Author
|
Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 1/8/2007 Publication Date: 3/2/2007 Citation: Purdy, P.H., Blackburn, H.D., Silverstein, J. 2007. Relationship of flow cytometric evaluations of cryopreserved rainbow trout Oncorhynchus mykiss sperm with fertility. Aquaculture 2007 Annual Meeting. p 734. Interpretive Summary: Cryopreserving rainbow trout milt enables breeders and germplasm repositories to maintain a secure reserve of genetic material from a large number of males for minimal costs, and for an indefinite amount of time. However, inseminations using cryopreserved milt generally result in lower fertility when compared with fresh milt. This reduction is due to physical and chemical damage inflicted on the sperm before, during, and after the cryopreservation process. Currently, little is known about the impact of cryopreservation on trout sperm physiology and how the viability of frozen-thawed sperm may affect fertility. Therefore, this study was conducted to determine how membrane integrity, intracellular calcium, and membrane quality influence the fertility of frozen-thawed rainbow trout sperm. Rainbow trout milt was collected and cryopreserved. After storage in liquid nitrogen, four insemination straws from each male were thawed and used for insemination and the fertility was determined. In addition, flow cytometry was used to evaluate plasma membrane integrity, intracellular calcium levels, and phosphatidylserine exposure in frozen-thawed sperm before and after activation. The post-thaw plasma membrane integrity was not correlated with fertility but the ability of trout sperm to incorporate calcium and minimize the exposure of the inner leaflet of the plasma membrane after activation were correlated with fertility. Together, these results imply that functional integrity of the sperm (ability to incorporate calcium intracellulary) and the membrane status (minimal exposure of phosphatidylserine which is indicative of programmed cell death) may play critical roles in trout sperm physiology and could potentially be used to predict the quality/fertility of a sample. Technical Abstract: The ability to cryopreserve rainbow trout milt enables breeders and germplasm repositories to maintain secure reserves of genetic material from large numbers of males with minimal costs, and the material can be maintained indefinitely. However, inseminations using cryopreserved milt generally result in lower fertility when compared with fresh milt. This reduced fertility is caused by physical and chemical damages the sperm experience before, during, and after the cryopreservation process. Currently, little is known about how cryopreservation impacts trout sperm physiology or how the viability of frozen-thawed sperm affects fertility. Therefore, this study was conducted to determine how membrane integrity, intracellular calcium levels, and membrane quality influence the fertility of frozen-thawed rainbow trout sperm. Rainbow trout milt was collected and maintained at 10ºC for 24 hr before being cryopreserved. Frozen sperm were maintained in liquid nitrogen when four insemination straws from each male (n = 8) were thawed and used to inseminate 60 mL of eggs (450 ± 35 eggs per male). The inseminated eggs were maintained at 12.5ºC for 18 days at which time egg fertility was determined. In addition, flow cytometry was used to evaluate plasma membrane integrity, intracellular calcium levels, and phosphatidylserine exposure in frozen-thawed sperm prior to and after sperm activation using the fluorescent stains SYBR-14, Fluo-3 AM, and Annexin V, respectively. Correlation and regression analyses were performed to determine the relationships between sperm characteristics (plasma membrane integrity, intracellular calcium levels, and phosphatidylserine exposure) and sperm fertilizing capacity. The plasma membrane integrity of frozen-thawed sperm was not correlated with fertility (P > 0.05), but the ability of trout sperm to incorporate calcium and maintain little phosphatidylserine exposure to the cell surface (Annexin V) after activation were correlated with sperm fertilizing potential (r = .73 and -.72, respectively; P < 0.05) and their regression models are presented in Figures 1 and 2, respectively. These results imply that sperm functional integrity (ability to elevate intracellular calcium) and the ability to maintain normal membrane status (minimizing sperm membrane rearrangement which is indicative of apoptosis) are critical elements in trout sperm physiology and can be used to evaluate the quality of cryopreserved trout sperm. |
