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Background: Centrifugation is a common procedure to improve the quality of chilled and frozen canine semen by removing debris and seminal plasma and adding semen extenders. The aim of this study was to evaluate the efficacy and influence of a second centrifugation after 48 h of storage at 5 °C on the sperm quality of canine semen. The ejaculates of 45 healthy male dogs, divided into three groups according to body weight, were analyzed for macro- and microparameters such as ejaculate volume, sperm concentration, kinematic parameters, morphology, and integrity of plasma membrane. Samples were analyzed at baseline conditions (T0), after 24 h (T24) and after 48 h (T48) to assess the effects of the different treatments on sperm quality. Results: The results showed a significant effect of a second centrifugation on the improvement of chilled sperm quality compared to the other techniques, especially up to 48 h. Conclusions: Analysis of the data showed that the semen samples centrifuged and then cooled at 5 °C had acceptable semen parameters, especially in terms of motility, with a gradual decrease in serial evaluations after 24 and 48 h. A second centrifugation after 48 h of storage may lead to better semen quality and improve the kinetics of sperm parameters, the percentage of morphologically normal sperm and the percentage of sperm with intact membranes. [ABSTRACT FROM AUTHOR]

Abstract Background Centrifugation is a common procedure to improve the quality of chilled and frozen canine semen by removing debris and seminal plasma and adding semen extenders. The aim of this study was to evaluate the efficacy and influence of a second centrifugation after 48 h of storage at 5 °C on the sperm quality of canine semen. The ejaculates of 45 healthy male dogs, divided into three groups according to body weight, were analyzed for macro- and microparameters such as ejaculate volume, sperm concentration, kinematic parameters, morphology, and integrity of plasma membrane. Samples were analyzed at baseline conditions (T0), after 24 h (T24) and after 48 h (T48) to assess the effects of the different treatments on sperm quality. Results The results showed a significant effect of a second centrifugation on the improvement of chilled sperm quality compared to the other techniques, especially up to 48 h. Conclusions Analysis of the data showed that the semen samples centrifuged and then cooled at 5 °C had acceptable semen parameters, especially in terms of motility, with a gradual decrease in serial evaluations after 24 and 48 h. A second centrifugation after 48 h of storage may lead to better semen quality and improve the kinetics of sperm parameters, the percentage of morphologically normal sperm and the percentage of sperm with intact membranes.

Simple Summary: This study focused on the importance of accurately assessing ram sperm quality for optimizing assisted reproductive technologies in sheep. Semen preservation can lead to sperm damage due to various stressors, including osmotic, biochemical, and thermal factors. To address this issue, the research aimed to determine the best time to evaluate ram sperm quality and identify the factor causing changes in sperm quality during liquid storage. In Experiment 1, ejaculated sperm were assessed for motility and functionality at different preservation times: 0, 3, 6, and 24 h. Both motility and functionality improved after 6 h. Experiment 2 delved into the responsible factor by using epididymal sperm. Results revealed that extender addition to the sperm caused altered motility at 0 and 24 h, and reduced functionality at 0 h. This suggests that the extender initially alters ram sperm, leading to sublethal damage that becomes reversible after 3 to 6 h of semen preservation. Thus, ram sperm require an adaptation period of 3 to 6 h to the extender before a precise quality assessment. This finding has practical implications for reproduction centers, allowing better workflow organization and optimal expression of ram sperm attributes at the time of cervical artificial insemination. Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed. [ABSTRACT FROM AUTHOR]

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.