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.     

Quantitative Aspects of Thyroid Scintigraphy With Pertechnetate (99mTcO4) in Cats

Thyroidal ^99mTcO₄(pertechnetate) uptake percentages were determined in unanesthetized euthyroid (n = 13) and hyperthyroid (n = 18) cats. Maximal uptakes were observed 60 minutes after IV injection of the radionuclide and ranged from 0.3 to 3.9% of the dose in euthyroid cats (median 2.23%) and from 5.2% to 23.9% of the dose in hyperthyroid cats (median 14.8%) ( P < .05). There were no overlaps in pertechnetate uptake percentages during any of the intervals evaluated. It is concluded that the optimal time for visualization of the thyroid by ^99mTcO₄-scanning is 60 minutes after IV injection of the radionuclide. Calculation of the percentage uptake is of additional diagnostic value.     

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)