Melatonin reduces apoptotic cells,SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts

Effects of adding different concentrations of melatonin (10^?7, 10^?9 and 10^?11 M) to maturation (Experiment 1; Control, IVM  + 10^?7, IVM  + 10^?9, IVM  + 10^?11) and culture media (Experiment 2; Control, IVC  + 10^?7, IVC  + 10^?9, IVC  + 10^?11) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10^?9 M) from Experiments 1–2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM  + 10^?9, IVC  + 10^?9, IVM /IVC  + 10^?9). In Experiment 1, maturated oocytes from Control and IVM  + 10^?9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC  + 10^?7 (43.5%; 56.7%) and IVC  + 10^?9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC  + 10^?9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC  + 10^?9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM /IVC  + 10^?9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC  + 10^?9 treatment also had a higher mRNA expression of antioxidant gene (SOD 2) compared to the Control, as well as the heat shock protein (HSPB 1) compared to the IVM  + 10^?9. Reactive oxygen species production was greater in the IVM /IVC  + 10^?9 treatment group. In conclusion, the 10^?9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.     

Comparative study of the effects of acepromazine and structurally related compounds on the TNF-α release by monocytes stimulated by Chlamydia pneumoniae

Acepromazine (ACP), a member of the phenothiazine family, has antioxidant properties and interacts with reactive oxygen species produced by stimulated neutrophils ( Serteyn et al. 1999 ). We found that ACP reduced the differentiation of monocytes induced by an overnight incubation with a crude Chlamydia pneumoniae extract ( Serteyn et al. 2001 ). The same model was used to test the effects of phenothiazines on the TNF‐α release by activated monocytes. A crude Chlamydia pneumoniae extract was obtained by mechanical disruption and centrifugation (1 minute, 1500 r.p.m.) of 78 hours infected McCoy cells. Monocytes (THP1 cell line; 2 × 10⁶ cells by assay) were incubated overnight with 30 µL of Chlamydia pneumoniae crude extract (equivalent to an endotoxin charge of 3.5 pg) in the presence or absence of phenothiazines (from 10^?6 to 10^?4 M) ( Mouithys‐Mickalad et al. 2001 ). For estimation of TNF‐α release, the supernatants were collected, centrifuged (to eliminate the undifferentiated monocytes) and used for TNF‐α measurements (n = 6) (Quantikine HS human TNF‐α, R&D Systems, UK). Acepromazine was compared to other phenothiazines (chlorpromazine, trifluoperazine) or to structural analogues of phenothiazines (phenoxazine, thioxanthen‐9‐one and methylene blue). For each assay, cytotoxicity was evaluated by microscopic examination and blue trypan exclusion method. Mean values of TNF‐α were compared by a Student t‐test (p < 0.05). TNF‐α release by Chlamydia‐treated THP1 was significantly decreased by ACP in a dose‐dependent manner, 378 ± 30, 209 ± 38 and 189 ± 35 ng mL^?1 for 10^?6, 10^?5 and 10^?4 M compared to the control values 385 ± 9 ng mL^?1. Similar inhibitions of TNF‐α release were obtained with trifluoperazine (313 ± 25 and 265 ± 14 ng mL^?1 at 10^?6 and 10^?5 M) and chlorpromazine (323 ± 29 and 227 ± 13 ng mL^?1 at 10^?6 and 10^?5 M), but at 10^?4 M, these two drugs were cytotoxic. The other structurally parent compounds increased significantly the TNF‐α production: 630 ± 46 and 468 ± 60 ng mL^?1 for thioxanthen‐9‐one and 547 ± 17 and 331 ± 111 ng mL^?1 for methylene blue at 10^?5 and 10^?6 (M). At 10^?4 M, the two compounds were cytotoxic. Phenoxazine increased the TNF‐α production, slightly at 10^?6 and 10^?5 M (444 ± 39 and 424 ± 16 ng mL^?1, respectively) and significantly at 10^?4 M (959 ± 30 ng mL^?1). Further studies are needed to verify if the inhibition of TNF‐α release by some phenothiazines could be linked to a reduction of the signal transduction, especially the NF‐κB pathway. These results could be interesting for the anaesthesia or treatment of animals suffering from a systemic inflammatory reaction.     

Concentration effect of prostaglandin E2 on the growth factor expression and cell proliferation in bovine endometrial explants and their kinetic characteristics

Bovine endometrium undergoes various physiological and histological changes that are necessary for blastocyst implantation during oestrous cycle. From pro‐oestrus to late‐oestrus, endometrium thickens gradually for implantation preparation and exhibits remarkable capacity for self‐repair after uterine lining shedding while implantation does not occur. The prostaglandin E₂ (PGE ₂) secretion pattern is synchronized with endometrial growth during oestrous cycles in bovine endometrium; however, limited information is available regarding the association between PGE ₂ secretion and endometrial growth. In this study, the concentration (10^?9 to 10^?5 M) and time effect (2–36 hr) of PGE ₂ treatment on a series of growth factors are essential for endometrial growth including connective tissue growth factor (CTGF ), fibroblast growth factor‐2 (FGF ‐2), interleukin‐8 (IL ‐8), transforming growth factor‐β1 (TGF ‐β1), matrix metalloproteinase‐2 (MMP ‐2), and vascular endothelial growth factor A (VEGFA ) mRNA and protein expression, and proliferation of epithelial and fibroblast cells was investigated in bovine endometrial explants in vitro. The results indicated that PGE ₂ at concentration about 10^?7 to 10^?5 M could up‐regulate CTGF , FGF ‐2, IL ‐8, MMP ‐2, TGF ‐β1, VEGFA mRNA and protein expression, and could induce the proliferation of epithelial and fibroblast cells and reduce the proapoptotic factor (caspase‐3) expression in bovine endometrial explants in vitro. These results collectively improved the possibility of PGE ₂ functions in endometrial growth during oestrous cycles.     

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10^?7 M melatonin, and (c) 10^?7 M melatonin +10^?7 M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5′‐triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM‐oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post‐fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.     

P‐glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity

The role of the transporter P‐glycoprotein (P‐gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo‐physiological features of the ruminant species, the constitutive expression of P‐gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real‐time quantitative PCR. P‐gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65‐fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6‐ and 120‐fold higher). The treatment with DEX did not elicit a significant effect on the P‐gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (P_eff S‐M = 3.99 × 10^?6 ± 2.07 × 10^?6) and DEX‐treated animals (P_eff S‐M = 6.00 × 10^?6 ± 2.5 × 10^?6). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

Contractile responses of isolated equine digital arteries under hypoxic or hyperoxic conditions in vitro: role of reactive oxygen species and Rho kinase

The underlying pathophysiological triggers for equine acute laminitis are unknown, although digital vasoconstriction, ischaemia, hypoxia and reperfusion injury may be involved. The contractile responses of isolated equine digital arteries (EDAs), harvested from the hindlimbs of normal horses postmortem at an abattoir, were studied acutely (up to 3 h) under hyperoxic (95% oxygen, 5% CO₂) and hypoxic (95% nitrogen, 5% CO₂) conditions in organ baths. Phenylephrine (PHE; 10^?6 m ), 5‐hydroxytryptamine (5‐HT; 10^?7 m ) and high potassium (K⁺; 118 mm ) caused contraction in EDAs which was significantly (P < 0.0001) enhanced under hypoxic conditions. In contrast, contraction stimulated by 9,11‐dideoxy‐9α,11α‐epoxymethanoprostaglandin F_2α (U44069; 3 × 10^?8 m ) was not significantly enhanced by hypoxia (P = 0.75). Hypoxia‐enhanced contraction in response to K⁺ was greater (P < 0.03) in vessels with a functional endothelium than in vessels in which the endothelium was removed by rubbing. Fasudil (10^?6 to 10^?5 m ), a Rho kinase inhibitor, and apocynin (10^?3 to 3 × 10^?3 m ), an NADPH oxidase inhibitor, significantly (P ≤ 0.05) inhibited hypoxia‐enhanced contraction in response to PHE and 5‐HT. In conclusion, hypoxia‐enhanced contraction occurred in EDAs. This appears to be partially mediated by reactive oxygen species produced by NAPDH oxidase, which activate Rho kinase to increase calcium sensitisation and enhance smooth muscle contraction.     

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs

The purposes of the present study were to examine the effect of naloxone, a mu‐opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M‐II) porcine oocytes, one‐, four‐cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10^?8 mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10^?4 mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10^?3 mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10^?8 mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10^?6 mol/L and 10^?8 mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.     

Hepatic tyrosine aminotransferase and glucocorticoid abuse in meat cattle

Bertarelli, D., Balbo, A., Carletti, M., Cannizzo, T., Girolami, F., Nebbia, C. Hepatic tyrosine aminotransferase and glucocorticoid abuse in meat cattle. J. vet. Pharmacol. Therap.  35 , 596–603. Besides being extensively applied as therapeutical remedies, glucocorticoids (GCs) – most notably dexamethasone or prednisolone – are also illegally used in livestock for growth‐promoting purposes. This study was designed to assess the suitability of liver tyrosine aminotransferase (TAT), a gluconeogenic enzyme known to be induced by GCs, to act as a reliable candidate biomarker to screen for GC abuse in cattle. Enzyme activity was measured spectrophotometrically in liver cytosols or in cell extracts, and TAT gene expression was determined by real‐time PCR. Compared with untreated veal calves, a notable scatter (20‐fold) and much higher median values (3‐fold) characterized TAT specific activity in liver samples from commercially farmed veal calves. A time‐related increase in both enzyme activity and gene expression was detected in rat hepatoma cell lines treated with dexamethasone concentrations (10^?8 or 10^?9 m ) in the range of those recorded in noncompliant samples from EU official controls. In experimental studies in which finishing bulls were administered GCs at growth‐promoting dosages, however, no such changes were recorded in dexamethasone‐treated animals; a statistically significant rise in liver TAT activity (+95%) only occurred in prednisolone‐treated bulls. Although further research is needed to characterize the GC‐mediated response in cattle liver, TAT does not appear to be a specific and sensitive biomarker of GC abuse in the bovine species.     

Intravenous administration of 9-aminocamptothecin to dogs with lymphoma

A colloidal dispersion formulation of 9‐aminocamptothecin (9‐AC) was administered intravenously to 10 dogs with previously untreated, spontaneously occurring, multicentric lymphoma. The dogs received a 72‐h infusion of 9‐AC at a rate of 46.5–51.25 µg m^?2 h^?1 (total dose range 3.35–3.69 mg m^?2). This dose range was associated with myelosuppression, consisting principally of neutropenia with a nadir at 7 days following the start of infusion. Neutropenia and thrombocytopenia were the most common toxicoses and are most likely to be dose‐limiting toxicities; low‐grade gastrointestinal signs were rarely seen. Concentrations of 9‐AC lactone, as well as clinical toxicities, compare favourably with those found in humans. Tumour responses were seen in all treated dogs. Response to other chemotherapy, following cessation of 9‐AC treatment, was not obviously compromised even in dogs clinically resistant to 9‐AC. 9‐AC is a novel treatment drug for canine lymphoma, which appears to show great promise.     

The Content of β-endorphin-like Immunoreactivity in Porcine Corpus Luteum and the Potential Roles of Progesterone, Oxytocin and Prolactin in the Regulation of β-endorphin Release from Luteal Cells in vitro

The amount of β‐endorphin‐like immunoreactivity (β‐END‐LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on β‐END‐LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1–5, 6–10, 11–13, 14–18, and 19–21 of the cycle were used to prepare extracts for β‐END‐LI determination. Additionally, corpora lutea from days 11–13 and 14–18 were enzymatically dissociated and isolated luteal cells were used for further study of β‐endorphin secretion in vitro. Cells were cultured in serum‐free defined M 199 medium (10⁶ cells/ml) at 37°C under 5% CO₂ in air, for 12 h. The influences of the following factors on β‐END‐LI secretion by luteal cells were tested: progesterone (10^–9, 10^–7 and 10^–5M ), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The β‐END‐LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of β‐END‐LI was lowest on days 1–5 of the cycle (0.35 ± 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14–18 (16.58 ± 0.52 ng/g wet tissue) and on days 19–21 it declined (11.10 ± 0.52 ng/g wet tissue). Progesterone at a low dose (10^–9 M ) resulted in significant (p < 0.05) increases and decreases in β‐END‐LI secretion by luteal cells from days 11–13 and 14–18, respectively. Higher doses of progesterone (10^–7 and 10^–5 M ) had no effect on β‐END‐LI release, compared with the control group. All dose‐levels of oxytocin used decreased β‐END‐LI secretion by luteal cells on days 11–13 and 14–18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11–13, and all doses tested on days 14–18 resulted in decreases in β‐END‐LI release from luteal cells. These results document evident changes in β‐END‐LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of β‐END‐LI.     

Cardiorespiratory effects of a cardioselective muscarinic antagonist in anesthetized horses

Treatment of bradycardia in horses has been historically ignored because of the motility depressant effects of nonselective antimuscarinics. This study evaluated the cardiopulmonary effects of a cardioselective (M2) muscarinic antagonist, methoctramine (MET), in anesthetized horses. In a previous in vitro study, we determined that supraphysiological doses of MET were necessary to inhibit acetylcholine‐induced longitudinal jejunal smooth muscle contractions in this species. Six adult horses were allocated to two treatments in a randomized complete block design. Anesthesia was induced with xylazine/ketamine, and maintained with halothane (1% end‐tidal) and a constant infusion of xylazine (1 mg kg^?1 hour^?1) under mechanical ventilation. Invasive hemodynamic variables were monitored at baseline (approximately 45 minutes after induction) and for 120 minutes after MET or saline (control) had been injected. MET was titrated at 10‐minute intervals (10 µg kg^?1 IV) until the heart rate (HR) increased at least 30% above the baseline, or a maximum cumulative dose of 30 µg kg^?1 had been injected. A person blinded to the treatment evaluated recovery scores and monitored intestinal auscultation until 24 hours after the end of anesthesia using previously published methods. Cardiovascular parameters were analyzed by anova followed by a Dunnet's test, and nonparametrical data were analyzed by a Mann–Whitney U‐test (p < 0.05). Values were mean ± SEM unless otherwise stated. MET significantly increased HR from baseline to 120 minutes post‐injection (from 29 ± 1 to 36 ± 2 beats minute^?1 at 20 minutes). Thermodilution cardiac output (CO) and mean arterial pressure (MAP) were increased from baseline to 75 minutes post‐MET injection (from 13.9 ± 0.8 to 19.4 ± 2.0 L minute^?1 for CO at 20 minutes, and from 82 ± 3 to 103 ± 5 mm Hg for MAP at 20 minutes). Recovery characteristics and bowel auscultation scores did not differ among the groups. The return to at least 75% of the maximum auscultation score occurred at 10 (8–18) hours [median (range)] for controls and at 9 (8–12) hours for MET. It was concluded that MET increased HR and improved hemodynamic function during halothane/xylazine anesthesia with no apparent effect on return to full‐bowel motility, as assessed by auscultation. Accordingly, M2 muscarinic antagonists might be represented as a safer alternative to treat intraoperative bradycardia in horse.     

N‐Acetyl‐d‐galactosamine prevents soya bean agglutinin‐induced intestinal barrier dysfunction in intestinal porcine epithelial cells

Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10^?4, 0.25 × 10^?4, 0.5 × 10^?4, 1.0 × 10^?4 and 2.0 × 10^?4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.     

The effects of pre-anesthetic administration of xylazine on the cardiovascular responses to dobutamine in halothane anaesthetized horses

Studies evaluating the effects of dobutamine in horses do not consistently report increases in cardiac output despite increases in arterial blood pressure. The concurrent administration of the α₂ agonist clonidine, in people, inhibited the chronotropic effects of dobutamine and increased left ventricular stroke work ( Zimpfer et al. 1982 ). Our study was performed to determine if pre‐medication with an α₂ agonist affects the response to dobutamine in anaesthetized horses. Eleven horses were anaesthetized on four separate occasions for one of four randomly assigned treatments; (I) no xylazine, no dobutamine (II) xylazine, no dobutamine (III) no xylazine, dobutamine, and (IV) xylazine, dobutamine. Horses received 0.02 mg kg^?1 of butorphanol IV 10 minutes prior to anesthetic induction. Two minutes prior to induction, groups II and IV received 0.5 mg kg^?1 of IV xylazine. Anaesthesia was induced with 6–7 mg kg^?1 of thiopental and maintained with halothane. End‐tidal halothane concentrations were maintained between 1.1 and 1.2% in groups I and III, and 0.9–1.0% for groups II and IV. Heart rate, cardiac output, right atrial pressure, and systolic (SAP), diastolic (DAP) and mean (MAP) arterial pressure were recorded 30 minutes after beginning halothane anaesthesia (T10). Cardiac output was estimated using Lithium dilution ( Linton et al. 2000 ). Baseline measurements were repeated twice, at 5‐minute intervals (T5 and T0). At time 0 (T0), an IV infusion of either saline (100 mL hour^?1) or dobutamine (0.001 mg kg^?1 minute^?1) was started and data recorded at 5‐minute intervals for 30 minutes (T5 – T30). Stroke volume and systemic vascular resistance (SVR) were calculated. Data were analysed using repeated measures anova (p < 0.01 significant) and Newman–Keuls for multiple comparisons. Cardiac output and stroke volume increased over time in groups III and IV. Cardiac index was higher in groups III and IV than in groups I and II from T10 until completion of the study. Estimates of cardiac index at T30 for groups I–IV were 45 ± 9, 46 ± 11, 71 ± 11, and 78 ± 19 mL kg^?1 minute^?1, respectively (mean ± SD). Stroke index was higher in groups III and IV than in groups I and II from T15 to T30. Values for stroke index at T30 for groups I–IV were 0.98 ± 0.19, 1.11 ± 0.18, 1.46 ± 0.21, 1.74 ± 0.33 mL kg^?1. Heart rate decreased from T10–T30 in groups I and II. Heart rate was greater in groups I and III than in groups II and IV at T5 and T0. Values for heart rate at T0 for groups I–IV were 48 ± 5, 42 ± 5, 50 ± 4, 43 ± 4 beats minute^?1. Systolic arterial pressure, DAP and MAP were higher in groups III and IV than in groups I and II from T5 to T30. There were no differences in SVR between groups. Dobutamine at 0.001 mg kg^?1 minute^?1 increased cardiac output, blood pressure, and stroke volume. Premedication with xylazine at 0.5 mg kg^?1 did not appear to affect the response to dobutamine.     

Nitric Oxide and Superoxide Anion Production During Heparin‐Induced Capacitation in Cryopreserved Bovine Spermatozoa

The aim of this work was to quantify NO, O₂^? and ONOO^? production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O₂ uptake, and NO production. Conversely, O₂^? production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O₂ consumption (5.9 ± 0.6 nmol/min × 10⁷ cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10⁷ cells), and a five‐fold increase in O₂^? production (1.3 ± 0.07 nmol/min × 10⁷ cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O₂^? generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O₂ uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O₂^? production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.     

Effects of dietary corn oil and vitamin E supplementation on fatty acid profiles and expression of acetyl CoA carboxylase and stearoyl‐CoA desaturase gene in Hu sheep

This study was conducted to assess the effects of dietary corn oil and vitamin E supplementation on fatty acid (FA) profiles and abundances of acetyl‐CoA carboxylase (ACC) and Δ⁹ stearoyl‐CoA desaturase (SCD) mRNA of Hu sheep. Animals were allocated to three dietary treatments: basal and supplemented with 3% corn oil (CNO), or CNO plus 500 mg/kg vitamin E (COE). The experiment lasted for 10 weeks. No differences were observed in growth performance and carcass qualities among the three treatments (P > 0.05). Feeding CNO and COE diets increased polyunsaturated FAs including cis 9 trans 11 conjugated linoleic acid, and decreased saturated FA in longissimus muscle (P < 0.05). The mRNA abundances of ACC and SCD as detected by real‐time PCR were reduced (P < 0.05) in liver and subcutaneous fat by supplementary oil, while the SCD mRNA level in longissimus muscle was also reduced (P < 0.05). Inclusion of vitamin E did not have further effects on mRNA abundances of these two enzymes. It is suggested that dietary corn oil supplementation may reduce FA biosynthesis and influence FA profiles in Hu sheep through decreased expression of both ACC and SCD genes.     

Urinary aldosterone to creatinine ratio in cats before and after suppression with salt or fludrocortisone acetate

Background: The endocrine diagnosis of primary hyperaldosteronism in cats currently is based on an increased plasma aldosterone to renin ratio, which has several disadvantages for use in veterinary practice. Objectives: To establish a reference range for the urinary aldosterone to creatinine ratio (UACR) and to determine whether oral administration of either sodium chloride or fludrocortisone acetate is effective for use in a suppression test. Animals: Forty‐two healthy cats from an animal shelter and 1 cat with primary hyperaldosteronism from a veterinary teaching hospital. Methods: Morning urine samples for determination of the basal UACR were collected from 42 healthy cats. For the suppression tests, urine samples for the UACR were collected after twice daily oral administration for 4 consecutive days of either sodium chloride, 0.25 g/kg body weight (n = 22) or fludrocortisone acetate, 0.05 mg/kg body weight (n = 15). Results: The median basal UACR was 7.2 × 10^?9 (range, 1.8–52.3 × 10^?9), with a calculated reference range of <46.5 × 10^?9. Administration of sodium chloride resulted in adequate salt loading in 10 of 22 cats, but without significant reduction in the UACR. Administration of fludrocortisone resulted in a significant decrease in the UACR (median, 78%; range, 44–97%; P < .001) in healthy cats. In the cat with an aldosterone‐producing adrenocortical carcinoma, the basal UACR and the UACR after fludrocortisone administration were 32 × 10^?9 and 36 × 10^?9, respectively. Conclusions and Clinical Importance: Using the UACR for an oral fludrocortisone suppression test may be useful for the diagnosis of primary hyperaldosteronism in cats.