There is an increased demand for artificial insemination and shipping canine semen in clinical practice. However, we need to process the semen samples using centrifugation and dilution with extenders to help preserve the breeding dose and semen quality. Our objective was to determine a clinically relevant range of centrifugation and concentration parameters for processing canine semen. In the first experiment, we hypothesized that higher g force and longer treatment improves sperm recovery rates yet causes greater decline in semen parameters over a 48-hour cooling period. Our study design used the raw semen evaluations which served as each dog's own control. Sperm RR (%) was calculated post-centrifugation, and sperm viability (%, Nucleocounter® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (NM%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 (T2) and 48 hours (T3) after cooling.
Sperm losses were minimal and similar for all treatment groups (median >98%, P≥0.062). Spermatozoa viability was not different between centrifugation groups at any time point (P≥0.38) but declined significantly during cooling (T1 vs. T2/T3, P≤0.001). Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 (P≤0.02). In conclusion, our study showed that centrifugation within a range of 400g-900g for 5-10 minutes is appropriate for processing canine semen.
In the second phase, we compared different sperm concentrations for cooled canine semen storage and hypothesized that lower concentrations would result in better semen quality. Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 x106 sperm/ml (C200), then serially diluted to 100, 50, and 25 x106 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24h, then centrifuged and re-extended. Parameters were assessed in raw semen (T0), post-extension (T1), after 24h of cooling (T2), and after processing at 24h (T3). Cooling resulted in significant declines in STM and NM for all groups, and in decreased PMI for CON and C25-50. After cooling (at T2), PMI was significantly lower for C25 compared to all groups and higher for CON compared to C25-100 (p≤0.038). For the motility parameters and NM, C25 performed worse than all or most of the other groups. Comparing CON at T3 with C25-200 at T2, PMI, STM and NM for CON were significantly lower than C25-200, C200, and C100-200, respectively. In conclusion, our results show that cooling canine semen for 24h at 200 x106 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on highest PMI. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24h of cooled storage. / Master of Science / We need to process canine semen using centrifugation and dilution for cooled shipments or cryopreservation. This is due to the increased demand for shipping canine semen for artificial insemination. Our goal was to define an acceptable range of centrifugation and concentration parameters (gravitational (g) force and time and sperm/ml) without severe negative impact on semen quality. In the first experiment, we hypothesized that higher g force (900g vs. 400g or 720g) and longer treatment (10 min. vs. 5 min.) improves sperm recovery rates yet causes greater decline in semen parameters over a 48-hour cooling period. Initial raw semen evaluations served as each dog's own control. Sperm recovery rates post-centrifugation were similar between treatment groups. Sperm viability, motility and morphology were not different between centrifugation treatment groups but declined over time. In conclusion, our range of 400-900g for 5-10 minutes centrifugation provides clinically viable semen quality after up to 48 hours of cooled storage in dogs.
In the second phase, we compared different sperm concentrations for cooled canine semen storage and hypothesized that lower concentrations would result in better semen quality. Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 x106 sperm/ml (C200), then serially diluted to 100, 50, and 25 x106 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24h, then centrifuged and re-extended. Cooling resulted in significant declines in subjective total motility and normal morphology (NM, %) for all groups, and in decreased viability for CON and C25-50. After cooling, viability of the sperm cells was significantly lower for C25 compared to all other groups, and higher for CON compared to C25-100 (P≤0.038). For motility parameters and NM, C25 performed worse than all or most of the other groups. In conclusion, our results show that cooling canine semen for 24h at 200 x106 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on highest plasma membrane integrity (PMI) or sperm cell viability. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24h of cooled storage.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/118608 |
| Date | 21 March 2024 |
| Creators | Sugai, Nicole J. |
| Contributors | Biomedical and Veterinary Sciences, Balogh, Orsolya, Cecere, Julie Tucker, Clark-Deener, Sherrie Gayle, Stewart, Jamie L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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