Early transcriptomic changes in peripheral blood 7 days after embryo transfer in dairy cattle.

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt re-insemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1, CXCL3, CCL4, and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15, MX2, OASY1, and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immune-related genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

The relationship between sucrose intake in coffee or tea, and root or coronal caries in an elderly Japanese population.

OBJECTIVES: Few studies have examined the effect of sucrose intake in coffee or tea (SCT) on dental caries, especially root caries, in elderly people. The purpose of the present study was to examine the associations between SCT and the prevalence of root or coronal caries in community-dwelling elderly Japanese. METHODS: Participants were 370 elders aged 75 years from a larger cohort study of elders in Niigata City, Japan. Dietary habits during the preceding month were assessed with a validated brief self-administered diet history questionnaire (BDHQ). The caries examination was conducted using mirror and ball-pointed periodontal probes under artificial light. Poisson regression analysis was performed to analyze the prevalence-rate ratios (PRRs) between SCT and the root or coronal caries. The number of root or coronal caries was selected as the dependent variable for the analysis. Then, SCT (tertiles labeled low, medium and high) and other 4 variables were selected as independent variables. RESULTS: The PRRs between the number of root caries lesions and SCT were signifi cantly higher in the second (medium intake, 1.38, p ⟨ 0.001) and third tertiles (high intake, 2.07, p ⟨ 0.001). Similar tendencies were seen for the number of coronal caries lesions (PRRs in the second and third tertiles 1.74 and 2.46, respectively, both p ⟨ 0.001). CONCLUSION: A significant positive relationship was observed between sucrose in coffee and tea and the number of coronal or root caries lesions in community-dwelling elderly Japanese.

Lack of ethnic differences of moxifloxacin and metabolite pharmacokinetics in East Asian men.

This study was designed to investigate ethnic differences in the pharmacokinetics (PKs) of moxifloxacin and its metabolites, M1 (sulfo conjugate) and M2 (acyl-glucuronate), among Japanese, Chinese, and Korean populations, following oral administration. We used a population PK modeling approach using data from a clinical study involving 79 healthy male volunteers. A comprehensive population PK model considering the PK mechanism of moxifloxacin and its metabolites was newly built. The structures of the final model were two-compartment for moxifloxacin and one-compartment for M1 and M2, with first-order absorption with lag time for all three compounds. The formation of M1 and M2 from moxifloxacin via a first-pass effect and subsequent metabolic clearance in the system were also modeled. Lean body mass on the central volume of distribution (V c ) and estimated glomerular filtration rate on renal clearance (CL r ) were identified as covariates of PKs of moxifloxacin. Additionally, bioavailability was slightly higher in Koreans, whereas CL r , non-renal clearance (CL nr ), and V c were slightly lower. Regarding M1 and M2, body surface area on CL r of M2 and UGT1A1*6 on F of M2 were modeled. Korean ethnicity was observed to influence CL nr of M2, F of M2, and the metabolic clearance of moxifloxacin to M2. However, the exposure levels of moxifloxacin, M1, and M2 in Koreans were comparable to those in Japanese and Chinese because the effects of Korean ethnicity on some PK parameters were counterbalanced. These results suggest that PKs for moxifloxacin and its metabolites among East Asian populations are essentially similar.

Specific HLA genotypes confer susceptibility to acute necrotizing encephalopathy.

Acute necrotizing encephalopathy (ANE) is a rare and severe syndrome of acute encephalopathy triggered by viral infections. Cytokine storm is considered as the main pathogenetic mechanism of ANE. ANE is prevalent in East Asia, suggesting the association of host genetic factors. To elucidate the genetic background of Japanese ANE, we examined genotypes of human leukocyte antigen (HLA)-A, C, B, DRB1, DQB1 and DPB1 in 31 patients. Significant positive association was observed in both the allele frequency and positivity of DRB1*09:01 (P=0.043 and 0.025, respectively), as well as those of DQB1*03:03 (P=0.034 and 0.026, respectively). The carrier frequency of DRB1*09:01 and DQB1*03:03 alleles was higher in the patients (45.16%) than in controls (28.57%). These alleles are more common in East Asian than in European populations, and are reportedly associated with various autoimmune diseases in Japanese patients. Our data provide further evidence that altered immune response based on individual HLA genotypes may contribute to ANE pathogenesis.

Lipopolysaccharide enhances apoptosis of corpus luteum in isolated perfused bovine ovaries in vitro.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL) in vivo. The objective was to investigate whether these effects were mediated directly by LPS or via LPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 µg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2α were measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2, -4) and LH (LHCGR); the cytokine TNFA; steroidogenic (STAR, HSD3B), angiogenic (VEGFA121, FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES, PGFS, PTGFR) and apoptosis (CASP3, -8, -9). Treatment with LPS abolished the hCG-induced increase in P4 (P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2α at 70 min after LPS challenge. Furthermore, mRNA abundance of TLR2, TNFA, CASP3, CASP8, PGES, PGFS, and VEGFA121 increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4 responsiveness to hCG in LPS-treated ovaries in vitro was not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2α could not be excluded.

Short communication: Urea induces T helper 2 (Th2) type environment at transcriptional level and prostaglandin E2 secretion in bovine oviduct epithelial cells in culture.

Excess dietary protein intake in early lactation dairy cows resulting in blood urea nitrogen of greater than 19 to 20mg/dL is associated with decreased fertility. Little is known about the local interference of urea in the normal immunological environment of the oviduct that provides optimal conditions for early reproductive events. A bovine oviduct epithelial cell (BOEC) culture was used to determine how urea influences immune environment. The BOEC monolayer was supplemented with low (20mg/dL) and high (40mg/dL) concentrations of urea together with ovarian steroids, estradiol (1ng/mL) and progesterone (1ng/mL), and LH (10ng/mL) at concentrations observed during the preovulatory period. The urea values used in this study were equivalent to 9.3 and 18.7mg/dL of blood urea nitrogen, which are typically common in lactating dairy cows with low or high protein intake, respectively. Stimulation of BOEC with 40mg/dL of urea induced gene expression of IL10 and IL4, epithelial-derived T helper type 2-driving (anti-inflammatory) cytokines as well as mPGES-1 expression and prostaglandin E2 (PGE2) secretion. However, urea concentrations of both 20 and 40mg/dL failed to alter the expression of IL1B and TNFA, Th1-driving cytokines, and the gene expression of TLR4. However, a concentration of 40mg/dL of urea stimulated α 1-acid glycoprotein expression, an acute phase protein. Data from this in vitro study suggest that urea, at least in part, contributes to influence the expression of some immune-related genes toward T helper type 2 type and prostaglandin E2 secretion, leading to disruption in local environment for fertilization and early embryonic development.

Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin-like growth factor-1 in pre-pubertal Holstein heifers.

Insulin-like growth factor-1 (IGF-1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR-SNPs were shown to be related to plasma IGF-1 concentration in cattle. Hence, the capacity to IGF-1 production in the liver might be affected by GHR-SNP and associated with performance in the future. This study examined whether GHR-SNP is associated with IGF-1 production in the liver of pre-pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR-SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre-injection than 24 h post-injection (p < 0.01). Moreover, plasma GH concentrations in AG post-injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF-1 concentrations in pre-injection than post-injection (p < 0.01), although oestradiol did not change IGF-1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF-1 production in the liver of cattle.

Antepartal insulin-like growth factor 1 and insulin-like growth factor binding protein 2 concentrations are indicative of ketosis in dairy cows.

A study involving a small number of cows found that the concentrations of insulin-like growth hormone 1 (IGF1) may be a useful predictor of metabolic disease. Further, IGF1 may provide also a pathophysiological link to metabolic diseases such as ketosis. The objective of the current study was to test whether the low antepartal total IGF1 or IGF1 binding protein (IGFBP) concentrations might predict ketosis under field conditions. Clinical examinations and blood sampling were performed antepartum (262-270 d after artificial insemination) on 377 pluriparous pregnant Holstein Friesian cows. The presence of postpartum diseases were recorded (ketosis, fatty liver, displacement of the abomasum, hypocalcemia, mastitis, retention of fetal membranes, and clinical metritis or endometritis), and the concentrations of IGF1, IGFBP2, IGFBP3, and nonesterified fatty acids were measured. Cows with postpartum clinical ketosis had lower IGF1 concentrations antepartum than healthy cows. The sensitivity of antepartal IGF1 as a marker for postpartum ketosis was 0.87, and the specificity was 0.43; a positive predictive value of 0.91 and a negative predictive value of 0.35 were calculated. The cows with ketosis and retained fetal membranes had lower IGFBP2 concentrations compared with the healthy cows. It can be speculated that lower IGF1 production in the liver during late pregnancy may increase growth hormone secretions and lipolysis, thereby increasing the risk of ketosis. Lower IGFBP2 concentrations may reflect the suppression of IGFBP2 levels through higher growth hormone secretion. In conclusion, compared with nonesterified fatty acids as a predictive parameter, IGF1 and IGFBP2 may represent earlier biomarkers of inadequate metabolic adaptation to the high energy demand required postpartum.

Evidence for a novel, local acute-phase response in the bovine oviduct: progesterone and lipopolysaccharide up-regulate alpha 1-acid-glycoprotein expression in epithelial cells in vitro.

Little is known about the local production and function of alpha 1-acid glycoprotein (AGP), an acute-phase protein, in the female reproductive tract. This study aimed to investigate the regulation and immune function of AGP in cultured bovine oviduct epithelial cells. Analysis by Western blotting and immunohistochemistry revealed that bovine oviduct tissue expresses AGP protein in epithelial cells and the smooth muscle layer. Stimulation of bovine oviduct epithelial cells in culture with either progesterone (1 ng/ml) or lipopolysaccharide (LPS, 10 ng/ml) induced both mRNA expression and secretion of AGP. Estradiol (1 ng/ml), progesterone (1 ng/ml), and luteinizing hormone (10 ng/ml), which are observed during the peri-ovulatory period in oviduct tissues (steroids) or in circulation (luteinizing hormone), suppressed LPS-induced expression and secretion of AGP, which in turn induced the expression of Toll-like receptor-4 (TLR-4) and interleukin-1ß (IL-1B), but suppressed TLR-2 and tumor necrosis factor-α (TNFA) expression. AGP also inhibited LPS-induced TLR-2 and TNFA expression, but had no effect on LPS-induced TLR-4 and IL-1B expression. These findings suggest that oviductal epithelial cells can participate in antimicrobial processes through the secretion of AGP, which is partly regulated by ovarian steroids. Moreover, oviductal AGP may regulate the response of epithelial cells, thereby reducing the expression of the acute pro-inflammatory cytokine TNFA, which could contribute to the local homeostasis during the acute response to endotoxin release in the oviduct's anti-infection process.

A naturally occurring α(s1)-casein-derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum-free conditions.

Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α(s1)-CN (f16-23) (RPKHPIKH), α(s1)-CN (f16-24) (RPKHPIKHQ), α(s1)-CN (f17-25) (PKHPIKHQG), α(s1)-CN (f46-52) (VFGKEKV), α(s1)-CN (f94-105) (HIQKEDVPSER), and ß-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of αs1 -casein, varied structurally during early lactation: α(s1)-CN (f17-25) (PKHPIKHQG) and α(s1)-CN (f16-23) (RPKHPIKH)/α(s1)-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α(s1)-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium.

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig.

Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502-SLA-3*070102-SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701-SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

Do farm audits improve milk quality?

Milk quality is assessed using bulk milk analysis and by farm audits in the Netherlands. However, the extent of the effect that dairy farm audits have on milk quality is unknown. Data from over 13,000 audits performed on 12,855 dairy farms from February 2006 to April 2008 were merged with laboratory test results of 325,150 bulk milk samples collected 6 mo before and after the audit. A linear mixed model with the method of restricted maximum likelihood was conducted to study whether the total bacterial counts (TBC) of bulk milk were lower during the periods before and after the dairy farm audit. Results showed that TBC values were 2 to 6% lower (i.e., 0.010 to 0.026 log cfu/mL) for a period from 1.5 to at least 6 mo after an audit. Additionally, several variables were significantly associated with bulk milk TBC values: seasonality, total number of attention points (given if some checklist points were not appropriate), audit type, audit result, and the categories milking equipment maintenance, and utility room-tank maintenance. The TBC values increased with a higher level of attention points. Furthermore, the farms rejected based on the audit results had the highest average TBC values and the approved farms had the lowest values. If dairy farms had an overall negative audit assessment and consequently needed a re-audit in the following year, the TBC values of bulk milk were more likely to be higher. Auditing may provide dairy farmers the opportunity to receive advice about factors that influence bulk milk TBC values, for a period of at least 6 mo following the audit.

Diagnostic and prognostic significance of the alternatively spliced ACTN4 variant in high-grade neuroendocrine pulmonary tumours.

BACKGROUND: High-grade neuroendocrine tumours (HGNTs) of the lung manifest a wide spectrum of clinical behaviour, but no method for predicting their outcome has been established. MATERIALS AND METHODS: We newly established a monoclonal antibody specifically recognizing the product of the alternatively spliced ACTN4 transcript (namely, variant actinin-4), and used it to examine the expression of variant actinin-4 immunohistochemically in a total of 609 surgical specimens of various histological subtypes of lung cancer. RESULTS: Variant actinin-4 was expressed in 55% (96/176) of HGNTs, but in only 0.8% (3/378) of non-neuroendocrine (NE) lung cancers. The expression of variant actinin-4 was significantly associated with poorer overall survival in HGNT patients (P=0.00021, log-rank test). Multivariate analysis using the Cox proportional hazards model showed that the expression of variant actinin-4 was the most significant independent negative predictor of survival in HGNT patients (hazard ratio (HR), 2.15; P=0.00113) after the presence of lymph node metastasis (HR, 2.25; P=0.00023). CONCLUSIONS: The expression of variant actinin-4 is an independent prognostic factor for patients with HGNTs. This protein has a high affinity for filamentous actin polymers and likely promotes aggressive behaviour of cancer cells. The present clinical findings clearly support this notion.

Expression of cyclins and cyclin-dependent kinase inhibitors in granulosa cells from bovine ovary.

The regulation of granulosa cell proliferation is complex, and it is essential for normal follicular development in mammals. The aim of this study was to examine the expression of cyclins and their inhibitors in the granulosa cells of follicles at different developmental stages. Follicles were classified into three groups: oestrogen-inactive dominant follicles (EIDs), oestrogen-active dominant follicles (EADs) and pre-ovulatory follicles (POs). The expression of CCND2 (cyclin D2) mRNA was significantly higher in granulosa cells from EADs and POs than in those from EIDs. The expression of CCND3 (cyclin D3) mRNA was significantly higher in granulosa cells from EADs than in those from other follicles. CCND1 (cyclin D1), CCNE1 (cyclin E1) and CCNE2 (cyclin E2) mRNA expression did not differ among the different follicular stages. The expression of CDKN1A (p21(cip1) ) and CDKN1B (p27(kip1) ) mRNA was significantly higher in granulosa cells from EIDs and POs, respectively, than in those from other follicles. Expression of CDKN2D (p19(INK4d) ) mRNA did not differ among the different follicular stages. Taken together, our study suggested that cyclins and their inhibitors are associated with granulosa cell proliferation at specific follicular developmental stages.

Association of SET domain and myotubularin-related proteins modulates growth control.

Several proteins that contribute to epigenetic mechanisms of gene regulation contain a characteristic motif of unknown function called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. We have demonstrated that SET domains mediate highly conserved interactions with a specific family of proteins that display similarity with dual-specificity phosphatases (dsPTPases). These include myotubularin, the gene of which is mutated in a subset of patients with X-linked myotubular myopathy, and Sbf1, a newly isolated homologue of myotubularin. In contrast with myotubularin, Sbf1 lacks a functional catalytic domain which dephosphorylates phospho-tyrosine and serine-containing peptides in vitro. Competitive interference of endogenous SET domain-dsPTPase interactions by forced expression of Sbf1 induced oncogenic transformation of NIH 3T3 fibroblasts and impaired the in vitro differentiation of C2 myoblast cells. We conclude that myotubularin-type phosphatases link SET-domain containing components of the epigenetic regulatory machinery with signalling pathways involved in growth and differentiation.

FOXG1 mutations in Japanese patients with the congenital variant of Rett syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by microcephaly, psychomotor regression, seizures and stereotypical hand movements. Recently, deletions and inactivating mutations in FOXG1, encoding a brain-specific transcription factor that is critical for forebrain development, have been found to be associated with the congenital variant of RTT. Here we report the clinical features and molecular characteristics of two cases of the congenital variant of RTT. We conducted mutation screenings of FOXG1 in a cohort of 15 Japanese patients with a clinical diagnosis of atypical RTT but without MECP2 and CDKL5 mutations. Two unrelated female patients had heterozygous mutations (c.256dupC, p.Gln86ProfsX35 and c.689G>A, pArg230His). Both showed neurological symptoms from the neonatal period, including hypotonia, irritability and severe microcephaly. Further, their psychomotor development was severely impaired, as indicated by their inability to sit unaided or acquire speech sounds, and they had a hyperkinetic movement disorder, because both displayed hand stereotypies and jerky movements of the upper limbs. Brain magnetic resonance imaging scans revealed delayed myelination with hypoplasia of the corpus callosum and frontal lobe. These cases confirm the involvement of FOXG1 in the molecular etiology of the congenital variant of RTT and show the characteristic features of FOXG1-related disorder.

Escherichia coli lipopolysaccharide administration transiently suppresses luteal structure and function in diestrous cows.

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1 ± 0.8 years old (mean ± s.e.m.), were given 10  ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 µg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm², P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P4) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P4 concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Metabolic and energy status during the dry period is crucial for the resumption of ovarian activity postpartum in dairy cows.

It is well known that the degree of negative energy balance in high-producing dairy cows is the major cause of delayed resumption of the ovarian cyclicity that closely relates to fertility. Recent evidence suggests that the energetic situation during early lactation critically affects nutrient partitioning, metabolism, and the reproductive axis, whereas the effect of energy status during the dry period is widely unknown. The aim of the present study was to investigate the effect of energy status throughout the entire dry period until early lactation on the onset of the ovarian cyclicity. Blood samples were taken in 23 cows from dry off at 8 wk before expected parturition to 8 wk postpartum for the analyses of metabolites and hormones, and milk samples were obtained 3 times weekly from d 7 of lactation onward to confirm luteal activity and pregnancy by milk progesterone analysis. Energy balance (EB) was measured weekly during the last 6 wk of the dry period and every other week after parturition. Liver biopsies were obtained at 8 wk before expected calving, within 1 d after calving, and at 4 wk postpartum to measure the mRNA abundance of various gluconeogenic enzymes and metabolic hormone receptors. Cows showing luteal activity within 3 wk postpartum were defined as ovulatory during the first follicular wave postpartum (OC), whereas cows without luteal activity within 3 wk postpartum were defined as anovulatory (AC). Energy balance and, concomitantly, plasma concentrations of glucose, insulin, insulin-like growth factor-1, 3,5,3'-triiodothyronine, and thyroxine were higher in OC than in AC during the dry period. Plasma thyroxine concentrations and body condition score during the postpartum period were higher in OC than in AC. At the mRNA level (19 cows), hepatic insulin receptor decreased from dry off to early lactation, and mRNA of pyruvate carboxylase was highest at parturition and decreased in early lactation in AC only, whereas both parameters remained unchanged in OC. The mRNA abundance of phosphoenolpyruvate carboxykinase-mitochondrial increased from dry off to parturition in both groups, remained high in OC, and decreased again in early lactation in AC. However, none of the investigated gene transcripts differed between OC and AC cows. Thus, ovarian function postpartum appears to be crucially influenced by the energy status during the dry period, which is reflected by timely changes in hepatic mRNA abundance of only a few key metabolic factors in the liver.

Characterization of 5-hydroxytryptamine-induced contraction and acetylcholine-induced relaxation in isolated chicken basilar artery.

The aim of the present study was to clarify the responsiveness of the chicken basilar artery to 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) and to characterize the related receptor subtypes in vitro. Basilar arteries were obtained from freshly slaughtered broiler chickens. The 5-HT induced concentration-dependent contraction of the arteries. The concentration-response curves for 5-HT were shifted 30-fold to the right by methiothepin (a 5-HT(1) and 5-HT(2) receptor antagonist) and 3-fold to the right by ketanserin (a 5-HT(2) receptor antagonist). In the presence of ketanserin, the concentration-response curve for 5-HT was shifted 10-fold to the right by methiothepin. The pA(2) value for methiothepin was 8.26. The ACh induced concentration-dependent relaxation under conditions of precontraction by 5-HT. The concentration-response curve for ACh was shifted to the right by atropine [a nonselective muscarinic (M) receptor antagonist] and hexahydro-sila-difenidol hydrochloride, a p-fluoroanalog (pFHHSiD, an M(3) receptor antagonist), but not by pirenzepine (an M(1) receptor antagonist) or methoctramine (an M(2) receptor antagonist). The pA(2) value for pFHHSiD was 7.55. Nω-Nitro-l-arginine (a nitric oxide synthase inhibitor) inhibited ACh-induced relaxation by approximately 50%. These results suggest that 5-HT induces contraction via activation of 5-HT(1) and 5-HT(2) receptors and that ACh induces relaxation via activation of the M(3) receptor. The 5-HT(1) receptor might play a dominant role in 5-HT-induced contraction. One of the factors involved in ACh-induced relaxation is probably nitric oxide released from endothelial cells.