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Previous studies have shown that stored ram semen (ram spermatozoa kept in contact with seminal plasma for 4½ hr at 30° C) is less likely to cause conception when inseminated into a ewe than fresh semen with a lower impedance change frequency (Dott, 1961). In the same paper it was shown that epididymal semen could be stored at 30° C and ejaculated semen at 20° C for 4½ hr without causing a serious drop in the conception rate obtained with them. It could be concluded from these experiments that some factor in the seminal plasma adversely affected the fertilizing ability of ram spermatozoa. This effect is dependent on time and temperature but it is not known whether it is dependent on the continued presence of seminal plasma. Ewes have been inseminated with ejaculated ram spermatozoa separated from the

Summary.

Bovine seminal plasma was separated into a number of fractions by dialysis, ethanol extraction or gel chromatography. Bovine spermatozoa suspended in the non-dialysable fraction or the ethanolinsoluble fraction or in fractions either excluded from, or retained by, the Sephadex gel, all reduced the impedance change frequency and viability of the spermatozoa. The fractions separated by gel chromatography also caused the spermatozoa to use more glucose and rendered them eosinophilic sooner than spermatozoa suspended in Ringer or in fresh whole seminal plasma.

The stereoscan electron microscope (Cambridge Instrument Co.) enables the surface of objects to be examined over a wide range of magnifications, but the object must be carefully washed to remove substances which might obscure its outlines, and when the object is biological the substances washed away may be an important part of the surface.

With this limitation in mind, samples of ejaculated bull and rabbit, and testicular ram spermatozoa were prepared in the following way: (1) Seminal plasma (or testicular fluid) was removed from semen by centrifuging at 20,000 g for 20 min. (2) Spermatozoa were resuspended in 40·0 ml phosphate-buffered saline (0·125 m-NaCl, 0·02 m-phosphate buffer, pH 7·4) and centrifuged at 1000 g for 10 min; the supernatant was removed. This was repeated four times. (3) The spermatozoa were fixed by adding 0·1 ml of sperm suspension to 0·9 ml 2·5% glutaraldehyde at pH 7·0 and keeping at 4·0° C for 1 hr. (4) The fixative was removed by centrifuging at 1000 g for 10 min. The spermatozoa were resuspended in distilled water. This process was repeated twice. (5) A drop containing fixed spermatozoa in distilled water was allowed to evaporate on a glass cover slip and examined in vacuo with the stereoscan microscope.

Summary.

The proportion of motile spermatozoa in bull semen diluted with phosphate-buffered saline, citrate or milk has been estimated both photographically and with an image-analysing computer. The two methods produced similar estimates. The proportion of noneosinophilic spermatozoa was higher than the proportion of immotile spermatozoa, particularly when fluoride or formaldehyde was added to the diluents. The proportion of non-eosinophilic spermatozoa in diluents containing formaldehyde was the same as in the original semen.

Summary. By 3 weeks after castration the majority of spermatozoa in the ligated cauda epididymidis were dead and decapitated and there was a 46% reduction in the total number of spermatozoa. By 5 weeks after castration >99% of spermatozoa had disappeared. Electrophoresis of cauda epididymal plasma (CEP) on polyacrylamide gels revealed that after castration the protein composition of CEP changed from a pattern specific to the epididymis to one similar to blood serum. These changes in the luminal content could be prevented by administering testosterone. When spermatozoa from the cauda epididymidis were diluted in CEP from normal rabbits they remained motile for several hours at room temperature. Dilution in CEP from 3- or 5-week castrated animals stimulated vigorous forward motility but this declined to very low levels after 40 min. The evidence indicates that dead spermatozoa can be removed from the epididymis after castration by mechanisms not involving granuloma formation.

Summary.

A 'flow dialysis' technique is described which enables spermatozoa to be maintained in an active state for prolonged periods of time, under conditions of constant temperature, gaseous partial pressure, pH, osmotic pressure and sperm density. The impedance-change frequency is used to assess sperm activity. The effects of centrifugation, dilution and washing of ram spermatozoa on their activity have been studied. Washing has been shown to produce a detrimental effect on sperm activity. This effect was most pronounced when a high degree of dilution was employed at each stage of the washing procedure. Washing produced a greater decrease in the activity due to aerobic endogenous respiration than in that due to anaerobic fructolysis. Other effects of washing included an increase in the proportion of 'eosinophilic' spermatozoa and an agglutination of motile spermatozoa. An attempt was made to assess the efficiency of the washing procedure by the use of fructose, [³²P]phosphate and iodinated [¹³¹I]protein as indicators. The effect of dilution on spermatozoa depends on at least three factors: (i) the degree of dilution, (ii) the rate of dilution, and (iii) the composition of the diluent.

Summary.

The continuous flow dialysis apparatus (cfda) has been used to investigate the importance of potassium for the maintenance of the activity of ram spermatozoa.

Ram spermatozoa contain 136 mg K+/100 g and the seminal plasma 78 mg K+/100 g. When semen is diluted one in three with a phosphate diluent, there is a rapid fall in the potassium concentration within the spermatozoa. Dialysis in the cfda causes a further exponential fall unless potassium is included in the dialysis fluid. Unless the phosphate buffer used in the cfda contains potassium ions, there is a sudden decrease in the impedance-change frequency (icf) between 3 and 5 hr from the start of dialysis. Washing or keeping unwashed spermatozoa for 2 hr prior to dialysis does not alter this response or the time of dialysis at which it takes place.

Semen diluted one in three with fresh seminal plasma or with plasma obtained from stored semen gave the same response (fall in icf). The decline in icf due to the absence of potassium was less rapid when undiluted semen was used.

Semen washed with a bicarbonate buffer had a lower initial icf than semen washed with phosphate, but the icf declined more slowly, and in the absence of potassium there was no sudden drop in the icf. Samples with potassium maintained a higher icf than those without. The most marked potassium effect was obtained under aerobic conditions using a phosphate fluid containing fructose, lactate or acetate. Under anaerobic conditions the presence of potassium ions in the dialysis fluid had little or no effect on icf.

A.R.C. Unit of Reproductive Physiology and Biochemistry, University of Cambridge, U.K.

A defect of the sperm acrosome, called 'knobbing' has been associated with sterility in the bull (Teunissen, 1946; Blom, 1948; Hancock, 1952; Donald & Hancock, 1953; Blom & Birch-Andersen, 1962; Blom, 1972), and boar (Bane, 1961), and has been seen in stallion (Dott, 1975), ram, dog and impala (unpublished).

Donald & Hancock (1953) came to the conclusion that this was an autosomal sex-linked recessive characteristic in Friesian bulls. At the level of the light microscope it is seen as an apical, often eccentric swelling which folds back over the head. Blom & Birch-Andersen (1962) described this character as a swelling of the apical body to six to eight times normal. Much of the region was cystic and contained vesicles with inclusions. The ultrastructure of this defect in Friesian bulls is described in the present paper.

Semen was collected in

Summary.

Epididymal contents were collected from a bull with an epididymal fistula and from normal bulls after slaughter. When this material was diluted with seminal plasma collected by means of an artificial vagina from either the bull with a fistula or from normal bulls, the impedance change frequency due to the motility of the spermatozoa was reduced as was the length of time for which it could be detected. The fluid obtained from the penis of the bull during electric stimulation with a low voltage did not produce the same effect. The latter fluid had no fructose, a low protein content and a different pattern of inorganic ion concentration from the seminal plasma; the agar gel electrophoresis pattern was also different.

Dilution of epididymal contents with normal seminal plasma did not affect the morphology of spermatozoa or their response to differential staining. Furthermore, neither these characteristics nor the impedance change frequency were affected by a mild degree of temperature shock produced by sudden cooling from 20° C to 10° C.

The purpose of a differential stain is to colour the cells concerned in such a way that two populations can be distinguished from each other and from the background. This is certainly true of staining mixtures used to differentiate `live' and `dead' mammalian spermatozoa. In practice, the distinction is between eosinophilic and non-eosinophilic cells with a secondary stain (e.g. nigrosin) which appears only to provide a background.

The degree of background staining can be varied from none to intense by varying the concentration of the secondary stain. At very low concentration, the non-eosinophilic spermatozoa tend to merge with the background, their outline is difficult to distinguish clearly and, consequently, they may be overlooked in a count. As the concentration of the background stain is increased,