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Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa.Six sperm categories of each stallion (n= 4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-).Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw;and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa.This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa.We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.

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The aim of this study was to determine if the addition of equine seminal plasma to epididymal semen enhances its fertility before or after freezing. Thirty-two mares were randomly assigned to 5 stallions; 3 stallions were kept in Pretoria, each having 7 mares, and 2 stallions were kept at Cornell, one having 6 mares and the other 5. Mares were synchronized using 10 daily IM progesterone and estradiol injections; an Ovuplant® implant (26 mg of deslorelin; Peptech Animal Health, Sydney, NSW, Australia) was inserted under the mucosa of the vaginal vestibulum once a follicle reached a diameter of 35 mm; implants were removed after ovulation. Mares were inseminated 30 h after implant insertion. Each insemination dose consisted of 200 million progressively motile sperm and was deposited into the uterine body. Following insemination, mares were examined for ovulation at 6 hourly intervals. Fourteen days after ovulation, mares were examined for pregnancy by transrectal ultrasonography and treated with PGF2α to induce the next estrus. Seminal plasma was collected from the stallions used in the trial prior to castration, frozen, and stored. In Pretoria, stallions were castrated and one epididymal tail was flushed with seminal plasma and the other with skim milk extender; in the first cycle, half of the mares were inseminated with one of the two sperm samples. In Cornell, testes of each stallion were removed 3 weeks apart, and all mares were inseminated first with one and 3 weeks later with the other semen sample. Mares were inseminated during consecutive estrous cycles using the following sperm types: fresh epididymal sperm that had been exposed to seminal plasma (G1: 4 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); fresh epididymal sperm that had never been exposed to seminal plasma (G2: 3 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); frozen–thawed ejaculated sperm (G3); frozen–thawed epididymal sperm that had been exposed to seminal plasma prior to freezing (G4); and frozen–thawed epididymal sperm that had never been exposed to seminal plasma (G5). The results of inseminations with fresh epididymal semen (G1–2) of 5 stallions and the preliminary results of inseminations with frozen–thawed epididymal semen (G3–5) of 2 stallions are summarized in the Table 1. Cycles where ovulation did not occur within 12 h after insemination were excluded. The pregnancy rate of mares inseminated with fresh epididymal sperm of G1 was significantly higher (chi-square test; P < 0.05) than that of mares of G2. The pregnancy rate of mares inseminated with frozen–thawed ejaculated semen (G3) was similar to that of mares inseminated with frozen–thawed epididymal semen of G4 and G5 (P = 0.3). Based on these preliminary results, we conclude that the fertility of fresh epididymal sperm can be enhanced by exposure to equine seminal plasma. To determine if the same holds true for frozen–thawed epididymal sperm, more inseminations must be performed. Table 1.Results of inseminations with various semen types