CHAPTER 50Low-Dose Insemination Techniques

BACKGROUND

To obtain acceptable per cycle pregnancy rates, the minimum recommended number of spermatozoa contained within a conventional insemination dose is generally >300 × 10⁶progressively motile spermatozoa for fresh semen ¹^–⁴ and >200 × 10⁶progressively motile spermatozoa for frozen semen.⁵ It has been shown that conventional insemination of mares with ≤100 × 10⁶spermatozoa results in unsatisfactory per cycle pregnancy rates.^6,⁷

Since the advent of fluorescence-activated cell separation technology ⁸ to sort spermatozoa into their X- and Y-chromosome-bearing populations at flow rates of 1000 to 5000 spermatozoa per second,^9,¹⁰ there has been renewed interest in the fertility of low numbers of spermatozoa. Consequently, this interest in low-dose insemination has fueled research and clinical trials aimed to reduce the minimum recommended dose of conventionally frozen stallion spermatozoa. Scott et al¹¹ observed that there is an aggregation of spermatozoa on the uterine side of the uterotubal junction, and subsequent research has focused on how few sperm may actually be necessary to result in fertilization when placed onto the papilla of the oviduct or within the oviduct.^12,¹³ These fertility investigations and novel methods of low-dose insemination have contributed to the investigation of the fertility of epididymal spermatozoa¹⁴ and the potential to improve the fertility of subfertile stallions and mares.¹⁵

Several studies have explored different low-dose insemination techniques, which can be grouped into two general methods: rectally guided insemination techniques ^13,¹⁶ and hysteroscopic techniques in which the papilla of the oviduct is actually visualized and becomes the target for semen deposition.^12,^17,¹⁸ Both insemination techniques have been developed to minimize sperm wastage and deposit a small number of spermatozoa closer to the site of the sperm reservoir in the mare. These techniques have been used to examine the fertility of fresh, chilled or frozen, and sorted and nonsorted spermatozoa. The sperm numbers have varied dramatically but have always been lower than the conventional insemination dose of 200 to 500 × 10⁶spermatozoa.

For practical reasons and based on the insemination regimens outlined in the preliminary studies,^12,¹⁹ most practitioners have used a “timed insemination” technique that is aimed at depositing the spermatozoa deep within the uterus before ovulation. This has been made possible by the use of ovulation-induction agents that facilitate the ability to predict ovulation in estrous mares and optimize fertility.²⁰

HYSTEROSCOPIC INSEMINATION PROCEDURE

Equipment

Expensive endoscopic equipment and a minimum of two people are required to perform the hysteroscopic insemination procedure. A variety of endoscopes are available for veterinary use that are suitable for hysteroscopic insemination. The researchers of the original studies ^9,^12,^19,²¹ used a Pentax EPM 3000 videoendoscope. Since then Minitube (Germany) has designed a purpose-built hysteroscope for mares. For hysteroscopic insemination, the scope should have a diameter of 11 to 13 mm, a channel for the insemination catheter, and a channel to allow the insufflation of the uterus with air. One of these channels should allow extraction of air from the uterus at the end of the procedure. A wider diameter scope (11 to 13 mm) is preferable over one that is more narrow (7 to 9 mm) to minimize the risk of the scope “doubling-back” on itself during its advancement through the uterine lumen. The ideal scope length is 1.4 to 1.6 m to facilitate the insemination of larger breed mares. To minimize the duration of the procedure and improve the accuracy of semen deposition directly onto the uterotubal junction, it is desirable to visualize the images on a monitor through either a videoendoscope or a camera attached to a normal endoscope.

Insufflation of the uterine lumen with air during passage of the scope reduces any damage to the endometrium and minimizes the duration of the procedure. Most videoendoscopes pump sufficient air through an air channel, but a nonvideoendoscope requires an auxiliary pump to supply air through the working channel. By simple reversal of the hosing, surgical suction pumps have been employed successfully for this purpose. These same pumps can be used to aspirate the air from the uterine lumen after the semen has been deposited.

An insemination catheter must be made out of a nonspermicidal material, be of sufficient length to pass through the endoscope, and include an appropriate attachment on one end for a syringe. The original double-lumen catheter was made from polythene (V-EFIS-2-200, COOK, Brisbane, Australia), and since then suitable single-lumen endoscopy catheters, 1.7 m long and with a 2.6 mm diameter, have been used (Product 64835, Har-Vet, Spring Valley, USA). A 5 to 60 ml syringe is attached to the proximal end of the catheter. The syringe is used to draw semen into the catheter, and sufficient air is needed within the syringe to push the semen from the catheter. During the insemination procedure it is important to ensure that the semen remains located in the distal end of the catheter.

Proper cleaning or sterilization of the endoscope is required after use and between mares to remove infectious organisms and to prevent cross-contamination of spermatozoa. However, the cleaning solution or disinfectant must be well rinsed from the exterior of the scope, as well as from the flush and biopsy channels, so that it does not provide a source of irritation to the endometrium. Water, detergent, or disinfectant solutions are spermicidal and need to be rinsed and removed from the scope. Ideally, disinfectants that contain glutaraldehyde should be avoided unless they can be thoroughly removed from the scope. Once any protein material has been rinsed from the scope, the working channels should be rinsed with 70% alcohol before being air-dried. Sterilization of the scope with ethylene oxide is also possible, but this process takes time and severely limits the number of procedures that can be performed in a given time frame.