PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder

To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K⁺ or carbachol (CCh) and with and without hypoxia (achieved by bubbling N₂ instead of O₂) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K⁺) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K⁺ and CCh. However, hypoxia significantly attenuated H-65K⁺- and CCh-induced contraction. H-65K⁺ and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K⁺- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K⁺, hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K⁺- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder.     

Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

In order to investigate the effects of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC₅₀ broth value of 0.025 µg ml⁻¹ and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC₅₀ of 0.025 µg ml⁻¹ and 0.1 µg per disc, and for M. dispar with 0.05 µg ml⁻¹ and 0.03 µg per disc.Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml⁻¹. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

Evidence for functional G-coupled protein receptors 43 and 120 in subcutaneous and intramuscular adipose tissue of Angus crossbred steers

We conducted 3 independent experiments to demonstrate functional G-coupled protein receptor 43 (GPR43) and GPR120 in bovine intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues. We hypothesized that media volatile fatty acids and long-chain fatty acids would affect cAMP-activated protein kinase-alpha (AMPKα) protein expression and cAMP concentrations differently in i.m. and s.c. adipose tissue. Experiment 1: oleic acid (18:1n-9) decreased phosphorylated AMPKα protein (p-AMPKα) and the p-AMPKα/AMPKα protein ratio in i.m. preadipocytes, increased the p-AMPKα/AMPKα protein ratio in bovine satellite cells, and had no effect in s.c. preadipocytes. Experment 2: ex vivo explants from the 5th to 8th longissimus thoracic rib muscle section of Angus crossbred steers were cultured 48 hr in media containing 0.25 µM ciglitizone, 5 mM glucose, and 5 mM acetate, in the absence or the presence of 100 µM oleic acid. Oleic acid increased acetate incorporation into fatty acids and GPR43 gene expression in i.m. adipose tissue (P < 0.05), but oleic acid had no effect on fatty acid synthesis or GPR43 expression in s.c. adipose tissue. Experiment 3: fresh s.c. and i.m. adipose tissue from the 5th to 8th longissimus thoracic rib muscle section of Angus crossbred steers was transferred immediately to 6-well culture plates containing 3 mL of KHB/Hepes/5 mM glucose. Samples were preincubated with 0.5 mM theophylline plus 10 μM forskolin for 30 min, after which increasing concentrations of acetate or propionate (0, 10⁻³, 10^−2.3, and 10⁻³ M) in the absence or the presence of 100 μM oleic acid or 100 µM palmitic acid (16:0) were added to the incubation media. Acetate had no effect on forskolin-stimulated cAMP production in s.c. adipose tissue but decreased cAMP in i.m. adipose tissue (P < 0.05); this indicates a functional GPR43 receptor in i.m. adipose tissue. The combination of 10⁻² M acetate and oleic acid decrease cAMP production in s.c. adipose tissue, consistent with GPR120 receptor activity, but oleic acid and palmitic acid attenuated the depression of cAMP production caused by acetate in i.m. adipose tissue. Palmitic acid depressed cAMP production in s.c. adipose tissue, and increased cAMP production in i.m. adipose tissue (P < 0.05). Propionate had no effect on cAMP production in s.c. or i.m. adipose tissue. These results provide evidence for functional GPR43 receptors in i.m. adipose tissue and GPR120 receptors in s.c. adipose tissue, both of which would suppress lipolysis.     

Effects of various selective phosphodiesterase inhibitors on carbachol-induced contraction and cyclic nucleotide contents in the guinea pig gall bladder

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.     

Glycated Tryptophan PHP-THβC Incorporated into Various Chicken Embryonic Cells Constitutes Cellular Proteins

Glycation is a non-enzymatic reaction, and amino acids are glycated by glucose in vivo. Tryptophan is glycated with glucose to form two types of glycated compounds, tryptophan-Amadori product and (1R, 3S)-1-(D-gluco-1, 2, 3, 4, 5-pentahydroxypentyl)-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC). Although PHP-THβC can be incorporated into various chicken embryonic cells, the mechanism of its incorporation into intracellular fluids has not been clarified. In this study, we examined whether PHP-THβC once incorporated into various chicken embryonic cells can combine with proteins. Embryonic cells from the breast muscle, liver, spleen, kidney, proventriculus, gizzard, and skin were prepared and ³H-PHP-THβC was added to the culture medium at final concentrations of 0, 200, 400, 600, and 800 µM to examine the incorporation of PHP-THβC. After 18 h of incubation, radioactivity was measured in the whole-cell and protein fractions of the chicken embryonic cells. As PHP-THβC concentration increased from 0 to 600 µM, its accumulation in the whole-cell fractions of all types of chicken embryonic cells linearly increased and reached the maximum level. The saturated PHP-THβC accumulation in the whole-cell fractions suggests that PHP-THβC could be incorporated into intracellular fluids across cellular membranes by some transporter proteins. As PHP-THβC concentration increased from 0 to 800 µM, its accumulation in the protein fractions of all types of chicken embryonic cells increased in a linear manner and reached a maximum level in the 800 µM PHPTHβC treatment group. This is the first study to indicate that a part of PHP-THβC incorporated into the whole-cell fraction was detected in the protein fraction of various chicken embryonic cells.     

Intraoperative fluid therapy for video-assisted ovariohysterectomy in dogs

BackgroundIntraoperative fluids are still poorly studied in veterinary medicine. In humans the dosage is associated with significant differences in postoperative outcomes.ObjectivesThe aim of this study is to verify the influence of three different fluid therapy rates in dogs undergoing video-assisted ovariohysterectomy.MethodsTwenty-four female dogs were distributed into three groups: G5, G10, and G20. Each group was given 5, 10, and 20 mL·kg⁻¹·h⁻¹ of Lactate Ringer, respectively. This study evaluated the following parameters: central venous pressure, arterial blood pressure, heart rate, respiratory rate, temperature, acid-base balance, and serum lactate levels. Additionally, this study evaluated the following urinary variables: urea, creatinine, protein to creatinine ratio, urine output, and urine specific gravity. The dogs were evaluated up to 26 h after the procedure.ResultsAll animals presented respiratory acidosis during the intraoperative period. The G5 group evidenced intraoperative oliguria (0.80 ± 0.38 mL·kg⁻¹·h⁻¹), differing from the G20 group (2.17 ± 0.52 mL·kg⁻¹·h⁻¹) (p = 0.001). Serum lactate was different between groups during extubation (p = 0.036), with higher values being recorded in the G5 group (2.19 ± 1.65 mmol/L). Animals from the G20 group presented more severe hypothermia at the end of the procedure (35.93 ± 0.61°C) (p = 0.032). Only the members of the G20 group presented mean potassium values below the reference for the species. Anion gap values were lower in the G20 group when compared to the G5 and G10 groups (p = 0.017).ConclusionsThe use of lactated Ringer''s solution at the rate of 10 mL·kg⁻¹·h⁻¹ seems to be beneficial in the elective laparoscopic procedures over the 5 or 20 mL·kg⁻¹·h⁻¹ rates of infusion.     

Antibiotic resistance,antimicrobial residues,and bacterial community diversity in pasture-raised poultry,swine, and beef cattle manures

Animal manure can be a source of antibiotic-resistant genes (ARGs) and pharmaceutical residues; however, few studies have evaluated the presence of ARG in pasture-raised animal production systems. The objective of this study was to examine changes in microbiome diversity and the presence of antibiotic residues (ABRs) on three farms that contained a diverse range of animal species: pasture-raised poultry (broiler and layer), swine, and beef cattle. Total bacterial communities were determined using 16S rRNA microbiome analysis, while specific ARGs (sulfonamide [Sul; Sul1] and tetracycline [Tet; TetA]) were enumerated by qPCR (real-time PCR). Results indicated that the ARG abundances (Sul1 [P < 0.05] and TetA [P < 0.001]) were higher in layer hen manures (16.5 × 10⁻⁴ and 1.4 × 10⁻⁴ µg kg⁻¹, respectively) followed by broiler chickens (2.9 × 10⁻⁴ and 1.7 × 10⁻⁴ µg kg⁻¹, respectively), swine (0.22 × 10⁻⁴ and 0.20 × 10⁻⁴ µg kg⁻¹, respectively) and beef cattle (0.19 × 10⁻⁴ and 0.02 × 10⁻⁴ µg kg⁻¹, respectively). Average fecal TetA ABR tended to be greater (P = 0.09) for broiler chickens (11.4 µg kg⁻¹) than for other animal species (1.8 to 0.06 µg kg⁻¹), while chlortetracycline, lincomycin, and oxytetracycline ABRs were similar among animal species. Furthermore, fecal microbial richness and abundances differed significantly (P < 0.01) both among farms and specific species of animal. This study indicated that the microbial diversity, ABR, ARG concentrations, and types in feces varied from farm-to-farm and from animal species-to-animal species. Future studies are necessary to perform detailed investigations of the horizontal transfer mechanism of antibiotic-resistant microorganisms (ARMs) and ARG.     

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H₂O₂ and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H₂O₂ (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.     

Thermoregulatory and physiological responses of nonpregnant,mid-gestation,and late-gestation sows exposed to incrementally increasing dry bulb temperature

Gestating sows may be more susceptible to increasing dry bulb temperatures (T_DB) due to greater metabolic heat production and increased body mass, especially as gestation advances. However, there are few studies on the thermoregulatory and physiological responses of sows at differing gestation stages exposed to gradually increasing temperatures. The study objective was to determine the thermoregulatory and physiological responses of nonpregnant (n = 12; parity 3.27 ± 0.86), mid-gestation (59.7 ± 9.6 d pregnant, n = 12; parity 3.25 ± 0.83), and late-gestation (99.0 ± 4.8 d pregnant, n = 12; parity 3.33 ± 0.75) sows exposed to increasing T_DB. Prior to the experiment (5.0 ± 0.7 d), jugular catheters were placed in all sows. During the experiment, the T_DB was increased incrementally by 2.45 ± 0.43 °C every 60 min from 19.84 ± 2.15 to 35.54 ± 0.43 °C over 400 min, and relative humidity was recorded at 40.49 ± 18.57%. Respiration rate (RR), heart rate (HR), skin temperature, and vaginal temperature were measured, and blood samples were obtained via the jugular catheter every 20 min. Data were analyzed using PROC MIXED in SAS 9.4. RR increased at a lower T_DB (P < 0.01) in late-gestation sows compared with mid-gestation and nonpregnant sows, but no differences were detected between mid-gestation and nonpregnant sows. Overall, late-gestation sows had greater RR (P < 0.01; 23 ± 2 breaths per min [brpm]) compared with mid-gestation (16 ± 2 brpm) and nonpregnant (15 ± 2 brpm) sows. Late-gestation sows had an overall greater HR (P < 0.01; 84 ± 5 beats per min [bpm]) than mid-gestation (76 ± 5 bpm) and nonpregnant (69 ± 5 bpm) sows. Late-gestation sows had overall reduced bicarbonate and total carbon dioxide levels (P = 0.02; 23.89 ± 1.97 and 25.41 ± 2.07 mmol/L, respectively) compared with mid-gestation (27.03 ± 1.97 and 28.58 ± 2.07 mmol/L, respectively) and nonpregnant (26.08 ± 1.97 and 27.58 ± 2.07 mmol/L, respectively) sows. Moreover, late-gestation sows had overall greater nitric oxide levels (P < 0.01; 248.82 ± 34.54 µM) compared with mid-gestation (110.47 ± 34.54 µM) and nonpregnant (41.55 ± 34.54 µM) sows. In summary, late-gestation sows appear to be more sensitive to increasing T_DB as indicated by thermoregulatory and physiological responses when compared with mid-gestation or nonpregnant sows. The results from this study provide valuable information regarding thermoregulatory thresholds of sows at differing gestation stages.     

5-Aminolevulinic acid combined with sodium ferrous citrate mitigates effects of heat stress on bovine oocyte developmental competence

High summer temperatures have deleterious effects on oocyte developmental competence. The antioxidant and autophagy-related properties of 5-aminolevulinic acid (5-ALA) gives the compound a broad range of biological activities. This study aimed to evaluate the effects of: 1) a high temperature-humidity index (THI) on the developmental competence of bovine oocytes, and 2) 5-ALA administration in combination with sodium ferrous citrate (SFC) during in vitro maturation (IVM) on bovine oocyte developmental competence evaluated at high THI. Bovine ovaries were collected from a local slaughterhouse at moderate environmental temperature (MT; THI of 56.2) and high environmental temperature (HT; THI of 76.7) periods; cumulus-oocyte complexes (COCs) were aspirated from medium-sized follicles, matured in vitro for 22 h, fertilized, and cultured for 10 days. For COCs collected during the HT period, 0 (control), 0.01, 0.1, 0.5, or 1 µM 5-ALA was added to the maturation medium in combination with SFC at a molar ratio of 1:0.125. The results showed that HT adversely affected blastocyst and hatching rates compared with MT. Adding 5-ALA/SFC (1 µM/0.125 µM) to the maturation medium of oocytes collected during the HT period improved cumulus cell expansion and blastocyst rates compared with the no-addition control. In conclusion, this study showed that high THI can disrupt bovine oocyte developmental competence. Adding 5-ALA to SFC ameliorates this negative effect of heat stress and improves subsequent embryo development.     

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.     

Cardiovascular effects of total intravenous anesthesia using ketamine-medetomidine-propofol (KMP-TIVA) in horses undergoing surgery

Cardiovascular effects of total intravenous anesthesia using ketamine-medetomidine-propofol drug combination (KMP-TIVA) were determined in 5 Thoroughbred horses undergoing surgery. The horses were anesthetized with intravenous administration (IV) of ketamine (2.5 mg/kg) and midazolam (0.04 mg/kg) following premedication with medetomidne (5 µg/kg, IV) and artificially ventilated. Surgical anesthesia was maintained by controlling propofol infusion rate (initially 0.20 mg/kg/min following an IV loading dose of 0.5 mg/kg) and constant rate infusions of ketamine (1 mg/kg/hr) and medetomidine (1.25 µg/kg/hr). The horses were anesthetized for 175 ± 14 min (range from 160 to 197 min). Propofol infusion rates ranged from 0.13 to 0.17 mg/kg/min, and plasma concentration (Cpl) of propofol ranged from 11.4 to 13.3 µg/ml during surgery. Cardiovascular measurements during surgery remained within clinically acceptable ranges in the horses (heart rate: 33 to 37 beats/min, mean arterial blood pressure: 111 to 119 mmHg, cardiac index: 48 to 53 ml/kg/min, stroke volume: 650 to 800 ml/beat and systemic vascular resistance: 311 to 398 dynes/sec/cm⁵). The propofol Cpl declined rapidly after the cessation of propofol infusion and was significantly lower at 10 min (4.5 ± 1.5 µg/ml), extubation (4.0 ± 1.2 µg/ml) and standing (2.4 ± 0.9 µg/ml) compared with the Cpl at the end of propofol administration (11.4 ± 2.7 µg/ml). All the horses recovered uneventfully and stood at 74 ± 28 min after the cessation of anesthesia. KMP-TIVA provided satisfactory quality and control of anesthesia with minimum cardiovascular depression in horses undergoing surgery.     

β-carotene and retinol contents in the meat of herbivorous ungulates with a special reference to their public health importance

The aim of this study was to estimate total carotenoids, β-carotene and retinol concentrations in the livers and muscles of some ungulates (cattle, buffalo, sheep, goats and horses) in comparison with the Wistar rats as a control. Cattle and horses had the highest contents of total carotenoids and β-carotene. Unexpectedly, sheep was the highest accumulator of retinol with a mean concentration of 203 ± 23.34 µg/g, while the least accumulator was buffalo, having a mean value of 58.28 ± 13.77 µg/g. Livers had higher contents of the examined phytochemicals than muscles. Consumption of these tissues may provide human with some needs from these important phytochemicals, though ingestion of livers, especially that of the sheep, is not advisable for the pregnant women.     

In vitro effects of meloxicam on the number,Foxp3 expression,production of selected cytokines,and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

The purpose of this study was to evaluate the effect of meloxicam (MEL) on selected immune parameters of bovine CD25^highCD4+, CD25^lowCD4+, and CD25-CD4+ cells. Peripheral blood mononuclear cells (PBMCs) collected from 12-month-old heifers were treated with MEL at a concentration corresponding to the serum level of this medication following administration at the recommended dose (MEL 5 × 10^-6 M) and at a concentration 10 times lower (MEL 5 × 10^-7 M). After 12 and 24 h of incubation with the drug, the percentage of CD25^highCD4+ cells decreased; however, this disturbance was quickly reversed. Furthermore, the absolute number of CD25^highCD4+ cells in the PBMC populations treated with MEL 5 × 10^-6 M for 48 and 168 h was increased. Prolonged (168 h) exposure to the drug increased the percentage of Foxp3+ cells in the CD25^highCD4+ cell subpopulation. The higher dose of MEL was found to significantly increase the percentage of IFN-γ+ cells among the CD25-CD4+ cells. These results indicated that MEL does not exert an immunosuppressive effect by depleting CD4+ cells and suppression of IFN-γ+ production by these cells. Furthermore, IL-10 and TGF-β production was not changed following exposure to MEL.     

Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation

Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol’s anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein.