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Frozen Semen— Assessing and Minimizing Sperm Damage
EL Squires
Animal Reproduction and Biotechnology Laboratory
Colorado State University, Fort Collins, Colorado USA

        With the recent acceptance of frozen semen by the American Quarter Horse Association and the possibility of acceptance by the Paint Horse Association, the use of frozen semen in the United States is likely to expand dramatically in the next decade.  Most of the semen frozen from stallions in these two breeds will be used internationally.  Thus, the decisions made in the USA may impact the horse industry worldwide.  Although frozen semen has been used in Europe and other countries for decades, there do not appear to be any major breakthroughs in this technology.

            One of the major drawbacks for implementation of frozen semen as a standard breeding method is the inability to assess fertility of the frozen-thawed sperm prior to insemination.  This sometimes results in disastrous fertility and discouragement with the use of frozen semen.  Progressive motility is a reasonable parameter for evaluating fertility of fresh or cooled semen, but is relatively useless in predicting fertility of frozen semen.  Stallions with acceptable post-thaw sperm motilities can have great differences in fertility.  The only advantage of assessing post-thaw motility is to eliminate stallions with very low motility.

        The poor correlation between motility and fertility of frozen-thawed spermatozoa is not surprising since other attributes of the sperm are alsodamaged during freezing and thawing.  This emphasizes the need for other tests that can be used to evaluate sperm damage due to freezing and thawing.  Use of fluorescent dyes to evaluate plasma membrane integrity, acrosomal status, mitochondrial function and sperm chromatin configuration are now being reported.  Flow cytometry is a powerful tool that allows thousands of sperm to be evaluated in a matter of seconds.  Problems that need to be addressed are validation of these staining techniques and interference of freezing extender components in the ability to stain and evaluate sperm.

        Once methods are available to properly evaluate frozen-thawed sperm and these procedures are shown to be correlated with fertility, then progress can be made in improving methods for freezing and thawing.  Studies are needed to determine the advantages of cryoprotectants other than glycerol.  Preliminary studies suggest dimethyl formamide may be an effective alternative cryoprotectant. One major cause of sperm damage may be swelling of the sperm after thawing and rupture of the plasma membrane.  Serial dilution of cryoprotectants from the sperm after thawing may be helpful in preventing membrane damage.  Antioxidants have been shown to prolong survival of sperm cooled to 5EC and held for 2 to 3 days.  Their role in freezing extenders also should be evaluated.

Key issues to be discussed in this section on frozen semen include:
Development of methods for evaluating sperm damage.
Why do sperm of some stallions freeze much better than that of others?
Is cooling a good “stress test” for identifying stallions whose sperm freeze well?
What changes in our freezing protocols will result in a dramatic increase in fertility?
Can fertility trials with frozen semen be organized on a worldwide basis?
What sperm numbers should we use in breeding mares with frozen semen?

What is the best breeding strategy for frozen semen?

A discussion of these questions is likely to create research ideas for the next decade.