The distribution of spermatozoa in the genital tract was determined in ewes killed 4 hr or 24 hr after cervical insemination with 100 million live spermatozoa from either freshly ejaculated or deep.frozen ram semen. At 4 hr, greater numbers of spermatozoa were present in the cervices, uteri, and fallopian tubes in ewes inseminated with fresh semen than in ewes inseminated with frozen semen. At 24 hr, the numbers of spermatozoa in the uteri and fallopian tubes of ewes inseminated with fresh semen had increased relative to the numbers at 4 hr but no spermatozoa were present in the uterus and fallopian tubes in ewes inseminated with frozen semen.

Egg yolk dispersed at levels between 0�1 and 3� 75% (v/v) in a diluent used for the preservation of ram spermatozoa at 5�C was shown to affect the structure and survival of spermatozoa.

SummaryRam semen was diluted 40-to 50-fold in various synthetic diluents and the motility of the spermatozoa scored after incubation at 37°C and after slow cooling to, and storage at, 5°C. Levels of potassium above 10 m-equiv. were deleterious at both temperatures. the addition of 5 m-equiv. calcium depressed motility at 37°C but improved survival rates of spermatozoa stored at 5°C. The inclusion of 6 m-equiv. magnesium in diluents had no detectable effect on motility scores.Replacement of the sodium chloride component (123 mM) of a diluent buffered with 20 mM phosphate buffer with an isosmotic lactose solution showed that diluents with a content of this sugar ranging from 61 to 246 mM were superior at both 37 and 5°C to that containing 123 mM sodium chloride and no lactose. Diluents containing 184 mM fructose, glucose, lactose, or sucrose and 31 mM sodium chloride were all better for the storage of ram spermatozoa at 5°C than the diluent containing 123 mM sodium chloride.Motility scores of spermatozoa incubated at 37°C were slightly, but significantly, lower in diluents containing 2·0 % casein than in those with 0·5 % casein. Scores at 5°C showed that 2· 0% casein gave much better survival than O· 5% casein.

Incidence of pregnancy in artificially inseminated Merino ewes was not significantly affected by dilution of semen with buffered glucose so as to present equal numbers of spermatozoa in different volumes (50 or 200 µI) or by reducing number of spermatozoa per insemination from 100 to 50 million. Respective pregnancy percentages for various volumes and concentrations of semen were: 50 µl, 50 x l06, 48.7%; 200 µl, 50 x l06, 57.1%; 50 µl,100 x l06, 53.8%; 200 µl , 100 x 106 52.9%; 31 µl, 100 x l06 (undiluted control), 60.2%. The percentage of pregnancies was significantly higher among ewes with clear or heavy (v. light) raddle marks left by the teaser rams used to detect oestrus, and among those with a copious flow of vaginal mucus at insemination. The fertility of all semen fell as the interval between collection and insemination increased to 1 hr.

The addition of 6% (vJv) egg yolk to a synthetic diluent [247 mM glucose, 49 mM NaCI, 5 mM KCI, 5 mM phosphate buffer, and 7·5% (vJv) glycerol] improved the survival of ram spermatozoa after deep·freezing. Judging by the activity of spermatozoa after thawing, a single addition of glycerol to the diluted semen at 5°C was as effective as multiple additions giving a graded increase of glycerol concentration over a period of 20 min. Reciprocal replacement of the sodium chloride of the diluent by an increase in the concentration of the phosphate buffer showed that motility of the spermatozoa after thawing was depressed when the level of phosphate exceeded 10 mM.A rapid rate of freezing of l·ml aliquots of semen in glass ampoules in the neck of a liquid nitrogen container gave lower survival of spermatozoa than a slower rate in dry ice (average rates to -40°C of 1 and 2 degrees Celsius per minute respectively).Spermatozoa survived a period of 2 weeks of storage in liquid nitrogen better than when stored in dry ice. There was a significant decline in the revival rate of samples prepared and frozen from four successive ejaculates collected by electro· ejaculation at intervals of 3 days.