Progesterone-induced acrosome reaction
- In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review
Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of stimuli, e.g. progesterone. Progesterone in mare follicular fluid induces the acrosome reaction in capacitated stallion spermatozoa. The reaction is mediated by a plasma membrane progesterone receptor . The percentage of spermatozoa with exposed progesterone receptors was highly correlated to fertility of stallions . Sperm from stallions classified as fertile on the basis of breeding history had higher percentages of porgesterone-induced acrosome reactions in comparison with stallions classified as subfertile . The test has not been applied to frozen semen.
In humans, the cervical mucus penetration test  and the microelectrophoretic motility test  have been applied to study quality of fresh and frozen semen, but neither of these test have been used in horses.
Sperm chromatin structure assay (SCSA)
Chromatin in a spermatozoon is condensed to one-sixth of the volume in somatic cells. Normal sperm development leads to a chromatin structure in which the DNA in situ is resistant to denaturation, whereas the DNA of spermatozoa with an abnormal chromatin structure is susceptible to denaturation in situ. The percentage of cells with abnormal chromatin and the extent of the abnormality can be detected by the SCSA carried out in flow cytometry . Subfertile stallions had a higher percentage of cells outside the main population (COM-Pαt) than normal stallions . In swine, SCSA correctly predicted both high- and low-fertility boars based on a ratio of offspring as deviated from the theoretical percentage . However, the boar sperm chromatin structure was unaltered by direct freezing on dry ice or in liquid nitrogen with or without different types of extenders. The loss of sperm fertility potential after freezing/thawing is due to factors other than damage to sperm chromatin structure, which is very resistant . Thus, SCSA is not suitable for evaluation of effects of freezing on sperm, but can be used to evaluate the COMPα of the DNA of frozen semen.
Resazurin reduction test
A resazurin reduction test has been useful in determining the fertility potential of bovine spermatozoa. Metabolically active sperm reduce resazurin (blue) to pink and upon further reduction to white. Assessment of the reduction from blue to pink allowed for the identification of 88% of the potentially low and 94% of the high-ertility samples . The resazurin reduction test performed immediately after thawing of frozen stallion semen had no correlation with fertility .
The transmigration rate (TMR%) has been used to compare motility of fresh and frozen-thawed stallion sperm. The rate is defined as the percentage of spermatozoa which within 3 min will migrate from the sample chamber through a membrane having pores of 8 mm against a flow (5 ml/h) of buffer medium to the goal chamber. There was a highly significant correlation between TMR, motility and the percentage of viable cells . However, since TMR was not reported for fresh semen, it is not possible to know how well the test would reflect changes which have taken place during the freezing and thawing processes. The test has not been correlated with fertility.
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