Low Dose Insemination
Dickson D. Varner, DVM, MS, Diplomate, ACT
Terry L. Blanchard, DVM, MS, Diplomate ACT
Steven P. Brinsko, DVM, Ph.D., Diplomate ACT
Charles C. Love, DVM, Ph.D., Diplomate ACT
Department of Large Animal Clinical Sciences, College of Veterinary Medicine,
Texas A&M University, College Station

Introduction
Many advances have been made in assisted reproduction for horses in recent years, and several breed registries in the United States have adopted many of these newly-found techniques.  The result has been an age of "mail order" semen and, more recently, "sexed" semen and low-dose insemination strategies.  Where will we go from here? On the horizon is commercial application of a procedure called intracytoplasmic sperm injection (ICSI).  With this technique, a single sperm from a stallion is injected into an egg in a laboratory setting, and the developing embryo is then transferred (implanted) into the reproductive tract of a surrogate (or recipient) mare. The emergence of new reproductive technologies is limited only by the imagination of investigators coupled with practical applications in the horse industry.

One such consideration is low-dose insemination of mares. What exactly does this procedure entail?  Is it practical?  How will this technique benefit the equine breeding industry? In what setting is there a tactical advantage for using low-dose insemination over traditional insemination methods?  As the technique is relatively new on the horse-breeding scene, many of the queries have not been thoroughly pondered, and only time will determine if it will gain a place in the armamentarium of horse breeders and veterinarians.

Considerations
Potential advantages and disadvantages are considered in the table below.
 

The perceived advantages of low-dose insemination are primarily to allow popular stallions to inseminate more mares with an ejaculate, and to improve reproductive performance of stallions with fertility problems. Although not critically tested to date, these two attributes of low-dose insemination are worthy of consideration.  Recent studies have revealed that, using low-dose insemination, mares can be bred with a sperm number over 100 times less than that of a traditional insemination dose (ie, 1 to 5 million sperm as opposed to 500 million sperm), with good pregnancy rates resulting.  It may also be possible to improve the fertility of some stallions by first concentrating the semen or removing a high percentage of the deformed sperm prior to low-dose insemination. Is this an irrefutable revelation or do we first need to consider the potential down side of the technique.  The procedure will undoubtedly result in added expenses for equipment and personnel, and those involved with providing this service will require intensive education on the procedural details. Will the technique lead to a reduction in the gene pool because of increased availability of semen from more popular stallions?  Will the technique lead to propagation of subfertile offspring, as a result of using assisted reproductive technologies to improve fertility of some stallions whos subfertility may be heritable?  As demonstrated here, we cannot simply adopt new techniques such as this without considering potential detrimental ramifications of the technique.

The Technique
Mares are usually bred with 250 million to 1 billion progressively motile sperm in a volume of 10 to 100 cc placed in the uterine body.  With a low-dose insemination technique, the sperm number can be reduced to 1 to 20 million sperm, and the insemination dose can be reduced to 0.1 to 0.2 cc. Using this approach, semen is placed in the tip of the uterine horn, on or near a structure called the oviductal papilla (Figure 1). This structure represents the opening from the uterus into the oviduct (or Fallopian tube), where fertilization occurs. Two techniques have been used to deliver the semen to this important location.  One technique utilizes a videoendoscope that is placed in the uterus to visualize proper placement of semen (Figure 1).  Another technique uses rectally-guided direction of a flexible insemination catheter to the tip of the uterine horn prior to insemination of semen (Figure 2).

Figure 1 Figure 2

Recent Studies
Recently, studies have been conducted at Texas A&M Univeristy's College of Veterinary Medicine to address the issues of deep uterine horn insemination and low-dose insemination strategies.

One study was conducted to determine the effect of insemination location on sperm numbers in the oviduct. Twenty mares in estrus were inseminated once with 500 million total sperm in a 10 -ml volume in the uterine body or in the tip of the uterine horn (on the side of the dominant pre-ovulatory follicle).  Ovariohysterectomies were performed on all mares at 18 hours following insemination, and the sperm numbers within the oviducts were determined. Regardless of the technique used for insemination, less than 0.0007% of the sperm actually gained access into the oviducts.  The relative numbers of oviductal sperm recovered from both oviducts of mares were compared. When mares were inseminated in the uterine body, an average of 44% of the sperm were in the oviduct in which fertilization would occur (mean of 1416 sperm in the oviduct on the side of a preovulatory follicle versus mean of 1801 in oviduct opposite to the ovulatory follicle). In comparison, an average of 84% of the sperm were in the oviduct in which fertilization would occur when inseminated in the tip of the uterine horn (mean of 3000 sperm in the oviduct on the side of a preovulatory follicle versus mean of 576 in oviduct opposite to the ovulatory follicle). These data indicate that more sperm gain access into the oviduct of fertilization when the insemination location is the tip of the uterine horn, as opposed to the uterine body.

Another study was performed to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, on or around the oviductal papilla.  In this study, 36 mares were inseminated with 5 million total sperm that had previously been stored in an extender at refrigerated temperature for 24 hours. Semen (0.2 cc volume) was inseminated on or near the oviductal papilla using a hysteoscopic insemination technique or using a rectally-guided technique with a newly-designed flexible insemination catheter. When mares were inseminated with the hysteroscopic technique, 67% (12/18) became pregnant, whereas 56% (10/18) became pregnant when the rectally-guided technique was used.  This work indicates that mares will become pregnant at an acceptable rate with low-dose insemination when using either a hysteroscopic or a rectally-guided approach.

A study was also conducted to determine if a low-dose insemination technique could be used to improve pregnancy rates in a stallion with reduced fertility.  In this study, semen from a fertile and a subfertile stallion were processed separately by centrifugation in a special medium as an effort to improve semen quality. The technique resulted in a significant increase in the semen quality of the subfertile stallion.  The processed semen of the subfertile stallion appeared to be of similar quality to that of the fertile control stallion, in that both had approximately 90% motile sperm in the processed sample. Subsequently, 40 mares in estrus were inseminated with 20 million progressively motile sperm in a 0.1 cc volume, using a hysteroscopic insemination technique. Pregnancy rates for the fertile stallion and subfertile stallion were 75% (15/20) and 35% (7/20), respectively.  In this trial, the processing technique improved the quality of the semen of the subfertile stallion, but it did not yield a pregnancy rate equivalent to the fertile stallion.  The technique may still have value for the subfertile stallion, because he historically had a pregnancy rate per cycle of approximately 20% with conventional breeding techniques.

The Future
As evidenced by the studies presented, low-dose insemination is a viable technique.  Indeed, it can add a unique dimension to equine breeding programs, but it does come with a relatively steep learning curve.  Utilization of assisted reproductive techniques such as this must be preceded by thoughtful deliberation with individual stallion owners regarding procedural costs and expectations. 

Acknowledgements:  Financial support for these studies was provided by the American Quarter Horse Association and the Link Equine Research Endowment Fund, Texas A&M University. EquiPure was provided by NidaCon International AB, G`teborg, Sweden