Artificial Insemination with Fresh, Chilled, and Frozen Semen
World Small Animal Veterinary Association World Congress Proceedings, 2013
Philip Thomas, BVSc, PhD, FACVSc, DACT
Queensland Veterinary Specialists, Stafford Heights, Queensland, Australia

Evidence-Based Canine Reproduction

Canine reproduction features 1/5th of the volume of literature of bovine reproduction. Of trials in canine reproduction, 41% had a control group and 7% were considered adequate to draw sound conclusions (Simoneit et al. 2011).

Semen for transvaginal insemination

Size of dog

Prostate fluid/extender volume

Less than 10 kg

5 mL

10–25 kg

10–15 mL

25–50 kg

20–30 mL

Semen gross appearance and approximate sperm concentration

Creamy, grainy semen

750–1500 x 106 sperm/mL

Full cream milk-like semen

400–750 x 106 sperm/mL

Skim milk-like semen

250–400 x 106 sperm/mL

Translucent semen

< 250 x 106 sperm/mL

Spermatozoa Numbers for Insemination

There are several ways to calculate an inseminate. For our purposes, an inseminate will be:

 Inseminate = total sperm x % normal morphology x % progressive motility

 Minimum inseminate for vaginal insemination = 200–400 million

 Minimum inseminate for intrauterine insemination = 100–200 million

* For processed semen, progressive motility is evaluated post-chilling or thawing.

Semen Processing

Chilled semen has been collected, possibly centrifuged, extended in chilling extender, chilled to 4°C, shipped, and stored for up to 10 days. Frozen semen has been collected, possibly centrifuged, extended in freezing extender, chilled, packaged, frozen at -196°C, and stored for days to many years, then thawed - sometimes in thaw medium - prior to insemination. Semen may also have been concentrated with swim-up or differential centrifugation techniques.

Reservoirs of Spermatozoa in the Female Tubular Tract

After mating or insemination, it is believed that spermatozoa form a sperm reservoir in any or all of the uterus, uterine glands, uterotubal junction and oviduct. Fresh ejaculated sperm survival in the tubular tract is routinely 5 days and may be longer. It is likely that the longevity of sperm in the tubular tract is reduced when sperm are chilled or frozen. There are not good data to support these contentions, but anecdote suggests chilled semen has a life of 12 to 36 hours and frozen semen up to 12 hours in the tubular tract.

Sperm Transport

At natural mating, the penile urethral meatus is located in the vaginal fornix and semen is ejaculated into the uterus (Pollitt, personal communication), filling the uterine horns. The male achieves erection only when the glans penis is within the vagina and the erectile tissue of the glans fills so the penis occludes and distends the vagina, with the bulbus glandis 'locked' into the vestibule and the pars longa glandis distending and occluding the vagina - this process may trigger local factors promoting sperm transport including oxytocin and/or prostaglandins. It seems intuitive that effective insemination techniques replicate this anatomic outcome.

Coitus (England et al. 2006) and mechanical stimulation of the vagina (England et al. 2012) cause an increase in uterine contractions, which may be involved in sperm transport. Semen components, especially prostatic fluid, are involved in uterine perfusion, sperm binding, uterine contractions, and fertility (England et al. 2012).

Transvaginal Insemination: Catheters

The Osiris catheter is a rigid catheter with an inflatable balloon with a syringe port to blow up the balloon and a separate port for insemination. The main limitation to Osiris catheters is that they only come in one size and so are a 'one size fits all.' They are adequate for small and medium breeds; however, they are inadequate for toy, large, and giant breeds.

Foley catheters are flexible catheters in a range of sizes and with an inflatable cuff. A separate sterile stylet provides rigidity but uses the insemination port and tunnel so the stylet has to be removed for insemination and the catheter cannot be maintained at the vaginal fornix.

Bovine/Equine insemination pipettes are commonly used for canine artificial insemination and are adequate but not optimal. The catheter is rigid, which allows placement at the fornix; however, the vagina cannot be occluded or distended.

The Mavic catheter (Minitube) is a plastic catheter with a malleable permanent stylet and a cuff located at the rostral end of the catheter, which is inflated with air once in situ. A separate insemination channel is provided, which has a valve to prevent backflow of semen. Mavic catheters come in 3 different sizes and most accurately emulate natural mating.

Transvaginal Insemination: Technique

The bitch should be in standing position. An assistant ensures she doesn't sit or collapse. Digital vaginal examination ensures no anatomic anomaly or obstruction to catheter passage, and provides a small amount of water-soluble lubrication. The catheter is passed along the dorsal surface of the caudal tubular tract, initially sharply dorsally through the vulva to avoid the clitoral fossa and the urethra, and then slightly ventrally as the catheter is advanced through the vestibulovaginal junction (the singulum) into the vagina with the tip of the catheter passing under the dorsal median post-cervical folds, coming to rest as close as possible to the vaginal fornix and the cervical opening.

With the Mavic catheter, once the catheter is in position, the cuff is inflated to occlude and distend the vagina, emulating the glans penis. Semen can be inseminated as an extended total volume or by inseminating the sperm-rich fraction first, followed by prostate fluid/semen extender. Collectively, the insemination should occur slowly over 10 to 20 minutes. Rapid insemination distends the anterior vagina, which results in contractions of the abdomen and/or vagina and expulsion of the inseminate.

Intrauterine Insemination: Indication

Intrauterine insemination is indicated for poor-quality semen, chilled semen, frozen semen, if progesterone is > 90 nmol/L, and for some obstructions to the female tubular tract.

Intrauterine Insemination: Transcervical Insemination (TCI), Norwegian Method

Andersen (1975) describes a technique using a rigid catheter, passed transvaginally into the vaginal fornix. The cervix is palpable and fixed by transabdominal palpation, and the catheter is then threaded through the cervix using manipulation of both the rigid catheter and the cervix. This technique requires skill, practice, and dogs with the appropriately sized abdomen and is used in Scandinavian countries.

Intrauterine Insemination: Transcervical Insemination (TCI), Endoscopy

TCI with a rigid endoscope is performed with a cystoscope or a human utero-endoscope. This allows the vaginal mucosa and the cervical opening to be seen prior to the passage of TCI catheter. The bitch is standing and, if receptive, is usually not sedated. The vagina is insufflated. A cuffed device can be used to occlude the caudal tubular tract and to hold the endoscope. The technique requires practice and skill and expensive equipment but is minimally invasive, allows visualization of the uterine lumen, and is predictable.

Intrauterine Insemination: Surgical Insemination

First reported by Smith (1985), under general anaesthesia, the bitch is in dorsal recumbency, and a midline exploratory laparotomy incision is made to permit exteriorization of the uterus. Each uterine horn is catheterized with 22-gauge catheters. The inseminate is inseminated into each of the catheters and the laparotomy incision is closed. The site of insemination does not seem to affect sperm distribution in the uterus (Fukushima et al. 2010). The technique requires minimal practice and skill, uses equipment available in any veterinary hospital, allows visualization of the ovaries and uterine surface, but requires anesthesia and is painful and invasive. Surgical insemination is illegal in some European countries.

The first report of a large data set (Burgess et al. 2012) of 238 surgical inseminations reveals:

 Whelping rate of 73.1% for all bitches

 No difference in whelping rate between parous and nulliparous

 Higher whelping rate for fresh (83.7%) than frozen (70.8%) semen

 Breed effect

 Higher whelping rate for 1–3 yo (80%) than > 3 yo (68.3%)

 Higher whelping rate for progressive motility > 75% (100%) than < 75% (47.6 to 82%)

 Higher whelping rate for inseminate of > 200 million progressively motile (87.5%) vs < 100 million (64.1–68%)

References

1.  Andersen K. Insemination with frozen dog semen based on a new insemination technique. Zuchthygiene. 1975;10:1–4.

2.  Burgess DE, Mitchell KE, Thomas PGA. Coeliotomy-assisted intrauterine insemination in dogs: a study of 238 inseminations. Aust Vet J. 2012;90:283–290.

3.  England GCW, Burgess CM, Freeman SL, et al. Relationship between the fertile period and sperm transport in the bitch. Theriogenology. 2006;66:1410–1418.

4.  England GCW, Moxon R, Freeman SL. Normal and abnormal uterine response to sperm deposition in the bitch. In: Proceedings of the 7th International Symposium Canine Feline Reproduction. Whistler, Canada; 2012.

5.  Fukushima FB, Malm C, Henry M, et al. Site of intrauterine artificial insemination in the bitch does not affect sperm distribution within the uterus. Reprod Dom Anim. 2010;45:1059–1064.

6.  Simoneit C, Heuwieser W, Arlt S. Evidence-based medicine in bovine, equine and canine reproduction: quality of current literature. Theriogenology. 2011;76:1042–1050.

7.  Smith FO. Cryopreservation of canine semen: technique and performance. Diss Abstr Int B Sci Eng. 1985;45:3441.

8.  Wilson MS. Nonsurgical intrauterine artificial insemination in bitches using frozen semen. J Reprod Fertil. 1993;47:307–311.

  

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Philip Thomas, BVSc, PhD, FACVSc, DACT
Queensland Veterinary Specialists
Stafford Heights, Queensland, Australia


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