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.     

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 Integration of Fertilizer and Green Manure Nitrogen on Yield Attributes, Nitrogen Uptake and Yield of Lowland Rice (Oryza sativa L.)

Field experiments were conducted at Tamil Nadu Rice Research Institute, Aduthurai, India, during the wet seasons of 1992 and 1993 to study the effect of full and partial substitution of fertiliser N with green manure N (Sesbania rostrata) on nitrogen uptake, yield attributes and yield of rice. The experiment consisted of eight treatments with two levels of N (100 and 200 kg ha⁻¹) and three sources of N application viz., fertilizer, integrated (1:1 fertilizer and green manure N) and green manure N compared to the recommended practice (150 kg fertilizer plus 6.25 t ha⁻¹ (72 kg N) green manure) and a no N control. Nitrogen application markedly increased the N uptake. Combined use of the two N sources at 200 and 222 kg N ha⁻¹ and of single fertilizer N at 200 kg N ha⁻¹ recorded the maximum N uptake, increased the yield attributes such as number of panicles per unit area, weight per panicle, number of total and filled grains per panicle and test weight. At 200 kg N ha⁻¹ full substitution of N by green manure reduced the grain yield but only partial substitution of N by green manure resulted in almost similar yield as single fertilizer N. Thus 200 kg N ha⁻¹ applied in equal proportions of fertilizer and green manure N can be recommended for medium duration rice cultivars.     

Vererbung des β-Glucangehaltes der Haferkaryopse

Inheritance process for β-glucan content in oat caryopsis
Due to the fact that there are only few studies concerning β-glucans in oat caryopsis with regard to plant-breeding aspects, three oat crosses were performed (cross 19 = low × high, cross 86 = low × low, cross 98 = high × high). In all of the three oat crosses, lower β-glucan content (% DM) was recorded in F, than in F, indicating that the β-glucan content (% DM) at a higher heterozygoty grade is lower than at a reduced heterozygoty grade. This points to a dominant inheritance of lower β-glucan content (% DM). A tendency towards the female β-glucan content (% DM) could not be proven. Thus, it can be started from a nuclear genetic inheritance of the β-glucan content (% DM). In most cases, the experiments showed a normal distribution of β-glucan content (% DM), indicating for several genes a quantitative inheritance of β-glucan content (% DM). By means of the regression of the F₃-lines to the F₂-plants, the narrow-sense heritability could be calculated for the cross 86 (h²= 0.51***) and the cross 98 (h²= 0.48***). The heritability for the cross 19 was not significant.