Studies on the freezing of stallion semen. II. Factors affectiing survival rates of stallion spermatozoa after freezing and thawing and results of a fertility trial
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The factorial experiment using pellet freezing techniques was made to test the effects of glycerol level, glycerol equillibration time and sugars on survival of stallion spermatozoa after freezing and thawing. <BR>The semen collected using artificial vagina and removed "gel" portion by cotton gauze was dilut-ed 1: 1 with yolk (10 parts)-5% glucose (90 parts) solution and then centrifuged (1300 × g, 20 min.) <BR>The semen concentrated by removal of the supernatant was diluted 1: 4 with yolk (10 parts)-sugar media containing glycerol and cooled to 5°C. <BR>After various (glycerol) equillibration time, 0.2 ml of the diluted semen was frozen in pelleted. form. (Fig. 1) <BR>1-5 hrs arter freezing, a pellet frozen semen was thawed on matal plate kept 40°C and the motility was examined of the semen thawed with and without the thawing media containing non glycerol. <BR>The samples whitch was added the thawing media to the thawed semen glycerolated at 3.5% showed better surviaval rates of the spermatozoa than those of the semen glycerolated at 1.75% and 7.0%, especially in 5 hrs equillibration time. <BR>The spermatozoan survivals of thawed semen without the thawing media reduced when glycerol equillibration time was longer. <BR>Survival rates of the spermatozoa in the raffinose solution was superior to that in sucrose at the longer glycerol equillibration time. <BR>In the fertility trial, the concentrated semen was diluted 1: 2 with yolk sugar glycerol diluters which was composed of 10 parts of egg yolk, 5.5 parts of glycerol and 84.5 parts of mixed sugar solution (glucose 1.8 gm, lactose 4.4 gm, and raffinose 5.4 gm in 100 m<I>l</I>). <BR>0.2 m<I>l</I> of the diluted, cooled and glycerol equillibrated (3-5 hrs) semen was frozen in the pellet form and preserved in the glass test tubes with crushed dry ice. <BR>At thawing, 10-30 pellets of the frozen semen were added to the 30 ml of the thawing media kept 30-35°C. <BR>The thawing media was composed of the same volumes of skim milk and buffer solution. Ingradients of the buffer solution were as follows; <BR>Sodium citrate dihydrate……1.57 gm, Sodium carbonate……0.075 gm, Potassium carbonate……0.085 gm, Glucose……2.7gm, P aminomethylbenzensulfonamide (Homosulfamine)……0.1 gm, Distilled water…………up to 100 m<I>l</I> <BR>pH of the buffer solution war adjusted to 7.0 using 5.5% solution of citric acid. <BR>Inseminations were made immediately after thawing. <BR>14 conceptions resulted from 42 inseminations, the fertility was 33.3 per cent. Fertility rates of No. 1, 2 and 3 of the stallions were 47.3, 26.8 and 12.5 per cent, respectively. <BR>Storage periods of dig semen at the conceptions were 1-28 days. <BR>Fertility trials using liquid nitrogen are continued at present in larger scales.
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