One of the great advantages of frozen semen, is that it can basically be stored forever, as long as it is kept in liquid nitrogen at a temperature of -196oC! In this way it is still today possible to use genetics of stallions that might have died many years ago!
Are you involved in a breed that has a limited genetic pool in South Africa? Then frozen semen is a fantastic opportunity to access a wider genetic pool from stallions standing abroad.
Using frozen semen has even more added benefits: Accessibility to semen from stallions in competition or stallions that become ill, injured or overbooked during the breeding season. Using frozen semen, you eliminate the need to organise stallion availability at the optimum time for breeding of the mare and disease transmission and chances for injury are decreased as direct mare-stallion contact is avoided.
Some disadvantages
There are, however, some disadvantages to frozen semen. The pregnancy rate per cycle for frozen semen is on average 30-40% compared to 60-70% for fresh semen. The management of mares during oestrus is more intense, which increases the costs involved in frozen semen inseminations.
This is, however, necessary as inseminations with frozen semen should be done as close to ovulation as possible, since frozen-thawed sperm cells have a shortened lifespan. Frozen-thawed sperm cells survive for about 12 hours in the reproductive tract of mares, whereas fresh semen can survive for 48-72 hours!
Why the difference in life-span?
The freezing of semen is a very complex process, during which the sperm cells undergo a series of physical and chemical changes that include partial dehydration, cryoprotectant (substances that protect the spermatozoa during freezing) penetration of sperm, reorganisation of membrane lipids and proteins, exposure to high salt concentrations and exposure to inter- and intracellular ice crystal formation.
Freezing protocols are designed to minimise the negative effects of these stresses, but the lifespan of sperm cells is decreased after freezing and thawing.
So, how are oestrus cycles for frozen semen inseminations managed? Management of the oestrus cycle is aimed at making sure that live sperm cells are available in the female reproductive tract at the time of ovulation. So what is ovulation, the major event of the oestrus cycle?
Ovulation is the physiological event when the dominant follicle (which is the structure on the ovary that contains the oocyte or "egg") releases the oocyte and changes in appearance. Ovulation can also be induced by certain drugs and is confirmed by ultrasound. Images 1 and 2 show the changes on the ovary as seen on ultrasound.
Protocol for fresh semen
If fresh semen is used to inseminate a mare, the oestrus cycle of the mare is monitored by rectal palpation and ultrasound until the dominant follicle is large enough for induction of ovulation. Insemination can be performed at the same time or 24 hours later. Mares will usually ovulate within 48 hours after induction of ovulation. This is confirmed by another ultrasound examination. As most fresh semen survives for 48-72 hours there will be sperm cells available at ovulation to fertilise the oocyte.
Protocol for frozen semen
For frozen semen, it is generally recommended that insemination is performed within 12 hours before, or up to six hours after, ovulation. Therefore, frequent rectal and ultrasound examinations of the reproductive tract are necessary to ensure that insemination is done within that period of time.
If only one dose of frozen semen is available, a protocol for frozen semen insemination would be to induce ovulation once the dominant follicle is large enough, and start 24 hours later to monitor the follicle by transrectal ultrasound examinations every 6-8 hours. Insemination is performed as soon as ovulation is detected. That ensures that sperm cells are available within six hours of ovulation. If two doses of frozen semen are available, ovulation is induced and the first insemination is performed around 36 hours later.
If ovulation has occurred about 12 hours after the first insemination, the second dose of semen is inseminated. If ovulation has not occurred, the follicle will be monitored every 6-8 hours by ultrasound and the second insemination will be done as soon as ovulation is confirmed.
How much is needed?
A minimum of 250-300 million live sperm cells, swimming in a straight line, are generally accepted as one insemination dose for frozen-thawed semen.
How many straws?
This depends on several factors: Volume per straw, sperm concentration per ml and percentage of straight-forward swimming sperm cells per straw after thawing. Usually 0,5 ml straws are used to freeze stallion semen and the concentration per ml varies according to freezing method.
As an example: If the semen is frozen at 500 million sperm cells per ml, there would be 250 million sperm cells in a 0,5 ml straw. If the percentage of straight-forward swimming sperm cells after thawing is 30%, there would be 75 million straight-forward swimming sperm cells in a straw (30% of 250 = 75).
In this case, four straws would be needed to get to an insemination dose of 300 million straight-forward swimming sperm cells (75 x 4 = 300). If, for this same case, the percentage of straight-forward swimming sperm cells would be 40% instead of 30%, only three straws would be one insemination dose (40% of 250 = 100 million straight-forward swimming sperm cells per straw, 3 x 100 = 300 million straight forward swimming sperm cells). Similarly, the number of straws needed per insemination would also change with increasing or decreasing sperm concentration per straw. The percentage of straight-forward swimming sperm cells as well as the concentration, can vary for the same stallion from ejaculate to ejaculate. It is normal and called inter-batch variation. It explains why sometimes more or less straws are needed for an insemination if the semen is from the same stallion, but frozen from different ejaculates.
Is post-thaw quality always the same?
No! It is important to know that semen quality can change due to a lot of reasons, e.g. increasing age, certain systemic diseases, fever reactions, long-term administration of certain medications and overuse. These factors would also affect the post-thaw semen quality.
Tips for buying frozen semen
Always buy insemination doses instead of "a number" of straws. Also consider that more than one oestrus cycle might be necessary to inseminate the mare due to the 30-40% pregnancy rate per cycle.
Frozen semen should also always be accompanied by a frozen semen report! These reports should clearly identify the straws (straw and plug colour, inscription on the straws) and give information on concentration of sperm cells per straw, percentage of straight-forward swimming sperm cells and how many straws are regarded as one insemination dose. Furthermore these reports should contain a thawing protocol as different freezing methods require different thawing methods.
Which mares are suitable?
Mare status is a major factor determining the pregnancy rate with frozen semen. Best suitable are young mares (younger than seven years old), followed by older mares (older than eight years old) that have had foals previously or that have foals at foot.
According to statistics, the lowest pregnancy rates with frozen semen have been seen in mares older than eight years that have never been pregnant before. The decreased chances of pregnancy establishment in older mares, can be due to age-related changes in the uterus. The rate of early embryonic loss is also higher in older mares compared to younger mares. Nevertheless, that does not mean that older mares cannot be used for frozen semen inseminations! Firstly, every mare is different and does not necessarily have to show these age-related changes as an individual. Secondly, a pre-breeding check can determine if a specific mare is likely to be suitable for frozen semen insemination.
Pre-breeding checks
Such pre-breeding checks would include a general clinical examination, a rectal and ultrasound examination of the reproductive tract to exclude any macroscopic abnormalities, an endometrial swab to exclude uterine infection (if regarded necessary) and a uterine biopsy (if regarded necessary) to evaluate the uterus at a microscopic level.
Such a pre-breeding check can then give you an estimate of what your mare’s chances are to fall pregnant and carry to term. It will also give you an idea on whether it is worthwhile to use frozen semen for that specific mare.
In brief
Buy insemination doses instead of "a number" of straws.
Frozen semen should always be accompanied by a frozen semen report.
Careful mare selection increases the chances of pregnancy using frozen semen.
Contact the authors on 072 835 7601 or 082 473 9969, or e-mail annett.heise@gmx.de
