Progress 01/01/04 to 12/31/05
Outputs
The long-term goals of the proposed studies are to gain insight into the molecular basis of boar sperm function and to utilize this information to improve semen storage techniques and hence improve sow fertility. In working toward this goal, the project addressed the following specific aims: We found that boar sperm samples incubated with both beta- cyclodextrin (HBCD) and cholesterol sulfate have decreased capacitation as assessed by less sperm undergoing the induced acrosome reaction, and less increase in protein tyrosine phosphorylation following incubation under conditions supporting capacitation. In contrast, sperm samples incubated in the presence of only HBCD are more capacitated, demonstrating a higher number of acrosome reactions and more protein tyrosine phosphorylation. These results indicate that manipulation of sperm plasma membrane cholesterol content does affect boar sperm capacitation status. Another objective was to determine the effects of HBCD and
cholesterol 3-sulfate (ChS), in a defined medium without egg yolk, on the viability and capacitation of porcine sperm following cold shock. We found that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock when compared to cold shocked sperm incubated without HBCD or ChS or with either component alone. Treatment with HBCD plus ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock and remained at basal levels. We have also determined that the addition of 40 or 60 mM HBCD to semen extender containing 20 percent egg yolk used for cryopreservation significantly improves post-thaw viability. Capacitation studies of porcine sperm cryopreserved with and without HBCD are
ongoing due to difficulty in performing capacitation assays in the presence of egg yolk. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation. We have determined that HBCD does improve the viability of frozen porcine sperm. In collaboration with Dr. Rebecca Krisher of Purdue University, we have initiated studies to determine the ability of porcine sperm cryopreserved in the presence of 0, 25 or 50 mM HBCD to participate in in vitro fertilization and embryo development.
Impacts
Artificial insemination with semen stored by freezing or cooling is of central importance in modern swine production. This project established the effect of sperm cholesterol manipulations during cooling and cryopreservation on sperm fertilizing capacity. It is expected to ultimately improve semen storage methods.
Publications
- GALANTINO-HOMER, H.L., ZENG, W., MEGEE, S.O., DALLMEYER, M., VOELKL, D., DOBRINSKI, I. (2005): beta-cyclodextrin plus cholesterol protects porcine sperm from the effects of cold shock. J. Androl. (Suppl.): 63.
- GALANTINO-HOMER, H.L., ZENG, W., MEGEE, S.O., MODELSKI, M., DOBRINSKI, I. (2006): Calcium removal increases the protective effects of beta-cyclodextrin plus cholesterol on porcine sperm during cold shock. Reprod. Fertil. Dev. 18 (1,2): 155 (abstract # 93)
- GALANTINO-HOMER, H.L., ZENG, W., MEGEE, S.O., MODELSKI, M., DALLMEYER, M., VOELKL, D., DOBRINSKI, I. (2005): Use of 2-hydroxypropyl beta cyclodextrin and cholesterol to improve porcine sperm viability following cryopreservation and cold shock. Theriogenology 64(3): 805.
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Progress 01/01/04 to 12/31/04
Outputs
A protocol for boar sperm preparation following collection was established to optimize viability with minimal capacitation at the initiation of the experiment. Initial experiments have determined that the optimal protocol for cold-shock using the defined medium is 10 C for 10 min following an equilibration for 15 min at 30 C. Our preliminary findings demonstrate a dose-dependant effect of cyclodextrins on viability with 0.8 mM HBCD resulting in the best viability at 0 h or incubation for 3 h at 39C in 5% CO2/95% air. This concentration of HBCD (0.8 mM) was therefore chosen for ongoing experiments. We are finding that boar sperm samples incubated with both HBCD and cholesterol sulfate have improved viability, less sperm undergoing the acrosome reaction, and less increase in protein tyrosine phosphorylation. In contrast, sperm samples incubated in the presence of only HBCD are more capacitated demonstrating a higher number of acrosome reactions and more protein tyrosine
phosphorylation. These results indicate that manipulation of sperm plasma membrane cholesterol content affects sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation. The objective was to determine the effects of HBCD and cholesterol 3-sulfate (ChS), in a defined medium without egg yolk, on the viability and capacitation of porcine sperm following cold shock (10C for 10 min). We found that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock when compared to cold shocked sperm incubated without HBCD or ChS or with either component alone. Immunoblots revealed that treatment with HBCD plus ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly
altered by HBCD and ChS following cold shock, although there was a trend towards less CTC B (capacitated) pattern sperm following incubation with HBCD plus ChS after cold shock. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation.
Impacts
Artificial insemination with semen stored by freezing or cooling is of central importance in modern swine production. The results of this project will establish the effect of sperm cholesterol manipulations during cooling and cryopreservation on sperm fertilizing capacity. Adding these novel components to long-term semen extenders is expected to ultimately improve semen storage methods.
Publications
- No publications reported this period
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