THE USE OF MONOSACCHARIDES, DISACCHARIDES, AND TRISACCHARIDES IN SYNTHETIC DILUENTS FOR THE STORAGE OF RAM SPERMATOZOA AT 37°C AND 5°C By K. R. LAPWOOD* and 1. C. A. MARTIN* [Manuscript received January 19, 1966] Summary Using synthetic semen diluents based on 20 mM phosphate buffer, 31 mM N aCl, 0·8 % non.dialysable skim milk solids, plus antibiotics, and 185 mM of the sugars ribose, arabinose, xylose, glucose, mannose, fructose, galactose, rhamnose, maltose, lactose, sucrose, or raffinose, it was found that ram spermatozoa survive best at 37°C in diluents containing glucose, mannose, fructose, or sucrose; however, at 5°C ribose, arabinose, xylose, and galactose were the sugars of choice. Increasing replacement of fructose in the diluent with up to 185 mM of these sugars resulted in increased survival at the respective temperatures. Part replacement of 185 mM fructose in the range 7·75-62 mM with the sugars most favourable at 5°C was of little benefit. No effect of changes in osmotic pressure was noted using varying concentrations of the sugars to give tonicities of 0·9, 1· 0, and 1 ·1 relative to 154 mM NaCI or 308 mM sugar. Increased motility scores and percentages of motile sperma· tozoa were observed when 17 mM fructose was added to ribose and arabinose diluents at 5°C, but not when added to diluents containing xylose, the hexoses, galactose, fructose, and glucose; nor for any sugar· containing diluent at 37°C. 1. INTRODUCTION Since the earlier work of Emmens and Blackshaw (1950), who found that the addition of sugars, particularly pentoses, improved revival of mammalian sperma­ tozoa after deep-freezing, viability and fertility studies have shown that the addition of various sugars to semen diluents improve spermatozoal survival at temperatures of approximately 37 and 5°C for the bull, ram, and cock. The most effective sugar varies with both storage temperature and species. Work with cock spermatozoa has shown that the hexoses are beneficial in semen diluents. Wales and White (1958a) found that with high pH levels, partial replacement of sodium chloride by glucose improved motility in hypotonic, isotonic, and hypertonic diluents at room temperature. Wilcox and Schaffner (1958) found that addition of 2 mg/ml fructose to a phosphate buffer diluent after storage at 10°C improved fertility, while Wilcox (1959) found that addition of 0 ·OllM fructose, glucose, mannose, and galactose, when added to phosphate buffer diluents, increased motility and fertility after 2 days at 10°C; the pentoses and disaccharides tested had no effect on motility. Subsequent work by Wilcox (1960) showed that increases in fertility when 2 or 4 mg/ml of fructose or glucose were added to a storage diluent * Department of Veterinary Physiology, University of Sydney. At!st. J. Biol. Sci., 1966, 19, 655-71 656 K. R. LAPWOOD AND 1. C. A. MARTIN containing egg white were not consistent. Several workers have found an increase in motility or fertility or both when glucose was added to, or partially replaced, diluents containing egg yolk for the storage of bull semen at temperatures from 46·5 to 5°C (Salisbury and Van Demark 1946; Ohms and Willett 1958; Foote and Bratton 1960). Under some conditions replacement of electrolytes by sugars in semen diluents has led to increased survival of spermatozoa (Emmens 1948; Kampschmidt, Mayer, and Herman 1953; Ohms and Willett 1958; Wales and White 1958a). Replacement of part or all of the sodium chloride content (123 mlVI) by lactose in a phosphate­ buffered saline diluent improved the survival of spermatozoa stored at 37 and 5°C, the optimum proportion of sugar and salt being 31 mlVI sodium chloride with 185 mlVI lactose (Martin 1966a, and unpublished data). Further experiments showed that although there was no difference in survival of spermatozoa in diluents containing 185 mlVI of fructose, glucose, lactose, or sucrose for storage at 5°C for 4 days, when fructose and lactose were compared as diluents at this temperature for storage for 6 days, fructose was just superior. However, later results showed that at this con­ tent of sugar, lactose was better than fructose as a component of a diluent for the deep-freezing of ram spermatozoa. Jones and Martin (1965) then demonstrated, in a factorial experiment comparing milk, yolk-citrate, and synthetic diluents con­ taining lactose or fructose, that although lactose was as satisfactory as milk for the preservation of spermatozoa during deep-freezing, spermatozoa frozen in the lactose diluent had to be resuspended in the fructose synthetic diluent for incubation at 37°C subsequent to thawing if survival rates were to remain as high as in those samples frozen and incubated in milk. Investigations reviewed by Mann (1964) show that spermatozoa can glycolyse the hexoses fructose, glucose, and mannose. White, Blackshaw, and Emmens (1954), and O'Dell, Almquist, and Flipse (1959) presented evidence that spermatozoa can oxidize arabinose. Vantienhoven et al. (1952) showed that bull spermatozoa meta­ bolize glucose in preference to fructose when incubated at 46· 5°C, while Lorenz (1958) found that cock spermatozoa convert glucose to fructose when incubated at 40°C. Choong and Wales (1963), Jones and Martin (1965), Jones (1965), and Martin (unpublished data) have shown the value of adding non-dialysable skim-milk solids to semen diluents. The present investigations were designed to look more fully into the use of sugars in synthetic diluents for storage of ram semen at 37 and 5°C, to find the most desirable sugar or sugar combination at these temperatures; the effect of varying tonicity; and the need to add fructose for metabolic purposes when it is not already present in the diluent. II. MATERIALS AND METHODS Semen was collected by electrical stimulation using a bipolar electrode (Black­ shaw 1954) and only samples of good motility were used. Except in experiment 5 unwashed spermatozoa were used, and dilution at the rate of approximately 70-fold was carried out at 37°C within 20 min of collection. In experiment 5 semen was twice washed, at a tenfold dilution, to remove seminal DILUENTS FOR RAM SPERMATOZOA 657 fructose, using a solution of 123 mM NaCl and 20 mM NaH2P04-Na2HP04 buffer, the supernatants being removed after centrifuging at 1500 r.p.m. (c. 300 g) for 7 min. Washed semen was then made up to its original volume with the washing diluent, before being diluted as above. In experiments at 5°C the diluted semen was cooled from 37 to 5°C in a refrigerated cabinet over a period of 2 hr, and was stored there for the duration of the experiment, which was 6 days except in experiments 1 and 5, where the periods were 8 and 3 days respectively. Semen in experiments at 37°C was incubated at this temperature in a warm bath, the experiments continuing for 6 hr except in experi­ ments 4 and 5, which lasted for 8 and 3 hr respectively. Thin films of diluted semen prepared between a microscope slide and coverslip were scored for motility (scale 0--4, Emmens 1947) and percentage of motile sperma­ tozoa. A low-power microscope fitted with a warm stage at 37°C was used. Diluents in these experiments consisted of 185 mM solution of one or more sugars (except in experiment 2 where the effect of varying this concentration was investigated), 31 mM NaCl, and 20 mM NaH2P04-Na2HP04 buffer. Non-dialysable milk solids (0·8% w/v), and antibiotics (500 i.u. penicillin/ml and 500 i.u. strepto­ mycin/ml) were present in all diluents, and fructose at the rate of 17 mM was included in the diluents in some experiments as explained below. The type and content of the sugars used in each experiment are shown in the tables of results. Four replicates were performed of each experiment, ejaculates from different rams being used for each replicate. Statistical Analyses Analyses of variance were performed on motility indices, and percentages of motile spermatozoa after angular transformation, for the final set of scores made in each experiment, except in experiment 1 at 5°C where results for both 6 and 8 days were analysed. A summary of the analyses of the data of experiment 1 is shown in Table 2. Contrasts between treatments were made using sets of orthogonal coefficients and Table 3 illustrates those used in experiment 1. A similar set of coefficients has been published (Martin 1965), and those used in the later experiments in this paper were designed on the same principles as these examples. The contrasts were designed to show differences between groups of sugars, and individual sugars within these groupings. For example, in Table 3 the groups are pentose, hexose, disaccharide, and trisaccharide, and the effect of the individual sugar is tested within each group. From these contrasts a ranking can be made of the effect of the sugars in maintaining activity of spermatozoa at 37 and 5°C. In Table 2 significant effects are shown, but to shorten the tables non-significant effects were pooled and termed "remainder". For experiments 2,3,4, and 5 mean scores of motility and percentage of motile spermatozoa for those contrasts which were significant for one or more scoring criteria are shown in Tables 5, 7, 9, and 11, respectively. Full details of the original analyses are available from the authors. For experiment 2 separate non­ orthogonal comparisons were also made to contrast the results obtained from using a particular sugar in a diluent with those obtained from diluents in which fructose alone was used, and these are described in detail in the results. 658 K. R. LAPWOOD AND 1. C. A. MARTIN III.