Methodologic studies for the measurement of kinetic parameters of the metabolism of whole body protein in association with measurements of energy metabolism in rats

In 3 successive experiments with growing rats the suitability of pulse labelling with [15N]glycine, linked with a 14C labelling by means of [14C]lysine (experiment 3), was tested for the determination of kinetic parameters of the protein metabolism of the whole body by the application of the compartment model in comparison with pulse labelling with a 15N amino acid mixture (experiment 2) and long-time labelling with 15N with 15N labelled wheat in the feed (experiment 1) under standardized experiment conditions. In simultaneously carried out measurings of energy metabolism with parallel groups of animals the comparability of the metabolic development was studied. The ascertained values of protein synthesis rate, protein catabolism rate and re-utilization rate showed insignificant differences only between the 3 15N tracer variants (with certain limitations for the 'protein turnover' (P)-group of experiment 2) in comparison with errors of the applied methods, from which conclusions can be drawn for the suitability of [15N] glycine as tracer, at least under the experiment conditions tested. The protein synthesis and degradation rates ascertained from 14CO2 excretion in experiment 3 were clearly below those average values ascertained with 15N. The differences in the average heat production between the main periods of the 3 experiments were statistically insignificant.     

Endogenous N-losses in piglets estimated by a [15N]-isotope dilution technique: effect of xylanase addition to a wheat and rye based diet.

The nitrogen pool of piglets weighing 19.4 +/- 1.4 kg at the beginning of the experiment was labeled with an oral application of ([15N]H4)2SO4 (1.26 [15N]-atom percent excess of dietary N) over a period of 7 d. The labeling period was followed by an equilibration period of 7 d without feeding the labeling compound. The two experimental diets were based on wheat (53%) and rye (25%) and were fed either with or without a xylanase containing enzyme preparation over both experimental periods. Additionally, diets were supplemented with an indigestible marker during the 2nd period of the experiment to allow the calculation of endogenous N-losses in subsequent segments of the digestive tract of the pigs. These endogenous N-losses were estimated at the end of the experiment by analyzing feces, ingesta and urine for [15N]-enrichment assuming that [15N]-enrichment of urine represents the [15N]-enrichment of the precursor pool. Endogenous N-losses were not significantly affected by xylanase addition at any measurement site (stomach, 3 sections of the small intestine, total digestive tract). Endogenous N-proportions of total nitrogen amounted on average for the six pigs to 42 +/- 11% and 56 +/- 5% at the last section of the small intestine and over the whole digestive tract, respectively, which corresponded to endogenous N-losses of 2.8 +/- 1.3 g N/kg DM and 2.0 +/- 0.3 g N/kg DM, respectively.     

Growth hormone increases whole-body protein turnover in growing pigs.

Ten pigs with an average initial live weight of 65 kg were used to investigate the effects of daily exogenous porcine pituitary growth hormone (pGH; .1 mg.kg-1.d-1) for a 13-d period on N retention and whole-body protein turnover. Feed intake was restricted to both the control (treated with excipient) and pGH-treated groups to ensure that animals in each group consumed equal amounts. Whole-body protein turnover was estimated from the excretion of 15N in urinary urea and ammonia after a single oral dose of [15N]glycine. Nitrogen balance and whole-body N flux were increased by 35 to 40% with pGH treatment (P less than .001). Protein synthesis and breakdown were increased by 56 and 59% (P less than .001), respectively, in pGH-treated pigs relative to controls. These higher rates of protein turnover seemed to lower slightly the efficiency of the metabolic process for protein deposition. However, the absolute increment in protein synthesis rate was greater than that for breakdown, leading to the increased net N retention. Thus, pGH treatment improved the utilization of dietary amino acids for protein deposition.     

Effect of colostomy on the occurrence of dietary [15N]urea in intestinal contents,blood, urine and tissues in chickens fed a low protein diet plus urea

1. The occurrence of ¹⁵N was examined in excreta for 10 h, and in intestinal contents, blood and tissues at 10 h after [¹⁵N]urea was fed to conventional and colostomised cockerels.

2. Total‐¹⁵N excretion and ¹⁵N‐balance in control chickens were 18.88 and 44.79 mg/kg body weight/10 h), respectively. The former was increased and the latter was decreased by colostomy by 10.75 mg (P<0.01).

3. Amounts of [¹⁵N]urea, [¹⁵N]ammonia and [¹⁵N]uric acid excreted by control birds were 13.78, 3.90 and 0.18 mg/kg body weight/10 h or 0.73, 0.21 and 0.01 of the total‐¹⁵N excreted respectively.

4. The [¹⁵N]urea, [¹⁵N]uric acid and total‐¹⁵N excreted were all increased after colostomy but [¹⁵N]ammonia was decreased (uric acid P<0.05, others P<0.01). The increase in total‐¹⁵N was mostly accounted for by [¹⁵N]urea.

5. Colostomy resulted in significantly less total‐¹⁵N in the contents of the whole intestine (P<0.01), less total‐¹⁵N, [¹⁵N]ammonia and [¹⁵N]urea in the contents of the co!o‐rectum (P<0.01) and less total‐¹⁵N and [¹⁵N]urea in the contents of the upper intestine (P< 0.05); it did not affect any in caecal contents.

6. [¹⁵N]Urea in blood, liver and kidney (blood P<0.01, others P< 0.05), and [¹⁵N]glutamine amide (P< 0.05) and [¹⁵N]uric acid (P< 0.01) in blood were significantly decreased after colostomy.

7. The results support the hypothesis that most of the dietary urea is utilised as the result of a back‐flow of ureteral urea into the caeca where it is rapidly converted into ammonia which is then metabolised to other compounds.     

Incorporation of nitrogen-15 from dietary [15N] diammonium citrate into amino acids of liver and muscle proteins in germfree and specific-pathogen-free neonatal pigs

Incorporation of 15N from dietary [15N]diammonium citrate ([15N]DAC) into amino acids isolated from hydrolyzed tissue proteins was investigated in the presence or absence of intestinal flora in neonatal pigs. The 15N was incorporated into all of the essential and nonessential amino acids from liver and muscles in one of two germfree pigs and two specific-pathogen-free pigs which were killed on the 5th day after the administration of [15N]DAC. But, a higher 15N concentration than natural abundance of 15N was not detected in histidine, lysine, and threonine from these tissues in another germfree pig killed the next day. Nitrogen transfer from DAC into all amino acids, including these three essential amino acids, may be possible in the specific-pathogen-free pig and even in the germfree pig.     

Threonine incorporation in tissues of growing broiler chickens using L-[15N] threonine

Most amino acid requirement trials appear for whole-body responses, but there is little information concerning amino acid incorporation in individual tissues, which may vary according to the age. L-[¹⁵N] threonine was used to evaluate its incorporation rate and distribution among broiler tissues in different ages. Seventy-two male broiler chickens were distributed into three different phases: starter (4 to 9 days old), grower (18 to 23 days old) and finisher phase (32 to 37 days old). L-[¹⁵N] threonine was added on balanced diets, and birds were fed for five days in each phase. Enriched samples of breast muscle, feathers, liver, jejunum and plasma were collected at 0, 6, 12, 24, 48, 72, 96 and 120 hr after fed birds with the tracer in each phase. In the tissues were analysed dry matter, nitrogen and stable nitrogen. The ¹⁵N isotope abundance according to the time was fitted into exponential or linear equations using a same intercept. The ratio of the steepness or slope coefficients was determined to compare the L-[¹⁵N] threonine incorporation according to the age. In addition, L-[¹⁵N] threonine mass balances were performed to assess the L-[¹⁵N] threonine distribution among the evaluated tissues. Except for feathers, the L-[¹⁵N] threonine incorporation rate decreased with ageing. Taking into account the L-[¹⁵N] threonine distribution in the tissues, only in the jejunum was not observed an increase as the broiler grew. The L-[¹⁵N] incorporation varied in each tissue and according to the age of the broiler chickens. These outcomes could be useful to comprehend changes in amino acid requirements tissue-specific according to age.     

酵母硒对乌珠穆沁羊免疫球蛋白及细胞因子的影响

[目的]通过饲养试验研究日粮中添加酵母硒对乌珠穆沁羊免疫球蛋白及关键细胞因子的影响,为合理确定内蒙古锡林郭勒地区乌珠穆沁羊日粮中酵母硒的适宜添加量以及酵母硒在该地区肉羊养殖中的推广使用提供理论依据。[方法]采用单因子随机试验设计,将240只健康乌珠穆沁羯羊随机分成4组（Ⅰ、Ⅱ、Ⅲ、Ⅳ组）。其中,Ⅰ组为对照组,日粮中添加无机硒（亚硒酸钠）0.3 mg/kg（DM基础）,其他3组为酵母硒组,日粮中分别添加0.3、0.6、0.9 mg/kg（DM基础）的酵母硒。预饲期15 d,正饲期60 d。[结果]在日粮中添加酵母硒能够显著提高乌珠穆沁羊血清中免疫球蛋白IgM、IgG、IgA以及细胞因子IL-1、IL-2及IL-6的含量。[结论]在乌珠穆沁羊日粮中添加酵母硒能显著提高乌珠穆沁羊的免疫水平,综合饲养成本及免疫增强效果,以添加0.6 mg/kg（DM基础）效果最佳。     

Determination of a prececal N-absorption from natural feed by 15N-labeled laboratory rats using the isotope dilution method

60 Wistar rats (5 animals/group) received 12 different feedstuffs over a period of 7 days and were simultaneously labelled with 15N (orally by means of 15NH4Cl in the feed). In the subsequent 5 days faeces were collected in order to determine the apparent and true digestibility of crude protein. On the 13th experimental day the animals were killed 3 hours after the intake of half the daily ration and the atom-% 15N excess (15N') was determined in the TCA-soluble and TCA-precipitable fractions of the blood plasma and the digesta of the 2nd and 3rd thirds of the small intestine. Precaecal N-absorption was calculated with the help of the quotient [formula: see text] the blood plasma and the digesta The following values (in %) were registered in comparison to true N-digestibility (in the following in brackets): casein = 95.8 (99.2), whole egg = 92.1 (97.5), fish meal = 85.8 (93.4), dried skimmed milk = 98.4 (96.1), soybean meal = 79.6 (90.6), assay protein = 94.2 (98.9), wheat = 92.9 (90.7), barley = 84.3 (84.8), yeast, grown on molasses = 85.6 (87.2), yeast, grown on whey = 86.1 (88.5), biomass of liquid manure = 43.6 (68.9), activated sludge = 54.4 (64.1). One can conclude that the isotope dilution technique demonstrated here as an evaluation method is very well suited for the characterization of the N-digestibility of a feedstuff in the small intestine.     

定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

为提高胰高血糖素样肽-2（GLP-2）生产效率以适应畜牧生产应用的需求,本试验利用基因工程技术表达出定点诱变的p[Gly2]GLP-2。用甘氨酸取代GLP-2氨基末端第2位的丙氨酸后,再根据大肠杆菌的偏好性对p[Gly2]GLP-2序列进行优化并在目标肽序列N-端添加肠激酶识别位点,合成基因序列,通过Kpn Ⅰ和Xho Ⅰ双酶切位点连接到pET-40b（+）构建原核重组表达载体p[Gly2]GLP-2-pET-40b（+）,重组质粒转化大肠杆菌BL21（DE3）感受态细胞,诱导表达重组蛋白。优化后最适表达条件为：菌液D_{600 nm}值为0.6时,用0.2 mmol/L IPTG在37℃诱导培养5 h;最终得到p[Gly2]GLP-2重组蛋白表达量较高的基因工程菌株。通过镍离子亲和层析柱纯化及分梯度洗脱获得纯度较高的p[Gly2]GLP-2融合蛋白,本结果为后续p[Gly2]GLP-2功能性的研究及其在畜牧生产的应用奠定了基础。     

15N transamination in the administration of various tracer substances. 1. Whole body studies in rats

4 groups of 3 growing Wistar rats each were orally given either 15N methionine, 15N lysine, 15N glycine or 15N ammonia sulphate over 10 days. By means of measuring 15N, the 15N accumulation in the amino acids (AA) of the body protein, statements were to be made on the transamination of the individual 15N substances and thus their suitability as tracer substances for studies of N metabolism. None of the tested 15N AA achieved a proportionate labelling of all AA of the body protein. The AA used as tracer in each case showed the highest 15N labelling of all AA in the body. Of the amino 15N detected in the animal body, ca. 19% were found in Met after a 15N Met application, ca. 88% in Lys after a 15N Lys application and ca. 50% in Gly after a 15N Gly application. After the application of 15N ammonia sulphate ca. 42% of the body amino 15N are apportioned to the essential and ca. 58% to the non-essential AA. Thus this substance produces a more proportional labelling of the essential and non-essential AA of the body protein than 15N Gly. The following quotas of the 15N amounts applied were found in the AA of the animal bodies: tracer substance lysine 52%, glycine 32%, ammonia sulphate 24%, methionine 21%. After summing up the amino acid 15N amounts in the animal body, eliminating in each case the tracer AA and taking into account the molecular weight of the AA, there was a good agreement of the intensity of the accumulation of 15N in the individual AA, irrespective of the applied tracer substance: arginine, glutamic acid, cysteine and aspartic acid highest, threonine, phenylalanine and lysine lowest accumulation.     

Utilization of 15N-labeled urea in laying hens. 8. 15N-incorporation in the amino acids of the oviduct

3 colostomized laying hybrids received orally with a conventional ration 1% urea with 96.06 atom-% 15N excess (15N'). over a period of 6 days. In the period of the experiment every hen consumed 2.87 g 15N'. After another 2 days, on which they received conventional feed urea, the animals were butchered. 15N' was determined in the total N and in 15 amino acids of the oviduct. Of the 15 amino acids the labelling of glutamic acid, glycine and serine was highest and on average amounted to 0.80, 0.66 and 0.67 atom-% 15N'. In lysine and arginine only 0.10 and 0.11 atom-% 15N' could be detected. The amino acid N with natural isotopic frequency amounted to a quarter for the basic amino acids, a tenth for the branched chain ones and for the non-essential ones (glutamic acid, aspartic acid, serine, glycine, alanine, proline) a third of the total oviduct 14N, The average quota of 15N' is only 3.6%, that of the branched chain amino acids 4.5 and that of the non-essential ones 21.1%. Consequently, the 15N' of the urea is mainly used for the synthesis of the non-essential amino acids of the oviduct.     

使用酵母培养物AYC-X6防治犊牛腹泻效果

[目的]犊牛腹泻是犊牛最常见的消化道疾病,是由饲养管理和多种感染性病原微生物共同作用引起。为研究在犊牛日粮中添加酵母培养物防治犊牛腹泻效果。[方法]在宁夏固原现代农业园区肉牛繁育基地随机选取120头2.5月以下犊牛作为试验牛群,对照组和试验组各60头,试验组犊牛每头每天添加30 g酵母培养物AYC-X6,对照组不添加。[结果]结果表明,试验组犊牛腹泻发病率减少35%,死亡率减少8%;平均每头治疗成本节省8.4元/头。[结论]犊牛日粮中添加酵母培养物AYC-X6,有利于建立健康稳定的瘤胃微生物菌群,对犊牛腹泻有很好的防治效果。     

N-ras mutation in a canine lymphoma: short communication

Lymphomas of dogs were investigated by molecular genetic methods. Regions of exon 1 and 2 of the N-ras gene, which harbours the mutation hot spots (codons 12, 13 and 61) were screened. A GGT [symbol: see text] GAT (glycine [symbol: see text] aspartic acid) mutation in codon 13 was present in a multicentric-type lymphoma of a 1-year-old male dog.     

Tracer studies of urea kinetics in growing pigs: I. The effect of intravenous infusion of urea on urea recycling and the site of urea secretion into the gastrointestinal tract.

Four gilts (average BW 80 kg) were used in the first experiment to study the effect of i.v. infusion of urea on urea kinetics by means of a radioisotope dilution technique. The pigs were fed twice daily 600 g of a cornstarch-based diet formulated to contain 16% CP by supplementation with isolated soy protein. Infusion of urea, compared with saline, increased (P < .05) plasma urea concentration, urea pool size, urea entry, urea excretion, and urea degradation rates; urea turnover rate and urea space were not affected (P > .05). Expressed as a percentage of the total entry rate, a lower (P < .05) percentage of urea was recycled in pigs infused with urea. The urea infused was almost completely excreted in urine, so there were no differences (P > .05) in N balance. In the second experiment, four gilts (average BW 40 kg), fitted with ileocecal reentrant cannulas, were used to determine whether the upper or the lower digestive tract represents the preferential site of urea secretion in pigs. Two pigs were fed twice daily 600 g of a cornstarch-based diet, formulated to contain 16% CP from soybean meal. The other two pigs were fed the same diet in which 15% cornstarch was replaced by beet pulp. After labeling the body urea pool of one pig on each treatment with [15N]urea, the reentrant cannulas were disconnected to prevent the flow of digesta from the small into the large intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

日粮中添加酵母培养物AYC-X6对犊牛生长发育的影响

[目的]为研究在犊牛日粮中添加酵母培养物对犊牛生长发育的影响。[方法]在宁夏固原现代农业园区肉牛繁育基地随机选取90头2.5月以上犊牛作为试验牛群,采用配对试验设计,试验分为2个处理组。[结果]结果表明,试验组犊牛比对照组犊牛平均干物质采食量提高了14.9%,日增重提高了24.6%,饲料消耗降低了0.08%。体重提高了24.1%,体高、胸围、腹围、体斜长分别提高了5.1%,11.3%,12.8%,24.9%。试验组粪便分离筛第1,2,3层筛上物分别为15%,20%,65%。试验组血清总蛋白(BSA)、球蛋白(BGG)含量均显著高于对照组,谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)含量均显著低于对照组。[结论]在犊牛日粮中添加酵母培养物AYC-X6,能提高饲料消化率,促进犊牛生长发育,提高犊牛平均日增重和生长速度。提高血清总蛋白、球蛋白含量,降低谷丙转氨酶、谷草转氨酶、碱性磷酸酶水平,改善犊牛免疫能力。     

Effects of protozoa on bacterial nitrogen recycling in the rumen

The effects of protozoa on ruminal NH3-N kinetics and bacterial N recycling were measured in five sheep (57.6+/-7.1 kg BW, x +/- SD) with ruminal and duodenal cannulas in naturally faunated, defaunated, and refaunated periods. The sheep were fed a diet of 239 g of alfalfa haylage and 814 g of barley concentrate per day (DM basis) divided into 12 equal portions and allocated at 2-h intervals. A pulse dose of 300 mg of 15N as [15N]NH4Cl was administered into the rumen (on d 1 and 15) and 300 mg of 15N as [15N]urea was administered intravenously to the blood (d 8). Enrichment of 15N was measured in ruminal NH3-N, bacterial N, and plasma urea N over a period of 35 h. Total collection of urine was made for 5 d and analyzed for purine derivatives to calculate the flow of microbial N. Ruminal parameters and nutrient digestibilities were also measured. Sheep were defaunated using a rumen washing procedure 50 d prior to measurements in the defaunated period. Sheep were refaunated with ruminal contents from a faunated sheep receiving the same diet. Measurements began 26 d following refaunation, at which time protozoal numbers had returned to those in the originally faunated sheep. Data reported in parentheses are for faunated, defaunated, and refaunated sheep, respectively. Total culturable and cellulolytic bacterial numbers were unaffected by defaunation, but there was an increase in flow of microbial N from the rumen (10.8, 17.3, and 11.1 g N/d; P < .05) in the defaunated period. Flux, irreversible loss, and intraruminal recycling of NH3-N and recycling of NH3-N from plasma urea N were not affected by defaunation. Defaunation had no effect on reducing the absolute amount (13.8, 10.0, and 11.3 g N/d; P > .20) of bacterial N recycling and the percentage of N flux through the bacterial N pool. Total-tract digestion was reduced in defaunated compared with faunated sheep by 8, 17, 15, and 32% for OM, N, NDF, and ADF, respectively. In conclusion, defaunation improved ruminal N metabolism through the enhancement of bacterial protein synthesis, and improvement in the flow of microbial protein to the host animal.     

Isolation and Identification of Highly Pathogenic PRRSV from a Pig Farm

[Objective] To identify suspected cases of highly pathogenic PRRSV from a pig farm. [Method] The suspected cases of highly pathogenic PRRSV were conducted pathologic anatomy, the gene of PRRSV N protein was amplified by RT-PCR and its sequences were determined and analyzed. [Result] The nucleotide sequence of N protein of N7/N8 isolate shared the consistency of 97.0% with highly pathogenic strains(JXA1, Hu N4, SX-1 and TJ), and that of isolate N9 shared the consistency of 97.3% with highly pathogenic strains, indicating the infected virus in the pig farm was highly pathogenic PRRSV. [Conclusion] This research provides a reference for the diagnosis of highly pathogenic PRRSV infec-tion in swine production.     

Differences between Brahman and Holstein cows in heat-shock induced alterations of protein synthesis and secretion by oviducts and uterine endometrium

Our objectives were to test differences in protein synthesis and secretion by cultured oviducts and endometrium from Brahman and Holstein cows and the response of those tissues to in vitro heat shock. Explants of oviductal tissue obtained at estrus from Holstein (n = 5) and Brahman (n = 6) cows were cultured at a homeothermic (39 degrees C) or heat shock (43 degrees C) temperature. At 6 h, cultures were pulse-chase labeled (2 h, L[4,5-3H]leucine; 2 h, L-leucine). Endometrial explants were cultured similarly except that pulse labeling was performed for the first 0 to 15, 0 to 30, 30 to 60 and 60 to 90 min following onset of heat shock. A temperature of 43 degrees C increased secretion of nondialyzable 3H-labeled macromolecules by both oviducts of Brahmans but depressed secretion by the oviduct ipsilateral to the side of ovulation of Holsteins. For both breeds, 43 degrees C decreased incorporation of [3H]leucine into trichloroacetic acid (TCA)-precipitable radioactivity in oviducts from the ipsilateral side. Secretion of 3H-labeled macromolecules by pulse-labeled endometrial explants increased at 43 degrees C. Heat shock caused an immediate increase in TCA-precipitable radioactivity in tissue during pulse labeling for Holstein tissues. Incorporation was decreased at 43 degrees C in tissue from Brahmans in the first 30 min and increased thereafter. Incorporation of [3H]thymidine by endometrial explants from Brahmans was increased at 43 degrees C, whereas it was suppressed at 43 degrees C in explants from Holstein cows. Heat shock proteins of 72,000 and 90,000 molecular weight were present in endometrial tissues. A major secretory product of endometrium had a molecular weight of 57,500 for Brahmans and a lower molecular weight (55,600) for Holsteins.     

Methodologic aspects of the determination of N-exchange parameters from 15N-tracer studies in swine on the basis of models of N-metabolism. 3. Calculation of protein synthesis and decomposition rate on the basis of lysine flux, ascertained from the amount of 15N in the urine

The final product method introduced here is based on the use of 15N L-Lysine as tracer amino acid for the determination of the protein synthesis and decomposition quotas of the total body with the flux of lysine. For this purpose it is necessary to complete the N flow and N compartments of the 3-pool model by the values of lysine content. The ascertained values of protein synthesis--8.4 g/d/kg--and protein decomposition--6.9 g/d/kg--agree very well with those determined with 15N glycine as tracer amino acid.     

Ammonia production from uric acid and its absorption from the caecum of the cockerel

1. Experiments were done in vitro and in situ in the caeca to measure ammonia production from uric acid and its absorption. 2. When uric acid was introduced into a caecal sac containing mixed caecal micro-organisms 0.77 disappeared in 1 h, with the concomitant appearance of ammonia. 3. Amounts of ammonia produced from added [15N] uric acid in the caeca in situ after 30 min and in caecal medium in vitro after 10 h, were 0.28 and 0.25 respectively of the added 15N. 4. About 0.92 of the ammonia introduced into a caecal sac disappeared from the lumen fluid in 30 min. 5. It is concluded that ammonia is produced from uric acid by caecal micro-organisms and rapidly absorbed.