Expression and function of Toll-like receptor 2 in canine blood phagocytes

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.     

Molecular mechanisms underlying pig oocyte maturation and fertilization

Since the pig is not only an important farm animal, but also a model animal for biomedical applications, the development of reproductive technologies in this species has been very important. In vitro oocyte maturation and fertilization (IVM-IVF) are basic techniques for a number of oocyte- or embryo-related technologies. The practical aspects for pig oocyte IVM-IVF have been reviewed, while the molecular mechanisms underlying oocyte meiotic maturation and fertilization have not been well summarized, although accumulating data have been obtained in recent one decade. This review will focus on what is known about the molecular mechanisms of porcine oocyte maturation and fertilization such as first meiosis resumption, meiotic spindle assembly, second meiosis metaphase (MII) arrest during oocyte maturation, sperm-egg recognition and fusion, sperm acrosome reaction, second meiosis resumption, sperm chromatin decondensation, and pronucleus formation during fertilization, as well as the establishment of polyspermy block.     

Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

Age-dependent changes of proinflammatory cytokine production by porcine peripheral blood phagocytes

The aim of the present study was to determine postnatal ontogeny of proinflammatory cytokines IL-1beta, IL-8 and TNF-alpha production by in vitro stimulated porcine blood leukocytes. Four age categories of pigs were chosen. Cytokine production was determined using intracellular flow cytometry. It was found that IL-8 and TNF-alpha production by blood monocytes significantly increased during the postnatal period while production of IL-1beta remained unchanged. In blood neutrophils, the IL-8 production increased only during the postnatal period, while the levels of TNF-alpha and IL-1beta were undetectable during the whole postnatal period. Generally, the most intensive changes in cytokine production occurred before weaning. The production of low levels of cytokines by monocytes and neutrophils from young pigs was not caused by a delayed cytokine response because the cytokine production after 8-h stimulation was lower than that after 4-h stimulation in all age categories. The ontogenetical changes showed the same trends when two different stimulators (LPS, heat-inactivated E. coli) were used, suggesting that the ontogenetical changes are not caused by a simple defect in one signalling pathway, but it is probably a more complex process. No differences in cytokine production between the whole blood and the isolated cells supplemented with newborn or adult serum were found. Thus the ability of newborn monocytes and neutrophils to produce proinflammatory cytokines was not decreased due to the influence of composition of the microenvironment, where the cells were present. In conclusion, the ability of porcine blood leukocytes to produce cytokines develops during postnatal life.     

Effects of 17beta-estradiol on in vitro maturation of pig oocytes in protein-free medium

The present study examined the effects of 17 beta-estradiol (E(2)) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E(2) (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E(2) added). Although oocytes matured in the presence of E(2) were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E(2) were prevented when the medium was supplemented with E(2) together with its antagonist, ICI 182,780 (1 microg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E(2) had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E(2). When cumulus-enclosed oocytes were cultured in the presence of progesterone (P(4); 600 ng/ml) alone or together with E(2), no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P(4) in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E(2) than in medium without E(2). These results indicate that E(2) inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E(2) antagonist or P(4). This E(2) inhibition may occur indirectly via the cumulus cells and inhibition of P(4) synthesis.     

血浆蛋白粉在仔猪饲料中的应用

血浆蛋白粉是一种新型蛋白质饲料,是将在屠宰场收集的新鲜全血(牛血或猪血),立即加上抗凝剂(如柠檬酸钠),然后将血泵入连续转动的离心机,使血细胞和血浆分离,通过超微过滤,将血浆从8%浓缩到20%,并随即冷却至1～2℃,然后对血浆进行喷雾干燥,而制成粉状浅棕褐色产品。为了提高血浆蛋白粉的质量,现在多采用喷雾干燥技术,因此其产品通常也叫做喷雾干燥血浆蛋白粉(ＳＤＰＰ)。由于它含有大量的功能性蛋白质,如免疫球蛋白和较理想的氨基酸组成,近几年在仔猪饲料中被越来越多的应用。本文就血浆蛋白粉的营养价值、应用效果、最适添加比例、作用方式…     

Phenotypic and functional characterization of pig lymphocyte populations

Monoclonal antibodies reactive with swine lymphoid cell subpopulation antigens and with the swine major histocompatibility complex, the SLA complex, antigens are summarized. The mAb have been used to analyze swine peripheral T cell populations, the CD4+ helper T cells and the CD8+ cytotoxic/suppressor T cells. Three unique properties of swine T cells have been described. 1) There is an unusually high (25%) number of CD4+ CD8+ dual expressor T cells. 2) The ratio of CD4+ to CD8+ T cells is lower than expected; the ratio of 0.6, which is normal for pigs, only occurs in pathological conditions in humans. 3) Resting CD8+ cells preferentially express class II SLA antigens. The significance of these unusual properties of swine T cells is currently under study.     

感染球虫的肉鸡日粮中添加甜菜碱能够增加十二指肠上皮淋巴细胞数量并增强吞噬细胞功能

甜菜碱通常用于细胞渗透压的调节,本试验的目的在于探索甜菜碱、渗透压、球虫病之间的联系。试验一,肉鸡饲喂玉米-大豆型日粮,分为3个处理,基础日粮中分别添加甜菜碱0、0.5、1.0g/kg,其中一半肉鸡感染堆型艾美尔球虫。球虫感染降低了肉鸡增重和饲料效率,并且增加了十二指肠、空场黏膜的渗透压(P0.01)。甜菜碱降低了十二指肠渗透压(P0.01),特别是有球虫感染的时候。球虫感染增加了十二指肠固有层厚度(P=0.04)和白细胞的数目(P0.01),特别是在高甜菜碱水平时(互作p=0.05)。球虫可以使绒毛高度降低(p=0.05),但当添加1.0g/kg甜菜碱时情况得以改善(互作p=0.04)。试验二,将腹膜巨噬细胞和外周血异嗜细胞在渗透压分别为200、310、600或900mOsmol,甜菜碱含量分别为0、0.1、0.5或1.5mmol/L的(4×4因子)介质中培养6h,然后加入堆型艾美尔球虫。从结果来看,高渗透压介质与等渗透压介质比较,吞噬作用与NO的释放受到了抑制,而IL-1、IL-6的含量升高。0.1mmol/L甜菜碱组显著(p=0.04)增加异嗜细胞NO的产生,并具有增加巨噬细胞NO产生的趋势(p0.1)。甜菜碱可以促进异嗜细胞分泌趋化因子增强单核白细胞趋化性。除了增加白细胞的数目外,巨噬细胞增强单核白细胞趋化性和NO的释放或许可以解释感染球虫时添加甜菜碱能够降低肠道损伤的现象。     

新鲜和冷冻猪卵子体外成熟培养对纤溶酶原激活物存在的影响

对体外成熟培养不同时间段猪的新鲜和冷冻的卵子中是否能够释放纤溶酶原激活物(PAs)的情况进行了研究。被卵丘细胞包裹的卵子取自于囊状卵泡,用NUSU-23培养液进行体外培养。OPS冷冻法冷冻卵子。在成熟培养液的卵丘细胞通过用细管不断地吸吐的方法去除。用SDS和酶谱法对猪的卵丘卵母细胞和裸卵里的纤溶酶原激活物进行密度测定。结果显示:在猪新鲜的卵丘卵母细胞中能够检测出Upa、tPA和tPA-PAI。体外培养24h后,在裸卵中也可以检测出uPA的活性;体外成熟培养48h后,在卵丘卵母细胞中能够检测出tPA和tPA-PAI,但在裸卵中没有检测出PAs。在所有冷冻组的试验中,没有检测出PAs活性。     

Differential electrocardiographic study on Iberica and Duroc breeds of pig during physical maturation

Sequential electrocardiograms were taken of 50 pigs (25 Iberica and 25 Duroc), from the ages of 5 days to 205 days. The records were analysed to establish the normal values of the different electrocardiographic intervals (RR, QRS, QT, TQ and ST intervals), the diastole/systole quotient and heart score in the first 7 months of life, as well as age-related electrocardiographic variations. In addition, it was intended to determine which of these two breeds showed the greater heart recovery capacity. Finally, positive or negative correlations between the RR interval and the electrocardiographic incidents studied were analysed. The mean values obtained for the electrocardiographic parameters were similar in the two breeds and increased with their physical maturity. Analysis of the correlation between the duration of the heart cycle and the different electrocardiographic incidents showed a positive and significant correlation, the r-values being higher for the RR interval-TQ interval correlation. There was very little correlation between RR interval and heart score in Duroc pigs and practically no correlation between the RR interval and the QRS interval in either breed.     

Survival and infectivity of Trypanosoma brucei in refrigerated pig blood

Trypanosoma brucei parasites survived for 72 h or longer in refrigerated pig blood. The survival period was directly proportional to the initial parasite concentration of the sample. Infectivity of the parasites declined faster than survival, being less than 1 per 10(5) motile organisms at 72 h. The stage of infection in the pig (early vs. late) did not appear to influence subsequent survival periods or infectivity of the parasites in vitro.     

Red blood cell volume of the pig fetus

A non-radioactive fluorescent excitation analysis technique was used to measure total red blood cell volume in 31 unanaesthetised pig fetuses in utero. Red blood cell volume (y in ml) was closely related (r = 0.94) to fetal bodyweight (x in g): where y = 2.92 + 0.0291x. Average red blood cell volume was 34 +/- 1 ml kg-1 fetal bodyweight. Average estimated (total) blood volume was 117 +/- 3 ml kg-1 fetal bodyweight. It was concluded that this non-radioactive indicator dilution measurement of red blood cell volume is a significant advance over the established 51Cr method, and that measurement of red blood cell volume may be used to estimate fetal bodyweight in utero.