The sperm mitochondrion (pictured left) is one of the most damaged organelles during cryopreservation and is likely responsible for the majority of loss in motility and fertility after a freeze-thaw cycle. Our laboratory at UC Davis has focused on developing an increased understanding of mitochondrial bioenergetics in sperm that could provide strong rationale for marked improvement in preservation and assisted reproduction techniques.
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The Sperm Mitochondrion: Organelle of Many Functions
Fertility of Equine Frozen-Thawed Semen Stored Cooled After Thawing
There are numerous advantages of frozen semen. If maintained properly in liquid nitrogen, frozen semen will last indefinitely. This allows frozen semen to be shipped to the mare and the mare bred at the most opportune time, close to the time of ovulation. One other use of frozen semen that seems to be gaining in favor is for farms to ship frozen semen to mares when there is a shortage of cooled semen. Alternatively, the breeding farm uses frozen semen to breed mares housed at the farm and ships out the cooled semen. Thus, frozen semen becomes the "backup plan" for breeding mares. You can find additional information to consider when deciding if frozen semen is a fit in your breeding program by reading these articles on the SBS Website: ‘Why Freeze Stallion Semen?’, ‘The Pros and Cons of Equine Frozen Semen’ and ‘The Hidden Value of Frozen Semen’.
The Major Causes of Damage to Sperm During Freezing…water and salts and ice, oh my!
I have always been fascinated by the exquisite design of biological systems. The more we humans understand about biology, the more we realize we don’t know. The process of mammalian fertilization is one of these complex biological systems that in nature requires the proper coordination of so many factors ranging from the behavior of male and female to biochemical changes at the cellular and molecular level. Defined as: “A process in sexual reproduction that involves the union of male (sperm) and female (ovum) gametes (each with a single, haploid set of chromosomes) to produce a diploid zygote”, fertilization requires that functionally viable sperm, at the right stage of maturity, are present in the oviduct of the mare during a brief window of time when a functionally viable oocyte is present.
Shuttle Stallions - Frozen Semen to the Rescue
Shuttle or Dual-Hemisphere Stallions are terms used to describe breeding stallions which travel between the Northern and Southern Hemispheres for their respective breeding season. Several questions arise when discussing shuttle stallions and may include: Why do stallion owners incur the costs and risks of sending their stallions thousands of miles away? Does the lack of sexual rest affect their fertility? Is there a way to service mares in a different hemisphere without sending the stallion so many miles away?
Equine Seminal Plasma: The Good, The Bad and The Ugly
To understand why seminal plasma is beneficial in some situations and potentially harmful in others we must first review what it is, where it comes from and what we think are its roles in reproduction. The term seminal plasma refers to the fluid portion of ejaculated semen in which the spermatozoa are suspended. This fluid consists of secretions from the accessory sex glands in the stallion’s reproductive tract. These include the ampullae, the vesicular glands (seminal vesicles), the prostate gland and the bulbourethral glands (figure 1).
Analysis of Frozen-Thawed Equine Semen
Once equine semen is frozen it is recommended that a post-thaw analysis be performed in order to ascertain the semen quality. There is variability in how well sperm from different stallions respond to the cellular stress of freezing and thawing. It is important to determine how well the sperm from a particular stallion withstood the stress of cryopreservation in order to make informed decisions regarding the management of their frozen semen inventory and breedings. Also, when we freeze a stallion for the first time we do a test freeze comparing several different protocols. We then use the post-thaw motility to select the freezing protocol for subsequent collections which gives the best post-thaw result. There are industry recommended minimums for commercial distribution of equine frozen semen and therefore it is pertinent to know if your stallion’s frozen semen would qualify. Consequently, the analysis of frozen-thawed semen is a valuable tool for the semen freezing lab, but also for the veterinarian receiving the semen. Your vet may base the breeding management of your mare upon the post-thaw quality of the semen. Also, many vets take a look at the motility of the frozen semen at the time of insemination, for future reference if there are concerns should the mare not check in foal.
Contract Considerations for Exporting Equine Frozen Semen
The tests have been done and the frozen semen is ready for export…Now what? In our article, Exporting Frozen Semen from the United States, we covered topics ranging from determining if there is a foreign market for your stallion, to how to export the semen once it’s been frozen. Stallion owners/agents also need to consider how they are going to offer the frozen semen to a foreign market in the form of their breeding contract. In this article, we will discuss ways to sell the semen (by the dose or with a live foal guarantee), responsibility of shipping fees, what is to be done with leftover doses as well as who covers the costs of health testing and freezing, plus more.
Should Frozen-Thawed Semen Be Diluted Prior to Insemination?
For veterinarians and technicians accustomed to inseminating fresh or cooled semen in large (20-60 ml) volumes, the idea of inseminating 0.5 to 4 ml of thawed frozen semen can be intimidating. During processing, frozen semen is concentrated by centrifugation and is typically packaged in small 0.5 ml straws at a sperm concentration that is often as much as 5 to 10 times greater than cooled semen. Therefore, a full insemination dose of frozen semen may be contained in just a few milliliters of volume whereas the same number of sperm extended for cooling may require 30-40 ml of volume.
Influence of Mare Status When Breeding with Frozen Semen
Whether a mare is maiden, recently foaled, or barren can influence her ability to conceive when breeding her with frozen semen. The first pregnancy obtained by using frozen-thawed semen in the equine species dates back to 1957 and was obtained using epididymal semen. However, for many years this reproductive technology has achieved limited progress in the horse compared with other species such as cattle. This was mainly because for a very long period of time only a few horse registries allowed the use of frozen-thawed semen. Hence, economic interests and resources allocated to research have always been minimal and as a result obstructing advances in this area.
Ovum Pickup in the Mare
Breeding horses has come a long ways in the last 50 years. Veterinarians have been able to overcome many of the obstacles presented when breeding horses with advancements in artificial insemination and embryo transfer. However, there are still times when infertility of either the mare, stallion or both prevent getting foals on the ground. The most recent advances in assisted reproductive technologies, Ovum Pickup and Intra-Cytoplasmic Sperm Injection (ICSI), have allowed us at Weatherford Equine Breeding Center (WEBC) to take the next step in overcoming these obstacles with some promising results.