Equine semen cryopreservation

Incredible biotechnology limited by misinformation

Cryopreservation of equine semen suffers from misinformation in the field about its efficiency, applicability, and the prerequisites required for its final adoption in reproductive strategies and operations. Unfortunately, misinformation generates a range of inaccurate conclusions and discredits cryopreservation technology. In the midst of the many “myths” linked to frozen semen, some deserve to be highlighted and clarified.
 
1) “Ultrasound control of the mare needs to be done every four hours until ovulation” 
This statement is as common as it is inaccurate. There is certainly no way to control the ovarian activity of a pre-ovulatory mare only at intervals of 24-48 hours as performed in natural breeding regimes. However, it is enough to consider and associate a minimum viability of 8 hours of thawed semen after insemination with the minimum 8-hour durability of oocytes after ovulation to conclude that it is viable and does not imply damage to the control every 8 hours or more.
 
2) “Insemination with frozen semen requires previous induction of ovulation, something that makes the procedure more expensive”
The application of ovulation inducers (hCG or deslorelin) is not mandatory, as the mare will naturally ovulate, and this is the “landmark” where insemination occurs. However, the use of hormone therapy is a tool to control the time when ovulation will occur, usually between 30-42 hours after the medication is applied. In addition, these medications are already in common use in routines with fresh or refrigerated semen, so ovulation inducers would not add an extra expense. The determination of the “ovulatory window” between 30-42 hours in association with the durations of semen and the oocytes already mentioned shows, once again, that there is no reason for follicular control with such short intervals.
 
3) "After several applications, ovulation inducers stop working and the follicles do not ovulate"
This is another empiricism that has already been scientifically overturned, regardless of the ovulation inducer. The absence of an ovulatory response is not associated with the “resistance” developed to the medication, but with the application of these in the wrong amounts or times of the year, by inadequate routes and, mainly, at the wrong time of the mare's estrous cycle, when a follicle despite having reached a certain diameter, it may not yet be able to induce or even not be a dominant follicle with ovulatory potential. The decision to induce a follicle must be based on several factors such as: the day of the estrous cycle, the development of the follicular wave, the ultrasound characteristics of the follicular wall, and the evolution of uterine edema, not only by the follicular diameter when it reaches 35 mm.
 
4) “Insemination with frozen semen needs to be deep, making it more difficult to perform” 
Insemination at the tip of the uterine horn is not exclusive to cryopreserved semen, being a technique that seeks effectiveness regardless of the semen type. It aims at better reproductive rates by depositing semen close to the fertilization site, resulting in fewer inseminations per breeding season. A flexible and slightly longer pipette is necessary, but irrelevant considering the benefits. The AI technique at the apex of the uterine horn is simple to perform for any veterinarian with good training and provides better rates. Therefore, it is indicated to be adopted in all inseminations when seeking maximum efficiency, by reducing costs and rework.
 
5) “The fertility of frozen semen is inferior”
Quality semen does not depend on whether it is being fresh, chilled, or thawed, but in the different ways of working with each type. Considering concentration, total number of sperm, form of insemination, intrauterine longevity and other points is what reflects on fertility.  The World Breeding Federation for Sport Horses (WBFSH) recommends a minimum of 300 million progressively motile sperm cells per dose for chilled/transported semen. 1-8 straws are used to make up a semen dose of frozen/thawed equine semen. The WBFSH recommends 250 million progressively motile sperm cells per insemination. The minimum post-thaw motility is 35%. This results in approximately 700 million sperm cells in total. This number is above the minimum required and consistent with the inseminating dose used in breeding farms, whether it is for thawed, chilled or fresh semen. If the fertility of the cryopreserved doses were actually bad, would it make sense to use a lower number of sperm as is done in many reliable places? Certainly not. The difference lies in determining the timing of insemination, its technique, and the quality of the inseminating dose.
 
6) “My stallion's semen does not freeze well”
This erroneous belief often labels a breeder as bad without exhausting all resources, as there are several freezing curves, cryopreserving diluents and handling methodologies that can be used to improve the final thawed dose. It is not consistent to conclude that all stallions respond in the same way to the same freezing technique, since there is great individual variability. Many breeding programs end up compromising themselves by using cryopreserved doses inappropriately, often using bad doses of a stallion despite their full capacity to produce doses with superior quality. Better post thaw results can be achieved by testing a range of extenders with different ingredients, cryoprotectants and freezing protocols for each stallion. 
 
7) “The cryopreserved semen is a genetic reserve of the stallion” 
Undoubtedly, cryopreserved genetic material lasts for generations when frozen. However, this thought leads many to believe that cryopreservation is something that can be used only when situations compromise the breeder. Semen cryopreservation should become a multiplier of the stallion's reproductive reach, making the stallion’s genetics available in several places while he is still at the height of recognition by the market. This can facilitate access to semen for breeders who would not be interested in refrigerated doses due to logistical difficulties, for example. In this way, while the stallion is still "in fashion" the breeder will be able to generate many more products and, consequently, more income with the sale of pregnancies.
 
8) "Insemination with frozen semen is more expensive compared to chilled" 
Another incorrect statement prevalent in many conversations between breeders and some veterinarians. The irrelevance of longer pipette costs and the need for ovulation induction have been discussed previously; however, the clearest advantage of cryopreservation is low-cost insemination, mainly logistical. For each ejaculate many more doses are produced if the semen is frozen, serving more mares and with the potential to generate more foals if the cryopreservation is done well with the appropriate thawing and insemination processes performed with precision. The dose:ejaculate ratio is superior in comparison to fresh and refrigerated semen as they are used in the current routine. In addition, refrigerated semen demands a large expense in air and land transport several times a week, something that does not exist with frozen semen. This methodology is economically viable even considering the cost of cryopreservation and a single displacement of the cylinder to the target region, where the doses will be available to serve the nearest farms. Do the calculations!

Now that we have the myths about frozen semen clarified and the benefits of this technique exposed, what are the main bottlenecks that may be contributing to the slow adoption of this methodology and feeding many of these incorrect beliefs? Certainly, there are many realities and particularities, but four points are mentioned whenever this discussion comes up, namely:
 
1) Mares' reproductive ultrasound
The ease of purchasing ultrasound equipment has made this examination common, but not necessarily more accurate or best accomplished. The reproductive ultrasound performed by many professionals is limited to the measurement of the follicular diameter and the evaluation of uterine edema; in addition to pregnancy diagnoses, these limited observations are basically what used to be obtained by good professionals during times where only transrectal palpation was performed. As already mentioned, the success of AI with frozen semen demands accuracy in the interpretation of ultrasound findings and an accurate reproductive follow-up for success, otherwise the merit of the technique is lost in the professional's malpractice, especially regarding the prediction of the ovulatory moment.
 
2) The quality of frozen semen
The freezing of semen from stallions with the appeal of genetic guarantee is the practice that grows most in the sperm cryopreservation field, as it represents a good source of income in the low season. Also, the “cake recipe” methodology that has spread in the field, allows many to be able to manipulate ejaculates for this purpose. At the same time, there are also reports of unsuccessful pregnancies because of low-quality post-thaw doses, taking the credibility of cryopreservation with it. Interestingly, the use of frozen doses does not keep pace with the cryopreserved ejaculates, making it possible to conclude that there is interest in offering cryopreservation, but at the same time there is insecurity for the use of these doses. It is necessary that the evaluation and the report of each frozen batch are required and, if possible, some test inseminations are made with these straws to resolve any doubts about their fertility.
 
3) The cost of pregnancies with frozen semen
For several reasons already highlighted here, the successful veterinarian inseminating with cryopreserved semen is better prepared than the professional who only indicates insemination/coverage on alternate days until ovulation, deserving remuneration differentiated from the others for its better qualification, as it happens with any other professional. It must be made clear that this professional is a “sniper of reproduction” and not an infantryman throwing grenades without seeing the target, as he strives for precision, and consequently, reducing unnecessary costs! When three inseminations are carried out on a mare in the same cycle until ovulation occurs it means that two of them were unnecessary, meaning lost work. Loss of refrigerated doses, time and money spent on logistics, generating rework, and consequent loss. There are cryopreserved and certified fertility doses that cost 1,500 EUR (1,820.65 USD) each reed, for example, so the question is: Which professional would you trust this asset with when inseminating? Reliability requires training and always the frustration with failure represents a much higher cost. It is necessary for the breeder to calculate the cost of rework and conclude on the advantages of a good professional, paying him in recognition of the security that this represents for his own investment. At the same time, this change is an incentive to the good result obtained, as this result in the methodology with cryopreserved semen represents time dedicated compared to other forms of insemination.
 
4) The work routine
This issue mainly concerns the employees of the farms, as a restricted work routine from 7 am to 6 pm will eventually be changed by a later insemination, however it is possible to establish a work limit until 11 pm, for example. In this way, for the sake of your diagnosis, the veterinarian can determine the proximity of ovulation and decide whether to perform the procedure at night, pre-ovulatory insemination, or in the morning, during ovulation or a few hours after ovulation has occurred. It is also worth noting another great advantage of this insemination format: planning. Induction makes it possible to establish the “ovulatory window” at the most comfortable time and does not require the collection of stallions at extreme times, as the semen is available 24 hours in the canister, in addition to eliminating the loss of ovulations due to unavailability of chilled doses due to logistical problems.

Finally, the keywords for the frozen semen process are TRAINING, ACCURACY, and COST. Only with these three factors within the semen cryopreservation process will it be possible to reach another technological and professional level; thus, reducing rework, improving the quality of life of veterinarians and collaborators, eliminating unnecessary expenses, and finally, obtaining an effective, high quality breeding program that focuses on results and profitability.
 
Special thanks to Márcio Menezes Nunes, DVM, MSC
Equine semen cryopreservation