 6 d periods between receiving the experimental diets. The pigs received the experimental diets for 8 d periods, with continuous collection of digesta for 24 h on each of the fifth and eighth days. The endogenous ileal amino acid flows were determined with reference to recovery of the marker, Cr, directly for pigs receiving the protein-free diet or after centrifugation and ultrafiltration (10,000 Da molecular mass cut-off) for pigs on the EHC-based diets. Mean endogenous ileal N flows were 1753, 1948, 2851 and 5743 micrograms/g DM intake when the pigs received diets containing 0, 50, 100 and 200 g EHC/kg respectively. There was a significant (P < 0.05) effect of dietary peptide concentration on the endogenous ileal flows of N and all of the amino acids, with an increase in endogenous ileal amino flow with increasing dietary EHC concentration.     

The binding of dietary protein by sorghum tannins in the digestive tract of pigs

The effects of reconstitution of sorghums on dry matter, energy, protein and amino acid digestibilities, and the nature of protein binding by sorghum tannins during digestion were investigated. Grains from a high and a low tannin sorghum were reconstituted by adding 30% (wt/wt) distilled water to the grain and stored at 25 degrees C for 20 days with an acetic-propionic acid mixture added to deter fungal growth. Another lot of grain from the same sources was untreated and used as control. The sorghum grains were then incorporated at an 85% level in diets for a digestibility trial with pigs cannulated at the terminal ileum. The digestibilities of dry matter, protein and amino acids at the terminal ileum were lower than the corresponding values measured over the total digestive tract. Reconstitution improved the digestibilities of dry matter, energy, protein and amino acids in the high but not low tannin sorghum diets. The improvement in digestibility of individual amino acids ranged from 7.5 to 23.5%. The tannin-associated proteins were more hydrophobic than the dietary protein. The results suggest that hydrophobic bonding is important in the formation of tannin-protein complexes in the digestive tract of pigs.     

The effects of feed intake and purified cellulose on the endogenous ileal amino acid flow in growing pigs.

The effects of level of feed intake (0.8, 1.2 and 1.6 kg/d) and body-weight of the pig (49 and 92 kg) in Expt 1, and dietary neutral-detergent fibre (NDF; 30, 60, 90, 120 and 150 g/kg) in Expt 2 on the endogenous ileal flow of amino acids (AA) and nitrogen were studied with protein-free diets into which purified wood cellulose was incorporated at the expense of maize starch. In Expt 1, one of the protein-free diets containing 90 g NDF/kg was used. Female pigs were fitted with a simple 'T' cannula at the terminal ileum. In Expt 1, the endogenous ileal AA and N flow, expressed as g/kg dry matter (DM) intake, decreased significantly (P < 0.05) with increasing DM intake, except for proline. By contrast, the values expressed as g/d remained constant. There was no significant difference in endogenous ileal flow (P > 0.05) between initial body-weights of 45 and 90 kg except for histidine, isoleucine, phenylalanine, threonine, valine and serine, in which the ileal flow determined at the higher body-weight was significantly higher (P < 0.05). In Expt 2, the effects of dietary cellulose levels on the endogenous ileal flow of AA and N were not significant (P > 0.05), although the values tended to increase as dietary cellulose levels increased for most AA and for N. It is concluded that the daily endogenous ileal flow of AA and N remains relatively similar at different DM intake and cellulose levels. Therefore, correction of apparent ileal digestibility of AA and N to the true ileal digestibility should be made with the endogenous ileal flow values expressed on a daily amount basis, not the values expressed on a DM intake basis.     

液质联用多肽识别技术用于龟甲的鉴别研究

目的建立龟甲(乌龟)专属性特征肽鉴别方法,用于区分常见8种混淆品(黄喉拟水龟、黄缘闭壳龟、中华花龟、巴西龟、小鳄龟、缅甸陆龟、马来闭壳龟和印度楞背龟)。方法样品以水提法提取,胰蛋白酶酶解,采用超高效液相色谱-四级杆飞行时间质谱(UPLC-Q-TOF-MS)和数据统计软件查找龟甲特征肽,并利用蛋白质搜库技术测序。利用找到的特征肽段,建立超高效液相色谱-串联四级杆质谱(UPLC-QQQ)的专属性龟甲特征肽鉴别方法。结果建立了以m/z 442.7(双电荷)→560.0,631.0为特征肽离子对的UPLC-QQQ专属性鉴别方法,阴性样品无干扰,最低检出浓度为0.4 g/L。对市售44批样品进行检测,其中仅14批样品检出龟甲特征肽。结论该方法的专属性强,符合分析检测的方法技术要求,可用于龟甲与混淆品的鉴别。     

Protein digestion in weanling pigs: effect of dietary protein source

The digestion and absorption of dietary protein was measured in weanling pigs (5 wk of age). Diets containing corn and either dried skim milk (DSM), soybean meal (SBM) or corn gluten meal (CGM) were fed for 7 d, and the contents of the stomach and six segments of the small intestine were collected. Nitrogen digestibility increased linearly from the proximal duodenum to the distal ileum, and was highest for DSM, intermediate for SBM and lowest for CGM diets at the distal ileum. The content of free amino acids in digesta increased 8-fold (SBM and CGM) to 12-fold (DSM) between the stomach and the proximal duodenum and reached a maximum concentration in the distal jejunum. Digestion of DSM was more proximal than was that of SBM. Although there was some accumulation of small peptides in the duodenum with a subsequent decrease in the jejunum, the molecular weight profiles of the soluble proteins were relatively constant throughout the small intestine. Protein solubility and the rate of proteolysis in the stomach and upper small intestine were the primary factors that limited the digestion of SBM and CGM.     

Utilization of ileal digestible amino acids by growing pigs: effect of dietary lysine concentration on efficiency of lysine retention

Diets were formulated using sugar, soya-bean meal and free amino acids to contain 0.1-0.8 g lysine/MJ digestible energy (DE) and offered at three times maintenance to male and female pigs from 20 to 45 kg live weight. Growth responses and retentions of protein, fat, energy and lysine were assessed. Increasing the dietary lysine concentration resulted in significant (P less than 0.001) linear and curvilinear increases in growth rates and decreases in food conversion ratios. There was only a small effect of lysine concentration on total energy retention, but a substantial effect on the partitioning of energy deposition, with increases in the rate of protein deposition and decreases in fat retention. There was no difference in the efficiency of protein deposition between male and female pigs but males responded more to higher lysine concentrations than females (estimated 0.93 and 0.74 g lysine/MJ DE for males and females respectively). Lysine concentration in the protein deposited by the pigs increased linearly and curvilinearly (P less than 0.01) from 5.8 to 6.6 g lysine/16 g N with increasing dietary lysine concentration. There was a linear and quadratic response (P less than 0.001) in retention of ileal digestible lysine, with the minimum retention of 0.16 occurring at 0.1 g lysine/MJ DE and increasing to a maximum retention of 0.73 at a dietary concentration of 0.47 g lysine/MJ DE. The efficiency of lysine retained/ileal digestible lysine intake was 0.86 and the endogenous lysine loss was estimated at 0.94 g/d.     

Utilization of ileal digestible amino acids by pigs: lysine

Two experiments were conducted to determine the utilization of ileal digestible lysine by pigs. In the first, the apparent ileal digestibility of amino acids in cottonseed meal, meat-and-bone meal and soya-bean meal was determined in pigs fitted with 'T'-shaped cannulas. In the second experiment, three lysine-deficient diets were formulated to 0.36 g ileal digestible lysine/MJ digestible energy (DE), with lysine contributed from the three protein concentrates as the only source of lysine in sugar-based diets. An additional three diets were formulated with supplements of lysine to verify that lysine was limiting in the first three diets. The growth performance and retention of lysine by pigs given the six diets over the 20-45 kg growth phase were then determined. The apparent ileal digestibility of lysine in the three protein concentrates (proportion of total) was: cottonseed meal 0.74, meat-and-bone meal 0.78, soya-bean meal 0.89. Growth rates (g/d) of the pigs given the three diets formulated to 0.36 g ileal digestible lysine/MJ DE were significantly different (P less than 0.001): cottonseed meal 377, meat-and-bone meal 492, soya-bean meal 541. The response of pigs to the addition of lysine confirmed that lysine was limiting in these diets. Crude protein (nitrogen x 6.25) deposited by the pigs was significantly higher (P less than 0.001) for those given soya-bean meal (77 g/d), relative to meat-and-bone meal (66 g/d) and cottonseed meal (38 g/d). The proportion of ileal digestible lysine retained by pigs given the three protein concentrates was: cottonseed meal 0.36, meat-and-bone meal 0.60, soya-bean meal 0.75. The results indicate that values for the ileal digestibility of lysine in protein concentrates are unsuitable in dietary formulations as the assay does not reflect the proportion of lysine that can be utilized by the pig. It appears that, with heat-processed meals, a considerable proportion of the lysine is absorbed in a form(s) that is (are) inefficiently utilized.     

The digestion of protein in young pigs and the utilization of dietary methionine

Forty pigs between 23 and 51 d of age were given ad lib. diets containing wheat and one of five protein concentrates: meat meal A, meat meal B, soya-bean meal, milk and lupins (Lupinus augustifolius). Twenty of these pigs were given indigestible markers from 51 to 56 d of age and were killed at 56 d of age. The diets containing meat meals A and B, soya-bean meal and milk contained 2.3 g total methionine/kg and the diet containing lupins contained 2.1 g/kg. A further forty pigs of the same age were given the same diets supplemented with 1 g synthetic methionine/kg. The weight gains and feed conversion ratios of the pigs given the diets containing 2.1-2.3 g methionine and 3.1-3.3 g methionine/kg were not significantly different. The weight gains of the pigs given lupins (2.1 g methionine/kg) were less than those of the pigs given the diets containing 2.3 g methionine/kg. The apparent digestibility of dry matter (DM) and nitrogen was less for the diets containing the meat meals (0.75 and 0.78 respectively) than for those containing the other protein concentrates (0.80 and 0.84). The retention times in the large intestine of the diets containing soya-bean meal and lupins were 965 and 1083 min which were greater than those of the diets containing the other protein concentrates, mean 732 min. The major site of N digestion and absorption for the diet containing milk was the area of the small intestine 25-50% of total length from the pylorus, while for the other protein concentrates the major site was 50-75% of its total length from the pylorus. The digestion and absorption of N in the large intestine was less (3.4%) for the diet containing milk than for those containing the other protein concentrates (7.5-11.3%). The apparent digestibility of the methionine to the ileum for the five diets ranged from 0.74 to 0.86 while the calculated retention of the apparently-absorbed methionine was 1.00. It was suggested that methionine digestibility could be used as an indicator of availability. The calculated retention of apparently absorbed N in the carcass was 0.71 for the pigs given the diet containing milk and 0.51-0.58 for the pigs given the other diets.     

Effect of dietary protein and zinc on anti-mycobacterial antibody responses in guinea pigs

An enzyme immunoassay was developed to quantify the levels of antibodies reactive with purified protein derivative (PPD) at 3, 4, 5 and 6 wks following vaccination with Mycobacterium bovis BCG and initiation of diets deficient in either protein (LP) or zinc (LZ). Significant antibody levels were detected in all groups as early as 3 wks post-vaccination compared to non-vaccinated animals. Both LP and LZ guinea pigs had higher levels of antibody than animals consuming an adequate diet at 3 and 4 wks, respectively. By 6 wks post-vaccination, animals on the low protein diet had the highest antibody titers, followed by control, chow and LZ guinea pigs in order of decreasing titers. A highly significant positive correlation was detected between the levels of anti-mycobacterial antibodies and the number of viable BCG recovered from the lymph nodes draining the vaccination site. A significant inverse correlation was seen between antibody levels and PPD skin reactions. High-titer sera from protein-deficient, but not zinc-deficient, BCG-vaccinated pigs significantly suppressed the tuberculin reactions when passively transferred into normal, PPD-positive recipients.     

Protein digestion in weanling pigs: effect of feeding regimen and endogenous protein secretion

Protein digestion and absorption were measured in weanling pigs (5 wk of age) using a total digesta collection procedure. In the first experiment, the objective was to measure the fate of nitrogenous digesta as it passed from the stomach to the terminal ileum when pigs were either allowed ad libitum access to feed or were fed four equal meals per day. Pigs were fed a standard corn-soybean meal diet in this experiment. There were no differences in flow rate or retention time of dry matter or nitrogen, and no differences in apparent nitrogen digestibility between the two feeding regimens. More than 80% of the soluble protein in the stomach and 65% in the small intestine was between 2,000 and 15,000 molecular weight. The molecular weight profiles of the soluble proteins were relatively constant throughout the small intestine, indicating that the products of protein hydrolysis were absorbed rapidly. The objective of the second experiment was to compare protein digestion and absorption in pigs fed a corn-soybean meal diet with those fed a protein-free diet. The molecular weight profiles of soluble protein were similar for the two groups of pigs. The data indicate that most of the endogenous enzymes were hydrolyzed rapidly, since little soluble protein corresponding to the molecular weight of pancreatic enzymes was detected.     

Utilization of ileal digestible amino acids by growing pigs: threonine

An experiment was conducted to determine the utilization of ileal digestible threonine by growing pigs. Three threonine-deficient diets (0.22 g ileal digestible threonine/MJ digestible energy (DE] were formulated using cottonseed meal, meat-and-bone meal and soya-bean meal respectively, as the only source of threonine in the diet. An additional three diets were formulated with supplements of threonine to confirm that threonine was limiting in the first three diets. The growth performance and retention of threonine by pigs given the six diets over the 20-45 kg growth phase was then determined. Growth rates (g/d) of the pigs given the three diets formulated to 0.22 g ileal digestible threonine/MJ DE were significantly different (P less than 0.001): cottonseed meal 417, meat-and-bone meal 452, soya-bean meal 524 (SED 13.6). The response of pigs to the addition of threonine confirmed that threonine was limiting in these diets. Crude protein (nitrogen x 6.25) deposited by the pigs (g/d) was significantly higher (P less than 0.001) for those given soya-bean meal (75), relative to meat-and-bone meal (62) and cottonseed meal (47) (SED 3.3). The proportion of ileal digestible threonine retained by pigs given the three protein concentrates was: cottonseed meal 0.44, meat-and-bone meal 0.59, soya-bean meal 0.64 (SED 0.024). These results indicate that values for the ileal digestibility of threonine in protein concentrates are unsuitable in dietary formulations as the assay does not reflect the proportion of threonine that can be utilized by the pig. It appears that, with heat-processed meals, a considerable proportion of the threonine is absorbed in a form(s) that is (are) inefficiently utilized.     

Endogenous gut nitrogen losses in growing pigs are not caused by increased protein synthesis rates in the small intestine

The purpose of this study was to establish, using a flooding dose of L-[ring 2, 6-(3)H] phenylalanine, whether feeding pigs diets that induce high endogenous gut nitrogen losses (ENL) also increases protein synthesis rates in (PSR) the visceral organs. Twelve 18-kg Yorkshire barrows with catheters in the right and left jugular veins were fed for 3 wk either casein-cornstarch- (CC) or barley-canola meal- (BCM) based diets formulated to a similar digestible energy /crude protein ratio and designed to induce either low or high ENL, respectively. Pigs were infused with 10 mL/kg body weight of a 150 mmol. L(-1) phenylalanine solution containing 230 MBq. L(-1) labeled phenylalanine for 12 min and killed 20 min later. Plasma phenylalanine specific radioactivity (SRA) rose to a plateau value within 3 min of starting the infusion and did not change (P > 0.10) thereafter. Fractional rates of protein synthesis (K(s), %/d) based on SRA in plasma- or intracellular-free phenylalanine did not differ (P > 0.10) in all tissues except pancreas (P < 0.05). Diet affected K(s )in liver (P < 0.01) and colon (P < 0.05) but not in pancreas, duodenum, jejunum and cecum. Based on plasma-free phenylalanine SRA, liver K(s)were 85.4 +/- 11.0 vs. 60.5 +/- 5.2 (mean +/- SEM) in CC- and BCM-fed pigs, respectively; these values were 82.3 +/- 4.7 vs. 98.2 +/- 5.8 in the colon. The absolute amount of protein synthesis (g.d(-1)) was higher in the liver (P < 0.05) and pancreas (P < 0. 05) of the CC pigs compared to BCM pigs. No dietary effects were observed in all other organs (P > 0.10). The present results suggest that feeding growing pigs a BCM diet that induces high ENL does not affect PSR in the small intestine of growing pigs from which >50% of ENL originates.