Detection of β2-agonists in milk replacer

₂-Agonist drugs may be illegally used as growth promoters for feedlot calves, when mixed into milk replacer immediately before feeding. To check for the presence of clenbuterol, salbutamol and terbutaline in such food, an analytical system was established using a screening method based on two commercial qualitative competitive ELISA tests, with antibodies raised against the arylamino group and thet-butyl group. The extraction procedure was based on precipitation of the milk samples with acetonitrile followed by filtration. The absence of any significant interference by other substances in the filtrate allowed detection of ₂-agonist drugs in spiked samples at the lowest concentration having a repartitioning effect (50 ppb for clenbuterol, mabuterol and terbutaline, 500 ppb for salbutamol). In view of a false positive response with tetracycline in milk samples and a cross-reaction between clenbuterol and mabuterol, an HPLC-MS technique was developed which, after extraction and purification of the samples with SPE C18 Polar Plus, was able to confirm the presence of these drugs. The good recovery after extraction (ranging from 84% to 90.2%) and the low detection limit with this method (250 ng/ml for clenbuterol, mabuterol and terbutaline, and 2.5 µg/ml for salbutamol) allowed easy confirmation and simultaneous detection of the four ₂-agonists at the lowest concentrations at which they are used in adulterated milk for calves.Abbreviations B optical density of the sample - B maximal optical density in total absence of competition - %B/B ₀ percentage of inhibition - ELISA enzyme-linked immunosorbent assay - EIA enzyme immunoassay - HPLC-MS high-performance liquid chromatography-mass spectrometry - m/z mass to charge ratio - ppb parts per billion - ppm parts per million - SPE solid-phase extraction     

Adverse reactions of α2-adrenoceptor agonists in cats reported in 2003–2013 in Finland

Objective

To describe suspected adverse drug reactions in cats associated with use of α₂-adrenoceptor agonists.

The Effect of PGF2α-Induction of Estrus on Pregnancy Rates in Mares

In mares, the onset of estrus is routinely induced after a luteolytic dose of a prostaglandin F2alpha analogue. Mares in diestrus with a mature, functional corpus luteum will respond by coming into estrus 3 to 4 days after induction¹. Shortening the interestrous interval in mares has an important economic impact on the equine breeding industry. Because the breeding season of the mare is short, timing of insemination and appropriate coordination of endocrine events is critical to pregnancy success and ultimate foaling rates. Several recent studies have reported that the use of prostaglandin analogues is associated with lower pregnancy rates. In this study the induction of estrus with cloprostonol (125-250 ug IM) did not affect pregnancy rates in mares that have ovulatory cycles (n = 461).     

Relationship between contractions of the uterus and concentration of PGF2α in uterine venous blood after luteolysis in gilts

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.     

Treatment of dairy cows with PGF2α or NSAID,in combination with antibiotics,in cases of postpartum uterine inflammation

Background

The aim of the study was to test the effect of two treatments in cases of acute puerperal metritis (APM) and clinical metritis (CM).

Methods

Cows with APM and CM (n = 40)) were matched according to plasma fibrinogen levels (Fb) into three groups. Two negative control groups D (n = 11) and E (n = 17) were composed of healthy cows. The proportion of animals with APM and CM was similar within the groups. Treatment was started on the 3rd day postpartum (PP). In group A (n = 15), intramuscular (i.m.) administration of ceftiofur was used for five days in combination with flunixin for three days. Group B (n = 15) received i.m. administration of ceftiofur for five days followed by two injections of prostaglandin F2_α, with an interval of 8 h, on the 8th day PP. Group C (n = 10) served as a control group with no treatment. The general health status, body temperature (BT) and vaginal discharge were evaluated daily. Endometrial biopsies for bacteriology were taken once a week for seven weeks PP. Blood samples for the analysis of acute phase proteins were collected once a week for six weeks PP. Samples for progesterone analysis were taken twice a week for seven weeks PP. Fertility performance data were recorded.

Results

The area under the curve of BT was higher in group B than in group D cows (P < 0.05). No differences were found for vaginal discharge. There were no differences in bacterial growth, start of ovarian activity or serum amyloid-A or fibrinogen levels among the groups. The haptoglobin concentration was higher in the first and second weeks PP in group B compared with the other groups (P < 0.05). The number of days open was higher in group A than in both groups B and D (P < 0.05). The pregnancy rate after the first two services was higher (P < 0.05) in groups B and D than in groups A and C. The number of services per pregnancy was lower in group B than in group C (P < 0.05).

Conclusions

Regardless of more severe uterine inflammation found in animals from group B, these cows showed the same fertility parameters as healthy animals.     

Seroprevalence of toxoplasmosis in horses in Niğde Province of Turkey

The present study was conducted to investigate the prevalence of Toxoplasma gondii specific antibodies in local horses from four districts of Niğde in the middle of Turkey, between April–June 2004. Serum samples were obtained a total of 125 horses which consisted of 81 (50 female, 31 male) 1–10 years old and 44 (25 female, 19 male) 11–20 years old and tested for antibodies to T. gondii using the Sabin Feldman Dye Test (SFDT). According to the results of the SFDT, antibodies to T. gondii were found by the SFDT in 9 (7.2%) of 125 sera with the titers of 1:16 (8 horses) and 1:64 (1 horse). Antibodies to T. gondii were found in 6 (7.40%) of 81 horses (1–10 years old) and 3 (6.81%) of 44 horses (11–20 years old). From the 5 (10%) out of 50 male horses and the 4 (5.33%) out of 75 female horses were detected anti-T. gondii antibodies. No statistically significant difference in age groups (p > 0.01) and genders (p > 0.005) were observed between the seropositive and seronegative horses using the x² test. Seropositivity rates ranged from 2.85% to 11.42%, depending on the study sites. In regard to study sites, there was no statistically significant difference was found (p > 0.005). This is the first serological report on toxoplasmosis in horses from Niğde of Turkey.     

Prediction and Analysis of Functional SNP on Exon Region of Insulin-like Growth Factor 2 Gene (IGF2) in Chickens

The aim of this study was to use bioinformatics methods to screen non-synonymous single nucleotide polymorphisms (nsSNPs) sites with potential biological functions in chicken IGF2 gene and provide a theoretical basis for carrying out marker-assisted selection to improve important economic traits of chicken. Twelve nsSNPs sites of chicken IGF2 gene were retrieved from dbSNP database. SIFT, Polyphen-2, PhD-SNP, and SNAP were used to predict the possible functional SNPs. The amino acid stability of mutation sites was analyzed by I-Mutant3.0 and Mupro methods. Multiple sequence alignment and conserved prediction of evolutionary sites were carried out on the amino acid sequence encoded by chicken IGF2 gene, and combined with MutPred2 to predict the possible functional consequences caused by mutations. Finally, Sopma was used to predict the secondary structure of IGF2 wild-type and mutant proteins, and I-TASSER was used to construct their tertiary structure. The results showed that rs740391349 (E29G), rs735633122 (T30P), rs739078786 (L31P), rs736255842 (E35G) and rs736800980 (V37G) might affect the protein function of chicken IGF2, and all the mutations reduced the protein stability of IGF2. Multiple sequence alignment and conservative analysis showed that rs740391349 (E29G), rs735633122 (T30P), and rs736255842 (E35G) were highly conserved and exposed functional residues. MutPred2 and secondary structural analysis showed that mutations at rs735633122 (T30P) and rs736255842 (E35G) both led to a decreased percentage of α-helix. Tertiary structural analysis showed that rs735633122 (T30P) and rs736255842 (E35G) could change the spatial structure of IGF2 protein. In summary, mutations at T30P and E35G seriously affect the structure of chicken IGF2 protein and may be the significant functional SNPs affecting chicken growth and body composition traits.     

The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F2α release from endometrium of pigs

The mechanism for prostaglandin (PG) F_2α release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP₃) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP₃ levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 μM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF_2α secretion observed after OT treatment (P < 0.001), PGF_2α release was increased (P < 0.01) after treatment with phorbol-12myristate-l3-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF_2α secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF_2α secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP₃ production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP₃ in resting cells was consistent with this conclusion.     

Cloning and Sequence Analysis of MHC Ⅰ β2m Gene in Chicken

In order to further understand molecular characteristics and immune mechanisms of chicken major histocompatibility complex class Ⅰ(MHCⅠ) β2m gene,the 15 sequence of chicken MHC β2m gene from three local breeds was cloned by RT-PCR. We compared these sequences with seven MHCⅠβ2m genes of human,mouse and other animals in the GenBank database. The results showed that the amino acid homology among chicken MHCⅠβ2m ranged from 98.0% to 100%,the most sequences were identical. Chicken MHCⅠβ2m shared a 60.6% amino acid homology with duck,the highest degree of homology indicated a close genetic relationship,and the grasscarp had the lowest homology,33.3%. It could be further confirmed by phylogenetic analysis. Moreover,alignment of 10 β2m sequences of mature protein,two cysteines were located in sites 24 and 79,and there was the "YXCXVXH" Ig-motif character between the sites 77 and 83 of chicken β2m. The results showed that MHCⅠβ2m of chicken and other species had the same basic unit of immune function,and they had the unique structural features.     

The influence of clenbuterol on prostaglandin F2α-induced dyspnoea in calves

Pulmonary function tests were performed in six healthy calves. Prostaglandin F₂ causes severe narrowing of both upper and lower airways (total lung resistance increased, dynamic compliance decreased). Clenbuterol administered intravenously fifteen minutes prior to prostaglandin F₂ aerosol, and in increasing doses (0, 0.4, 0.8, 1.2 g/kg), on days 1, 2, 4 and 6 of the experiment, effectively but not entirely suppressed these responses.These data indicate that -adrenergic receptors are present in the bovine airways and that the use of clenbuterol (0.8 g/kg) may be effective in treating clinical respiratory disease such as bronchopneumonia in calves.     

CYP2D-related Metabolism in Animals of the Canoidea Superfamily – Species Differences

CYP2D-related drug metabolism in liver microsomes from animals of the Canoidea super family, i.e. mink (Mustela vison), bears (Ursus arctos), foxes (Vulpes vulpes) and dogs, were investigated. Propranolol, bunitrolol and imipramine, which are typically substrates of CYP2D subfamilies, were used in the experiment. All the animals of the Canoidea superfamily that were tested lacked the ability to catalyse 7-hydroxylation of propranolol, which is one of the major metabolic pathways in rats. Stereoselectivity of propranolol metabolism was towards (S)-propranolol in all the reactions of the animals tested with the exception of mink, which showed a selective tendency towards (R)-propranolol in N-dealkylation. As far as metabolic patterns of (R)- and (S)-propranolol are concerned, bears, foxes and dogs are alike, but minks are somewhat different. Liver microsomes from mink showed, among the animals of the Canoidea superfamily, the lowest propranolol hydroxylase activity at 4- and 5-positions and imipramine 2-hydroxylation and {N-}demethylation activities. We could not detect bunitrolol 4-hydroxylation in mink liver microsomes at the low substrate concentration used. We conclude that mink have the lowest activity of CYP2D-related xenobiotic metabolism among the Canoidea superfamily.     

Effects of endotoxin and influence of cyclooxygenase-2 on β-adrenergic mediated relaxation in isolated equine digital artery

The effects of endotoxin on β-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA β-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of β-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA β-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.     

The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the β2-toxin in enteric disease of domestic animals,wild animals and humans

The virulence of Clostridium perfringens, a bacterium causing enteritis and enterotoxaemia in domestic and wild animals and humans, results largely from its ability to produce toxins. In 1997, an unknown toxin of C. perfringens, the β2-toxin, and its encoding gene cpb2 were described. Since that time numerous studies have been published dealing with a possible association of cpb2-harbouring strains of C. perfringens and the occurrence of enteric disease in domestic and wild animals and humans. This article offers an overview of the current literature on the spread and pathological significance of cpb2-harbouring C. perfringens. Unambiguous conclusions on the prevalence of cpb2 and the contribution of β2-toxin to the disease cannot be drawn from existing studies but in some animal species a strong correlation between the presence of cpb2-harbouring C. perfringens, the β2-toxin and enteric disease has been reported.     

Individual and combined effects of t‐2 toxin and das in laying hens

1. The individual and combined effects of T‐2 toxin and 4,15‐diacetoxyscirpenol (DAS) on laying hens were investigated in an experiment consisting of a 2 X 2 completely randomised factorial design with dietary concentrations of 0 and 2 mg/kg T‐2 toxin and 0 and 2 mg/kg DAS.

2. Individually, T‐2 toxin and DAS induced oral lesions in half of the hens and decreased significantly egg production and food intake.

3. The effects of T‐2 toxin and DAS were additive for reduced food consumption and incidence of oral lesions. However, a synergism for reduced egg production was observed during the last experimental period.

4. No effects on body weight were observed during this study. Mild changes in selected plasma enzymes activities and no change in liver malondialde‐hyde content were detected.

5. The combination of T‐2 toxin and DAS was more toxic than the single mycotoxins, for some parameters, and therefore, may pose a greater economic threat to the poultry industry than either of the toxins individually.     

Expression and Localization of Integrin αv in Different Parts of the Fallopian Tube in Reproduction Cycle of Yak

The objective of this study was to explore the expression differences of integrin αv in different parts of the fallopian tube at different reproduction stages of yak, and provide some important theoretical foundations for understanding the effect of integrin αv on the reproductive performance of yak. The samples of the tubal umbrella, ampulla, and isthmus of healthy female yaks (3-6 years old) during follicular, luteal and pregnancy stages were collected, the fallopian tubes of the follicular phase, the luteal phase and the pregnancy phase were divided into 9 groups according to the umbrella, ampulla and isthmus. Real-time PCR (qRT-PCR) and Western-blotting (WB) were used to detect the expression of integrin αv gene and protein. The expression of integrin αv protein was localized by immunohistochemistry. Results: 1) qRT-PCR results showed that, during the follicular phase, the expression of the integrin αv gene in the umbrella of the fallopian tube was the highest and extremely significantly higher than those in the ampulla and isthmus (P<0.01). During the luteal phase, the expression of the integrin αv gene in the isthmus of the fallopian tube was the highest and extremely significantly higher than those in the umbrella and ampulla (P<0.01). During the pregnancy phase, the expression of the integrin αv gene in the umbrella of the fallopian tube was the highest and extremely significantly higher than those in the ampulla and isthmus (P<0.01). 2) Western-blotting results showed that, during the follicular phase, the expression of integrin αv protein in the umbrella of the fallopian tube was the highest and extremely significantly higher than those in the ampulla and isthmus (P<0.01). During the luteal phase, the expression of integrin αv protein in the isthmus of the fallopian tube was the highest and extremely significantly higher than those in the umbrella and ampulla (P<0.01). During the pregnancy phase, the expression of integrin αv protein in tubal isthmus and umbrella was not significantly different, but these were extremely significantly higher than that in the ampulla (P<0.01). 3) The results of immunohistochemistry showed that integrin αv protein was positively expressed in ciliated cells, secretory cells, basal cells, muscular layers and serous glands of the fallopian tube umbrella, ampulla and isthmus. The results indicated that the expression of integrin αv in different parts of the fallopian tube at different reproduction stages of the yak were significantly different, which showed that αv might be involved in a series of reproductive processes such as fertilization and early embryonic development, the results provided basic information for the study of yak's reproductive performance.