Determination of acrosin amidase activity in equine spermatozoa.
Abstract: Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (microIU acrosin/10(6) sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r(2) = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 +/- 49 microU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.
Publication Date: 2006-05-27 PubMed ID: 16728208DOI: 10.1016/s0093-691x(97)00352-xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research article investigates the activity of the enzyme acrosin amidase in horse sperm, using a specific process which involves hydrolysing an arginine-based substance. It implies that the evaluation of this enzyme’s activity could offer a new way to assess sperm function.
Theoretical Significance of Acrosin Amidase
- Acrosin amidase activity in sperm cells has been linked to successful in vitro fertilization in human studies.
- The research proposes the use of acrosin amidase activity as an alternative method for evaluating sperm function in test-tube settings.
Procedure of the Study
- The acrosin amidase activity in horse sperm was determined by hydrolysing an optional substrate known as an arginine amide.
- The researchers used a special detergent to release acrosomal enzymes into a medium of basic pH. Here, the enzyme proacrosin was activated to acrosin, which then hydrolysed a specific compound to create a coloured product.
- Sperm samples from four individual stallions were collected and washed to be free from seminal plasma. These were then mixed with a detergent-substrate mixture and incubated for three hours.
- To halt the reaction, a compound called benzamidine was added and the sperm cells were then removed through centrifugation.
- The team then measured the absorbance at a specific nanometer value to determine the acrosin amidase activity.
Findings of the Study
- The study discovered a correlation between the concentration of sperm and the acrosin amidase activity.
- Significant differences in acrosin activity were found between stallions and within ejaculations from the same stallion.
- The researchers found that the acrosin activity detectable in horse seminal plasma was 312 +/- 49 microU/ml.
- Adding a unique cryopreservation medium to the horse spermatozoa and subsequent cryopreservation significantly increased acrosin amidase activity in comparison to spermatozoa from raw semen, a finding contrary to what was previously reported for frozen-thawed human spermatozoa.
Conclusions and Future Research Directions
- The study concludes that assessing acrosin amidase activity in horse spermatozoa could provide a new way of evaluating sperm function in vitro.
- However, the team acknowledges that further research is needed to understand the relationship between acrosin activity and fertility in horses.
Cite This Article
APA
Ball BA, Fagnan MS, Dobrinski I.
(2006).
Determination of acrosin amidase activity in equine spermatozoa.
Theriogenology, 48(7), 1191-1198.
https://doi.org/10.1016/s0093-691x(97)00352-x Publication
Researcher Affiliations
- Department of Clinical Sciences Cornell University, Ithaca, NY 14853, USA.
Citations
This article has been cited 1 times.- Katila T. In vitro evaluation of frozen-thawed stallion semen: a review. Acta Vet Scand 2001;42(2):199-217.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
