Freezing of stallion semen for AI of mares; Information for the breeder

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The freezing of stallion semen for storage and subsequent usage to AI mares is a common practice in many horse breeds. The idea behind semen freezing is to allow the movement of a stallion’s genetics through both time and distance.  After being frozen (or cryopreserved) semen can be stored essentially indefinitely until its retrieval and thawing before being used for the artificial insemination (AI) of mares.

Collection of an ejaculate from a stallion
As a first step frozen semen requires the collection of an ejaculate before processing to prepare it for freezing and storage. The stallion must first be trained by experienced operators to safely mount a phantom mare (“dummy”) to allow the successful collection of his semen. Most stallions can be trained to mount a phantom mare with or without the presence of a “teaser” mare in oestrus (“on heat”). This training is critical for the future success of an individual stallion as a semen donor.  Once encouraged to mount, the collector deflects the erect penis into an artificial vagina (AV). The temperature and pressure exerted by this AV should help to stimulate ejaculation. The collected semen is then removed to a suitable laboratory for further processing. An essential feature of this procedure is strict attention to biosecurity to prevent transmission of bacteria and other microorganisms from, and between, stallions. This transmission can occur at the time of semen collection via contact with contaminated equipment such as the phantom and AV or even from clothing or hands of the operators and handlers. Most of these micro-organisms survive the freezing process as well as, or even better, than do the sperm! Thereafter these unwanted passengers can be easily transferred with the dose of semen at AI. All stallions for semen collection should be tested annually for venereal diseases that may be transmitted in this way before any semen collection. Current South African legislation requires annual testing of all breeding stallions for both dourine (a blood sample) and CEM (genital swabs).

Processing the ejaculate
The collected semen is filtered to remove the excessive fluid and debris before careful dilution with extenders that protect the sperm in the semen from the extreme environmental changes that it will be exposed to during the procedure. The diluted semen is then centrifuged. This process removes the fluid component of the ejaculated semen and additionally concentrates the sperm to allow it to be re-suspended in a freezing diluent and to be packaged into a small volume in a straw. Sperm would be unable to survive the enormous physical stresses of freezing and thawing without addition of this freezing diluent.

Packaging and freezing the sperm
Several hundred million sperm are packaged in a single straw. Currently, the preferred packaging method is a 0.5-ml PVC straw. The straws are all individually labelled with information essential for correct identification of the semen. This must include: the stallion’s ID, the individual ejaculate number or date, the operator, and the place of processing. The straws are then sealed for freezing by being exposed to liquid nitrogen at a final temperature of –196 ºC.

Storage of frozen semen
The frozen straws are transferred to a liquid nitrogen canister for storage at –196 ºC until their removal for thawing and insemination. Frozen semen must be held in a properly maintained nitrogen storage flask at all times until thawing for AI. Exposure (even for seconds) to environmental temperatures exceeding that of liquid nitrogen can be disastrous.

Evaluating the frozen semen
It is essential to thaw and evaluate straws from each individual frozen ejaculate, as this provides the information which will determine the necessary dosage which will optimise the chances of obtaining a pregnancy with the semen. The evaluation must include determining the numbers of live sperm and percentage of progressive sperm motility post-thaw. This enables the calculation of the correct “dose” of the semen. This will be variable between different stallions but will determine the number of straws that must be thawed for each dose. It is preferable that each batch of frozen semen is accompanied by a veterinarian’s certification of its post-thaw quality.

Bio-security
It is also vital to ensure “bio-security” as the frozen-thawed semen must not include microbial pathogens (e.g. viruses, fungi and bacteria) that may reduce fertility and cause disease. Safety in terms of potential transmission of infectious agents with frozen semen must never be compromised. This can be achieved by a combination of swabbing the genitals, culture of the semen and by serology from blood samples collected from the stallion at the time of collection.

RELEVANT INFORMATION
There are numerous factors (and idiosyncrasies) that need to be considered when answering questions regarding the freezing and AI of stallion semen. Here are a selection of frequently asked questions.

1. What results are obtainable with frozen semen AI?
The AI of reproductively normal mares with frozen semen obtained from a particular stallion are unlikely to produce the same pregnancy rates obtainable by natural covering or AI with fresh (or chilled) semen from the same stallion. This difference is difficult to quantify as numerous factors play a role. Studies comparing the pregnancy rates resulting per oestrus (heat) cycle using semen from individual stallions showed these results:
• fresh, raw semen =  60-70%
• diluted, chilled semen = 50-60%
• frozen-thawed semen = 30-50%

2. The stallion’s “freezability”?
Not all stallions produce ejaculates that freeze successfully. The tremendous variation in this “freezability” is one of the major limitations of stallion semen cryopreservation. This variation occurs between both individual stallions and to an extent between ejaculates from the same stallion. This may be independent of their inherent fertility achieved by natural covering or even AI of their fresh or chilled semen.
Current data suggest the following:
• 30% of stallions are “good freezers”
• 40% are “satisfactory”
• 30% of stallions fail to produce acceptable frozen-thawed semen

3. What is an insemination “dose”?
The term “dose” as it relates to frozen-thawed semen is defined in terms of using a minimum number of live and progressively moving sperm post-thaw, that is associated with an acceptable pregnancy rate per cycle. The term “dose” is independent of both the number of straws and the volume of the fluid deposited in the mare’s uterus at time of AI. The sperm numbers associated with an effective dose may be packaged in anything from 1 to >12 straws. The current recommendations for standard methods of frozen semen AI suggest an AI dose in the range of 200-500 million progressively motile sperm.

4. How many “doses” are obtainable from collecting an individual ejaculate?

This number varies with both the individual animal and with each ejaculate obtained from the same animal. The number of doses is obviously directly related to the numbers of sperm produced per ejaculate. This relates to each stallion’s inherent sperm production capacity. This in turn depends on numerous factors, including his:
• age
• body mass
• breed
• testicular size
• ejaculation frequency
• collection method.
The average stallion, collected every other day, should in most cases produce 12-15 doses of frozen semen. This number in the same individual can vary by as much as 2-40 doses processed for every ejaculate collected. It may be more useful to think in terms of potential numbers of pregnancies rather than doses of semen. The calculation that should be considered is that typically, a pregnancy rate per cycle of 30-50% is achieved using 1-2 doses per AI cycle. Thus, as a minimum, approximately 2-4 pregnancies should be obtained per collection.

5. How long is the life span of frozen semen?
With correct storage and handling the frozen semen should retain its viability for many years. Pregnancies with stallion semen are regularly achieved more than 10 -20 years after collection and processing. It is vital to ensure proper maintenance of nitrogen storage flasks. Transfer of the semen, once frozen, to a commercial frozen semen “bank” until usage is recommended in order to achieve this.

6. Where is stallion semen frozen?
Acceptable results may be obtained, collecting and processing stallion ejaculates both at dedicated semen freezing centres or veterinary practices as well as “in the field”. The advantages of a centre include access to a phantom, trained personnel and a dedicated laboratory.

7. How can results be optimised with frozen semen AI?
Acceptable pregnancy rates are achievable using frozen-thawed semen to AI mares, despite the lower pregnancy rates per cycle when compared with other methods of breeding. A comprehensive study in Italy (Barbacini et al) over several years, involving the AI of several hundred mares utilising frozen semen concluded that the most important aspects for consideration are:
• comprehensive evaluation of stallions and frozen-thawed ejaculates by suitably-qualified veterinarians
• utilisation of semen only if meeting the minimum criteria for post-thaw quality
• intensive reproductive management of mares by experienced veterinarians.

Fourways Equine Clinic offers comprehensive reproductive services through our consulting reproductive specialist.
For further information or any queries, please contact us at the clinic on 011 4683393.

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