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Title: OSMOTIC TOLERANCE LIMITS AND EFFECTS OF CRYOPROTECTANTS ON MOTILITY OF BOVINE SPERMATOZOA

Author
item Guthrie, Howard
item LIU, J - INDIANA UNIVERSITY
item CRITSER, J - INDIANA UNIVERSITY

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Cryopreserved bull spermatozoa have been used many years for insemination of cattle. However up to 60-70% of sperm are killed by current methodologies. In addition, spermatozoa of many bulls are not cryopreserved because their spermatozoa do not survive well using current protocols. In order to improve cryopreservation of bull spermatozoa more information is required regarding their biophysical characteristics as related to membran permeability to water and permeating cryoprotective agents. Semen from beef bulls was collected by electroejaculation and extended in TL-Hepes 1:3. Over the range of 150 to 1200 mOsmolality/kg (mOsm), bull spermatozoa behaved as linear osmometers (r2 = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 um. Retention of at least 90% of isosmotic motility could be maintained only between 270-360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92 to 103% of their isosmotic cell volume. Motility following the one- step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31, 90, and 6%, respectively, compared to CPA addition. These results indicate that scientists should develop new cryopreservation protocols for bull spermatozoa which maintain cell volume within a very narrow range and test ethylene glycol as a cryoprotectant.

Technical Abstract: This study was conducted to determine how motility was affected by osmotic stress, by the addition and removal of cryoprotectants (CPA), and to determine isosmotic cell volume and the osmotically inactive cell fraction. Semen was collected by electroejaculation and extended 3:1 in TL-Hepes medium containing 6 mg/ml BSA and 100 ug/ml pyruvate. Osmotic tolerance limits for maintenance of motility at 90% or more of the isosmotic value ranged from 270 to 360 mOsmol/kg. Five min exposure to either 0, 1, or 2 molar (M) CPA: glycerol (Gly), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) (n=4) decreased (p=.0001) motility as a function of CPA concentration (1 or 2M) by 25 and 38%, respectively, compared to no CPA. When sperm cells in 1 M CPA were returned to isosmotic conditions, motility was decreased (p=.0001) by 30, 90, and 5% for Gly, DMSO, and EG, respectively; compared to no CPA (n=5). Analysis of volume change in anisosmotic conditions using a Coulter Counter (n=4) indicated that bull sperm were linear osmometers in the range of 150 to 1200 mOsmol/kg and that 61% of total cell volume is osmotically inactive. These results indicate that bull spermatozoa should be maintained within 92% to 103% of their isosmotic cell volume during cryopreservation to retain 90% of pretreatment motility. In addition, EG should be evaluated as a CPA for bull sperm because one-step addition and removal of EG is less damaging to bull sperm motility than either Gly or DMSO.