Examination and preventive medicine protocols in psittacines.

Preventive medicine is the best insurance for effective avian health maintenance and infectious disease control. Psittacine birds should have a yearly physical examination including a thorough history. Additionally, these animals require adequate management and husbandry including a commercial bird food diet supplemented with fresh fruits and vegetables. A complete preventive medicine program should include diagnostic tests as deemed necessary by the avian veterinarian in addition to strict quarantine, vaccination, and disinfection. Necropsy and histopathology are also important components of an effective preventive medicine program.

Direct preparations from chorionic villi--relationship between villous morphology and mitotic index.

It has been postulated that chorionic villi with abundant sprouts have a higher mitotic index and are therefore preferable for obtaining direct chromosome preparations from chorionic villus samples. This theory was tested by correlating villous morphology with mitotic index. Surprisingly, no statistically significant relationship was found. Choice of culture medium, however, was found to be important, with serum-free RPMI yielding a higher mitotic index than 40 per cent FCS in MEM. We conclude that villous morphology, as assessed in this study, is not a major factor in determining the success of direct chromosome preparations.

Idiograms of horse chromosomes at prometaphase, early metaphase, and midmetaphase after R-banding by BrdU incorporation followed by the fluorochrome-photolysis-Giemsa technique.

We present three idiograms of equine chromosomes, R-banded after BrdU incorporation and stained by the fluorochrome-photolysis-Giemsa technique. The haploid set of prometaphasic chromosomes shows 591 bands (range 7-38 per chromosome), the early metaphasic set 404 (range 5-26), and the midmetaphasic set 272 (range 3-18). Following cell synchronization with thymidine, more than twice as many R-bands were revealed on the resulting prometaphasic chromosomes, making possible the establishment of a very accurate and characteristic representation of this banding pattern in the domestic horse.

Light and electron microscopy of Ag-NORs in domestic horse chromosomes identified after R-banding.

Silver staining shows the presence in the domestic horse of six NORs located on chromosomes 1, 26 and 31 as identified after R-banding. Following electron microscopy, the argyrophilic material was observed outside the terminal secondary constrictions (satellite stalks) on the terminal portion of the short arm of chromosome 1, outside the secondary constrictions on the proximal region of the long arms of chromosome 31, and beside the proximal region of the long arms of chromosome 26. Satellite staining applied to these chromosomes appears to reveal only the active NORs.

Analysis of X-chromosome inactivation in horse embryos.

To define the time of X-chromosome inactivation in the horse, 122 conceptuses were collected transcervically between Days 6 and 28 (ovulation = Day 0) and subjected to cytogenetic analysis: 59 of the embryos were divided and in 41 of these separate cytogenetic analysis of the embryonic disc and remaining tissues was possible. Conceptuses were measured and photographed before capsule removal, culture in the presence of 5-bromodeoxyuridine and subsequent fixation for cytogenetic analysis. On average, 15 slides were prepared per conceptus. C-banding was used to determine the sex of each conceptus and, in the females, the effects of 5-bromodeoxyuridine incorporation were revealed by u.v. irradiation which produced R-bands and allowed identification of the inactive X-chromosome. Inactivation of the X chromosome began gradually in the trophoblastic (+/- attached endoderm and mesoderm) cells around Day 7.5 and in the embryonic disc around Day 11.5. Cells with an inactive X-chromosome predominated in the trophoblast by Day 10.5 and in the disc by Day 12.5.

Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine.

Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: initially the BrdU is incorporated during the first part of the S-phase and then it blocks the cells at mid-S-phase. Within the cell cycle, mid-S-phase appears to be the most vulnerable time to various blocking agents. To differentiate the regions of BrdU incorporation from those that have not been substituted, the fluorescence-photolysis-Giemsa (FPG) technique was applied as modified for horse chromosomes. This dynamic technique, which produces many prometaphase and prophase chromosomes showing very sharp G-bands, is certain to enhance the accuracy of cytogenetic analysis and aid in the standardization of equine chromosomes.

High resolution R-bands produced in equine chromosomes after incorporation of bromodeoxyuridine.

Cell synchronization was used to obtain an adequate percentage of very long chromosomes in equine mitotic spreads. Reported here is our variation, adapted to horse chromosomes, of a method using excess thymidine followed by bromodeoxyuridine incorporation. This technique routinely yields excellent quality cells, predominantly in prometaphase and prophase. Among other differences with the standard technique, this method does not use Colcemid, which, in addition to inhibiting spindle fiber formation, also increases chromosome contraction resulting in thicker and thus fewer bands. Consequently, horse prometaphase chromosomes, which have incorporated BrdU in the late-S-phase, are very long and display a large number of R-bands after the fluorescence-photolysis-Giemsa method. This technique should definitely be useful for the analysis of structural anomalies and the standardization of equine R-bands.

A direct technique for the preparation of chromosomes from early equine embryos.

A technique is described for the preparation of banded chromosomes from early equine embryos cultured for less than 10 h in a medium containing bromodeoxyuridine. In addition to standard Giemsa staining and C-banding, chromosomes thus prepared can also be R-banded by either the RBA or the RB-FPG methods. This technique is rapid, repeatable, and limits cell loss, making it ideal for the preparation of early embryos.