When the cooled semen arrives for insemination of the mare, current recommendations are to:

• Prepare the mare for breeding. The mare should be adequately restrained with her tail wrapped and held to the side. The perineal area is thoroughly scrubbed and rinsed, paying particular attention to the vulva. Any dirt or fecal material within the caudal vestibule should be removed during the washing process to prevent contamination of the anterior reproductive tract during insemination. Two to three scrubs with a non-irritating soap (e.g., Ivory Soap) or a surgical scrub are recommended, followed by thorough rinsing to eliminate residual soap that may be spermicidal or irritating to mucous membranes. The perineal and vulvar area should be thoroughly dried prior to breeding.
• Open the shipping container and confirm the identity of the stallion using the accompanying form. Remove the chilled semen, gently mix it, aspirate the semen into a syringe, and attach an insemination pipette. Sterile nontoxic disposable equipment is recommended for AI procedures. Syringes with non-spermicidal, plastic plungers (Air-tite, Vineland, NJ) are preferable for AI since rubber plungers may possess spermicidal properties.
• Inseminate the semen into the uterus of the mare. To inseminate the mare, a sterile shoulder-length plastic sleeve is first placed over the arm used for insemination. The tip of a 20 to 22-inch insemination pipette is then positioned in the cupped hand and a small amount of sterile, non-spermicidal lubricant is applied to the back of the hand. Just prior to passing the hand into the cranial vaginal vault a syringe containing extended semen is attached to the insemination pipette then gently press the plunger on the syringe until the extended semen fills the pipette. Then the covered hand and insemination pipette are passed into the cranial vaginal vault where the index finger identifies and penetrates the cervix. The insemination pipette is then advanced through the cervix to the mid-body of the uterus. Slowly deposit the entire syringe of extended semen into the uterine lumen. Remove the syringe while leaving the pipette in the uterus draw approximately 5 mL of air into the syringe, re-attach the syringe to the pippette and plunge the remaining extended semen left in the pipette into the uterus... (Note:The volume of the pipette is approximately 5-6 mL & you do not want to waste 25% of your breeding dose) An alternate, but equally satisfactory, method of insemination is to pass the insemination pipette through the cervix into the body of the uterus using a lighted speculum preplaced in the vagina.
• A small aliquot of the extended semen should be kept and warmed to 37oC, and sperm motility should be assessed and recorded for quality control purposes. If sperm motility is poor, inquiries can be made in an effort to determine if this was an unexpected problem. It is sometimes helpful to prepare a specimen for assessment of sperm morphology. A high percentage of morphologically abnormal sperm would be expected to yield a low precentage of progressively motile sperm following cooling. If sperm motility is consistently poor and the mare fails to conceive on repeated breedings, it is possible the stallion is incapable of producing sperm which survive the cooling/transportation process. Alternatively, use of a different semen extender or dilution ratio may prove beneficial to semen harvested from that particular stallion.

Overseas workers often recommend warming the cooled semen to body temperature prior to insemination of the mare. In the U.S., the cooled semen is usually inseminated directly into the uterus as soon as possible after opening the shipping container. Since no detrimental effects on pregnancy rate have yet been determined by inseminating mares with cooled semen, at present we see no reason to prewarm the cooled semen prior to insemination unless a cream-gel extender is used. This extender forms a gel at refrigerated temperature, so it must be warmed prior to insemination. The packaged sample can be placed in an incubator or waterbath preset at 37-38oC. It is important to avoid contamination of the extended semen with water when warming the sample in a waterbath.

Double inseminations

It has become common practice for many stallion owners/managers to prepare two bags (insemination doses) of extended semen for shipment - one to be used for an initial insemination upon arrival, and one to be held for insemination again the next day. For many stallions, the longer the semen is held at refrigerated temperature, the poorer sperm motility becomes. However, if semen is to be held for breeding again the next day, precautions should be taken to maintain the cooled semen at 4-6oC until the time of the second insemination. Once a shipping container is opened for the first breeding, temperature of the semen left in the container is likely to rise above 10oC, causing premature sperm death. The remaining semen dose can be immediately placed in an insulated box within a refigerator (4-6oC) - until it is to be opened for breeding the next day. This would be especially critical for semen cooled in some "disposable" type shipping containers which may only maintain semen at < 10oC for 27.5 to 33.5 hours.