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Modification of the ethidium bromide exclusion procedure for evaluation of turkey semen
M R Bakst1, H C Cecil, T J Sexton
1USDA, Avian Physiology Laboratory, Beltsville, Maryland 20705.
Abstract:
The objective of the present study was to modify the ethidium bromide exclusion procedure in order to detect dead and labile turkey spermatozoa after 24 h storage at 7 C. In Experiment 1, fresh and stored spermatozoa were suspended in phosphate-buffered saline (PBS) ranging in osmolarity from 296 to 3 mOsm/kg H2O, each solution containing the same concentration of the nuclear fluorochrome ethidium bromide. In the 3 mOsm/kg H2O PBS, nearly all the fresh (98.3%) and stored (96.7%) spermatozoa were stained with ethidium bromide, indicating they were nonviable. Fresh spermatozoa revealed minor variation in the percentage of nonviable spermatozoa in the ethidium bromide exclusion procedure (ethidium bromide in PBS at 296 mOsm/kg H2O) (20.9%) and the remaining hypotonic PBS solutions (range 20.4 to 18.9%). In the stored semen samples a previously undetected subpopulation of more labile spermatozoa became apparent in hypotonic solutions with osmolarities of 122 and 56 mOsm/kg H2O. In Experiment 2, hen fertility was determined using fresh and stored semen. All hens were inseminated with 100 x 10(6) viable spermatozoa. Viability of stored semen was determined either by the original ethidium bromide exclusion procedure or using the sperm-stress test (ethidium bromide in PBS at 56 mOsm/kg H2O). The observations showed that adjusting the number of viable spermatozoa inseminated based on the percentage of viable sperm estimated from the sperm-stress test did not improve hen fertility over a 15-wk egg production period.
Insights
A modified ethidium bromide staining method helps identify dead and fragile turkey sperm after storage. This sperm-stress test, using hypotonic phosphate-buffered saline (PBS), did not improve hen fertility when adjusting insemination doses.
Area of Science:
- Animal Science
- Reproductive Biology
- Spermatozoa Viability Assessment
Background:
- Assessing turkey spermatozoa viability after storage is crucial for artificial insemination success.
- The standard ethidium bromide exclusion procedure may not fully detect all nonviable or labile sperm populations.
Purpose of the Study:
- To modify the ethidium bromide exclusion procedure for detecting dead and labile turkey spermatozoa after cold storage.
- To evaluate the efficacy of a modified sperm-stress test in predicting fertility outcomes.
Main Methods:
- Fresh and stored turkey spermatozoa were exposed to phosphate-buffered saline (PBS) of varying osmolarities (296 to 3 mOsm/kg H2O) with ethidium bromide.
- Hen fertility was assessed using fresh and stored semen, with viable sperm counts determined by the original method or the sperm-stress test (56 mOsm/kg H2O).
Main Results:
- Nearly all spermatozoa were stained at 3 mOsm/kg H2O PBS, indicating nonviability.
- A subpopulation of labile spermatozoa, undetected by the standard method, became apparent in hypotonic solutions (122 and 56 mOsm/kg H2O).
- Adjusting insemination doses based on the sperm-stress test did not enhance hen fertility over a 15-week period.
Conclusions:
- The modified ethidium bromide exclusion procedure, particularly the sperm-stress test, can identify labile turkey spermatozoa missed by standard methods.
- Fertility prediction using the sperm-stress test did not improve outcomes compared to the original viability assessment in this study.