How to carry out artificial insemination using fresh semen

01 November 2012

8 mins read

02 November 2012

Volume 3 · Issue 9

ISSN (print): 2044-0065

ISSN (online): 2052-2959

Abstract

Artificial insemination can provide many benefits to breeding both in canines and other species. It allows the use of semen from stud dogs around the world without the requirement to transport the dogs, thereby opening up the possibilities of genetic diversity within a breed. The techniques required to perform insemination are complex but an invaluable tool in the breeding world.

Artificial insemination (AI) is a method of introducing semen, previously collected from the male, and depositing it into the bitch's vagina or uterus to achieve a pregnancy.

There are many reasons that AI in the dog may be required (Figure 1). For example, it could be that a planned mating has not resulted in a ‘tie’, the male or female are inexperienced or the male, while still fertile, is not physically able to mate the bitch, or it could simply be that semen has been imported from another country to enable breeders to keep their gene pools diverse.

Figure 1. Guide dogs will use artificial insemination as a method of conception only when all attempts to achieve a normal mating have been tried. Conception rates are higher when a normal mating has been achieved. This litter will be monitored for their health and temperament throughout the first 12–14 months of their puppy training, to assess their suitability to become a guide dog, at which point, all being well, they will enter their formal training period.

Occasionally, and particularly if the reason for insemination is due to the inexperience of the male, it may not prove straight forward to collect a good semen sample. Therefore, ideally, before insemination is considered, the male should be proven fertile either by means of a fertility test or from a previous successful mating, and the dog should have been collected from at least once so that the process of collection is familiar to him. Unfortunately, fertility testing is not usually offered as a service in private veterinary practice, therefore, if a fertility test is required it is likely this would need to be arranged with a reproductive veterinary specialist.

AI must always be performed by an experienced professional. Incorrect collection techniques could cause pain to the male and could prevent a collection being successful or future collections being successful. A painful experience could also lead to difficulties in future matings. Improper insemination technique could cause pain to the bitch or result in damage to her vaginal tract. Improper insemination techniques could also result in failure to conceive in that breeding season. In the UK, in order to register a litter born from AI, the Royal College of Veterinary Surgeons have advised that AI is a veterinary procedure and therefore should only be performed by a veterinary surgeon (Kennel Club, 2012).

Any breeder wishing to register a litter of puppies as the result of AI should first refer to the Kennel Club's regulations surrounding the use of AI. The Kennel Club will not register a litter resulting from AI unless certain criteria are met. For example, the General Committee will not normally accept an application to register a litter if the donor male is alive and domiciled in the UK, with one exception, namely that Irish Wolfhounds of 8.5 years or older and domiciled in the UK may be used as donors in AI (Kennel Club, 2012). Refer to the Kennel Club's website for further information (http://www.thekennelclub.org.uk/item/478).

Methods of AI

There are three main methods of AI: vaginal insemination; trans-cervical insemination [TCI] also commonly known as uterine insemination; and surgical insemination. In addition, and depending on the time delay between collection and insemination, the semen may need to be preserved. Therefore, there are also three semen types: fresh semen; chilled semen (preserved with an extender and kept chilled); and frozen semen (preserved with an extender and cryoprotect-ants and stored, frozen in liquid nitrogen) (Table 1). The extender is a liquid that contains buffers, sugars and cryoprotectants to nourish and protect the sperm while they are kept chilled or frozen.

Table 1. Survival time of semen once collected and where applicable, stored with an extender.

Semen type	Survival time (approximately)
Fresh semen	12 hours at room temperature
Chilled semen	4 days (kept chilled at 5^oC)
Frozen semen	Theoretical indefinite life at a temperature of -196^oC

Due to the varying survival times of sperm within the semen types (Table 2), some methods of insemination will be unsuitable (Table 3).

Table 2. Survival time of semen in the reproductive tract after insemination

Semen type	Survival time In reproductive tract (approximately)
Fresh semen	7 days
Chilled semen	2 days
Frozen semen	(post thaw) 6 hours

Table 3. Type of Insemination suitable depending on semen type.

Semen type	Type of insemination suitable
Fresh semen	Vaginal insemination
Chilled semen	Vaginal insemination
Frozen semen	Trans-cervical/ uterine or surgical insemination

The timing of the insemination should also be carefully considered depending on the type of semen used and the method of insemination chosen. For example, due to the relatively short survival time of chilled and frozen semen, it is imperative that the bitch has already ovulated within the preceding 24–48 hours, thus ensuring the ova are available to be fertilized immediately. There are several methods available to enable the optimum mating time to be detected. One of these methods is blood hormone levels, the most common type of testing available is serum progesterone levels, most commonly the qualitative test. This test detects a colour change in the sample which gives the user a result of low progesterone, intermediate progesterone or high progesterone levels. These will give an indication of the level of progesterone in the blood and therefore detect ovu-lation — at high progesterone levels (usually 4–8 ng/ml) ovulation occurs (two matings are preferable, 48 hours apart). Vaginal cytology is another method available. By looking directly at the cells that line the vagina and the changes that happen, it is possible to detect the optimum mating time.

Semen

A dog ejaculates in three distinct fractions. The first fraction is clear fluid and originates from the prostate gland; its function is to flush any urine or cell debris from the male's urethra (Simpson et al, 1998).

The second fraction is the sperm rich fraction, usually cream in colour, containing millions of sperm and the fraction that is required for insemination (Simpson et al, 1998).

The third fraction is again clear fluid, originating from the prostate gland and its function is to wash the sperm through the bitch's cervix into her uterus and forwards into her uterine tubes where fertilization will take place (Simpson et al, 1998). However, while the second fraction is used for insemination, the third fraction is not used as a flushing fraction. This is replaced with sterile saline, to ensure there is no chance of any detriment to the sperm.

For this practical article, AI using fresh semen and the vaginal insemination method is discussed.

Things to remember

There are a number of things to remember when carrying out AI:

The chemicals present in tap water can be toxic to canine sperm and will kill them. Ensure any equipment is free from contamination with tap water.

Syringes with rubber stoppers should be avoided since some types of rubber can be toxic to sperm.

All equipment should be warmed to body temperature. Sperm are susceptible to heat changes and their motility will slow as semen cools.

After collection, handle the semen carefully but inseminate as soon as possible (see Table 1) to prevent cooling of the semen and subsequent slowing/ reduced motility of the sperm, thus ensuring the semen is in the best possible condition.

One ejaculate is required for each insemination.

Step-by-step guide to artificial insemination

Method

Prior to semen collection, pre prepare all required insemination equipment. You will need a syringe of sterile saline (typically for a medium to large dog, 3–5 mls of saline are used), a cotton swab with surgical spirit on it, an insemination catheter, semen sample (after collection) (Photo 1).

NB all syringes, semen collection equipment and insemination catheters should be placed either on a heated plate or in an incubator set at body temperature.

Collect the semen sample from the male. (Photo 2 and Photo 3).

The collected sample should be evaluated to ensure the male has good fertility. Ideally the dog should have had regular semen evaluations prior to being considered for AI due to the time required to assess a semen sample for fertility. If the dog has had regular semen evaluations, then the sample can simply be checked under the microscope to ensure the motility is sufficient for this insemination (Photo 4).

Using a syringe and quill (a hollow plastic tube that will enable the user to aspirate the semen from the test tube and into the syringe), aspirate the semen from the test tube into the syringe, ensuring the quill is emptied of semen (Photo 5)

Discard the quill

Place the semen loaded syringe back on the heated plate.

With all equipment and samples prepared, the bitch should be ready for insemination. (Photo 6)

Prepare the bitch for insemination. The bitch can either be stood on an examination table, or if preferred, allowed to remain in a standing position on the floor. If the latter method is favoured, it is recommended that a stool is available to sit on as the bitch will need to be supported during and after insemination. Her vulva should be swabbed this will ensure the vulva is clean prior to introducing the insemination catheter.

If allowing the bitch to remain standing on the floor, gently lift her back legs, so she is slightly tilted forwards. Her hind legs can be rested on the inseminator.

Elevate the vulva and carefully introduce the catheter into the bitch's vulva and gently work the catheter forwards into the vagina as close to the cervix as possible (Photo 7 and Photo 8). NB the cervix is located at the cranial end of the vagina, the vagina is long, up to 20 cm (England, 1998). In large dogs it is expected that an insemination catheter should be up to 30 cm in length to achieve an insemination close to the cervix.

Once the catheter is in position, attach the syringe hub to the catheter

Slowly expel the contents of the syringe into the vagina, ensuring the syringe has been emptied (Photo 9).

While holding the catheter in place, remove the syringe and place a finger over the catheter end to prevent the semen from flowing back out of the catheter.

Attach the second syringe containing sterile saline and slowly expel its contents. This ensures all semen has been expelled from the catheter and acts to replace the third fraction of semen, the flushing fluid (Photo 10).

Remove the syringe from the catheter, placing a finger over the end of the catheter as before.

Draw a small quantity of air into the syringe, 3 mls is sufficient

Replace the syringe onto the end of the catheter and slowly expel the air. This will ensure the catheter is completely empty of semen before its removal.

Carefully withdraw the catheter from the bitch.

It is then advised that the inseminator should insert his finger into the vulva to stimulate the bitch. This mimics a ‘tie’ situation, and stimulates the bitch to start contracting her pelvic muscles. This helps move the semen through into the cervix, maximizing the chances of conception. This process is often referred to as ‘feathering’ (Van Hutchinson, 2012).

The bitch should remain in her elevated or standing position for at least 10 mins.

She should be discouraged from spending (toileting) for at least an hour after insemination

Both the male and female, if living in a pack, should be carefully reintroduced to their pack.

Equipment required

The following equipment are required for AI:

Heated plate

Semen collection vessels (clean, dry funnels and test tubes)

Syringes, 5 ml x 2

Sterile quill

Insemination catheter

Sterile saline

Surgical spirit on cotton wool swab

Gloves.

The procedure

See Step-by-Step guide for detail on carrying out AI.

Insemination should be repeated after 48 hours, providing the bitch is still in oestrus. This will maximize the coverage of the fertile period in the bitch and maximize the chances of conception (Simpson et al, 1998).

Conclusion

AI is used for a number of reasons, and it is essential that it is carried out by an experienced professional/practitioner in order to be successful and without problems. The correct timing is key to the success of AI, and survival times of semen and timing of ovula-tion should be carefully coordinated.