Effects of genetic variants and sialylation on in vitro digestibility of purified κ-casein.

Milk with different κ-casein (CN) phenotypes has previously been found to influence its gastric digestion rate. Therefore, the aim of the present study is to disentangle contributions of genetic variation and its related sialylation on the in vitro digestion process of κ-CN. Accordingly, κ-CN was purified from milk representing homozygous cows with κ-CN phenotypes AA, BB, or EE and used as substrate molecules in model studies using the INFOGEST 2.0 in vitro static digestion model. Furthermore, the effect of removal of the terminal sialic acids present on the O-linked oligosaccharides of the purified κ-CN A, B, and E protein variants were studied by desialylation enzymatic assays. The κ-CN proteins were purified by reducing anion exchange chromatography with purities of variants A, B, and E of 93.0, 97.1, and 90.0%, respectively. Protein degradations of native and desialylated κ-CN isolates in gastric and intestinal phases were investigated by sodium dodecyl sulfate-PAGE, degree of hydrolysis (DH), and liquid chromatography electrospray ionization mass spectrometry. It was shown that after purification, the κ-CN molecules reassembled into multimer states, which then constituted the basis for the digestion studies. As assessed by DH, purified variants A and E were found to exhibit faster in vitro digestion rates in both gastric and intestinal phases compared with variant B. Desialylation increased both gastric and intestinal digestion rates for all variants, as measured by DH. In the gastric phase, desialylation promoted digestion of variant B at a rate comparable with native variants A and E, whereas in the intestinal phase, desialylation of variant B promoted better digestion than native A or E. Taken together, the results confirm that low glycosylation degree of purified κ-CN promotes faster in vitro digestion rates, and that desialylation of the O-linked oligosaccharides further promotes digestion. This finding could be applied to produce dairy products with enhanced digestibility.

Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry.

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ∼70%; where P indicates phosphorylation from single to triple phosphorylation (1-3P), and G indicates glycosylation from single to triple glycosylation (1-3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ∼12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ∼0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.

Identification of rare genetic variants of the αS-caseins in milk from native Norwegian dairy breeds and comparison of protein composition with milk from high-yielding Norwegian Red cows.

Several factors influence the composition of milk. Among these, genetic variation within and between cattle breeds influences milk protein composition, protein heterogeneity, and their posttranslational modifications. Such variations may further influence technological properties, which are of importance for the utilization of milk into dairy products. Furthermore, these potential variations may also facilitate the production of differentiated products (e.g., related to specific breeds or specific genetic variants). The objective of this study was to investigate the genetic variation and relative protein composition of the major proteins in milk from 6 native Norwegian dairy breeds representing heterogeneity in geographical origin, using the modern Norwegian breed, Norwegian Red, as reference. In total, milk samples from 144 individual cows were collected and subjected to liquid chromatography-electrospray ionization/mass spectrometry-based proteomics for identification of genetic and posttranslational modification isoforms of the 4 caseins (αS1-CN, αS2-CN, ß-CN, κ-CN) and the 2 most abundant whey proteins (α-lactalbumin and ß-lactoglobulin). Relative quantification of these proteins and their major isoforms, including phosphorylations of αS1-CN and glycosylation of κ-CN, were determined based on UV absorbance. The presence and frequency of genetic variants of the breeds were found to be very diverse and it was possible to identify rare variants of the CN, which, to our knowledge, have not been identified in these breeds before. Thus, αS1-CN variant D was identified in low frequency in 3 of the 6 native Norwegian breeds. In general, αS1-CN was found to be quite diverse between the native breeds, and the even less frequent A and C variants were furthermore detected in 1 and 5 of the native breeds, respectively. The αS1-CN variant C was also identified in samples from the Norwegian Red cattle. The variant E of κ-CN was identified in 2 of the native Norwegian breeds. Another interesting finding was the identification of αS2-CN variant D, which was found in relatively high frequencies in the native breeds. Diversity in more common protein genetic variants were furthermore observed in the protein profiles of the native breeds compared with milk from the high-yielding Norwegian Reds, probably reflecting the more diverse genetic background between the native breeds.

Account and Utilization of Blood Lipid Profiles: Lipid Levels Predicted Hemosporidian Infection in Migrating Northern Saw-Whet Owls of Eastern North America.

Noting lipidomic changes following the parasitism of migrating birds, the metabolic needs of which are primarily fueled by lipids, can deepen our understanding of host-parasite interactions. We identified lipids of migrating Northern saw-whet owls (Aegolius acadicus) using collision-induced dissociation mass spectrometry, compared the lipidomic signatures of hemoparasite-infected and noninfected individuals, and performed cross-validation analyses to reveal associations between parasite infection and lipid levels. We found significantly lower levels of lipid classes phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) in infected Northern saw-whet owls than in the noninfected individuals. Conversely, we found higher levels for certain lysoPS and lysoPE species, and variable lipid level changes for free fatty acid (FFA) species. Reporting lipidomic changes observed between hemosporidian-infected and noninfected Northern saw-whet owls can strengthen our understanding of the mechanisms governing parasite proliferation in this species. Furthermore, our analysis indicated that lipidomic signatures are better predictors of parasite infection than the log-adjusted mass/wing chord body index, a metric commonly used to assess the influence of hemosporidia infection on the health of birds. Establishing a lipidomic profile for Northern saw-whet owls that provides baseline lipid levels during fall migration may assist future studies assessing causes of reductions in breeding brought about from subtle differences in behaviors such as delayed migration.

Application of a non-target variable data independent workflow (vDIA) for the screening of prohibited substances in doping control testing.

A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits of the workflow. Additional targets fulfilling the above requirements can be added without changing any settings. The performance of the vDIA workflow was illustrated by applying it to two screening methods in horse urine, with one workflow covering 331 basic drugs and the other covering 45 quaternary ammonium drugs (QADs). Both screening methods have good detection sensitivity with 84% of the basic drugs having Limits of Detection (LoDs) of ≤ 1 ng/mL and 84% of the QADs having LoDs of ≤ 0.4 ng/mL. Other method characteristics including retention reproducibility, method precision and false hit rate will also be presented.

Quantitative analysis of paracetamol, metacetamol, and orthocetamol in equine urine from racehorses in Japan using liquid chromatography-electrospray ionization-tandem mass spectrometry.

Paracetamol is commonly used as an over-the-counter analgesic and antipyretic medication for humans, but not sold as a legitimate therapeutic medication for horses in Japan. However, paracetamol is commonly found in horses together with its two isomers, metacetamol and orthocetamol. We previously reported that paracetamol and orthocetamol were both present in selected feed consumed by Japanese racehorses. For the purpose of the doping control of paracetamol in local Japanese horses, we proposed establishing residue limits (Japanese residue limits, JRLs) to minimize the risk of reporting paracetamol from environmental contributions and to differentiate its presence from active administration. Recently, we proposed a preliminary JRL for paracetamol in equine plasma based on a population study of more than 300 Japanese racehorses. In this paper, we will present our studies on the urinary concentrations of paracetamol, metacetamol, and orthocetamol in postrace samples collected from 403 Japanese racehorses over a 1 year period, detected using liquid chromatography-electrospray ionization-tandem mass spectrometry. Our results revealed that the hydrolyzed urinary concentrations of paracetamol, metacetamol, and orthocetamol were in the range 15.7-2,360 ng/mL (median 363 ng/mL), 8.07-382 ng/mL (84.5 ng/mL), and 919-74,418 ng/mL (13,475 ng/mL), respectively. Based on our statistical model, the preliminary JRL of hydrolyzed paracetamol in equine urine was determined to be 7,400 ng/mL, at a risk factor of 1 in 10,000. Further investigations will be required to demonstrate the applicability and validity of our preliminary plasma and urine JRLs to local Japanese racehorses.

Investigation of plasma concentrations of paracetamol, metacetamol, and orthocetamol in Japanese racehorses using liquid chromatography-electrospray ionisation-tandem mass spectrometry.

Paracetamol is used widely as an over-the-counter analgesic and antipyretic medication for humans, but not for Japanese racehorses. Paracetamol can be an environmental substance, and is found together with its two isomers, metacetamol and orthocetamol, in equine urine. However, the sources and routes of paracetamol exposure remain unclear. To control the misuse of paracetamol, it is appropriate to establish residue limits for paracetamol to differentiate the administration of paracetamol from its environmental levels. In this study, we developed and validated a quantitative method for paracetamol, metacetamol, and orthocetamol in equine plasma using liquid chromatography-electrospray ionization-tandem mass spectrometry and applied it to postrace samples from 320 Japanese racehorses for approximately 1 year. In addition, we conducted feed analysis and related pharmacokinetics simulations to evaluate the contributions from exposure via feed. The hydrolyzed plasma concentrations of paracetamol, metacetamol, and orthocetamol ranged from 0.787 to 39.8 ng/mL (median 5.87 ng/mL), 0 to 2.13 ng/mL (0.347 ng/mL), and 1.98 to 82.8 ng/mL (16.6 ng/mL), respectively. Such low concentrations of paracetamol were deemed irrelevant to therapeutic effect. Based on statistical analysis, the preliminary Japanese residue limits of unhydrolyzed and hydrolyzed paracetamol were determined to be 70.5 ng/mL and 112 ng/mL, respectively, in plasma, at a risk factor of 1 in 10,000. Furthermore, we detected paracetamol and orthocetamol in feed samples. A pharmacokinetics simulation showed that the presence of orthocetamol is most probably related to daily feed rations. As for paracetamol, feed can be one of the sources and other possible sources require further investigation.

Comprehensive analysis of the ubiquitome in rabies virus-infected brain tissue of Mus musculus.

Ubiquitination is an important post-translational modification (PTM) that plays a key role in almost every aspect of cellular processes and many signaling pathways in eukaryotes. In this study, we performed a quantitative ubiquitome study to identify the global change of ubiquitination induced by rabies virus (RABV) infection in the mouse brain tissue. 4,243 ubiquitinated sites, mapping to 1,626 proteins were identified; using a cutoff of fold change >2, 644 and 70 ubiquitinated proteins were up- and down-regulated, respectively. GO analysis indicated that the differentially ubiquitinated proteins (DUPs) were significantly enriched in the myelin sheath of cells and binding activity. KEGG pathway analysis indicated that the identified proteins were related to biosynthesis of amino acids. Of note, ubiquitination was observed on all five RABV proteins by both proteomics and biochemical approaches. Our study revealed the global ubiquitome of RABV-infected mice and provides a valuable resource for investigating the pathogenic mechanisms of RABV.

Electrospray ionization mass spectrometry characterization of ubiquitous minor lipids and oligosaccharides in milk of the camel (Camelus dromedarius) and their inhibition of oxidative stress in human plasma.

The aim of this study was to characterize minor lipids in methanol fraction extracted from raw camel milk after loading it on a water-preconditioned short C18 open column and fractionating with a gradient of methanol/water. The C18 column showed high fractionation efficiency of minor lipids, such as glycosphingolipids, lipopolysaccharides, or oligosaccharides, when compared with other constituents, in particular polysaccharides, proteins, and free fatty acids. Liquid chromatography electrospray ionization tandem mass spectrometry in negative ion mode was used to identify 21 new glycosphingolipids, lipopolysaccharides, and oligosaccharides. Electrospray ionization tandem mass spectrometry was qualified to provide relevant data for recognizing the molecular mass, glycosylation sequences, and structure of saccharide moieties for the revealed compounds. The sequence of combinations of one selected lipopolysaccharide, which was considered the backbone of the remaining lipopolysaccharides, was confirmed in a density functional theory study. The obtained results showed that the tested fraction is a rich source of glycosphingolipids, lipopolysaccharides, and oligosaccharides with antioxidant activity.

Mass spectrometric determination of disulfide bonds and free cysteine in grass carp IgM isoforms.

Disulfide bonds are fundamental in establishing Ig structure and maintaining Ig biological function. Here, we analysed disulfide bonds and free cysteine in three grass carp IgM isoforms (monomeric, dimeric/trimeric, and tetrameric IgM) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results revealed that Cys574 residue status at the C-terminal tail differed substantially in monomeric IgM in comparison with polymeric IgM, Cys574 was found as free thiol in monomeric IgM, while it formed disulfide linkages in dimeric/trimeric and tetrameric IgM. Five intra-chain disulfide bonds in the CH1~CH4 and CL1 domains, as well as one H-H and one H-L inter-chain disulfide linkages, were also observed and shown identical connectivity in monomeric, dimeric/trimeric, and tetrameric IgM. These findings represent the first experimental assignments of disulfide linkages of grass carp IgM and reveal that grass carp IgM isoform formation is due to alternative disulfide bonds connecting the Cys574 residue at the C-terminal tail.

Simultaneous quantification of free curcuminoids and their metabolites in equine plasma by LC-ESI-MS/MS.

The human health benefits attributed to turmeric/curcumin spice has resulted in its wide utilization as a dietary supplement for companion pets and other animals including horses. While the quantification of free curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and their phase-2 metabolites (curcumin-O-sulfate, curcumin-O-glucuronide) have been extensively investigated in human and rodent biological samples (primarily plasma and serum), there is lack of similar data for horses. Herein, we report a validated LC-ESI-MS/MS method for the simultaneous quantification of the aforementioned free curcuminoids and their metabolites in equine plasma. The linearity of the aforementioned curcuminoids and curcumin-O-sulfate was in the range of 0.5-1000 ng/mL and 1-1000 ng/mL for curcumin-O-glucuronide with 85-115% accuracy and <15% precision in equine plasma. The method was validated based on US FDA criteria and applied to characterize the pharmacokinetics of curcumin-O-sulfate in equine plasma.

A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry.

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of 'principal component analysis (PCA)'. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.

Proteome of equine oviducal fluid: effects of ovulation and pregnancy.

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC-tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.

Integrative transcriptomics and proteomics analysis of longissimus dorsi muscles of Canadian double-muscled Large White pigs.

Canadian double-muscled Large White pigs are characterized by notable muscle mass, showing high daily gain and lean rate and good meat quality. In order to identify the major genes or proteins involved in muscle hyperplasia and hypertrophy, three pairs of full-sib pigs with extreme muscle mass difference from Canadian Large White were selected as experimental animals at 3 months age. The phenotypic differences of longissimus dorsi muscles (LD) were investigated with microarray and proteomics (2-DE, MALDI-TOF-MS), and results were verified by real-time PCR and western bolting respectively. The gene expressing profiling identified 57 and 260 and 147 differently expressed genes (DEGs) from the three pairs respectively with Bayesian statistics and significant analysis of microarrays (SAM) (p<0.05, q<0.05, fold>2). From the network of these DEGs, some major genes were displayed, such as EGF, PPARG, FN1, SERPINE1, MYC, JUN, involved in Wnt, MAPK and TGF-ß signal pathway respectively, which mainly participated in cell differentiation and proliferation. In parallel, proteomics analyses revealed 50 differently expressed protein (DEP) spots with mass spectrum, and 33 spots of them were found annotated, which took part in energy metabolism and the structure and contraction of muscle fiber. In brief, our integrated study provides a good foundation for the further study on the genetic mechanism of the double muscle traits in pigs.

Buffalo cervico-vaginal fluid proteomics with special reference to estrous cycle: heat shock protein (HSP)-70 appears to be an estrus indicator.

Cervico-vaginal fluid (CVF) plays significant roles in coitus, sperm transport, and implantation. It is believed to be a good noninvasive biomarker for various diagnostic purposes. In this study, a comprehensive proteomic analysis of buffalo CVF was performed during the estrous cycle in order to document the protein expressions, utilizing SDS-PAGE, mass spectrometry, and immunoblot. The main objective was to screen the CVF of buffalo for one or more estrus-specific proteins. A total of 416 proteins were identified in the CVF of both estrus and diestrus phases. Out of these proteins, 68 estrus-specific proteins have been extensively reviewed in the protein database. The major physiological functions of estrus CVF proteins appeared to be stress response, immune response, and metabolic. Eventually, the expression level of heat shock protein-70 in the CVF during the estrus phase, as revealed in SDS-PAGE analysis, was higher than during diestrus. The identity of the protein was confirmed by immunoblot analysis as heat shock protein-70. The findings provide a potential lead for the evaluation of these proteins for estrus detection in buffalo because CVF biomarker detection is a noninvasive technique. The mass spectrometric data of identified proteins have been deposited at the ProteomeXchange with the identifier PXD000620.

Quantification of malachite green in fish feed utilising liquid chromatography-tandem mass spectrometry with a monolithic column.

The purpose of this study was to develop a rapid and sensitive method for the quantification of malachite green (MG) in fish feed using LC-ESI-MS/MS with a monolithic column as stationary phase. Fish feed was cleaned using ultrasonic assisted liquid-liquid extraction. The separation was achieved on a Chromolith® Performance RP-18e column (100 × 4.6 mm) using gradient mobile phase composition of methanol and 0.1% formic acid at the flow rate of 1.0 ml min⁻¹. The analyte was ionised using electrospray ionisation in positive mode. Mass spectral transitions were recorded in selected reaction monitoring (SRM) mode at m/z 329.78 → m/z 314.75 with a collision energy (CE) of 52% for MG. The system suitability responses were calculated for reproducibility tests of the retention time, number of theoretical plates and capacity factor. System validation was evaluated for precision, specificity and linearity of MG. The linearity and calibration graph was plotted in the range of 15.0-250 ng ml⁻¹ with the regression coefficient of >0.997. The lower limits of detection and quantification for MG were 0.55 and 1.44 ng ml⁻¹, respectively, allowing easy determination in fish feed with accuracy evaluated as a percentage recovery of 92.1% and precision determined as % CV of < 5. The method was also extended to the determination of MG in an actual fish feed. The sensitivity and selectivity of LC-ESI-MS/MS using monolithic column offers a valuable alternative to the methodologies currently employed for the quantification of MG in fish feeds.

Comparative metabolism of PGFM (13,14-dihydro-15-keto-PGF2α) in feces of felids.

Methods for monitoring endocrine activities are useful tools for reproduction management. In particular, captive breeding of endangered felid species is considered to be an important part of the species conservation efforts. Within breeding programs, reliable methods for pregnancy diagnosis are highly demanded to prevent peri- and postpartal losses, but pregnancy diagnosis based on gestagen metabolites in felids is hampered by pseudopregnancies. Recently, we described fecal PGFM as an indicator for pregnancy in several feline species, but peak levels of PGFM secretion differed dramatically between species. It is believed that prostaglandin composition and metabolism pathways may differ as well. Therefore, a study was devised to both compare various fecal immunoreactive PGFM metabolites and to identify prostaglandins in fecal extracts by liquid chromatography-mass spectrometry (LCMS). Our results confirmed that fecal metabolite patterns differ between feline species. The identity of PGFM was confirmed in six of eight felids. In Iberian lynx and the Sumatran tiger, PGFM did not exceed 5% of all immunoreactivities. The total number of immunoreactivities varied between two (e.g., domestic cat) and four (e.g., oncilla). Several prostaglandins were identified by LCMS; apart from PGFM, all LCMS-identified prostaglandins, including tetranor-PGFM, did not show any cross-reactivity with our PGFM-specific antibody. This indicates the existence of still unknown eicosanoids and further studies are needed to clarify the origin of the different metabolites. Although differing stages of pregnancy did not reveal significant differences in the composition of metabolites, we could not exclude the possibility that metabolites from other prostaglandins (e.g. PGE2) contributed to the fecal metabolite patterns.

Global proteomic characterization of uterine histotroph recovered from beef heifers yielding good quality and degenerate day 7 embryos.

The objective was to analyze the proteomic composition of uterine flushes collected from beef heifers on day 7 after insemination. Estrus was synchronized in crossbred beef heifers by using a protocol with a controlled intravaginal drug releasing device. Heifers detected in standing estrus (within 24-48 h after removal of controlled intravaginal drug releasing device) were inseminated (estrus = day 0) with frozen-thawed semen from a single ejaculate of a bull with proven fertility. Heifers from which an embryo was recovered (after slaughter on day 7) were classified as either having a viable embryo (morula/blastocyst stage) or a degenerate embryo (arrested at the 2- to 16-cell stage). The overall recovery rate (viable and degenerate combined) was 64%. Global liquid chromatography coupled to tandem mass spectrometry proteomic analysis of the histotroph collected identified 40 high-confidence proteins present on day 7; 26 proteins in the viable group, 10 in the degenerate group, and 4 shared between both groups. Five proteins (platelet-activating factor acetylhydrolase IB subunit γ [PAFAH1B3], tubulin α-1D chain, tubulin ß-4A chain, cytochrome C, and dihydropyrimidinase-related protein-2) were unique or more abundant in the histotroph collected from animals with a viable embryo, and 1 protein (S100-A4) was more abundant in the histotroph collected from animals with a degenerate embryo. Of interest, PAFAH1B3, detected only in histotroph from the group yielding viable embryos, belongs to the group of platelet-activating factors that are known to be important for the development of the pre-implantation embryo in other species. To our knowledge this is the first report of PAFAH1B3 in relation to bovine early embryonic development.

Pharmacokinetic/pharmacodynamic evaluation of the efficacy of flumequine in treating colibacillosis in turkeys.

Flumequine (FLU) is used in the treatment of systemic bacterial infections in poultry, including colibacillosis, which is a common disease in turkeys. The pharmacokinetic (PK) behavior of FLU administered to 32 healthy turkeys as an oral bolus via gavage or as 10-h pulsed administration in drinking water were compared, using the authorized dose of 15 mg/kg and the double dose of 30 mg/kg. The minimum inhibitory concentrations (MIC) of 235 Escherichia coli field strains isolated from poultry were determined for pharmacodynamics (PD) to develop a PK/PD model. Blood samples were collected at established times over 24 h, and the obtained plasma was analyzed using a liquid chromatography tandem mass spectrometry method that was validated in-house. A monocompartmental model and a noncompartmental model were applied to the data to obtain the PK results. For both types of administration and both dosages, the ratios of the maximum concentration (Cmax)/MIC50 and the area under the plasma concentration-time curve (AUC)/MIC50 achieved were considerably lower than the fluoroquinolone breakpoints usually adopted for efficacy. The Cmax/MIC50 and AUC0-24/MIC50 ratios were, respectively, 0.67 ± 0.09 and 4.76 ± 0.48 and 1.18 ± 0.35 and 7.05 ± 2.40 for the 15 and 30 mg/kg bolus doses, respectively. After 10-h pulsed administration of 15 mg/kg, values of Cmax/MIC50, 0.19 ± 0.02 on d 1 and 0.30 ± 0.08 on d 5 of therapy were obtained, the AUC/MIC50 ratios were 2.09 ± 0.29 and 3.22 ± 0.93 on d 1 and 5, respectively. Higher values were obtained with the doubled dose of 30 mg/kg: the Cmax/MIC50 ratios were 0.49 ± 0.11 on d 1 and 0.69 ± 0.18 on d 5; the AUC/MIC50 ratios were 5.15 ± 1.15 and 6.57 ± 1.92 on d 1 and 5, respectively. Based on these results, FLU administration should be adopted when specific diagnostic findings indicate its efficacy, and revising the dosage scheme to comply with the prudent and responsible use of antimicrobials in veterinary medicine is advisable.