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Cryopreservation of sperm

27 June 2019, at 9:00am

Learn about the history of freezing equine semen and the applications for artificial insemination today

Originally driven by the pressure to increase the speed of genetic improvement in farm animals, the availability of artificial insemination has expanded across many species, including the equid. The first recorded successful artificial insemination was in a canine in 1784 by the scientist Lazzaro Spallanzani (Italy). In 1776, it is also thought that Spallanzani was the first to record the effects of cooling sperm, rendering them motionless.

Around 100 years later, Ilya Ivanovich Ivanoff (1870 to 1932) was the next significant influence within the progression of equine artificial insemination. Ivanoff developed practical methods for insemination and advanced the production of superior progeny by crossing superior stallions. Techniques have been developed and refined since the first artificial insemination, but the principles remain the same. Today, frozen semen is increasingly used as a viable alternative to fresh or chilled semen, due to the difficulties in physically moving horses internationally and the disease risks associated with such transportation.

Another advantage of frozen semen is the ability to order and ship semen in advance of the breeding season, enabling a smooth insemination at the optimal ovulation time for the mare.

FIGURE (1) The number of frozen semen straws exported in 2018
FIGURE (1) The number of frozen semen straws exported in 2018

© Stallion AI Services, England

Why should we cryopreserve equine spermatozoa?

Freezing equine semen allows the production of elite performance horses and aids species preservation.

Using frozen semen as opposed to fresh or chilled allows the breeding of equids on an international scale (see Figure 1). This is because frozen semen straws can be transported further than chilled semen due to the infinite longevity of the sperm cells (if stored, handled and thawed correctly).

Due to the longevity, frozen semen also allows for preparation for the unexpected: injury, old age or death which could halt fresh or chilled semen production. Furthermore, frozen semen is helping to secure a future for endangered breeds. Cryopreserving semen provides a reserve of genetics for present and future use to conserve endangered breeds such as the Suffolk Punch horse.

An overview of cryopreservation

The process begins by collecting the sperm-rich fraction of the ejaculate from a stallion and extending it in a milk-based medium, which helps to increase the longevity of the spermatozoa. Centrifugation then separates the spermatozoa from the seminal plasma, which can have detrimental effects during the cooling process.

FIGURE (2) The process of cell band removal after centrifugation
FIGURE (2) The process of cell band removal after centrifugation

After centrifugation, the supernatant is then discarded; the cell band is removed (see Figure 2) and re-extended in a freezing extender containing a cryoprotective agent, to a concentration of between 100 to 400 x 106/ml. This value varies depending on the number of straws per dose and stallion fertility; in general, the lower the concentration, the higher the straws per dose. Some studies have found that the quality of semen frozen at lower concentrations is improved compared to those frozen at a higher concentration. However, there is ongoing research in this area.

FIGURE (3) Printed straws show the stallion name, date of collection, concentration and the centre where they were frozen
FIGURE (3) Printed straws show the stallion name, date of collection, concentration and the centre where they were frozen

Straws of a half-millilitre volume are generally used for the insemination of mares. The straws are printed with the stallion’s name, date of collection and the centre where they were frozen (see Figure 3). They are then filled, sealed and cooled to 4°C. After initial cooling, the straws are frozen and plunged into liquid nitrogen (-196°C). At this point, the semen is frozen and can be indefinitely stored. Freezing generally takes place rapidly to ensure the spermatozoa can withstand the dehydration and osmotic pressures and to reduce the formation of intracellular ice crystals during the process. Research into the optimal freezing method is still ongoing.

Advances in breeding technology

Artificial breeding techniques now include intracytoplasmic sperm injection (ICSI), embryo transfer (ET) and oocyte transfer. ICSI allows one sperm cell to be injected into an oocyte, thus vastly increasing the number of potential off-spring from one single ejaculate. ET and oocyte transfer enable a recipient mare to carry the foal to term, allowing the donor mare to be free from pregnancy. ET may be used so that the donor mare can continue competing, is not exposed to a potentially life-threatening foaling or in cases where the mare may not be physically able to carry a foal to full-term.

Due to the advances in technology and freezing techniques, the quality of frozen semen is closing the gap between frozen and chilled semen. Frozen semen is currently producing pregnancy rates of 30 to 70 percent per cycle. The major factor influencing this varied range is the individual sire. Therefore, ongoing investigation is needed to continue to improve pregnancy rates obtained from frozen semen.

Summary

From the first recording of cooling spermatozoa in 1776, artificial breeding technologies have vastly improved within the equine reproductive industry. The rapid freezing process enables the maintenance of high-quality spermatozoa. Frozen semen is also closing the gap between frozen and chilled semen, achieving pregnancy rates of 30 to 70 percent per cycle with ongoing research to further improve frozen semen standard. Overall, cryopreservation of equine spermatozoa allows the production of superior progeny marketed internationally and the preservation of our favoured rare breeds.

References
Author Year Title
Morris, G. J., Fraszer, K., Green, J. E., Draper, D., Grout, B. W. W. and Fonseca, F. 2007 Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology, 68, 804-812.
Ombelet, W. and Van Robays, J. 2015 Artificial insemination history: hurdles and milestones. Facts, Views & Vision in ObGyn, 7, 137-143
Gomez-Arrones, V., Carrasco, J. J., Gaitskell-Phillips, G., Pena, F. J. and Ortega Ferrusola, C. 2018 Effect of sperm concentration of the frozen ejaculate of donkeys on post thaw semen quality. The Journal of Equine Veterinary Science, 66, 60

Bethany Morse-Wolfe, BSc (Hons), is a Laboratory Technician at Stallion AI Services. Stallion AI Services is one of the UK’s leading equine semen collection centres. Based in Shropshire, the company specialises in processing of semen from sub fertile stallions and pioneering methods of semen collection and preservation.

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