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I. C. A. MARTIN

Summary.

With 1% lecithin and 1 ·0 m-glycerol, the activity of deep-frozen bull spermatozoa after thawing was better if they were frozen in an isotonic diluent than if frozen in a hypertonic ( 1 ·2 x ) solution (the solution referred to as isotonic was : 50 mm-sodium citrate, 62 mm-fructose and 20 mm-sodium phosphate buffer). There was no significant difference between the mean effects of addition of the glycerol either immediately the diluted semen had been cooled to 5° C (equilibration) or just prior to freezing after a period of storage at 5° C equivalent to the equilibration period (ageing). A period of 4 hr of storage at 5° C prior to freezing gave the best mean motility scores and percentage motile, but the percentage of spermatozoa unstained by congo-red-nigrosin solutions continued to rise over a period of 18 hr storage.

Yolk-containing diluents, with the tonicity range of 0·90, 1·05 and 1·20 x, were tested and hypotonic diluents were superior to hypertonic. Equilibration for up to 18 hr had no significant effect on the scores of activity of spermatozoa after thawing, but, again, there was a highly significant rise in the percentage of unstained spermatozoa.

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I. C. A. MARTIN

Summary.

Bull spermatozoa were deep-frozen after periods of 0, 1 and 4 hr of equilibration at 5° C prior to freezing. Their revival was scored by the motility score, the percentage motile and the percentage unstained by congo-red-nigrosin. By all scores the best results were seen after a 4-hr period and the highest numbers of motile spermatozoa were seen when 62 mm-fructose was mixed with the buffered sodium citrate diluent.

There were higher survival rates in diluents containing 1% (w/v) egg lecithin than in those with 25% (v/v) egg yolk, and best counts of unstained spermatozoa were found in lecithin-containing diluents after 4 hr storage at 5° C.

In a second experiment, after replacement of part of the citrate buffer by 62, 123 or 185 mm-fructose solution, there was no significant change in the activity of the thawed spermatozoa, but the percentage of unstained spermatozoa was significantly lower in diluents containing 185 mm-fructose than in those with 62 mm. One per cent lecithin gave better motility results than 0·5 or 2·0%, and the proportion of unstained spermatozoa was higher in 2·0% than in 0·5% lecithin.

Improvement in the percentage of unstained spermatozoa continued for up to 12 hr storage at 5° C and best results were scored from semen diluted and equilibrated for 12 hr in a mixture of 62 mm-fructose, 60 mmsodium citrate, 20 mm-phosphate buffer and 2·0 m-glycerol.

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I. C. A. MARTIN and C. S. WALLACE

Summary.

With the addition of a phase sensitive detector and a discriminator circuit to the impedance bridge (Rothschild, 1948), counts of the impedance change frequency (icf) of a semen sample can be recorded on a mechanical counter. Further separate circuits to indicate balance of the bridge have been added so that the cathode ray tube of the original instrument is no longer needed for balancing or counting icf.

Highest icf counts were recorded when the concentration of ram spermatozoa lay between 9 and 12 × 108 spermatozoa/ml. Activity fell rapidly in more concentrated samples tested at 38° C unless the sample was simultaneously dialysed against an isosmotic buffered saline diluent containing fructose.

There were no significant differences between the performance of four electrodes tested, but icf was depressed by the presence of an electrode in the sample for 10 min at 38° C before a reading was taken.

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I. C. A. MARTIN and C. W. EMMENS

Summary.

Four treatment methods for the preparation of deep-frozen bull semen were tested for fertility in a factorial design involving the artificial insemination of 1728 first-service cows with the following results :

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of non-returns to service at 3 months, with equal treatment groups of 432 cows. The improvement in fertility after equilibration in the presence of fructose was highly significant (P < 0·01), but not in its absence, when fertility was depressed even in equilibrated semen.

There was a higher revival rate of spermatozoa after the longer period of equilibration and the use of fructose in the diluent significantly improved the percentage of motile spermatozoa after thawing.

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R. C. JONES and I. C. A. MARTIN

Summary.

In a factorial experiment skim milk, egg-yolk-citrate and synthetic diluents composed of fructose or lactose, sodium chloride and phosphate buffer containing 3·0% w/v of a lyophilized preparation of non-dialysable solids from (cow) milk were used as diluents for deepfreezing ram spermatozoa and for incubating spermatozoa at 37° C after thawing. All samples of semen were diluted forty-fold before freezing. Egg-yolk-citrate was inferior to skim milk as a diluent for freezing spermatozoa and for incubating spermatozoa after thawing. Of the two synthetic diluents, lactose synthetic was better for freezing spermatozoa whilst fructose synthetic was better for incubating spermatozoa after thawing. Only spermatozoa frozen in the lactose synthetic diluent and resuspended after thawing in the fructose synthetic survived incubation at 37° C for 2 hr as well as spermatozoa frozen and incubated in skim milk diluents.

Two factorial experiments compared egg-yolk-citrate and skim milk as diluents for freezing semen diluted from ten- to forty-fold. At ten-fold dilution, revival was much the same after freezing in milk or egg-yolk-citrate. A twenty- or forty-fold dilution was better than a tenfold dilution in milk, but revival was depressed at these higher dilution rates after freezing in egg-yolk-citrate. When semen was frozen at a tenfold dilution it was advantageous to resuspend spermatozoa in a diluent free of glycerol after thawing. A diluent based on Krebs—Henseleit— Ringer solution containing 0·5% w/v of non-dialysable milk solids was better for incubating spermatozoa after thawing than egg-yolk-citrate or milk.

A period of 5 hr equilibration at 5° C before freezing was better than 30 min equilibration.

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R. C. JONES and I. C. A. MARTIN

Summary.

A transmission electron microscope was used to examine samples of ram semen after 30-fold dilution in a buffered glucose solution and storage at either 35° C or 5° C. The appearance of sections of heads and mid-pieces of spermatozoa was systematically scored so that the frequency of occurrence of several changes in ultrastructure in specimens could be compared between treatments.

Incubation of diluted semen at 35° C for 1 or 2 hr did not cause significant changes in acrosomes but a significant proportion of spermatozoa showed condensation of mitochondria or loss of material. Although the acrosomes of spermatozoa in undiluted semen were unaffected by incubation at 35° C for 2 hr, mitochondria had lost material and were scored as 'pale' in 80·5% of spermatozoa; only 13·5% of spermatozoa in the control samples were scored in this category.

Acrosomes and mid-pieces were affected by cooling to 5° C and storage at this temperature for up to 72 hr. The acrosome changes involved swelling and vacuolation or, in a small proportion of spermatozoa, swelling and vesiculation of the outer acrosomal membrane. Mid-pieces showed condensation of mitochondria and loss of material. The inclusion of 3% (v/v) egg yolk in the diluent reduced the frequency of these changes.

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I. C. A. Martin and P. F. Watson

A large amount of wool on the face (face cover) of Merino sheep has been regarded as a fault by Australian sheep breeders as it has been associated with poor reproduction rates. Fail & Dun (1956) and Drinan & Dun (1967) reported that the degree of face cover was negatively correlated with fertility, but Young, Turner & Dolling (1963) found no evidence of a genetic association. In a review of these factors, Turner & Young (1969) commented that degree of face cover had been related to reduced reproduction rates in various breeds of sheep in Australia, New Zealand and the United States but not in the Soviet Union. The degree of face cover of individual ewes changes with season, and during pregnancy and lactation, with pregnant and lactating ewes showing less face cover than their non-pregnant contemporaries in the same flock (Mullaney, 1965). Physiological status of the ewe could therefore influence phenotypic assessment and may have contributed to the conflict of opinion on the importance of face cover as a criterion of selection.

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P. F. WATSON and I. C. A. MARTIN

The results from the insemination of frozen-thawed bull semen approach or equal those expected from natural mating, and contrast with the poor results obtained using frozen-thawed ram semen (Emmens & Robinson, 1962). Healey (1969), describing the ultrastructural changes in spermatozoa resulting from freezing, noted differences between species in the degree of acrosomal deterioration.

This report summarizes the results of experiments on the cooling and deep-freezing of ram and bull spermatozoa. The degree of change in the acrosomes was estimated by means of light microscopy after the specimens had been stained with Giemsa, using the following scoring system: 0, normal acrosome; 1 and 2, stages of acrosomal damage; 3, acrosome entirely lost. A typical spermatozoon in each of the categories is shown in Text-fig. 1.

Ejaculates from fifteen bulls were examined in one experiment and from twelve rams in a second experiment. Semen was diluted tenfold at 30° C and

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J. E. Norman, L. M. Ward, W. Martin, A. D. Cameron, J. C. McGrath, I. A. Greer and I. T. Cameron

The purpose of this study was to determine the relaxant effects in vitro of two nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, which are currently available for use in vivo, on contractions of non-labouring myometrium from pregnant women. Since nitric oxide also mediates relaxation by increasing the concentration of cGMP, sensitivity to 8-bromo-cGMP (a cGMP analogue) was also determined. The effects of the K+-channel opener lemakalim and of the Ca2+-channel blocker nifedipine were studied for comparison. After the addition of glyceryl trinitrate (0.1–100 μmol l−1), sodium nitroprusside (0.1–100 μmol l−1) or 8-bromo-cGMP (0.001–3 mmol l−1), the spontaneous rhythmic contractility of myometrial strips was inhibited in a concentrationdependent manner: the maximum inhibition produced by the highest tested concentration of each drug was 40 ± 7%, 53 ± 8% and 39 ± 8% of the original degree of contraction, respectively. Myometrial contractions were completely abolished by lemakalim and by nifedipine and verapamil at concentrations of ≥ 10−5 mol l−1. The nitric oxide donors, glyceryl trinitrate and sodium nitroprusside, attenuate myometrial contractions and are therefore useful as tocolytic agents. However, at equimolar concentrations in vitro, the ability of glyceryl trinitrate and sodium nitroprusside to attenuate myometrial contractions is less than that of lemakalin, nifedipine and verapamil. Controlled trials are required to determine the side-effects and clinical efficacy of each of these agents in vivo.

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T. D. QUINLIVAN, C. A. MARTIN, W. B. TAYLOR and I. M. CAIRNEY

Summary.

Estimates of pre- and perinatal loss in parous and non-parous 2½-year-old ewes which conceived to a second or third mating are described. This paper is a continuation of the study on losses in New Zealand Romney Marsh ewes that conceived to one service (Quinlivan, Martin, Taylor & Cairney, 1966).

The data presented show the estimated mean prenatal loss (excluding non-fertilization) for parous and non-parous ewes, that conceived to second service, to be 25·7%. From a consideration of lambs born dead the total estimated pre- and perinatal loss amounts to 47·9%.

The percentage of ewes exhibiting genital tract abnormalities is considered and shows that 1% and 6·6% of the parous ewes in the 1963 and 1964 observations respectively, and 6% and 15% of the non-parous ewes in the two observations revealed some abnormality at autopsy.