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The fluorescent membrane marker, 1-anilinonaphthalene-8-sulphonate (ANS) was used to investigate the attachment of egg-yolk to the plasma membranes of ram spermatozoa. The degree of fluorescence was assessed using a subjective scoring system. It was found that egg yolk competes with ANS for sites on the plasma membrane. When the diluent contained 10% egg yolk, no ANS could be detected on the membranes. Egg yolk attached to the plasma membrane could be removed by washing twice with a yolk-free diluent.

Loss of sperm motility in the presence of ANS was observed but some spermatozoa remained motile after incubation at 37°C for 15 min with 2 mm-ANS. Egg yolk protected spermatozoa against this loss of motility. It is suggested that egg yolk protects spermatozoa during chilling and freezing by its attachment to the sperm plasma membrane.

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P. F. Watson

Summary. Both the low-density lipoprotein fraction of egg yolk (LDF) and sonicated lecithin liposomes provided an equal measure of protection for ram spermatozoa during cold shock, but LDF was superior to lecithin during cold storage. The protective activity of LDF during storage at 5°C was not altered by a subfractionation procedure which did not alter the molecular organization. Removal of the protein from the surface of LDF particles gave preparations with altered lipid:protein ratios. Neither the low-lipid fraction nor bovine plasma albumin protected spermatozoa but the high-lipid fraction was as protective as LDF. Survival of spermatozoa decreased as the lipid:protein ratio fell below 1·67 compared with a ratio of 4·76 for LDF. The absolute lipid content was more important than the ratio except at low ratios. Lipid-depleted preparations bound more effectively to the plasma membrane than did LDF whereas the lipid-enhanced preparation showed little binding capability.

It is concluded that the phospholipid of LDF provides the protection to the sperm cell membrane. The protein of LDF serves to solubilize the lipid and bind it to the cell membrane. The importance of the role of the protein during cold storage is discussed.

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P. F. Watson

Wellcome Institute of Comparative Physiology, The Zoological Society of London, Regent's Park, London NW1 4RY, U.K.

Interest in the application of the techniques of artificial breeding to non-domesticated mammals has been stimulated in recent years by the failure of some species to breed in captivity and by the decreasing availability of animals from the wild. Several authors have drawn attention to the need to develop techniques suitable for exotic animals (Jones, 1971;Francoeur, 1972; Rowlands, 1965,1974; Warner, Martin & Keeling, 1974), but as yet very few species have been studied.

A brindled gnu (Connochaetes taurinus) at The Zoological Society of London was sedated with Xylazine (Rompun, Bayer U.K. Ltd) and semen was collected by electroejaculation using finger ring electrodes and a sine wave stimulator (Watson & Hime, 1976). Stimulation for periods of 5 sec with 5-sec rest periods was commenced with a frequency of 10-15 Hz and a peak voltage of

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L. Robertson and P. F. Watson

Summary. Ram semen was subjected to various dilution rates and temperatures of dilution, and was also subjected to slow cooling and rewarming. Calcium ion movements across sperm membranes were measured using the radioisotope 45Ca2+. It was shown that even 2- to 4-fold dilution caused an increase in intracellular calcium content. An increase in intracellular calcium also occurred in proportion to a decreased temperature of dilution.

After an initial increase in intracellular calcium content, spermatozoa appeared able to restore a low intracellular calcium level over a period of 2 h at 22°C or above. This ability was lost at 16°C or below. However, if undiluted semen was slowly cooled (0·125°C/min) even to 5°C and rewarmed to 22°C before dilution, the spermatozoa regained this ability. In contrast, spermatozoa rapidly cooled to 5°C and rewarmed to 22°C before dilution were not able to restore the low intracellular calcium level.

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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 W. J. Anderson

Summary. Butylated hydroxytoluene (BHT) significantly reduced the degree of acrosome damage which occurred to ram spermatozoa during cold shock. A higher percentage of spermatozoa were motile after cold shock in the presence of BHT than in its absence, but it had little effect on the quality of motility or the percentage of cells which stained with eosin. A concentration of 2–4 mm-BHT provided the maximum response. No advantage was gained by using the solvent, dimethyl sulphoxide, as a vehicle to introduce BHT to the cells. An osmotic stress test after cold shock also failed to demonstrate any advantage of BHT. It is concluded that BHT provides little protection to the functional capacity of ram spermatozoa undergoing cold stress and is unlikely to be of benefit for the preservation of ram semen.

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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. M. Plummer and P. F. Watson

Summary. Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate—osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits.

In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock.

Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.

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M. R. Curry, J. D. Millar, and P. F. Watson

Ram and human spermatozoa have a high coefficient of osmotic water permeability (Pf) with a low activation energy (Ea), suggesting the presence of water channels within their plasma membranes. Sperm membranes were examined for the presence of two known water channel proteins, CHIP28 and glucose transporters belonging to the GLUT family of proteins. The water permeability of ram spermatozoa was not inhibited by mercuric chloride to which the CHIP28 channel is sensitive. The CHIP28 protein was not located in western blots of ram sperm membrane preparations that used an anti-CHIP28 antibody. The water permeability of ram and human spermatozoa was inhibited in the presence of phloretin, an inhibitor of glucose transport. Rabbit spermatozoa, which have a low Pf and a high Ea value, suggesting a non-porous membrane, were unaffected by phloretin. These results indicate that the erythrocyte and proximal tubule water channel, CHIP28, is not present in sperm membranes but that sperm membrane glucose transporters may have a secondary water channel function.

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L. M. Thurston, P. F. Watson, and W. V. Holt

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace–Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0–22.0 μm, 22.1–73.0 μm and 73.1–130.0 μm). The Landrace–Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1–130 μm long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean ± sd; 66.36 ± 24.70 μm) to the Landrace–Meishan introgression boar (mean ± sd; 67.09 ±21.80 μm). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.