Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.     

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.     

Use of Echinococcus granulosus worm antigens for immunodiagnosis of E. granulosus infection in dogs.

Echinococcus granulosus worm excretory/secretory antigens (WES) were used in ELISA for diagnosis of E. granulosus infection in dogs and compared with protoscolex somatic antigens (PSM). Sera from 224 dogs were tested. There was no correlation between ELISA absorbance values and E. granulosus worm burdens using either antigen. There was a significant linear relationship between absorbance values of sera tested in the ELISA using WES (W-ELISA) and the ELISA using PSM (P-ELISA). However, there was a small but significant difference between the absorbance values of the sera tested against the two antigens. Western blot analysis of WES using sera from E. granulosus-infected and uninfected dogs revealed antigenic components of relative molecular mass (Mr) larger than 94,000, Mr 94,000-68,000 and Mr 43,000-39,000 in worms, and these were specific for E. granulosus and not identified in PSM; these antigenic differences may be responsible for differences in reactivity in ELISA. The sensitivities of W-ELISA and P-ELISA were 80.8% and 75.6%, respectively. The specificities of W-ELISA and P-ELISA were 93.7% and 97.9%, respectively. The reduced specificity in W-ELISA was mainly attributable to increased background reactivity of sera from Taenia hydatigena-infected dogs. Despite the reduction in specificity, both ELISAs are valuable epidemiological tools to determine the prevalence of antibody to E. granulosus in dog populations and to monitor the success of hydatid control campaigns.     

Surgery of hepatic cysts in a cat.

A 2-year-old female Japanese domestic cat weighing 3.6 kg was presented due to marked abdominal distention and was diagnosed as hepatic cyst. The bile acid concentration of the hepatic cyst was as low as the serum bile acid concentration. The membrane of the cysts was excised from the edge of the compressed hepatic tissue and no other surgical manipulation was performed. This surgical procedure is simple and quick compared to hepatic lobectomy, however, it is necessary to evaluate the fluid of the cyst before performing this procedure.     

A Quantitative Study of Ganglion Cells in the German Shepherd Dog Retina

As in the number of mammals, the most prominent feature of the ganglion-cell layer in the retina of the German shepherd dog is the sharp increase in the density of ganglion cells in the central area. There is an area of maximum density and also a ‘cat-like’ visual streak, located dorsal to the optic disc. The isodensity lines of ganglion-cell distribution is roughly concentric. Their values vary from 5300-13 000 cells/mm² in the central area, with the cells densely packed, to 1000 cells/mm² or less in the periphery, where the cells are sparsely distributed. There were some individual differences amongst the animals studied, although all of them were pure-bred dogs. This suggests that the configuration of the retina in the canine species is not only dependent on the breed itself but also on some other parameters such as phylogenetic heritage, environment, aptitude, lifestyle, or even training.     

Dynamics of changes in the cytological picture of vaginal smears and circulating ovarian hormones during the puerperal period in ewes]

A postparturient period is characterized by low basal secretion of adenohypophysis gonadotropins with the following appropriate changes in ovarian hormones and their response to the morphology of vaginal epithelium. In this study the dynamics of the cytological picture of vaginal swabs and ovarian hormones 17 beta-oestradiol (E2) and progesterone was investigated in the puerpery of ewes. The objective was to obtain and extend the knowledge of cytological changes in vaginal epithelium and levels of ovarian hormones of ewes after parturition and of their relationships from the first several days after lambing until the 51st day of the period of observation. Vaginal swabs for vaginal cytology were taken from nine ewes on days 1, 4, 7, 14, 17, 21, 25, 34, 42 and 51 after parturition. These swabs were fixed in ether-alcohol 1:1, stained according to the Faltínová-Zidovsky method, embedded in Canada balsam and evaluated by differentiation of cells according to Luksh (1953). Blood samples for E2 and P4 determinations were taken from the jugular vein in the same intervals as vaginal swabs. The serum was centrifuged and stored at -18 degrees C until use. E2 and P4 concentrations were determined radioimmunologically, using kits RIA-test ESTRA and RIA-test PROG from URVJT Kosice. A statistically significant decline (P < 0.05) of percentual representation of basal and parabasal cells (Fig. 1, Tab. I) on day 7 after lambing was replaced by their multiplication from day 14 reaching the values of 66.07 +/- 3.95 on day 42. A statistically significant decrease in intermediary flat cells (Fig. 2, Tab. II) was observed on days 14 (P < 0.001), 34 and 42 (P < 0.01; P < 0.001), in comparison with the first day after lambing. An evaluation of intermediary convoluted cells revealed their highest percentage on days 1 and 17 after parturition (34.65 +/- 4.77-20.62 +/- 12.57) and their decline to values in the range of 6.77 +/- 1.46-7.66 +/- 2.25 on the remaining days of the period of observation. Percent occurrence of superficial flat cells (Fig. 3, Tab. I) ranged from 3.9 +/- 1.10 to 10.63 +/- 7.23 from day 1 to day 51 after lambing. The lowest percentual representation (1.32 +/- 0.79-4.10 +/- 1.89) was recorded for superficial convoluted cells. Multiplication of the evaluated cells was observed, reaching the highest but insignificant representation (P > 0.05) on day 25 of postparturient investigation: 4.10 +/- 1.89 (Fig. 3, Tab. I). 17 beta-oestradiol (E2) concentrations were compared to the -1st day before parturition, when its values varied at the level of 2.45 +/- 0.64 nmol/l serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effectiveness of the combined treatment of thrombocytic hemorrhagic diathesis of dogs with prednisolone and blood transfusion in the model of an aspirin-produced thrombocytopathy

The administration of 20 mg/kg of acetylsalicylic acid in 18 clinically healthy dogs resulted in a thrombocytopathy with lengthened capillary bleeding time and irreversible aggregation inhibition. Through the set up of individual dilution series, one could conclude the proportional percentage of aggregation functional transfused thrombocytes. The capillary bleeding time was shortened after the intravenous injection of prednisolone (5 mg/kg) without measurable influence on the thrombocytes. Compared to the singular use of cortisone or blood transfusion alone, the effect on capillary bleeding time became magnified when one combined transfusion and corticosteroids. The fresh blood conserves (12 hours) were, with respect to the haemostyptical properties, superior to blood stored for 5 days. The component of aggregation-functional thrombocytes was, due to the prophylactic cortisone application prior to transfusion, not increased.     

Residue management for reducing evaporation in relation to soil type and evaporativity1

Abstract. Usual residue-management options are to remove the residue, use it as mulch with or without undercutting or to incorporate it into the soil. While the role of surface mulch in evaporation has been widely studied, the information on the effect on evaporation of mulch with undercutting or residue incorporated into soil, particularly in relation to soil type and evaporativity (E_o) is lacking. We studied the effect of wheat straw used in various ways on the course of evaporation loss from soil columns with three soils at Ludhiana, India and one soil at Bushland, Texas, USA, under two E_o's Energy-limited evaporation rates under mulch (E_om) followed the soil-specific relation E_om/E_o= a e^(bRes+cEo), where Res is residue rate t/ha and a, b and c are constants; E_o, is expressed in mm/d. In an effort to model the total evaporation (CE) during the energy-limited stage ‘U’ was obtained from appropriate CE versus time curves and (CE-U) was regressed over (t - ti)^0.5 to obtain the slope ‘α’ (Ritchie 1972) for the soil-limited evaporation stage. The observed ‘U’ was independent of mulch rate and E_o but was strongly affected by soil type, Values of ‘α’ decreased with increase in mulch rate and decrease in E_o and coarseness of soil. The otherwise short lived benefit of evaporation reduction with mulch per se, which peaked after a few days was maintained when residue was mixed with soil at the stage when evaporation reduction reached a maximum; this benefit continued for several weeks. Cumulative evaporation values computed from ‘U’ and ‘α’ agreed closely with the observed values under straw mulch for loamy sand and clay loam soils and for ‘undercut’ and ‘residue mixed’ treatments on all soils regardless of E_o, and for all situations under small E_o. However, for sandy loam and silt loam soils under E_o of 10 mm/d, the modified square root of the time function of Jalota et al. (1988) gave a better fit.     

Effect of dietary copper and vitamin E supplementation, and extensive feeding with acorn and grass on longissimus muscle composition and susceptibility to oxidation in Iberian pigs

The objective of this study was to assess the effect of dietary copper and/or vitamin E supplementation on composition and oxidation of M. longissimus in Iberian pigs and to compare it with meat samples from pigs produced extensively and fed with acorn and grass. Grass had the highest α-tocopherol content (> 150 mg/kg DM), while acorns had the highest copper concentration (78.1 mg/kg DM). Dietary treatment did not affect copper composition in muscle and no interactions were detected between copper and α-tocopherol. The α-tocopherol content in muscle from pigs fed diets supplemented with α-tocopheryl acetate (100 mg/kg feed) was 1.5 times greater (p < 0.0001) than those from pigs receiving a basel diet. M. longisssimus dorsi from pigs fed extensively had a higher concentration of α-tocopherol than those fed in confinement with the basel level of α-tocopheryl acetate, but lower values than pigs fed supplemented levels. Total n -3 fatty acids (p < 0.02) and hematin (p < 0.0001) concentrations were significantly higher in muscle from pigs fed extensively than when fed in confinement. Muscle homogenates from Iberian pigs fed in extensive conditions showed significantly (p < 0.02) higher susceptibility to oxidation than those from pigs fed mixed diets. Dietary α-tocopheryl acetate supplementation (100 mg/kg feed) significantly (p < 0.05) reduced lipid oxidation of muscle, while dietary copper concentration did not modify susceptibility to lipid oxidation.