Sperm Sexing in the Dog by Fluorescence In Situ Hybridization.
Bibliographic record
Abstract
The specific production of male or female offspring by separation of X- or Y-bearing spermatozoa and artificial insemination is a long standing goal in breeding of domestic and other animals. For dogs, higher percentages of female puppies are desired in breeding of guide dogs for the blind as well as companion animals. Effective preselection of sex has been accomplished in several livestock and humans using flow-cytometric sperm sorting. Since a guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing, to be able to apply the specific production of male or female in dog, direct visualization of sex chromosomes in single sperm using fluorescence in situ hybridization (FISH) was developed in this study. The second fraction of ejaculates was collected from Labrador Retrievers (n=3). After washing with PBS, the sperms were fixed with methanol: acetic acid fixative and placed on a defatted glass slide. These preparations were dried and dehydrated on a hot plate at 70°C. For decondensation of sperm nuclei, the specimen was treated with 1M NaOH for 3, 4 or 5 min at room temperature, rinsed with PBS, and then washed with distilled water. After air-drying, the specimen was placed on a hot plate at 70°C. For a double staining of canine X- and Y-chromosome of sperm, sex chromosome probe: X-labelled with SpecrumGreen and Y-labelled with Cy3 were used. Hybridization was carried out in a moist chamber at 37°C overnight. FISH signals were analyzed by eye using an ocular grid counting random microscope fields and scoring 500-600 spermatozoa per sample. Spermatozoa were scored only if they were intact, non-overlapped, had a clearly defined border, and had not decondensed to more than twice the size of a non-decondensed sperm head. There were no significant difference at the P>0.05 level between the theoretical ratio (50:50) and the observed ratio in X- and Y-chromosome-bearing spermatozoa from all three dogs, when samples were treated with 1 M NaOH for 4 min. Hybridization efficiency was 98-99%. The established FISH protocol in this study might be suitable for reliable routine validation of sorted canine spermatozoa.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".