Distinct neutrophil C5a receptor inflammatory events in cows initiated by chemoattractant C5a and lipopolysaccharide around parturition and in mid lactation.

In neutrophils, toll-like receptor and complement component 5a (C5a) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C5a receptor, complement component 5a receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C5a and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C5a stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections.

F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Screening of ragdoll cats for kidney disease: a retrospective evaluation.

OBJECTIVES: To assess the prevalence of renal abnormalities in ragdoll cats. Ragdoll breeders often warn clients to watch for future renal problems, mainly due to chronic interstitial nephritis and polycystic kidney disease. Therefore, ragdoll screening by abdominal ultrasonography, measurement of serum creatinine and urea concentrations and genetic testing is often performed without documented scientific evidence of increased risk of renal disease. METHODS: Retrospective evaluation of ragdoll screening for renal disease at one institution over an eight-year period. RESULTS: Renal ultrasonography was performed in 244 healthy ragdoll cats. Seven cats were positive for polycystic kidney disease, 21 were suspected to have chronic kidney disease, 8 had abnormalities of unknown significance and 2 cats had only one visible kidney. Cats suspected to have chronic kidney disease were significantly older and had significantly higher serum urea and creatinine concentrations than cats with normal renal ultrasonography. All 125 genetically tested cats were negative for polycystic kidney disease. However, only one of the seven ultrasonographically positive cats underwent genetic testing for polycystic kidney disease. CLINICAL SIGNIFICANCE: Ultrasonographic findings compatible with chronic kidney disease were observed in almost 10% of cats, and polycystic kidney disease occurred at a low prevalence (<3%) in this ragdoll population. Further studies are required to elucidate if ragdoll cats are predisposed to chronic kidney disease.

The importance of adequate fixation for immunofluorescent staining of bovine embryos.

Immunofluorescent staining is often used to investigate the expression of specific proteins in pre-implantation embryos. The success of this method is determined by the specificity of the antibodies, but also by the protocol used for fixation and permeabilization of the samples. In this study, different fixatives are compared in combination with immunofluorescent staining of caudal-type homeobox 2 (CDX2), fibronectin 1 (FN1) and integrins (ITGs) on bovine blastocysts. For both CDX2 and the ITGs, the outcome of the staining was largely dependent on the fixation methods. Paraformaldehyde fixation was best for the intracellular CDX2 protein, whereas acetone fixation gave the best results for the transmembrane ITGs. No difference was observed for the FN1 staining between samples fixed with paraformaldehyde or acetone. These examples demonstrate that the choice of fixation and permeabilization agents is very important for the outcome of the experiment, and this choice is dictated by the (extra)cellular location of the protein under investigation. Inappropriate fixation and/or permeabilization methods can lead to erroneous conclusions regarding the site and amount of protein expression.

Differential gene expression of the toll-like receptor-4 cascade and neutrophil function in early- and mid-lactating dairy cows.

During early lactation, neutrophils display several reduced immune functions. Particularly, a delayed recruitment of neutrophils into the infected udder seems to be one of the underlying events involved in the severity of postpartum Escherichia coli intramammary infections. The purpose of this study was to analyze the effect of in vitro chemotaxis and diapedesis on the expression of toll-like receptor-4 (TLR4)-related genes in bovine blood neutrophils isolated from 10 early-lactating (EL) and 10 mid-lactating (ML) cows. Functional characterization of the neutrophil population was performed by measuring phagocytosis and production of reactive oxygen species (chemiluminescence). Messenger RNA was extracted from neutrophils, and the expression of TLR4 and associated genes in EL and ML cows was analyzed by reverse-transcription quantitative PCR. To study the effect of chemotaxis and diapedesis on the expression of genes of the TLR4 cascade, neutrophils were stimulated to (trans)migrate in response to C5a using in vitro models. Our salient findings were that both neutrophil migration in vitro and lactation stage induced significant changes in the expression of several genes of the TLR4 signaling cascade. Before migration, expression of TRAF6, ATF3, RELA, IL8, and C5aR were lower in EL than in ML cows. Diapedesis and chemotaxis induced an increase in expression of TLR4, ATF3, and IL8 in both EL and ML cows. Diapedesis resulted in a downregulation of Syk, a TLR4-associated gene, in ML cows. This study shows that the perturbations in neutrophil functions during EL are accompanied by modulation of TLR4 pathway genes. These data can contribute to the understanding of the mechanisms explaining the relationship between stage of lactation and risk of severe E. coli mastitis.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry.

Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.

Modeling the interaction of gametes and embryos with the maternal genital tract: from in vivo to in silico.

Understanding the complex interaction between gametes or embryos and the maternal genital tract requires the use of experimental models. The selection of the right model is an important task to undertake, and despite many new developments in this area, an ideal model system has not yet been developed. In this review article, we focus on how the most appropriate model species and model system can be selected, each with its particular advantages and disadvantages. Selection criteria need to be based on the evaluation of the aim of the experiment, the tools that are available to the scientist, and the ethics that are involved in working with particular animal species and model systems. Society and politics direct scientists to "Refine, Reduce, and Replace" the use of experimental animals, which means that the use of in vivo models is increasingly being discouraged. An in vivo model allows experimentation in the full biological environment of a living organism. In contrast with in vivo models, in vitro models are less complex and are abstracts of in vivo systems, leading often to results that are different from the in vivo situation. If an investigator could understand all the components of a complex biological system and re-create them as individual smaller models in a computer, he or she could create in silico models that would completely represent the complexity of in vivo models. We predict that in the future, in silico modeling will be the natural departure from in vivo, in situ, and in vitro modeling approaches. In addition to numerous advantages that this modeling approach can bring to studying maternal interaction with gametes and embryo, it is perhaps the only true alternative method to animal experimentation.

Identification of polymorphisms in the ovine Shadow of prion protein (SPRN) gene and assessment of their effect on promoter activity and susceptibility for classical scrapie.

Shadow of prion protein (SPRN) is an interesting candidate gene thought to be involved in prion pathogenesis. In humans, an association has already been discovered between mutations in SPRN and the incidence of variant and sporadic Creutzfeldt-Jakob disease. However, in sheep, the effect of mutations in SPRN is largely unknown. Therefore, we analysed the presence of mutations in the entire ovine SPRN gene, their association with scrapie susceptibility and their effect on SPRN promoter activity. In total, 26 mutations were found: seven in the promoter region, four in intron 1, seven in the coding sequence and eight in the 3' untranslated region. The mutations detected in the coding sequence and the promoter region were subsequently analysed in more detail. In the coding sequence, a polymorphism causing a deletion of two alanines was found to be associated with susceptibility for classical scrapie in sheep. Furthermore, a functional analysis of deletion constructs of the ovine SPRN promoter revealed that the region 464 to 230 bp upstream of exon 1 (containing a putative AP-2 and putative Sp1 binding sites) is of functional importance for SPRN transcription. Six mutations in the SPRN promoter were also found to alter the promoter activity in vitro. However, no association between any of these promoter mutations and susceptibility for classical scrapie was found.

Association analyses of candidate single nucleotide polymorphisms on reproductive traits in swine.

The ability to identify young females with superior reproduction would have a large economic impact on commercial swine production. Previous studies have discovered SNP associated with economically important traits such as litter size, growth rate, and feed intake. The objective of this study was to test for association of candidate SNP with sow prolificacy reproductive traits in gilts of a Landrace-Duroc-Yorkshire composite population. Association analyses regressed additive (A), dominant (D), and imprinting (I) SNP effects on each trait with an animal model. A carnitine palmitoyltransferase 1A SNP and a glycogen synthase 1 SNP were associated with age at puberty (AP; D = 10 d; P = 0. 0037 and A = 3.8 d; P = 0.0078, respectively). Four IGF2 SNP were associated with AP as well, having additive or dominant effects (3.2 to 5.8 d; P < or = 0.0052). Two mannosidase 2B2 SNP and 2 prolactin receptor (PRLR) SNP were also associated with AP. Solute carrier 22, subfamily member 5 SNP was weakly associated with AP (D = 3.9 d; P < 0.10). Polymorphisms within glycogen synthase 1 and protein kinase AMP-activated, gamma 3 noncatalytic subunit had associations with ovulation rate. Estrogen receptor (ESR) 1, ESR2, PPAR gamma coactivator 1, and IGFBP3 SNP were significantly associated with weaning-to-estrus interval. Two PRLR SNP were associated with total number of piglets born (A = 0.57 piglets; P = 0.0095 and D = 0.61 piglets; P = 0.0016, respectively). A SNP within PRLR was also associated with number of piglets born alive (D = 0.61; P = 0.0016). The PPAR gamma coactivator 1 SNP was associated with total number of piglets born (D = 0.38 piglets; P = 0.0391) and number of piglets born alive (D = 0.53 piglets; P = 0.0032). The SNP within ESR1 (A = 0.65 piglets; P = 0.0950), ESR2 (A = -0.33 piglets; P = 0.0176), IGF2 SNP (A = -0.26 piglets; P = 0.0032), and IGFBP3 SNP (D = 0.35 piglets; P = 0.0683) were associated with number of piglets born dead. A leptin SNP was associated with mummified fetuses (D = 0.09 piglets; P = 0.0978). Many of the SNP analyzed in this study are from genes involved in regulation of metabolism, suggesting that there is an important link between physiological events associated with reproduction and energy utilization. Furthermore, these production and growth trait SNP may serve to assist in selection of young females for superior reproductive performance.

Correlation between porcine PPARGC1A mRNA expression and its downstream target genes in backfat and longissimus dorsi muscle.

Knowledge of in vivo relationship between the coactivator PPARGC1A and its target genes is very limited, especially in the pig. In this study, a real-time PCR experiment was performed on longissimus dorsi muscle (MLD) and backfat with 10 presumed PPARGC1A downstream target genes, involved in energy and fat metabolism, to identify possible relationships with PPARGC1A mRNA expression in vivo in the pig (n = 20). Except for UCP3 and LPL, a very significant difference in expression was found between MLD and backfat for all genes (P < 0.01). Hierarchical cluster analysis and the significant pairing of mRNA expression data between sampling locations suggested a genetic regulation of the expression of several target genes. A positive correlation with PPARGC1A was found for CPT1B, GLUT4, PDK4, and TFAM (P < 0.0001). A negative correlation was found for UCP2, FABP4, LEP (P < 0.0001), and TNF (P = 0.0071). No significant correlation was detected for UCP3 and LPL. This study provides evidence for a clear difference in mRNA expression of crucial genes in fat and energy metabolism between 2 important tissues. Our data suggest a clear impact of PPARGC1A on energy and lipid metabolism in vivo in the pig, through several of these downstream target genes.

Porcine OGN and ASPN: mapping, polymorphisms and use for quantitative trait loci identification for growth and carcass traits in a Meishan x Piétrain intercross.

The porcine orthologues of human chromosome HSA9q22.31 genes osteoglycin (OGN) and asporin (ASPN) were mapped to porcine chromosome SSC3 using linkage analysis and a somatic cell hybrid panel. This mapping was refined to SSC3q11 using fluorescence in situ hybridization. These results confirm the existence of a small conserved synteny group between SSC3 and HSA9. Polymorphisms were revealed in both genes, including a pentanucleotide microsatellite (SCZ003) in OGN and two single nucleotide polymorphisms (AM181682.1:g.780G>T and AM181682.1:g.825T>C) in ASPN. The two genes were included in a set of markers for quantitative trait loci (QTL) mapping on SSC3 in the Hohenheim Meishan x Piétrain F2 family. Major QTL for growth and carcass traits were centred in the ASPN-SW902 region.

SNP identification, linkage and radiation hybrid mapping of the porcine lamin A/C (LMNA) gene to chromosome 4q.

The lamins are components of nuclear lamina and they have a profound influence on nuclear structure and functions. They are encoded by three genes, LMNA, LMNB1 and LMNB2. A genomic fragment of the porcine LMNA gene (822 bp; from exons 7 to 9) was amplified by polymerase chain reaction and comparatively sequenced. Four single nucleotide polymorphisms (SNPs) were identified in intronic sequences: G162A, G208A, T367G and C618T. The SNPs are within the restriction sites for enzymes Bsh1236I, HpaII, AluI and Bsh1236I respectively. Allele frequencies at SNPs G208A, T367G and C618T were determined by using eight pig breeds. Linkage analysis in the Hohenheim Meishan x Piétrain family placed the LMNA gene in the chromosome 4q linkage group, between MEF2D and GBA (MEF2D - 3.0 cM - LMNA - 0.2 cM - GBA). In radiation hybrid mapping LMNA was most significantly linked to SW270 on chromosome 4 (39 cR; LOD = 7.86). The LMNA gene is located in the quantitative trait loci region for some carcass traits on chromosome 4q.

Porcine PPARGC1A (peroxisome proliferative activated receptor gamma coactivator 1A): coding sequence, genomic organization, polymorphisms and mapping.

We report here the characterisation of porcine PPARGC1A. Primers based on human PPARGC1A were used to isolate two porcine BAC clones. Porcine coding sequences of PPARGC1A were sequenced together with the splice site regions and the 5' and 3' regions. Using direct sequencing nine SNPs were found. Allele frequencies were determined in unrelated animals of five different pig breeds. In the MARC Meishan-White Composite resource population, the polymorphism in exon 9 was significantly associated with leaf fat weight. PPARGC1A has been mapped by FISH to SSC8p21. A (CA)n microsatellite (SGU0001) has been localised near marker SWR1101 on chromosome 8 by RH mapping and at the same position as marker KS195 (32.5 cM) by linkage mapping. The AseI (nt857, Asn/Asn489) polymorphism in exon 8 was used to perform linkage analysis in the Hohenheim pedigrees and located the gene in the same genomic region. Transcription of the gene was detected in adipose, muscle, kidney, liver, brain, heart and adrenal gland tissues, which is in agreement with the function of PPARGC1A in adaptive thermogenesis.

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence.

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.