Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability

Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.     

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 μl of 500 μM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.     

Identification of the heat shock protein 70 (HLHsp70) in Haemaphysalis longicornis

A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311 bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal "KDEL" that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4-37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28 kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks.     

Relationships of barometric pressure and environmental temperature with incidence of parturition in beef cows

ABSTRACT:The relationship between barometric pressure (BARO) and maximum (MAX_T) and minimum (MIN_T) environmental temperatures with the incidence of parturition in beef cows was examined through exploratory data analysis. Spring- and fall-calving records from a 5-yr period (2005 through 2009) collected at the University of Arkansas, Livestock and Forestry Research Station (Batesville) and the Department of Animal Science Savoy Research Unit (Savoy, AR) were used. All cows were multiparous, predominantly Angus, and naturally bred. During this period, 2,210 calves were born over a cumulative 1,547 d. Local weather station BARO and MAX_T and MIN_T data were obtained from the Southern Regional Climate Center, Louisiana State University, Baton Rouge. The combined calving record and climate variables were used to determine differences in BARO, MAX_T, and MIN_T on d 0 (d of calving) and -1, -2, or -3 d, respectively, before calving occurred (CALFD) or did not occur (NOCALFD). Location and season also were included in the model. For fall-calving cows, BARO on d 0 and -1, -2, or -3 was not different between CALFD and NOCALFD (P > 0.10). For spring-calving cows, BARO on d 0, -1, -2, and -3 was greater (P < 0.05) for CALFD compared with NOCALFD. The MAX_T was greater on d -1 (24.4 vs. 22.9°C) and -3 (24.8 vs. 23.4°C) for CALFD in the fall compared with NOCALFD (P < 0.05). No differences were detected in the fall for MAX_T on d 0 or -2 (P > 0.10). In the spring, a decreased MAX_T was associated with CALFD. Maximum environmental temperatures on d 0 (14.7 vs. 16.0°C), -1 (14.4 vs. 16.0°C), and -3 (14.0 vs. 15.7°C) were less for CALFD compared with NOCALFD (P < 0.05). No difference was detected on d -2 (P > 0.10). For fall, MIN_T was greater on d -1 (12.8 vs. 11.3°C), -2 (13.0 vs. 11.4°C), and -3 (13.1 vs. 11.7°C) for CALFD compared with NOCALFD (P < 0.05). In spring, MIN_T for d 0 (2.6 vs. 3.9°C), -1 (2.5 vs. 3.7°C), -2 (2.1 vs. 3.7°C), and -3 (1.8 vs. 3.8°C) were lesser (P < 0.05) for CALFD vs. NOCALFD. These data indicate that for spring-calving cows, a greater BARO and decreased MAX_T and MIN_T were associated with CALFD, whereas for fall-calving cows, an increase in MAX_T and MIN_T was associated with CALFD. Therefore, monitoring weather conditions may assist producers in preparing for the obstetric assistance of beef cattle.     

Retrospective study of the prevalence of postanaesthetic hypothermia in cats

A retrospective study of 275 anaesthetic records of cats was undertaken to examine the prevalence of postanaesthetic hypothermia, its clinical predictors and consequences. Temperature was recorded throughout anaesthesia. The temperature reached at the end was classified as hyperthermia (>39.50 °C), normothermia (38.50 to 39.50 °C), slight hypothermia (38.49 to 36.50 °C), moderate hypothermia (36.49 to 34.00 °C) or severe hypothermia (<34.00 °C). Statistical analysis consisted of multiple regression to identify the factors that affect the temperature at the end of the procedure. Before premedication, the mean (sd) temperature was 38.2 (1.0) °C. At 60, 120 and 180 minutes from induction, the temperature was 35.4 (1.4) °C, 35.0 (1.5) °C and 34.6 (1.5) °C, respectively. The prevalence of hypothermia was slight 26.5 per cent (95 per cent CI 21.7 to 32.0 per cent), moderate 60.4 per cent (95 per cent CI 54.5 to 66.0 per cent) and severe 10.5 per cent (95 per cent CI 7.4 to 14.7 per cent). The variables associated with a decrease in the temperature recorded at the end of anaesthesia were the duration of anaesthesia, the reason for anaesthesia (abdominal and orthopaedic surgeries significantly reduced the temperature when compared with minor procedures) and the anaesthetic risk (high-risk cats showed lower temperatures than low-risk cats). The temperature before premedication was associated with an increase in the final temperature.     

Factors affecting storage stability of various commercial phytase sources

A 360-d study was performed to evaluate the effects of different environmental conditions on storage stability of exogenous phytases. Coated and uncoated products from 3 phytase sources [Ronozyme P (DSM Nutritional Products, Basel, Switzerland), OptiPhos (Phytex LLC, Sheridan, IN), and Phyzyme (Danisco Animal Nutrition, Marlborough, UK)] were stored as pure forms, in a vitamin premix, or in a vitamin and trace mineral (VTM) premix. Pure products were stored at -18, 5, 23, and 37°C (75% humidity). Premixes were stored at 23 and 37°C. Sampling was performed on d 0, 30, 60, 90, 120, 180, 270, and 360. Sampling of the pure products stored at -18 (lack of sample) and 5°C (because of mold growth) was discontinued after d 120. Stability was reported as the residual phytase activity (% of initial) at each sampling point. For the stability of the pure forms, all interactive and main effects of the phytase product, coating, time, and storage temperature were significant (P < 0.01), except for the time × coating interaction. When stored at 23°C or less, pure phytases retained at least 91, 85, 78, and 71% of their initial phytase activity at 30, 60, 90, and 120 d of storage, respectively. However, storing pure products at 37°C reduced (P < 0.01) phytase stability, with OptiPhos retaining the most (P < 0.01) activity. Coating mitigated (P < 0.01) the negative effects of high storage temperature for Ronozyme and OptiPhos (from d 90 onward), but not for Phyzyme. For the stability of phytase in different forms of storage, all interactive and main effects of phytase product, form, coating, time, and temperature of storage were significant (P < 0.01). When stored at room temperature (23°C), retained phytase activities for most the phytase sources were more than 85, 73, and 60% of the initial activity up to 180 d when stored as pure products, vitamin premixes, or VTM premixes, respectively. When stored at 37°C, pure phytase products had greater (P < 0.01) retention of initial phytase activity than when phytases were mixed with the vitamin or VTM premixes. Coated phytases stored in any form had greater (P < 0.01) activity retention than the uncoated phytases at all sampling periods. Results indicate that storage stability of commercially available phytases is affected by duration of storage, temperature, product form, coating, and phytase source. Pure products held at 23°C or less were the most stable. In premixes, longer storage times and higher temperatures reduced phytase activity, but coating mitigated some of these negative effects.     

Porcine somatotropin decreases acetyl-CoA carboxylase gene expression in porcine adipose tissue

Crossbred barrows were treated daily with porcine somatotropin (pST; 4 mg/d) from 79 to 127 kg BW to determine whether pST regulates the activity and gene expression of adipose tissue acetyl-CoA carboxylase (ACC), the rate limiting enzyme in de novo fatty acid synthesis. Administration of pST reduced ACC enzyme activity, protein content, and mRNA abundance in adipose tissue by 40 to 50%. When comparisons were made among all pigs, ACC enzyme activity and mRNA abundance were closely associated (r² = .94). In summary, our results indicate that pST decreases ACC enzyme activity and that this is associated with a significant reduction in ACC mRNA abundance. We speculate that decreased ACC enzyme activity results from a reduction in ACC protein and that this occurs because pST reduces the abundance of mRNA available for translation.     

Tolerance to low temperatures of Toxocara cati larvae in chicken muscle tissue

Infectivity of Toxocara cati larvae in muscle tissue of chickens after storage at 4°C and -25°C was assessed in a mouse bioassay to provide information on the risk of meat-borne toxocarosis. Muscle tissue samples of 30-day old T. cati infections were stored at 4°C for 14 and 28 days and at -25°C for 12, 24 and 48h, whereafter, larvae were released by digestion. For each experimental group, the released larvae were inoculated in six mice. After 15 days, mice were euthanized and larval burden was assessed by digestion. In the control group (no storage of the infected chicken meat), 47.9% of the inoculated larvae established in mice, whereas storage of meat at 4°C for 14 days or 28 days reduced the recovery to 24.1% or 3.3%, respectively. Muscle larvae exposed to -25°C for 12, 24 or 48h did not establish in the mice. The observation that larvae retain infective after refrigeration at exposure in 4°C for 28 days, emphasize the zoonotic potential of poultry meat as a causative agent of human toxocarosis.     

Proteolytic activity in salivary gland products of sheep bot fly (Oestrus ovis) larvae

This study identified and characterized hydrolytic enzymes in salivary gland products of Oestrus ovis larvae. Third instars were collected from the heads of slaughtered goats. Salivary glands were extracted, their products obtained by centrifugation and the enzymatic profile determined. Optimum pH, temperature of maximum proteolytic activity, thermal stability, and resistance of salivary gland products were determined on collagen and subclasses of proteases were identified using protease inhibitors. Zymograms were used to determine the molecular weight of proteases. Antigenic protein bands were revealed by immunoblotting using sera obtained from experimentally infested goats. Seven positive enzymatic activities were detected in salivary gland products: acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase (C4), esterase lipase (C8), leucine arylamidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Optimum pH for proteolytic activity was 8.0; proteolytic activity increased with temperature (10-50 degrees C) then drastically decreased at 60 degrees C. Proteases in O. ovis salivary gland products belong to the serine subclass. In Zymograms, bands of proteolytic activity were detected in the 20-63 kDa range; the immunoblot showed three antigenic bands, one of them related to a protease band (63 kDa). Serine proteases in O. ovis salivary gland products are most likely involved in larval nutrition and host immuno-modulation.     

玉米肽对黄鳝生长及肠道消化酶活性的影响

试验旨在研究玉米肽对黄鳝生长及肠道消化酶活性的影响。试验挑选600尾黄鳝随机分成4组,每组3个重复,每个重复50尾。对照组(GYF)黄鳝饲喂42%鱼粉的黄鳝商品饲料,处理组分别饲喂32%鱼粉(YM0)、32%鱼粉+1.5%玉米肽(YM1.5)和32%鱼粉+3.0%玉米肽(YM3.0),试验期8周。结果显示,与GYF组对比,YM3.0组的黄鳝增重率显著提高5.9%(P<0.05),肠道脂肪酶含量显著降低55.1%(P<0.05),肠道淀粉酶含量显著降低15.5%(P<0.05)。研究表明,以玉米肽替代饲料中10%的鱼粉可以改善黄鳝的生长性能、肉质品质及肠道消化酶活性。     

A Comparison of Three Methods for Extracting IgY from the Egg Yolk of Hens Immunized with Sendai Virus

Hens were immunized with partially purified Sendai virus that had been grown on chicken embryos. The titres of specific antibodies to Sendai virus varied from log₂ 13.0 to 15.5 during the 4 months after immunization and the immunoglobulin Y (IgY) concentration varied from 2 to 10 mg per ml of egg yolk. A method has been developed employing dextran blue to isolate IgY from the egg yolk. The dextran blue method was compared with two other methods (poly(ethylene glycol) and chloroform) in terms of yield, total protein content, IgY concentration, specific activity of IgY and SDS-PAGE analysis. The specific activity and the IgY content obtained by these three methods proved to be identical, in the range log₂ 12.9–14.1, and 4.6–12.8 mg/egg, respectively. However, the total protein content when purified by chloroform was 2-fold to 4-fold higher than that of the others. The analysis of IgY by SDS-PAGE demonstrated that IgY purified with dextran blue contained three major protein components with molecular weights of 34.7 kDa, 41 kDa and 66 kDa and one minor protein of 45 kDa. IgY that was extracted with chloroform contained two major proteins of 45.7 kDa and 75.2 kDa and that extracted with PEG-6000 contained only one major protein of 66 kDa. The IgY obtained by these latter methods also contained several minor proteins in the range 41–80 kDa.     

Relationship of ruminal temperature with parturition and estrus of beef cows

Spring-calving Angus cows (n = 30) were used to evaluate changes in ruminal temperature (RuT) related to parturition and estrus. Cows were synchronized and artificially inseminated with semen from a single sire. Temperature boluses were placed in the rumen at 7.0 ± 0.2 mo of gestation. Boluses were programmed to transmit RuT every 15 min. Cows (BW = 623 ± 44 kg, BCS = 4.9 ± 0.4) calved during 3 wk, and estrus was synchronized at 77 ± 7 d after calving with PGF(2α). Cows were observed every 12 h to detect estrus. Daily average ambient temperatures ranged from 2 to 22 °C during parturition (February to March) and 17 to 25 °C during estrus (May to June). Ruminal temperature from 7 d before to 3 d after parturition and 2 d before to 2 d after visual detection of estrus was analyzed using the MIXED procedure. Ruminal temperatures <37.72 °C were attributed to water consumption and excluded from analyses. Day did not influence (P = 0.36) RuT from d -2 to -7 before parturition (38.94 ± 0.05 °C). Ruminal temperature decreased (P < 0.001) from d -2 to d -1 before parturition (38.88 ± 0.05 to 38.55 ± 0.05 °C, respectively). Ruminal temperature was not influenced (P = 0.23) by day from 1 d before to 3 d after parturition (38.49 ± 0.05 °C). Ruminal temperature at 0 to 8 h after detection of estrus (38.98 ± 0.09 °C) was greater (P < 0.001) compared with RuT at the same daily hour of the day before (38.37 ± 0.11 °C) or the day after estrus (38.30 ± 0.09 °C). Ambient temperature did not influence (P > 0.30) RuT at parturition or estrus. Ruminal temperature decreased the day before parturition and increased at estrus in spring-calving beef cows and has potential use as a predictor of parturition and estrus.     

鸡血管生成素样蛋白4重组蛋白对肉鸡肝脏和胸肌脂肪代谢的影响

本试验旨在探究鸡血管生成素样蛋白4(ANGPTL4)重组蛋白对肉鸡肝脏和胸肌脂肪代谢的影响。分体内动物试验和体外细胞试验2部分进行。体内动物试验选用35日龄健康禁食状态下爱拔益加肉公鸡36只,随机分为6组,每组6个重复,每个重复1只鸡。对照组翅静脉注射灭菌的生理盐水,试验组分别翅静脉注射20、100、500、2 500、12 500 ng/kg BW鸡ANGPTL4重组蛋白,注射剂量均为550μL,试验期30 min。体外细胞试验设3个组,分别是生理盐水组、组氨酸-小分子泛素样修饰蛋白(His-SUMO)标签组和鸡ANGPTL4重组蛋白组,分别在细胞培养基中添加灭菌的生理盐水、His-SUMO标签蛋白(其含量与鸡ANGPTL4重组蛋白组中标签蛋白含量一致)和鸡ANGPTL4重组蛋白(250 pg/mL),添加剂量均为5μL,5%CO₂、37℃孵育24 h。结果发现:1)与对照组相比,20、100、500、2 500 ng/kg BW鸡ANGPTL4重组蛋白组的肝脏脂肪酸合成酶(FAS)mRNA相对表达量和500 ng/kg BW鸡ANGPTL4重组蛋白组的肝脏FAS活性均显著提高(P<0.05)。随着鸡ANGPTL4重组蛋白注射剂量增加,肝脏FAS mRNA相对表达量和活性均呈现一次线性和二次曲线增加的效应(P<0.05)。2)与对照组相比,100、500、2 500 ng/kg BW鸡ANGPTL4重组蛋白组的肝脏苹果酸酶(ME) mRNA相对表达量和12 500 ng/kg BW鸡ANGPTL4重组蛋白组的肝脏乙酰辅酶A羧化酶(ACC) mRNA相对表达量均显著降低(P<0.05)。随着鸡ANGPTL4重组蛋白注射剂量增加,肝脏ACC mRNA相对表达量呈现一次线性和二次曲线降低的效应(P<0.05)。不同注射剂量的鸡ANGPTL4重组蛋白对肉鸡肝脏ME和ACC活性均无显著影响(P>0.05)。3)与对照组相比,500 ng/kg BW鸡ANGPTL4重组蛋白组的胸肌脂蛋白脂酶(LPL)活性显著增加(P<0.05)。4)鸡ANGPTL4重组蛋白组的成肌细胞甘油三酯(TG)含量显著高于生理盐水组和His-SUMO标签组(P<0.05)。综上所述,鸡ANGPTL4重组蛋白具有调控肉鸡肝脏脂肪合成相关基因mRNA表达和酶活性以及促进肉鸡胸肌脂肪沉积的作用,以500 ng/kg BW注射剂量效果较好。     

Acetyl-CoA carboxylase and fatty acid synthase activity and immunodetectable protein in adipose tissues of ruminants: effect of temperature and feeding level

To gain insights into the regulation of fat synthesis, we have investigated the effect of cold environmental exposure and feed restriction of sheep on activity and immunodetectable protein content of acetyl-CoA carboxylase (ACC) and fatty acid synthase in adipose tissue. Subcutaneous and mesenteric adipose tissues were collected at slaughter from sheep exposed to either cold (0+/-2 degrees C) or warm (23+/-2 degrees C) environment, and given either ad libitum or restricted access to feed for three 5-wk periods. Acetyl-CoA carboxylase was isolated from frozen adipose tissue samples and activity determined as the rate of incorporation of H14CO3- into acid stable malonyl-CoA. Cold exposure and feed restriction reduced (P < .05) ACC activity in the two adipose tissue depots. Western blot analysis with peroxidase-conjugated streptavidin showed that both adipose tissue depots express a single isoform of ACC. In s.c. adipose tissue, cold exposure increased (P < .05) ACC protein abundance, which is opposite to the change in activity. However, feed restriction reduced immunodetectable ACC protein. There was no significant effect of environment or feeding level on ACC protein abundance in mesenteric tissue. Fatty acid synthase activity determined in ammonium sulfate extract by measuring the malonyl-CoA- and acetyl-CoA-dependent oxidation of NADPH was decreased (P < .05) by feed restriction in both s.c. and mesenteric tissues. Cold exposure reduced fatty acid synthase activity in s.c. but not in mesenteric tissue. There was no effect of environment on fatty acid synthase protein abundance in either adipose tissue depot. However, feed restriction significantly reduced fatty acid synthase protein abundance in the two depots. The data suggest that feed restriction and exposure of ruminants to cold environmental conditions may significantly down-regulate the activity of key lipogenic enzymes.     

Thermoregulation of the testicle in response to exercise and subsequent effects on semen characteristics of stallions

Stallions (n = 8) were implanted with a thermal sensory device in the muscle of the neck and the subcutaneous tissue of the scrotum and then assigned to either a nonexercise (Non-EX; n = 4) or exercise (EX; n = 4) group. A motorized equine exerciser was used to work EX stallions 30 min/d for 4 d/wk during a 12-wk period from July through October 2010. Temperatures (subcutaneous scrotal, intramuscular neck, and rectal) were recorded at 0, 22, and 30 min after the start of exercise, as well as 60 and 120 min post-exercise. Hourly ambient temperature and relative humidity data were also obtained. Semen was collected at 0, 4, 8, and 12 wk and analyzed for volume, sperm concentration, total sperm numbers, percentages of total and progressively motile sperm, sperm morphologic characteristics, and sperm DNA quality. No effect (P > 0.05) of exercise was observed on any of the measured semen variables. Implantation of thermal sensory devices had no demonstrable acute or chronic effects on the scrotal or neck tissue, indicating that the thermal sensory devices are a safe and effective way to measure subcutaneous scrotal and neck temperatures. At 22 and 30 min of exercise, rectal and neck temperatures increased (P < 0.0001) approximately 1.9 and 2.4°C, respectively, and scrotal temperatures simultaneously increased, although not significantly (P = 0.33), approximately 0.8°C. Correlations existed between scrotal, neck, rectal, and ambient temperatures, with the correlation between scrotal and rectal temperatures being greatest (r(s) = 0.76; P < 0.0001). Although moderate exercise for a short duration in extreme heat and humidity did significantly increase core body temperatures in stallions, scrotal temperatures did not significantly increase, and sperm parameters were unaffected.     

Purification and characterization of the subtilisin-like protease of Streptococcus suis that contributes to its virulence

Streptococcus suis is a major swine pathogen that is responsible for severe infections such as meningitis, endocarditis, and septicemia. S. suis is also recognized as a zoonotic agent and expresses several virulence factors. The recently identified subtilisin-like protease (SspA) of S. suis plays an important role in the pathogenicity of this bacterium in animal models. The objective of the present study was to clone, purify, and characterize the SspA of serotype 2 S. suis P1/7. The SSU0757 gene encoding SspA was amplified and a 4798-bp DNA fragment was obtained. It was cloned into the expression plasmid pBAD/HisB and then inserted into Escherichia coli to overproduce the protein. The recombinant protease was purified by chromatography procedures and showed a molecular weight of 170 kDa by SDS-PAGE. Its activity was optimal at pH 7 and at temperatures ranging from 25°C to 37°C. It had a high specificity for the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-pNa while specific inhibitors of serine proteases inhibited its activity. In addition to degrading gelatin, the protease hydrolyzed the Aα chain of fibrinogen, which prevented fibrin formation by thrombin. The recombinant subtilisin-like protease also showed toxicity towards brain microvascular endothelial cells. Lastly, sera from pigs infected with S. suis reacted with the recombinant SspA, indicating that it is produced during infections. In conclusion, the SspA of S. suis shared similarities with subtilisin-like proteases produced by other pathogenic streptococci and may contribute to the pathogenic process of S. suis infections.     

Amino acid digestibility in heated soybean meal fed to growing pigs

Heat treatment of soybean meal (SBM) is necessary to reduce the concentration of trypsin inhibitors, but excessive heat treatment may reduce AA concentration and digestibility because AA can be destroyed by the Maillard reaction. The objective of this experiment was to determine the effects of heat treatment of SBM on apparent ileal digestibility and standardized ileal digestibility (SID) of AA by growing pigs. A source of conventional dehulled SBM (48.5% CP) was divided into 4 batches. One batch was not additionally heated, 1 batch was autoclaved at 125°C for 15 min, 1 batch was autoclaved at 125°C for 30 min, and 1 batch was oven-dried at 125°C for 30 min. Four SBM-cornstarch diets were formulated, and each of the 4 batches of SBM was used as the sole source of dietary AA in 1 diet. A N-free diet was used to estimate basal endogenous losses of AA. Ten growing barrows with an initial BW of 25.3 ± 2.0 kg were individually fitted with a T-cannula in the distal ileum. Pigs were allotted to treatments in a replicated 5 × 5 balanced Latin square design with 5 diets and 5 periods. Each period lasted 7 d, and ileal digesta were collected on d 6 and 7 of each period. Results of the experiment indicated that the apparent ileal digestibility and SID of CP and all AA decreased linearly (P < 0.01) as the time of autoclaving increased from 0 to 30 min. The concentration of furosine and the color of samples of SBM indicated that autoclaving resulted in a Maillard reaction in the SBM. However, oven drying at 125°C for 30 min did not change (P > 0.10) the SID of CP and AA in the SBM or the furosine concentration, and the color in the oven-dried sample indicated that this sample was not heat damaged. In conclusion, the digestibility of all AA in autoclaved SBM is linearly reduced as the autoclaving time increases from 0 to 30 min. The reason for these changes is most likely that autoclaving at 125°C results in Maillard reactions in SBM.     

Comparative studies on temperature threshold for heat shock protein 70 induction in young and adult Murrah buffaloes

To know the temperature threshold for heat shock protein 70 (HSP70) induction in lymphocytes and to assess physiological changes, if any, in relation to HSP70 induction in young and adult Murrah buffaloes, this study was divided into two parts: I. In vivo study: where assay of HSP70 was performed in blood samples collected from acutely exposed young and adult Murrah buffaloes (n = 6) inside a climatic chamber at 40, 42 and 45 °C for 4 h and thermoneutral temperature (22 °C). Physiological parameters viz., rectal temperature, respiratory rate, pulse rate and skin temperature of different body parts were monitored to assess magnitude of stress in the animals owing to thermal exposure II. For in vitro study, equal numbers of lymphocyte cells were separated from blood collected from young and adult buffaloes and were subjected to four temperature treatments (38, 40, 42 and 45 °C) for 4 h. A significant increase (p < 0.05) in all the physiological parameters in both young and adult buffaloes was observed after exposure to 40, 42 and 45 °C for 4 h as compared to 38 °C. The average plasma HSP70 concentrations (ng/ml) were significantly higher (p < 0.05) at 40, 42 and 45 °C as compared to 38 °C in both young and adult and were higher in young than adult buffaloes at 38 and 45 °C. Heat shock protein 70 level in lymphocyte lysate showed highest concentration after 3-h exposure to all temperatures (40, 42 and 45 °C) in both young and adult buffaloes. The intensity of changes of all physiological parameters was more in young animals than in the adults indicating the greater susceptibility of younger animals to heat stress and was found to be changed at around 40 °C when animals were exposed to different temperatures, indicating the possibility that HSP70 production may be initiated at this temperature which is 2 or 3 °C higher than core body temperature.     

Maternal obesity upregulates fatty acid and glucose transporters and increases expression of enzymes mediating fatty acid biosynthesis in fetal adipose tissue depots

Maternal nutrient restriction leads to alteration in fetal adipose tissue, and offspring from obese mothers have an increased risk of developing obesity. We hypothesized that maternal obesity increases fetal adipogenesis. Multiparous ewes (Columbia/Rambouillet cross 3 to 5 yr of age) carrying twins were assigned to a diet of 100% (Control; CON; n = 4) or 150% (Obese; OB, n = 7) of NRC maintenance requirements from 60 d before conception until necropsy on d 135 of gestation. Maternal and fetal plasma were collected and stored at -80°C for glucose and hormone analyses. Fetal measurements were made at necropsy, and perirenal, pericardial, and subcutaneous adipose tissues were collected from 7 male twin fetuses per group and snap frozen at -80°C. Protein and mRNA expression of fatty acid translocase [cluster of differentiation (CD) 36], fatty acid transport proteins (FATP) 1 and 4, insulin-sensitive glucose transporter (GLUT-4), fatty acid synthase (FASN), and acetyl-coA carboxylase (ACC) was evaluated. Fetal weight was similar, but fetal carcass weight (FCW) was reduced (P < 0.05) in OB versus CON fetuses. Pericardial and perirenal adipose tissue weights were increased (P < 0.05) as a percentage of FCW in OB versus CON fetuses, as was subcutaneous fat thickness (P < 0.001). Average adipocyte diameter was greater (P < 0.01) in the perirenal fat and the pericardial fat (P = 0.06) in OB fetuses compared with CON fetuses. Maternal plasma showed no difference (P > 0.05) in glucose or other hormones, fetal plasma glucose was similar (P = 0.42), and cortisol, IGF-1, and thyroxine were reduced (P ≤ 0.05) in OB fetuses compared with CON fetuses. Protein and mRNA expression of CD 36, FATP 1 and 4, and GLUT-4 were increased (P ≤ 0.05) in all fetal adipose depots in OB versus CON fetuses. The mRNA expression of FASN and ACC was increased (P < 0.05) in OB vs. CON fetuses in all 3 fetal adipose tissue depots. Fatty acid concentrations were increased (P = 0.01) in the perirenal depot of OB versus CON fetuses, and specific fatty acid concentrations were altered (P < 0.05) in subcutaneous and pericardial adipose tissue because of maternal obesity. In conclusion, maternal obesity was associated with increased fetal adiposity, increased fatty acid and glucose transporters, and increased expression of enzymes mediating fatty acid biosynthesis in adipose depots. These alterations, if maintained into the postnatal period, could predispose the offspring to later obesity and metabolic disease.     

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.