![]() This study, however, used pooled canine semen and extended it prior to centrifugation, similar to stallion andrology work. Although higher speed settings of 1620 g and 2880 g allowed for better sperm pellet formation reducing total sperm losses, these treatments resulted in a significantly higher number of dead and moribund sperm cells with damaged plasma membranes. concluded that 720 g for 5 min gave the best spermatozoa recovery rate (RR) and membrane integrity combination, but they investigated a wide range of centrifugation speeds (180 g–2880 g). This study provided the foundation for optimizing centrifugation for canine semen processing. To date, in dogs, only one study specifically compared the effects of different centrifugation gravitational (g) forces on sperm losses and on in vitro semen quality during cooling. Therefore, it is critical to optimize this one step when processing canine semen. The disadvantages of centrifugation are the damage to spermatozoa, loss of spermatozoa into the supernatant, and risks of decreasing fertilizing ability due to damage from the procedure itself. Increased ROS levels can decrease sperm motility, DNA integrity and spermatozoa membrane integrity due to lipid peroxidation and may cause the apoptosis of spermatozoa. ![]() The presence of such contaminants can lead to increased ROS content, changes in osmolality and pH balance, and negatively affect semen quality. Therefore, centrifugation of canine semen prior to cooled shipment or cryopreservation is routinely performed in clinical practice to remove the potentially harmful effects of prostatic fluid or other contaminants such as urine. Additionally, the admixture of prostatic fluid and other seminal plasma factors in raw samples produces reactive oxygen species (ROS), which are known to be harmful to spermatozoa. These include routine processing methods that can negatively impact the viability, motility, and fertilizing capacity of the sperm cells. However, the gains may be countered by other biologic factors and handling conditions, which can reduce the quality of shipped cooled semen at the time of insemination. Comparable pregnancy rates achieved by cooled and fresh semen inseminations have added to the increase in popularity. The gain in popularity for shipping cooled canine semen is due to the relative ease, decreased expense in logistics compared to frozen semen, and the reduced strain for transporting live animals. The demand for insemination in dogs has significantly increased over the last few decades. The use of cooled and frozen semen for artificial insemination (AI) for canine breeding is vital for overcoming geographic and temporal barriers. In conclusion, our study showed that centrifugation within a range of 400 g–900 g for 5–10 min is appropriate for processing canine semen. Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 ( p ≤ 0.02). Spermatozoa membrane integrity was not different between centrifugation groups at any time point ( p ≥ 0.38) but declined significantly during cooling (T1 vs. ![]() Sperm losses were minimal, and RRs were similar across treatment groups (median >98%, p ≥ 0.062). ![]() Sperm RR (%) was calculated post-centrifugation, and plasma membrane integrity (%, Nucleocounter ® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 h (T2) and 48 h (T3) after cooling. Individual ejaculates collected from 14 healthy dogs were split into six treatment groups (400 g, 720 g, and 900 g for 5 or 10 min). Cooled storage under standard shipping conditions was used as a stressor to evaluate long-term treatment effects. We hypothesized that higher gravitational (g) force and longer time of centrifugation would result in improved spermatozoa recovery rate (RR) but poorer semen quality. Our objective was to determine a clinically relevant range of centrifugation parameters for processing canine semen.
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