Studies on the freezing storage of stallion semen. III. Freezing of pellet frozen semen preserved in liquid nitrogen

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Fertility trials using pellet frozen stallion semen preserved in liquid nitrogen were conducted in 1966. Semen, collected from 9 stallions (Percheron 3, Breton 4, Pony 2) with gel portion removed, was extended 1:1 with yolk (5 parts) -5% glucose (95 parts) solution and then centrifuged at 1, 300g, 20 min. for concentrate of semen. The concentrated semen had the supernatant removed and was extended again 1:23 with yolksugar-glycerol extender, which was composed of 10 parts of egg-yolk, 5.5 parts of glycerol and 84.5 parts of sugar media (glucose 1.8 gm, lactose 4.4 gm, raffinose 5.4 gm in 100 ml). The extended semen was cooled to 5°C at semi-rapid rate by keeping it in small test tubes which were kept standing directly in 5°C cold air room. The 0.2ml of the cooled semen was placed directly on a block of dry ice after 23 hours of glycerol equilibration. Semen frozen in pellet form on dry ice was transfered into plastic tubes (diameter 1.2cm, height 5.0 cm) which were made to stand in holes made in dry ice. Then the plastic tubes were sealed with plastic-aluminum film using the electric heat sealer. The small plastic tubes were stored in liquid nitrogen until use. The frozen semen was carried to the mares owned by farmers in crushed dry ice by A. I. technicians. Inseminations were carried out immediately after thawing, in which the 520 pellets of semen were added to 20ml of the thawing media in ampoules kept at 3035°C. The thawing media were a mixture of following solutions and were stored at 4°C or at room temperature after heating repeatedly in ampoules. 1) Skim milk 100ml 2) Buffer solution 77.0ml. Composed of; Sod. citrate 1.57gm, Sod. carbonate 0.075gm, Pot. carbonate 0.085 gm, Glucose 2.7 gm, P-aminomethylbenzensulfonamide 0.2gm, Distilled water up to 100ml. 3) Glycerol 3.0ml. The pH of the mixed media…7.0 (adjusted by 5.5% citric acid solution). The mares were inseminated 13 times per heat under conditions of the routine work and diagnoses to determine conceptions were performed by the rectal palpation. Inseminations to 93 mares, each of which was inseminated only during one heat, resulted in 43 conceptions. Fertility was 46.7 per cent, except one mare for which we could not know the result (Table 1). Semen conditions in successful inseminations were as follows ; Storage period. … 1110 days, Survival rates of sperm.…1565%, No. of pellets inseminated … 3-11, No. of sperms inseminated.…63-31.24 (×108)). Relationships of no. of spermatozoa inseminated and conceptions were as follows; 34 (×8))… 50% (2/4), 56…50% (5/10), 7-8…76.9% (10/13), 9-10…50% (1/2), 13-14…42.8% (3/7). Although the number of mares was too small and further observations should be needed, these results suggest that inseminations with 78 (×8)) spermatozoa would be enough to get high fertility when mare heat condition and insemination technique are good.
日本繁殖生物学会の論文

Studies on the freezing storage of stallion semen. III. Freezing of pellet frozen semen preserved in liquid nitrogen

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日本繁殖生物学会 | 論文

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