Thursday, June 06, 2013

Sperm from dead horses

The unexpected death of a prize stallion need not necessarily spell the end of his breeding career.  Obviously many breeding stallions will have had semen frozen and stored for future use. But what if that has not been done?

Or you may have a promising colt and be unable to decide between keeping him entire for breeding, or having him castrated. The answer may lie in cryopreservation of epididymal sperm.

It is now possible to salvage sperm from the tail of the epididymis – the long convoluted tube into which the sperm pass after being produced in the testis. As well as acting as a reservoir for the sperm, the epididymis provides an environment in which they can mature and become able to fertilise oocytes.

The procedure for harvesting and freezing the sperm is not widely available. So what can be done if the laboratory is a long way away? Will  delay adversely affect the viability of the sperm?

Research in the Department of Physiology at the University of Murcia in Spain suggests that spermatozoa stored in the epididymis for up to 96 hours at 4º C can be cryopreserved successfully and still retain the ability  to fertilise. 

In work published in the journal Animal Reproduction  Science, Luis Vieira and others  studied the viability of sperm stored  in the epididymis obtained from castrated horses.

Testes were transported to the laboratory in insulated  containers at ambient temperature within one hour of castration. At the laboratory, the epididymides were washed in physiological saline, wrapped in foil to prevent them drying out, and stored in a refrigerator at  4º C for up to 96 hours.

The scientists harvested the sperm by introducing a syringe and needle into the vas deferens and flushing out the epididymal contents. The sperm were mixed with extender before being frozen and stored in liquid nitrogen.

Measurements  of viability of sperm in the epididymal fluid were made at various stages in the procedure.

Viability was good (85%) if the sperm were harvested on the day of castration , and remained at more than 80% until 72 hours after castration. Sperm stored for 96 hours before harvesting were significantly less viable.

After dilution in the freezing media and storage at 4º C for 30 minutes, viability remained good for sperm harvested within 48 hours of castration.  Viability was lower in sperm collected 72 and 96 hours after castration.

On the other hand, time (up to 96 hrs) from castration to harvesting did not appear to affect the viability after freezing and thawing, which was almost 35%.

Other measures of sperm viability and tests of their ability to fertilise in vitro, were carried out, including chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate. These showed that epididymal stallion sperm stored for up to 72h in the epididymis at 4°C followed by cryopreservation, maintained both viability and ability to fertilize in vitro.

Although these were laboratory tests of viability, epididymal sperm have been used successfully.


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