Low-dose insemination: Pros and cons (Proceedings)

The minimum insemination dose of 500 million progressively motile spermatozoa for fresh semen and double that for cooled semen, established by Colorado workers in the mid 70s has been widely accepted as the industry standard. However clinical impressions would suggest that mares can be impregnated with fewer sperm but that there is a profound stallion effect. It is not uncommon to have high levels of fertility when mares are inseminated with less than the 500 million sperm.

The increasing use of frozen semen and the popularity of some stallions has provided has triggered an incentive to reduce the number of spermatozoa used per insemination to maximize the efficiency of this procedure. In 1998 in separate studies Vasquez and Manning provided information that suggested that deposition of relatively low numbers of spermatozoa at the uterotubal papilla with the use of an endoscope resulted in pregnancy of mares Although pregnancy rates were low (22 to 30%) after insemination of 1 to 3.8 million PMS, these studies demonstrated the feasibility of dramatically lowering the number of sperm inseminated by direct deposition of sperm at the uterotubal junction (UTJ) via hysteroscopically guided techniques.

Rigby and coworkers (2000) showed that when semen is deposited in the body of problem or normal mares that the distribution of semen in the oviducts was approximately 50% in to each oviduct. However when semen was deposited deep in the uterine horn ipsilateral to the ovary containing the pre-ovulatory follicle, the distribution of sperm was skewed significantly and 80% of the sperm were found in the oviduct closer to where the sperm were deposited regardless of whether the mare was normal or a problem breeder.

Since then other investigators have reported fertility rates of mares inseminated either by rectally guided or hysteroscopic inseminations using fresh or frozen semen at varying concentrations of sperm.

Low-dose insemination

In 2002 Morris et al reported on the fertility of mares inseminated with different numbers of sperm. In that experiment this group showed that inseminations with 1,5 or 10 million sperm resulted in pregnancy rates ranging from 60-75%. However when mares were inseminated with 500,000, 100,000 or 1,000 sperm pregnancy rates dropped significantly.

The great majority of experiments have been directed at depositing the sperm at the UTJ either by rectally guided or by hysteroscopic techniques. However, Morris et al in one study indicated that low dose insemination (14x106 motile frozen-thawed sperm) placed into the uterine body or at the UTJ resulted in similar and acceptable fertility. However pregnancy rates significantly decreased when 3 million sperm were inseminated in the uterine body but not at the UTJ.

Endoscopic or rectally guided: Which one is better?

The initial reports dealing with low-dose insemination techniques in the mare were based upon endoscopically guided procedures to deposit the sperm on the UTJ, other workers have used a transrectally guided technique to direct a flexible insemination catheter to the tip of the uterine horn ipsilateral to the preovulatory follicle. In a report by Brinsko et al., pregnancy rates after insemination with 5 x 106 motile sperm at the tip of the uterine horn by either pipette or endoscopic procedures were similar. Samper et al reported a small difference (4%) in pregnancy rates in favor of the endoscopic technique when mares were bred at in a commercial setting. These studies suggest that low insemination doses of approximately 1/4 of a standard dose of 250 x 106 motile sperm can achieve acceptable pregnancy rates when deposited by deep-intrauterine techniques without the need for endoscopic equipment.

The typical volume of the inseminate when using endoscopic insemination ranges between 20 uls to 1,000 uls with volume having little to no effect on pregnancy rates. Volumes of 500 to 1,500 uls have been used successfully when using the deep horn insemination technique. However when deep horn insemination is used volumes larger than 5 ms seem to have a detrimental effect on fertility.

As a result of the success of these techniques, the obvious question that has been raised is if we can improve the fertility of sub-fertile stallions by changing the site of insemination. The answer is starting to be elucidated and it appears that it could be stallion dependent. For stallions that have non-compensatory defects such as head or chromatin defects it does not appear to be helpful regardless of the number of sperm that are deposited. On the other hand a small but consistent increase in pregnancy rates is observed with other stallions.

Techniques and equipment for low dose AI

Endoscopic

Breeding management of all mares bred with either technique should consider a close selection of candidate as well as stallion fertility will influence the success of this procedure. Young, fertile mares with good reproductive histories are optimal candidates. Older mares with poor uterine clearance will be less desirable candidates and are more likely to have intrauterine fluid accumulation after insemination. In reported studies the incidence of intrauterine fluid accumulation varies. The use of a 1.6 M videoendoscope with an operating channel facilitates visualization of the UTJ in most mares. The small volumes of inseminate used in this procedure are extremely susceptible to contaminants remaining in the biopsy channel of the endoscope. The catheter length should be a minimum of 20 cm longer than the biopsy channel in order to provide adequate working length for insemination. A minimum of three personnel are typically needed to complete the procedure.

The mares are often inseminated 6 hrs on either side of ovulation. Most mares will be given an ovulatory inducing agent typically, either hCG, injectable deslorelin or deslorelin acetate (Ovuplant) and inseminated around 32-46 hrs after injection. For endoscopic insemination mares should be restrained in stocks and sedation with detomidiine and butorphanol may be required. The mare's rectum should be evacuated, and the location of the preovulatory follicle should be confirmed. Careful preparation of the vulva and perineum for the vaginal procedure should be performed. The endoscope can be introduced through the cervix and guided by manipulation per rectum up the uterine horn prior to insufflation. Once the UTJ is identified, the insemination catheter should be passed through the biopsy channel and the inseminate deposited directly onto the UTJ. Due to the low volume as well as the low number of sperm, it is important to insure that sperm are maintained at the appropriate temperature to avoid cold shock of the sperm. All catheters and lab-ware used for semen handling should be pre-warmed to body temperature. Once the endoscope is in place and the UTJ is identified, the inseminate should be drawn into the distal end of the insemination catheter.

Rectally guided

Mare preparation for this technique is similar to that used for endoscopic insemination. Palpation per rectum should confirm the location of the follicle and evacuate feces from the rectum. After preparation for the vaginal procedure, insemination is conducted by passing the insemination catheter through the cervix. After the catheter is introduced into the cervix, the catheter is guided per rectum to the tip of the uterine horn ipsilateral to the preovulatory follicle. Entry into the uterine horn can be facilitated by either grasping the base of the contralateral uterine horn while applying caudal traction, or by pre-bending the pipette and then giving it a 180 degree rotation so that the tip of the pipette is directed towards the desired horn.

The insemination catheter (65 cms) from Minitube is the preferred method. http://www.ivis.org/signin.asp?url=/advances/Reproduction_Ball/ball3/chapter_frm.asp?LA=1#footnotes#footnotes This catheter allows the introduction of a 0.5-ml straw for insemination and also allows the use of multiple 0.5-ml straws if needed without removing the catheter. However this procedure requires two operators.

Conclusion

Deep rectally guided or endoscopic insemination are techniques that should be considered for the breeding management of mares or stallions with fresh, frozen or cooled semen. For routine broodmare with frozen semen the techniques can maximize the use of frozen semen and can result in a reduction of inflammatory reactions in mares with the subsequent increase in pregnancy rates.

References

1. Householder DD, Pickett BW, Voss JL, et al. Effect of extender, number of spermatozoa and hcg on equine fertility. J Equine Vet Sci 1981; 1:9-13.

2. Pickett BW, Voss JL. The effect of semen extenders and sperm number on mare fertility. J Reprod Fertil Suppl 1975; 23:95-98.

3. Cooper WL. Artificial Breeding of Horses. Vet Clin North Am Large Anim Pract 1980; 2:267-275.

4. Vazquez JJ, Medina VM, Liu IKM, et al. Nonsurgical uterotubal insemination in the mare. In Proc Annu Conv Am Assoc Equine Pract 1998; 44:68-69.

5. Manning ST, Bowman PA, Fraser LM, et al. Development of hysteroscopic insemination of the uterine tube in the mare. In Proc Annu Conv Am Assoc Equine Pract 1998; 44:70-71.

6. Morris LH, Hunter RH, Allen WR. Hysteroscopic insemination of small numbers of spermatozoa at the uterotubal junction of preovulatory mares. J Reprod Fertil 2000; 118:95-100.

7. Brinsko SP, Rigby SL, Lindsey AC, et al. Pregnancy rates in mares following hysteroscopic or transrectally-guided insemination with low sperm numbers at the utero-tubal papilla. Theriogenology. 2003; 59:1001-1009.

8. Morris LHA, Tiplady C, Allen WR. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction. Equine Vet J 2003; 35:197-201.

9. Morris LH, Allen WR. An overview of low dose insemination in the mare. Reprod Domest Anim 2002; 37:206-210.

10. Rigby SL, Lindsey AC, Brinsko SP, et al. Pregnancy rates in mares following hysteroscopic or rectally-guided utero-tubal insemination with low sperm numbers. In: Proc 3rd Int Symp Stallion Reprod 2001; 49.

11. Dell'Aqua JA, Papa FO, Alvarenga FC, et al. Effects of warming rate on sperm parameters and of insemination and dose on the fertility of equine frozen semen. Anim Reprod Sci 2001; 68:344-346.

12. Alvarenga M, Trinque CC, Lima MM, et al. Utilisation of hysteroscopy for the application of stallion frozen semen in commercial programmes. In: Havemeyer Foundation Workshop on Transporting Gametes and Embryos 2003; 27.

13. Squires EL, Reger HP, Maclellan LJ, et al. Effect of time of insemination and site of insemination on pregnancy rates with frozen semen. Theriogenology 2002; 58:655-658.

14. Lindsey AC, Varner DD, Seidel GE Jr, et al. Hysteroscopic or rectally guided, deep-uterine insemination of mares with spermatozoa stored 18 h at either 5 or 15 °C prior to flow-cytometric sorting. Theriogenology 2002; 58:659-662.

15. Samper et al. Pregnancy rates after Rectally guided or endoscopic insemination with frozen semen. Proc. AAEP 2005