Uso da somatotropina recombinante bovina em búfalas leiteiras II: metabolismos energético e mineral / Use of recombinant bovine somatotropin (rbST) in dairy buffaloes II: energy and mineral metabolism

Avaliou-se a influência da somatotropina recombinante bovina (rbST) sobre os metabolismos energético e mineral de búfalas entre 63e 154 dias em lactação. Foram utilizadas 22 búfalas, distribuídas em dois grupos experimentais: grupo rbST - aplicação de 500mg de rbST a cada 14 dias; grupo Controle - sem aplicação de rbST. A cada sete dias, foram coletadas amostras de sangue para a determinação do perfil bioquímico e mensuraram-se a produção de leite e o escore de condição corporal dos animais. As médias dos parâmetros estudados para os grupos rbST e Controle foram, respectivamente: produção de leite (PL): 6,44kg vs. 6,68kg; escore de condição corporal-ECC (1-5): 3,51 vs. 3,57; glicose: 70,58 vs. 64,81mg/dL (P = 0,0003); colesterol: 132,38 vs. 133,40mg/dL; triglicérides: 29,18 vs. 28,32mg/dL; proteína total: 8,57 vs. 8,75g/dL; albumina: 3,47 vs. 3,60g/dL; ureia: 32,46 vs. 33,86mg/dL; creatinina: 1,27 vs. 1,39mg/dL; cálcio:10,25 vs. 10,73mg/dL; fósforo:5,76 vs. 5,62mg/dL; e magnésio:3,70 vs. 3,70mg/dL. O uso de 500mg de rbSTinfluenciou o metabolismo da glicose, porém não modificou a PL, o ECC e os níveis dos demais parâmetros metabólicos estudados.(AU)

The aim was to evaluate the influence of recombinant bovine somatotropin (rbST) on the energy and mineral metabolism of buffaloes between 63 - 154 days in milk. Twenty-two buffaloes distributed in two experimental groups were used: Group rbST (n= 11) - application of 500mg of rbST every 14 days; Control Group (n= 11) - no rbST. Every seven days, blood samples were taken to determine the biochemical profile, and milk production and body condition score were measured. The averages of the variables for rbST and Control groups were, respectively: milk yield (MY) - 6.44kg vs. 6.68kg; body condition score (BCS) - 3.51 vs 3.57 (1-5); glucose - 70.58 vs. 64.81mg/dL (P = 0.0003); cholesterol - 132.38 vs. 133.40mg/dL; triglycerides -29.18 vs. 28.32mg/dL; total protein - 8.57 vs. 8.75g/dL; albumin - 3.47 vs 3.60g/dL; urea - 32.46 vs 33.86mg/dL; creatinine - 1.27 vs 1.39mg/dL; calcium - 10.25 vs. 10.73mg/dL; phosphorus - 5.76 vs 5.62mg/dL; and magnesium - 3.70 vs 3.70mg/dL. Use of 500mg rbST influenced glucose metabolism, but did not modify the MY, BCS and the levels of the other metabolic parameters studied.(AU)

Intoxicação natural por Ipomoea asarifolia (Convolvulaceae) em búfalos na Ilha de Marajó, Pará / Natural poisoning by Ipomoea asarifolia (Convolvulaceae) in buffaloes on the Marajó Island, Pará, Brazil

Epidemiological aspects and clinical signs of natural poisoning by Ipomoea asarifolia in buffaloes are described. The poisoning was diagnosed in four buffaloes on three different farms, in the County of Cachoeira do Arari, Marajó Island, Pará, and occurred in November-December, a period of drought and severe pasture shortage. The clinical signs were of the central nervous system, as unsteady gait, hypermetria, severe muscular tremors, falling down in unusual positions, nystagmus and marked excitement, signs that turned more severe after movement. Based on the epidemic aspects, clinical signs and absence of histopatological changes, poisoning by Ipomoea asarifolia was diagnosed.

Descrevem-se os aspectos epidemiológicos e os sinais clínicos dos primeiros casos de intoxicação natural por Ipomoea asarifolia em búfalos. A doença foi diagnosticada em quatro bubalinos de três diferentes propriedades, no município de Cachoeira do Arari, Ilha de Marajó, PA, e ocorreu nos meses de novembro e dezembro, o período mais seco do ano nesta região e de escassez de alimento. Os sinais clínicos observados foram relacionados ao sistema nervoso central, como andar trôpego, hipermetria, acentuados tremores musculares, queda ao solo em posições incomuns, nistagmo e marcada excitação, sinais que se agravavam após movimentação. Baseado nos aspectos epidemiológicos, sinais clínicos e na ausência de leões histopatológicas, concluiu se tratar de intoxicação por Ipomoea asarifolia.

An enzyme-linked immunosorbent assay (ELISA) to measure growth hormone level in serum and milk of buffaloes (Bubalus bubalis).

An indirect Sandwich ELISA to measure growth hormone level in serum and milk of buffaloes was developed. The assay was based on purified anti rbST IgG raised in rabbits and chicken and rabbit anti chicken IgG horseradish peroxidase. The assay was validated in terms of sensitivity, specificity, precision and recovery. Parallelism was demonstrated between the standard curve and serially diluted serum, milk and pituitary derived growth hormone. Sensitivity of the assay was 0.1 ng/ml. Recovery of exogenous bovine somatotropin from serum and milk ranged from 90 to 102% and 96 to 108% respectively. The intra and inter assay variations to measure growth hormone in serum and milk were 3.36 to 8.81% and 6.01 to 12.31% respectively. Statistical analysis for parallelism and cross-reactivity of rbST with serum of other species confirmed the reproducibility of the assay.

Disposition kinetics and urinary excretion of cefpirome after intravenous injection in buffalo calves

We investigated the disposition kinetics and urinary excretion of cefpirome in buffalo calves after a single intravenous administration of 10 mg/kg. Also, an appropriate dosage regimen was calculated. At 1 min after injection, the concentration of cefpirome in the plasma was 57.4 +/- 0.72 microgram/ml, which declined to 0.22 +/- 0.01 microgram/ml at 24 h. The cefpirome was rapidly distributed from the blood to the tissue compartment as shown by the high distribution coefficient values (8.67 +/- 0.46/h), and by the drug's rate of transfer constant from the central to the peripheral compartment, K12 (4.94 +/- 0.31/h). The elimination halflife and the volume of distribution were 2.14 +/- 0.02 h and 0.42 +/- 0.005 l/kg, respectively. Once the distribution equilibrium was reached between the tissues and plasma, the total body clearance (ClB) and the ratio of the drug present in the peripheral to the central compartment (T/P ratio) were 0.14 +/- 0.002 l/kg/h and 1.73 +/- 0.06, respectively. Based on the pharmacokinetic parameters we obtained, an appropriate intravenous cefpirome dosage regimen for treating cefpiromesensitive bacteria in buffalo calves would be 8.0 mg/kg repeated at 12 h intervals for 5 days, or until persistence of the bacterial infection occurred.

Pharmacokinetics and dosage regimen of ceftriaxone in E. coli lipopolysaccharide induced fever in buffalo calves

The present study was planned to investigate the pharmacokinetics of ceftriaxone in experimentally induced febrile buffalo calves (n = 5). The fever was induced by intravenous injection of E.coli lipopolysaccaride (1 microgram/kg). To study the pharmacokinetics, ceftriaxone was administered at the dose rate of 10 mg/kg body wt. in all animals. At 1 min, the peak concentration of ceftriaxone was 79.4 +/- 2.37 microgram/ml and the drug was detected up to 6 h. The elimination rate constant was 0.35 +/- 0.02 /h and elimination half-life was 2.04 +/- 0.14 h. The apparent volume of distribution (Vd(area)) and total body clearance (ClB) were 1.21 +/- 0.15 l/kg and 0.41 +/- 0.03 l/kg/h, respectively. To maintain a minimum therapeutic concentration of 1 microgram/kg, a satisfactory dosage regimen of cefriaxone in febrile buffalo calves is 19 mg/kg followed by 18 mg/kg at 8 h intervals.

Equilibrium denaturation of buffalo pituitary growth hormone.

To understand the structural properties of buffalo growth hormone (buGH), the equilibrium denaturation using guanidinium chloride (GdmCl) was carried out and was monitored by ultraviolet absorption spectroscopy, intrinsic fluorescence spectroscopy, far UV-circular dichroism and size-exclusion chromatography. The normalized denaturation transition curves for each of the above methods were not coincident, showing that buGH does not follow a simple two state folding mechanism. Further, size-exclusion chromatography also showed the presence of an associated intermediate during the unfolding of buGH. It was observed that in buGH, denaturation resulted in an initial disruption of the tertiary structure, whereas the secondary structure and the degree of compactness were disrupted at a higher concentration of the denaturant. This suggests that buGH follows the hierarchical model of protein folding.

Inhibitory activity in buffalo follicular fluid and its elution pattern during different season.

Two pools of whole buffalo follicular fluid collected in winter (December, January) and spring (March, April) season were fractionated by Sephadex G-200 column chromatography. Follicular fluid collected in winter and spring eluted into different pattern resulted into four and three peaks respectively. Both the pools of follicular fluid were salted out with 18.5% ammonium sulphate. The salted out fraction of winter and spring season follicular fluid was again subjected to sephadex column chromatography which eluted into two and single peak respectively. Whole follicular fluid (0.6 ml) was administered into ovariectomized mice to see its inhibitory effect. The percentage of compensatory ovarian hypertrophy was 16.3 +/- 4.4 which was significantly different (P < 0.01) as compared to control. 200 micrograms material from peak 1 and peak 2 obtained after fractionation of salted out winter follicular fluid also had inhibitory effect on compensatory ovarian hypertrophy as compared to control group. It was 35.6 +/- 9.3 and 15.9 +/- 4.3% respectively. Thus, the variation in nature of buffalo follicular fluid and its inhibitory effect in ovariectomised mice have significant relation with anoestrus condition in summer and humid months in this species.

Purification and tissue/species dependence of the specificity of buffalo kidney cathepsin B.

A simple purification scheme was developed for isolation and purification of cathepsin B from buffalo kidney. The use of CM-Sephadex and chromatofocusing helped in better and simultaneous separation of cathepsin B, H and L. As judged by PAGE and SDS-PAGE studies, the enzyme was found to be pure on the basis of charge and had a molecular mass of 25.5 kDa. The amino acid composition, number of free sulfhydryl groups and other major physico-chemical properties of the purified enzyme were similar to the properties reported for cathepsin B from other sources/tissues. However, the NH2-terminal amino acid residue of the enzyme was found to be Ala as against Leu reported from other tissues/species. The total carbohydrate content was also found to be significantly lower (3.6%) as compared to 7.0-7.6% reported for the enzyme from other sources. Thiol reducing compounds activated the enzyme whereas thiol blocking compounds inhibited it. The buffalo kidney enzyme hydrolyzed Z-Phe-Arg-MCA (Vmax/K(m) = 17.1) as the most efficient substrate followed by Z-Arg-Arg-MCA, BANA and BAPNA. Among the protein substrates, goat hemoglobin (Vmax/K(m) = 874) was found to be the most preferred. Rabbit muscle aldolase, usually considered to be a good substrate for cathepsin B, proved to be a poor substrate for this enzyme; only 25-30% inactivation of aldolase was observed. Antibodies raised against the enzyme recognised only cathepsin B and did not have any cross reactivity with cathepsin H or L from the same or different sources. These differences in the properties of the buffalo kidney enzyme vis-a-vis the same enzyme from other tissue/species have been attributed to specialized function of cathepsin B in diversified tissues.

Isolation, purification and properties of cathepsin B from buffalo liver.

Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.

Inhibin in individual buffalo ovarian follicles in relation to size.

A sensitive and specific radioimmunoassay was validated and applied for measurement of inhibin in follicular fluid (bFF) obtained from individual buffalo ovarian follicles. Follicular size was measured with an ultrasound machine and follicles were categorized as small, medium and large. Presence of inhibin was detected in all the antral follicles above 3 mm diameter. Inhibin concentration showed a positive relationship (R = 0.27, P < 0.01) with follicular diameter and was significantly higher (P < 0.05) in bFF from medium and large follicles (8.44 +/- 0.54 and 7.70 +/- 0.45 micrograms/ml, respectively) in comparison to that from small follicles (5.74 +/- 0.80 micrograms/ml). Total inhibin content was highly correlated (R = 0.92, P < 0.001) with follicular diameter and the inhibin content was higher (P < 0.001) in large > medium > small follicles.

A possible urease-based homologous ELISA for buffalo prolactin.

The feasibility of a direct binding as well as competitive ELISA using urease labelled probe is described for buffalo pituitary prolactin. Rabbit anti-buPRL serum and goat anti-rabbit IgG conjugated to urease were used as primary and secondary antibodies respectively. Assay was performed in polystyrene wells pretreated with 3-amino-propyltriethoxysilane (APTES) compound. APTES pretreatment provided a more receptive surface for prolactin (PRL). In direct binding mode the assay could detect 24 pg of PRL per well. In competition mode the detection limit was less than 0.1 ng of competitor PRL per well. Urease-based ELISA was found highly sensitive when the substrate solution containing pH indicator, bromocresol purple, was used at lower pH (4-4.2). No false positive reactions occurred in the control wells. A good parallelism between buffalo serum sample inhibition curve and the standard PRL inhibition curve was observed.

Strategies for purification of four reproductive hormones from the same batch of buffalo (Bubalus bubalis) pituitaries.

The occurrence of different reproductive hormones like LH, FSH, TSH and prolactin, in different side fractions obtained during the extraction of buffalo pituitary glands either by the procedure of Papkoff et al. [Arch Biochem Biophys, 111 (1965) 431] or by that of Ellis [Endocrinology, 69 (1961) 554], was examined with the aid of antisera to respective heterologous hormones as well as bio-assays. Thus in the procedure of Papkoff et al., the SP-Sephadex fractions could be taken for purification of LH and TSH, while the acid pellet yielded prolactin. Further it was shown that 50% (NH4)2SO4 could be directly size fractionated and following cation exchange chromatography yields LH and TSH. FSH could be purified from 80% ammonium sulphate pellet. In another protocol of Ellis, differential extraction and chromatographic separation yielded all the four reproductive hormones. Some of the physico-chemical and immunobiological characteristics of these hormones are described.

Biochemical study of semen of buffaloes. I. Determinantions of fructose, calcium, GOT and GPT

Seis búfalos provenientes de cruzamentos entre as raças Jaffarabadi e Mediterrâneo, com aproximadamente 36 meses de idade, foram submetidos a 16 colheitas de sêmen, quinzenalmente, entre os meses de maio a agosto de 1990, por meio de eletroejaculaçäo ou vagina artificial. No sêmen total foram determinadas, em mg/ml, as concentraçöes de frutose e cálcio, enquanto que, na fraçäo rica em espermatozóides, fizeram-se dosagens de GOT e GPT, em ug/ml. Verificou-se que nas amostras colhidas com vagina artificial os valores de frutose foram significantemente maiores, ao passo que, naquelas obtidos por eletroejaculaçäo, predominaram valores mais altos de GOT e GPT. Relativamente ao cálcio, näo houve diferença significativa quanto aos métodos de colheita

Lipid peroxidation in buffalo (Bubalus bubalis L.) spermatozoa: effect of added vitamin C and glucose.

Addition of 2.5 mM vitamin C or 40 mM of glucose to washed buffalo spermatozoan suspensions in Ca2(+)-free Kreb's Ringer Hanseliet saline buffer (pH 7.0) resulted in significant lower malonaldehyde concentration and higher spermatozoan motility and liver spermatozoa compared to control levels after 45 min of aerobic incubation at 37 degrees C or pre-incubation levels.