Breeding

Deep-Horn Insemination

Does this technique improve horse-breeding success using frozen semen?

If your veterinarian mentions the use of deep-horn insemination, you may not know exactly what that entails. Here’s a look at the process and how it might help your mare get in foal. Deep-horn insemination is an artificial insemination technique whereby semen is deposited at the tip of the uterine horn on the same side of the body as the preovulatory follicle. It is not the magic bullet that guarantees frozen semen success.  It does, however, allow for more efficient use of semen without sacrificing and potentially improving fertility. The most commonly utilized deep-horn insemination techniques in the horse-breeding industry are the transrectally guided and hysteroscopic techniques.  Both techniques are minimally invasive and are similar to conventional uterine body insemination, except that a flexible insemination pipette or flexible endoscope is passed to the tip of the uterine horn with transrectal manipulation or with vaginal or transrectal manipulation.

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To optimize the benefits of deep-horn insemination, steps should then be taken to promote the pooling of semen at or adjacent to the oviductal papilla, the structure at the uterotubal junction.  With the hysteroscopic technique, semen is slowly dripped onto the oviductal papilla or applied in a froth-like manner.  With transrectally guided insemination, the tip of the uterine horn is held in a downward position during and for a few minutes after insemination. Inseminating 10 mL of semen containing 500 million sperm, Texas workers1 found that transrectally guided deep-horn insemination improved the percentage of sperm in the oviduct (site of fertilization and early embryonic development) by almost 25 percent over uterine body insemination.  Utilizing insemination volumes of 0.5 mL or less, workers in the United Kingdom2 obtained approximately 65 percent pregnancy rates utilizing 14 million frozen-thawed sperm from two extremely fertile stallions and uterine body insemination.  When the insemination dose was reduced by approximately 75 percent (3 million sperm), pregnancy rates significantly declined when semen was deposited into the uterine body but were maintained when hysteroscopic deep-horn insemination was utilized.  Comparison of the results of these studies suggests that smaller semen volumes may maximize the benefits of deep-horn insemination. However, studies directly comparing semen volume have not been performed. It is reasonable to postulate that smaller volumes stay closer to the site of insemination (i.e. have less backflow into the uterine body), maximizing the amount of sperm that enter the ipsilateral oviduct.  These results also suggest that the hysteroscopic technique may be superior to transrectally guided deep-horn insemination in maximizing the percentage of sperm in the oviduct adjacent to the preovulatory follicle, especially when the tip of the uterine horn is not held in a downward position during and for a few minutes post-insemination.  However, when held downward, my mentors and I3 found no difference in pregnancy rates between the two techniques when normal fertile mares were inseminated with 0.2 mL of semen containing 1 million (hysteroscopic 77 percent vs. transrectally guided 73 percent) and 0.5 million (hysteroscopic 20percent vs. transrectally guided insemination 31 percent) fresh sperm.  It is important to recognize that even when the insemination dose (0.5 million) resulted in subfertile pregnancy rates, neither technique could sustain the pregnancy rates seen with the 1 million insemination dose. Deep-horn insemination does have its limitations, most notably involving poor semen quality.  Texas workers4 could not improve the fresh semen pregnancy rates (29 percent) of a stallion with teratospermia (semen with a high percentage of morphologically abnormal sperm) simply by changing the insemination site from the uterine body to the tip of the uterine horn ipsilateral to the preovulatory follicle. Workers in Italy5, however, improved fresh semen pregnancy rates of a commercial breeding stallion with teratospermia following a sperm purification with density gradient (silicon-coated silica particles, RediGradTM; Amersham Biosciences, NJ, USA) followed by transrectally guided deep-horn insemination.  Specifically, these workers obtained 62 versus 42 percent pregnancy rates when mares were bred with 50 million morphologically normal, progressively motile sperm processed with routine cushion-centrifugation only (~500 million total sperm) or in combination with RediGradTM centrifugation (~130 million total sperm), respectively.  Two other commercial density gradient centrifugation colloids, EquiPureTM (silane-coated silica particle; Nidacon International AB, Mölndal, Sweden)6,7 and Androcoll-ETM (silane-coated silica particle in buffered salt solution; SLU, Uppsala, Sweden)8-10 have demonstrated an ability to improve post-thaw semen quality (DNA quality, sperm morphology and motility) primarily through removal of sperm with abnormal heads and midpieces, bent midpieces, bent tails, coiled tails and premature germ cells6-11.  EquiPureTM centrifugation has also anecdotally shown promise as a means to improve frozen semen fertility12.

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No minimum standards exist for commercial frozen semen in the United States.  Most dose recommendations are made based on conventional uterine body insemination; however, some now include recommendations for both uterine body and deep-horn insemination techniques.  Recommendations made for uterine body insemination typically range from 600 to 800 million (range = 400 million to 1 billion) total sperm or contain a minimum of 200 to 250 million progressively motile sperm.  Post-thaw progressive motility is ideally greater than 30 to 35 percent, but it is not uncommon to be breeding with semen below these parameters. 

Frozen semen certified for international exportation may have minimum quality and dose requirements, often following the World Breeding Federation of Sport Horses recommendations of a minimum of 250 million progressively motile sperm per insemination dose with post-thaw progressive motility of 35 percent or higher. The number of insemination doses required per mare cycle is typically one to three, depending on whether “timed” or a single post-ovulation breeding is utilized.  Based on the studies described earlier in this article, deep-horn insemination should allow a 25 to 75 percent reduction in sperm used per cycle.  In other words, if the recommended dose of semen for uterine body insemination is 250 million progressively motile sperm, the deep-horn insemination dose would range from 62.5 to 187.5 million progressively motile sperm.  These types of reductions have been successfully utilized with frozen semen from some commercial stallions but unsuccessful in others13,14.  Thus, reductions in the insemination dose should always be based on the fertility of an individual stallion or, more importantly, on the fertility of a specific ejaculate. If trying to improve upon previous per-cycle pregnancy rates utilizing uterine body insemination or using semen of unknown fertility, I recommend using deep-horn insemination but starting with the insemination dose recommended for uterine body insemination.  Once fertility is established for an individual stallion and ejaculate, it is reasonable to adjust the insemination dose accordingly.  Keep in mind that frozen semen per-cycle pregnancy rates average 30 to 55 percent (range = zero to greater than 70 percent). Deep-horn insemination is a valuable tool that should be considered whenever frozen semen is utilized, due to its potential to improve fertility over uterine body insemination.  I greatly prefer the transrectally guided technique over the hysteroscopic method, due to its practicality and its use of a sterile, disposable, inexpensive pipette for each insemination.  I always try to hold the uterine horn in a downward position during and for a few minutes after insemination to facilitate pooling of semen at or adjacent to the uterotubal junction when performing transrectally guided deep-horn insemination. Dr. Shelby Hayden is a clinical assistant professor of theriogenology at Oklahoma State University.

References

1.    Rigby S, Derczo S, Brinsko S, Blanchard T, Taylor T, Forrest DW, Varner D. Oviductal sperm numbers following proximal uterine horn or uterine body insemination. Proc 46th Ann Conv Amer Assoc Equine Pract 2000;46:332-4. 2.     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. 3.    Hayden SS, Blanchard TL, Brinsko SP, Varner DD, Hinrichs K, Love CC. Pregnancy rates in mares inseminated with 0.5 or 1 million sperm using hysteroscopic or transrectally guided deep-horn insemination techniques. Theriogenology 2012;78:914-20. 4.    Woods J, Rigby S, Brinsko S, Stephens R, Varner D, Blanchard T. Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body. Theriogenology 2000;53:1827-36. 5.    Mari G, Castagnetti C, Rizzato G, Mislei B, Iacono E, Merlo B. Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility. Anim Reprod Sci 2011;126:96-100. 6.    Mancill SS, Love CC, Brinsko SP, Edmond AJ, Foster ML, Teague SR, Waite JA, Varner DD. Effect of density gradient centrifugation on cryopreservation of equine spermatozoa. Anim Reprod Sci 2010;210S:S208-9. 7.    Stoll A, Love CC, Ball BA. Use of a single-layer density centrifugation method enhances sperm quality in cryopreserved-thawed equine spermatozoa. J Equine Vet Sci 2013;33:547-51. 8.    Macias Garcia B, Morrell JM, Ortega-Ferrusola C, Gonzales-Fernandez L, Tapia JA, Rodriguez-Martinez H, Peña FJ. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen. Anim Reprod Sci 2009;11:193-202. 9.    Macias Garcia B, Gonzalez Fernandez L, Morrell JM, Ortega Ferrusola C, Tapia JA, Rodriguez Martinez H, Pena FJ. Single-layer centrifugation through colloid positively modifies the sperm subpopulation structure of frozen-thawed stallion spermatozoa. Reprod Dom Anim 2009;44:523-6. 10.    Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, De Schauwer C, De Kruif A, De Vliegher S. Sperm selection using single layer centrifugation prior to crypreservation can increase thawed sperm quality in stallions. Equine Vet J 2011;43:35-41. 11.    Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD. Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm. Theriogenology 2012;77:959-66. 12.    Carroll BS. Personal communication. 2012. 13.    Samper JC, Sanchez R, Gomez I, Leite B. Artificial insemination with frozen semen: pregnancy rates after rectally guided or endoscopic deposition. Proc 51st Ann Conv Amer Assoc Equine Pract 2005:51;213-14. 14.    Samper JC, Gomez I, Sanchez R. Rectally guided or hysteroscopic insemination: Is there a difference? J Equine Vet Sci 2008;28:640-4.