THE USE of MONOSACCHARIDES, DISACCHARIDES, and TRISACCHARIDES in SYNTHETIC DILUENTS for the STORAGE of RAM SPERMATOZOA at 37°C and 5°C by K

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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. RESULTS Experiment 1 tested the replacement of 62 mM and 123 mM of the fructose content (185 mM) of the diluent with an equimolar amount of each of the 11 sugars: ribose, arabinose, xylose, glucose, mannose, galactose, rhamnose, maltose, lactose, sucrose, and raffinose. At 5°C the effect of time of incubation was investigated. Results, analyses of variance, and orthogonal coefficients are shown in Tables 1, 2, and 3, respectively.

TABLE 1 EXPERIMENT 1: MEAN MOTILITY AND MEAN PERCENTAGE OF MOTILE SPERMATOZOA

37°C 5°C Fructose Sugar Diluent Percentage Concn. Sugar Concn. Motility No. Percentage Motile (mM) (mM) Motility Motile 6 Days 18 Days 6 Days 8 Days

1 185 - - 2·00 30·0 2·75 1·87 47·5 40·0 2 185 - - 1·87 27·5 2·75 2 ·12 55·0 42·5 3 123 Ribose 62 1·37 22·5 2·87 2·37 52·5 42·5 4 62 Ribose 123 1·87 30·0 3·12 2·87 57·5 52·5 5 123 Arabinose 62 1·62 28·75 2·37 2·00 45·0 37·5 6 62 Arabinose 123 1·37 16·25 2·87 2·00 47·5 35·0 7 123 Xylose 62 1·75 27·5 2·75 2·37 55·0 45·0 8 62 Xylose 123 1·75 25·0 2·87 2·62 52·5 45·0 9 123 Glucose 62 2·12 30·0 1·37 0·50 20·0 7·5 10 62 Glucose 123 2·37 42·5 1·12 0·37 17·5 5·0 11 123 Mannose 62 1·62 27·5 2·00 1·25 31·25 20·0 12 62 Mannose 123 1·75 25·0 2·50 1·00 42·5 20·0 13 123 Galactose 62 2·00 27·5 2·37 2·25 42·5 40·0 14 62 Galactose 123 1·37 26·25 3·00 2·37 47·5 40·0 15 123 Rhamnose 62 1·75 27·5 2·25 2·00 37·5 30·0 16 62 Rhamnose 123 1·75 28·75 2·25 1·87 37·5 30·0 17 123 Maltose 62 0·87 15·0 2·62 1·50 42·5 27·5 18 62 Maltose 123 0·75 12·5 2 ·12 1·25 40·0 30·0 19 123 Lactose 62 2·12 31·25 2·62 2·12 45·0 30·0 20 62 Lactose 123 2·12 32·5 2·12 1·62 32·5 23·75 21 123 Sucrose 62 2·00 27·5 1·62 1·12 28·75 18·75 22 62 Sucrose 123 2·12 27·5 1·87 1·12 30·0 18·75 23 123 Raffinose 62 2·37 35·0 2·37 1·87 40·0 30·0 24 62 Raffinose 123 1·75 28·75 2 ·12 1·00 30·0 15·0 - Overall, the mean survival of spermatozoa at 37°C in the sugar diluents tested did not differ from that in the control diluents containing only fructose, but some individual sugars did have effects on the survival of spermatozoa. At 37°C significant results were: (1) Glucose was superior to mannose for maintaining both motility and per centage of motile spermatozoa. (2) Lactose and sucrose diluents maintained motility and percentage of motile spermatozoa at higher levels than did maltose diluents (contrast K, Table 3). DILUENTS FOR RAM SPERMATOZOA 659

At 5°C significant results were: (1) Largely due to poor results observed in glucose, mannose, and sucrose diluents, the percentage of motile spermatozoa was higher in the diluent containing fructose alone, than in the mean response calculated from all other diluents, and diluents containing pentoses were superior to those containing hexoses (contrasts A and B, Table 3). (2) The apparent superiority of mono- to disaccharides (contrast D, Table 3) was due to the beneficial effects of the pentoses.

TABLE 2

SUMMARY OF ANALYSES OF VARIANCE OF DATA IN EXPERIMENT

Degrees Variance Ratios, 37°C Variance Ratios, 5°C Source of Variation of Freedom Motility % Motile Motility % Motile

Sugars (a) Between sugars Fructose v. rest (A) 1 0·88 0·38 3·60 9'47** Pentoses v. hexoses (B) 1 3·34 1·29 34'65*** 42·75*** Monosaccharides v. di- and trisaccharides (D) 1 0·02 0·14 7'45** 7'07** Glucose v. mannose (G) 1 4'60* 4'43* 10'90** 14'12*** Glucose and mannose v. galactose (H) 1 1·55 0·69 31'10*** 28'54*** Glucose, mannose, and galactose v. rhamnose (I) 1 0-34 0-50 3-99* 3-00 Lactose v_ sucrose (J) 1 0-05 0·54 7-23** 3-79 Lactose and sucrose v. maltose (K) 1 31·84*** 13-44*** 0-18 1-65 Remainder 3 1-44 1-17 1-34 1-11 (b) Levels of replacement within sugars 12 0·74 0-68 0-58 0·80 Ejaculates 3 33-63*** 35-51*** 12'92*** 15-85*** Sugar X ejaculates 69 I-lOt 55-94t 2·09t 103 -28t Time 1 28'62*** 21-13*** Time X sugars 23 0-32 2 -18** Time X ejaculates 3 0-24 0-30 Time X sugars X ejaculates 69 2-69t 133-88t * P<0-05. ** P

(3) Galactose-containing diluents were better than mannose diluents, which in turn were superior to those containing glucose (contrasts G and H, Table 3). Although contrast I of Table 3 shows that the motility score of spermatozoa in rhamnose diluents was better than the mean score in diluents containing the above three sugars, in fact the scores in galactose diluents were slightly higher than those in rhamnose diluents. (4) Motility scores in lactose diluents were higher than in sucrose diluents_ (5) There was a significant decline of both motility and percentage of motile spermatozoa from 6 to 8 days_ For percentage of motile spermatozoa the interaction of time and sugar is significant, much of the variance of this 0> o0>

TABLE 3 ORTHOGONAL COEFFICIENTS USED FOR BETWEEN-SUGAR CONTRASTS IN ANALYSIS OF VARIANCE OF DATA IN EXPERIMENT 1 P1

~ Diluent No. Contrast 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 o~ o A Fructose v. rest +11 +11 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 tI B Pentoses v. hexoses o 0 +4 +4 +4 +4 +4 +4 -3 -3 -3 -3 -3 -3 -3 -3 0 0 0 0 0 0 0 0 o Disaccharides v. trisaccharide o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +1 +1 +1 +1 +1 +1 -3 -3 D Monosaccharides v. di- and trisaccharides o 0 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 -7 -7 -7 -7 -7 -7 -7 -7 ~ t-t E Ribose v. xylose o 0 +1 +1 0 0 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 F Ribose and xylose v. arabinose o 0 +1 +1 -2 -2 +1 +1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 G Glucose v. mannose o 0 0 0 0 0 0 0 +1 +1 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 !> H Glucose and mannose v. galactose o 0 0 0 0 0 0 0 +1 +1 +1 +1 -2 -2 0 0 0 0 0 0 0 0 0 0 I Glucose, mannose, and galactose v. rhamnose o 0 0 0 0 0 0 0 +1 +1 +1 +1 +1 +1 -3 -3 0 0 0 0 0 0 0 0 J Lactose v. sucrose o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +1 +1 -1 -1 0 0 K Lactose and sucrose v. maltose 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 I o +2 +2 -1 -1 -1 -1 DILUENTS FOR RAM SPERMATOZOA 661

interaction being due to mannose diluents in which there was a mean decline in motile spermatozoa of 16· 87%, considerably higher than in the other diluents. The effect of addition of 31, 62, and 93 mM each of the 11 sugars under test, to diluents containing 123 mM fructose, to give total sugar concentrations of 154, 185, TABLE 4 EXPERIMENT 2: MEAN MOTILITY AND MEAN PERCENTAGE OF MOTILE SPERMATOZOA IN A DILUENT CONTAINING 123'mM FRUCTOSE AND THE LEVELS OF SUGAR SHOWN BELOW

37°C 5°C Test Diluent Sugar Test Sugar No. Concn. Percentage Percentage Motility Motility (mM) Motile Motile

1 Ribose 31 1·87 32·5 2·00 40'0 2 Ribose 62 1·50 25·0 2·00 42·5 3 Ribose 93 1·62 27·5 2·25 42·5 4 Arabinose 31 2·00 37·5 2·12 42·5 5 Arabinose 62 1·75 27·5 2·25 47·5 6 Arabinose 93 1·75 27·5 2·12 47·5 7 Xylose 31 2·25 37·5 2·00 40·0 8 Xylose 62 2·12 35·0 1·87 40·0 9 Xylose 93 1·87 31·25 2·00 37·5 10 Glucose 31 2·50 40·0 1·00 17·5 11 Glucose 62 2·37 37·5 1·00 23·75 12 Glucose 93 2·75 45·0 1·00 16·25 13 Mannose 31 2·37 40·0 1·62 32·5 14 Mannose 62 2·37 37·5 1·37 25·0 15 Mannose 93 2·25 35·0 1·50 30·0 16 Galactose 31 1·87 30·0 2·12 47·5 17 Galactose 62 2·00 27·5 2·12 45·0 18 Galactose 93 2·12 35·0 1·87 42·5 19 Rhamnose 31 1·87 32·5 2·37 45·0 20 Rhamnose 62 2·12 35·0 2·50 37·5 21 Rhamnose 93 2·12 37·5 1·50 27·5 22 Maltose 31 1·50 25·0 1·87 42·5 23 Maltose 62 1·00 12·5 1·75 40·0 24 Maltose 93 0·25 5·0 2·00 42·5 25 Lactose 31 2·50 40·0 1·87 40·0 26 Lactose 62 2·12 35·0 1·87 42·5 27 Lactose 93 2·37 37·5 2·12 40·0 28 Sucrose 31 2·75 37·5 2·12 47·5 29 Sucrose 62 2·87 50·0 2·00 35·0 30 Sucrose 93 2·62 47·5 2·12 37·5 31 Raffinose 31 2·50 42·5 2·12 42·5 32 Raffinose 62 2·75 47·5 2·12 42·5 33 Raffinose 93 2·37 37·5 1·75 42·5 34 Fructose 62 2·30 37·5 1·83 33·3 and 216 mM, and theoretical relative tonicities of 0'90, 1'00, and l·lO, respectively, was then investigated in experiment 2. Results and mean scores for significant contrasts are shown in Tables 4 and 5. 662 K. R. LAPWOOD AND 1. C_ A_ MARTIN

Significant results at 37°0 were: (1) Both scoring criteria showed that the hexoses tested were superior to the pentoses for maintaining spermatozoal activity (contrast A, Table 5)_ (2) Motility scores were higher in xylose than in ribose and arabinose-containing diluents (contrast D, Table 5)_

TABLE 5

COMPARISONS OF SURVIVAL RATES OF SPERMATOZOA WHICH WERE SIGNIFICANTLY DIFFERENT IN ONE OR MORE SCORING CRITERIA IN EXPERIMENT 2

Mean Scores at 37°C Mean Scores at 5°C

Contrast Treatment Percentage Percentage Motility Motility Motile Motile

A Pentoses 1-S6 31-25 2-07 42-2 Hexoses 2-22*** 36-0* 1-66*** 32-5*** B Disaccharides 2-00 32-2 1-97 40-S Trisaccharide 2-54*** 42-5*** 2-00 42-5 G Monosaccharides 2-07 34-0 1-S4 36-7 Di- and trisaccharides 2-13 34-S 1-9S* 41-25** D Ribose and arabinose 1-74 29-6 2-12 43-75 Xylose 2-0S* 34-6 1-96 45-S E Glucose 2-54 40-S 1-00 19-2 Mannose 2-33 37-5 1-50** 29-2** F Glucose and mannose 2-43 39-2 1-25 24-2 Rhamnose 2-02** 35-0 2-12*** 36-7*** G Glucose, mannose, and rhamnose 2-30 37-S I-54 2S-3 Galactose 2-00* 30-S* 2-04*** 45-0*** H Lactose 2-33 37-5 1-95 40-S Sucrose 2-75* 45-0* 2-0S 40-0 I Lactose and sucrose 2-54 41-25 2-02 40-4 Maltose 0-92*** 14-2*** 1-S7 41-7 J Rhamnose, 31 mM 1-S7 32-5 2-37 45-0 Rhamnose, 93 mM 2-12 37-5 1-50*** 27-5* K Maltose, 31 mM I-50 25-0 1-S7 42-5 Maltose, 93 mM 0-25*** 5-0*** 2-00 42-5 - * P<0-05_ ** P

(3) Both indices of spermatozoal activity indicated that glucose, mannose, and rhamnose diluents were superior to those containing galactose (contrast G, Table 5), while motility scores were better maintained by glucose and mannose than by rhamnose (contrast F, Table 5)_ (4) Sucrose diluents were superior to those containing lactose, which in turn were better than those containing maltose (contrasts H and I, Table 5)_ (5) Increasing the concentration of maltose and the tonicity, decreased the motility and percentage of motile spermatozoa, and this was the main reason for the inferiority of the disaccharide-containing diluents as a whole, compared to raffinose diluents (contrasts K and B, Table 5)_ DILUENTS FOR RAM SPERMATOZOA 663

Separate tests of significance showed that diluents containing 62 mM ribose plus 123 mM fructose were inferior to those containing 185 mM fructose, for the maintenance of motility [t(14) = 3·23, P < 0·01]. Also diluents containing 62 mM maltose together with 123 mM fructose were inferior to those containing 185 mM fructose for maintaining both motility [t(14) = 4·06, P < 0·01], and percentage of motile spermatozoa [t(14) = 3·70, P < 0·01]. Significant results at 5°C were: (1) Contrasts E, F, and G in Table 5 indicate that the order of protectionj preservation of spermatozoa by hexoses was galactose followed by rham nose, mannose, and glucose; however, motility scores were maintained at a slightly higher level in rhamnose than in galactose diluents. (2) Low scores in diluents containing glucose and mannose were the main reason for the superiority on average of the pentoses over the hexoses (contrast A, Table 5). (3) Increasing the level of rhamnose from 31 to 93 mM resulted in lower scores for both motility and percentage of motile spermatozoa (contrast J, Table 5). TABLE 6 EXPERIMENT 3: MEAN MOTILITY AND MEAN PERCENTAGE OF MOTILE SPERMATOZOA

Sugar 37°C 5°C Diluent (cono. of each No. 185 mM) Percentage Percentage Motility Motility Motile Motile

1 Fructose 2·37 47·5 1·50 30·0 2 Ribose 1·25 30·0 2·50 47·5 3 Arabinose 2·00 37·5 2·37 45·0 4 Xylose 2·37 42·5 2·25 40·0 5 Glucose 2·75 47·5 0·62 8·75 6 Mannose 2·37 42·5 1·25 21·25 7 Galactose 2 ·12 42·5 2·00 37·5 8 Rhamnose 2·12 42·5 1·87 40·0 9 Maltose 1·37 30·0 1·37 31·25 lO Lactose 1·75 30·0 1·37 20·0 11 Sucrose 2·37 40·0 1·37 22·5 12 Raffinose 1·75 30·0 1·12 20·0 -- -- - Separate tests of significance using the motility scores showed that the diluents containing 185 mM fructose were inferior to those containing 123 mM fructose plus 62 mM of the sugars: arabinose [t(14) = 2·43, P < 0·05]; galactose [t(14) = 2·62, P < 0·05] ; ribose [t(14) = 2 ·18, P < 0·05] ; and lactose [t(14) = 2 ·18, P < 0·05]. However, the diluent containing 123 mM fructose plus 62 mM glucose was inferior, in terms of percentage of motile spermatozoa, to that containing 185 mM fructose rt(14) = 3·35, P

Significant results at 37°C were: (1) Generally survival of spermatozoa in hexose-containing diluents was superior to that in diluents containing 185 mM of a pentose (contrast A, Table 7), and the results from the use of monosaccharides were better than in diluents based on di- and trisaccharides (contrast B, Table 7). (2) Xylose diluents were better for maintaining motility than those containing arabinose, which were superior to those based on ribose (contrasts C and D, Table 7).

TABLE 7

COMPARISONS OF SURVIVAL RATES WHICH WERE SIGNIFICANTLY DIFFERENT IN ONE OR MORE SCORING CRITERIA IN EXPERIMENT 3

Mean Scores at 37°C Mean Scores at 5°C

Contrast Treatment Percentage Percentage Motility Motility Motile Motile

--- 1 Pentoscs 1·87 36·7 2·37 44·2 Hexoses 2·35** 44'5* 1'45*** 27'5*** B Monosaccharides 2·17 41·6 1·79 33·75 Di- and trisaccharides 1'81** 32'5** 1'31*** 23'4** (} Ribose 1·25 30·0 2·50 47·5 Arabinose 2'00* 37'0 2·37 45·0 D Ribose and arabinose 1·62 33·75 2·43 46·25 Xylose 2'37** 42·5 2·25 40·0 E Glucose 2·75 47·5 0·62 8·75 Mannose 2·37 42·5 1'25* 21·25 F Glucose and mannose 2·56 45·0 0·93 15·0 Galact.ose 2 ·12 42·5 2'00*** 37'0*** G Glucosc, mannose, and galactose 2·41 44·2 1·29 22·5 Rhamnose 2·12 42'0 1'87* 40'()** 11 Maltose and lactoso 1·56 30·0 11' 37 25· (j Sucrose 2'37** 40·0 1·37 22·5 * P<0·05. ** P

(3) Diluents which contained sucrose were better than maltose and lactose diluents for the maintenance of motility (contrast H, Table 7). Significant results at 5°C were: (1) Diluents containing a pentose again preserved spermatozoa more satis factorily than those with hexoses and were the reason for the superiority of the mono- over the di- and trisaccharides used (contrasts A and B, Table 7). (2) Contrasts E, F, and G in Table 7 indicate the hexose diluents in descending order of value were those containing rhamnose, galactose, mannose, and glucose; however, galactose diluents maintained slightly higher motility scores than did those containing rhamnose. DILUENTS FOR RAM SPERMATOZOA 665

Ribose, arabinose, xylose, and galactose were used in experiment 4 for the reciprocal replacement of 62,31,15'5, and 7 ·75 mM of either fructose or glucose, to test whether these low concentrations produced the effects seen in previous experi-

TABLE 8 EXPERIMENT 4: MEAN MOTILITY AND MEAN PERCENTAGE OF MOTILE SPERMATOZOA

37°C 5°C Diluent Sugar Constituents of Diluents and Concentrations (mM) Percentage Percentage No. Motility Motility Motile Motile

I Fructose 2·25 35·0 1·25 20·0 1 2·50 45·0 0·75 1l·25 2 Glucose 3 Ribose All 185 1·12 32·5 2·25 50·0 4 Xylose 2·12 37·5 2 ·12 35·0 5 Arabinose f 2·00 37·5 2·12 47·5 6 Galactose 2·12 32·5 2 ·12 37·5 7 Fructose 123 + ribose 62 2·00 37·5 1·50 25·0 8 154 + 31 2·12 37·5 1·25 20·0 9 169·5 + 15·5 2·12 32·5 1·37 22·5 10 177·25 + 7·75 2·37 37·5 1·87 30·0 11 Fructose 123 + xylose 62 2·12 35·0 1·50 30·0 12 154 + 31 2·25 35·0 1·12 27·5 13 169·5 + 15·5 2·25 42·5 1·37 27·5 14 177·25 + 7·75 2·25 32·5 1·25 22·5 15 Fructose 123 + arabinose 62 2·25 37·5 1·87 32·5 16 154 + 31 2·50 42·5 1·75 25·0 17 169·5 + 15·5 2·12 37·5 1·37 25·0 18 177·25 + 7·75 2·37 42·5 1·62 27·5 19 Fructose 123 + galactose 62 2·25 35·0 1·50 27·5 20 154 + 31 2·50 40·0 1·50 25·0 21 169·5 + 15·5 2·12 37·5 1·50 27·5 22 177·25 + 7·75 2·25 42·5 1·62 32·5 23 Glucose 123 + ribose 62 2·12 37·5 0·87 U·25 24 154 + 31 2·62 42·5 0·62 6·25 25 169·5 + 15·5 2·37 40·0 0·75 7·5 26 177·25 + 7·75 2·50 45·0 0·75 1l·25 27 Glucose 123 + xylose 62 2·12 45·0 0·75 1l·25 28 154 + 31 2·75 50·0 0·50 6·25 29 169·5 + 15·5 2·62 45·0 0·50 10·0 30 177·25 + 7·75 2·25 40·0 0·75 6·25 31 Glucose 123 + arabinose 62 2·25 45·0 1·00 10·0 32 154 + 31 2·25 37·5 1·00 13·25 33 169·5 + 15·5 2·50 45·0 0·87 8·75 34 177·25 + 7·75 2·62 55·0 0·75 12·5 35 Glucose 123 + galactose 62 2·50 37·5 0·87 12·5 36 154 + 31 2·75 47·5 0·87 13·25 37 169·5 + 15·5 2·25 42·5 1·25 16·25 38 177·25 + 7·75 2·50 45·0 0·87 12·5 ments where the degree of fructose replacement was greater. 17 mM fructose was added to all diluents. Results and mean scores for significant contrasts are shown in Tables 8 and 9. 666 K. R. LAPWOOD AND 1. C. A. MARTIN

Significant results at 37°C were: (1) Higher scores of spermatozoal viability were recorded from diluents in which fructose and glucose were partially replaced, than from diluents containing 185 mM of one sugar (contrast A, Table 9). (2) Viability scores for the diluents containing 185 mM of a single sugar were similar to those seen in experiment 3 (contrasts Band C, Table 9).

TABLE 9

COMPARISONS OF SURVIVAL RATES WHICH WERE SIGNIFICANTLY DIFFERENT IN ONE OR MORE SCORING CRITERIA IN EXPERIMENT 5

Mean Scores at 37°C Mean Scores at 5°C

Contrast Treatrnent Percentage Percentage Motility Motility Motile Motile

A Diluents containing one sugar 2·02 36·7 1·77 33·5 Diluents containing two sugars 2·34*** 40'8* 1·15*** 18'6*** B 185 mM ribose 1·12 32·5 2·25 50·0 185 mM xylose, arabinose, and galactose 2·08*** 35·8 2·12 40·0 C 185 mM ribose, xylose, arabinose, and galactose 1·84 35·0 2·15 42·5 185 mM fructose and 185 mM glucose 2·37** 40·0 1'00*** 15'6*** D Diluents containing fructose and another sugar 2·24 37·81 1·50 26·72 Diluents containing glucose and another sugar 2·43** 43'75*** 0'81*** 10'6*** E Diluents containing glucose, and ribose and xylose 2·42 43·1 0·69 8·75 Diluents containing glucose, and arabinose and galactose 2·45 44·4 0'93** 12'4* F 123 mM glucose + 62 mM xylose 2·12 45·0 0·75 11·25 154 mM glucose + 31 mM xylose 2·75* 50·0 0·50 6·25 G 123 mM glucose + 62 mM arabinose, and 154mM glucose + 31 mM arabinose 2·25 41·25 1·00 11·6 169·5 mM glucose + 15·5 mM arabi· nose, and 177·25 mM glucose + 7·75 mM arabinose 2·56 50'0* 0·81 10·6 - * P<0·05. ** P

(3) Diluents containing glucose and one of the four test sugars were, on average, better for maintenance of motility and percentage of motile spermatozoa than the matching diluents based on fructose (contrast D, Table 9). (4) Increasing the xylose content from 31 to 62 mM (with a corresponding reduction in the molarity of glucose in the diluent), gave a significant decrease in motility (contrast F, Table 9). DILUENTS FOR RAM SPERMATOZOA 667

(5) The mean percentage of motile spermatozoa was lower in diluents in which arabinose replaced 62 and 31 mM of the glucose, than where the replace ment was only 15·5 and 7·75 mM (contrast G, Table 9). Significant results at 5°C were: (1) Mean scores of spermatozoal survival were higher in diluents containing one sugar than in those containing two sugars (contrast A, Table 9). (2) Of the diluents containing only one sugar, those based on fructose and glucose were poorer than those containing ribose, xylose, arabinose, and galactose (contrast 0, Table 9).

TABLE 10

EXPERIMENT 5: ~IEAN MOTILITY AND MEAN PERCENTAGE OF MOTILE SPERMATOZOA

Presence or NaCl or Sugar 37°C 5°C Diluent Absence of Content of No. 17 mM Diluent Percentage Percentage Motility Motility Fructose (mM) Motile Motile

1 - NaCl92·5 0·75 27·5 0·37 7·5 2 + NaC192·5 1·25 35·0 0·50 15·0 3 - Ribose 185 1·37 40·0 2·00 57·5 4 + Ribose 185 1·50 32·5 2·25 62·5 5 - Xylose 185 2 ·12 45·0 2·12 50·0 6 + Xylose 185 1·87 50·0 2·12 57·5 7 - Arabinose 185 1·75 37·5 1·25 40·0 8 + Arabinose 185 1·87 42·5 2·00 57·5 9 - Galactose 185 2·00 45·0 2·12 57·5 10 + Galactose 185 2·25 47·5 2·12 55·0 11 - Fructose 185 2·37 52·5 1·50 37·5 12 + Fructose 185 2·12 52·5 1·50 37·5 13 - Glucose 185 2·75 57·5 0·87 20·0 14 + Glucose 185 2·62 55·0 0·75 12·5

(3) Both indices of spermatozoal viability were greater in diluents in which fructose, rather than glucose, was partially replaced (contrast D, Table 9). Partial replacement of glucose with arabinose and galactose resulted in higher scores of viability than was observed in glucose-based diluents con taining ribose and xylose (contrast E, Table 9). The effects of the presence of 17 mM fructose in diluents containing 92 mM NaCl or 185 mM ribose, arabinose, xylose, galactose, fructose, or glucose were measured in experiment 5. Washed spermatozoa were used in this experiment, to reduce the effects of fructose contained in the seminal plasma on the experiment. Results and mean scores for significant contrasts are shown in Tables 10 and 11. Significant results at 37°C were: (1) The saline diluents were poorer than those containing sugars for maintain ing motility and percentage of motile spermatozoa (contrast A, Table Il). 668 K. R. LAPWOOD AND 1. C. A. MARTIN

(2) Hexoses were better than pentoses, xylose was better than ribose, and glucose was better than fructose and galactose (contrasts B, C, and F. Table ll). Significant results at 5°C were: (I) Higher mean scores of motility and percentage of motile spermatozoa were seen in sugar diluents than in saline diluents (contrast A, Table ll). The pentoses were in turn superior to those containing hexoses largely due to poor survivals in the fructose- and glucose-containing diluents (contrast B, Table ll).

TABLE 11

COMPARISONS OF SURVIVAL RATES WHICH WERE SIGNIFICANTLY DIFFERENT IN ONE OR MORE SCORING CRITERIA IN EXPERIMENT 5

Mean Scores at 37°C Mean Scores at 5°C

Contrast Treatment Percentage Percentage Motility Motility Motile Motile

A Saline 1·00 31·25 0·42 11·25 Sugars 2'05*** 46'5*** 1'72*** 45'4*** B Pentoses 1·74 41·25 1·96 54·2 Hexoses 2'35*** 51'7** 1'47*** 36'7*** G Ribose 1·43 36·25 2·12 60·0 Xylose 2'00* 47·5 2·12 53·75 D Ribose and xylose 1·71 41·9 2·12 56·9 Arabinose 1·81 40·0 1'62** 48·75 E Fructose 2·25 52·5 1'50 37·5 Galactose 2·12 46·25 2'12*** 56'25** }i' Fructose and galactose 2·18 49·4 1·81 46·9 Glucose 2'68* 56·25 0'81*** 16'25*** ------* P<0·05. ** P

(2) Motility scores in ribose- and xylose-containing diluents were higher than those containing arabinose (contrast D, Table II). (3) Both scoring criteria showed that the hexoses could be ranked, in order of decreasing preference for inclusion in diluents for 5°C storage: galactose, fructose, and glucose (contrasts E and F, Table ll). (4) The interaction of addition of 17 mM fructose with sugar type showed that the improvement in survival when fructose was added to pentose containing diluents was greater than when added to hexose diluents, and this improvement in survivals was greater when fructose was added to arabinose diluents, than to those containing ribose and xylose.

IV. DISOUSSION The temperature of storage (incubation) dictates which sugar is to be used to give the greatest viability of ram spermatozoa in synthetic diluents as can be seen by comparing glucose and ribose at 37 and 5°C. The former is very good at the DILUENTS FOR RAM SPERMATOZOA 669 higher temperature but poor at 5°0, while the reverse of this relationship applies to ribose. Reasons for these effects are obscure. The functions which sugars may have in semen diluents are: (1) Provision of a readily metabolizable substrate; (2) facilitation of the metabolism of fructose; (3) maintenance of osmotic equilibria; or (4) an effect on the structure of the sperma tozoa reducing the rate at which spermatozoa are damaged by dilution and cold temperature (this may be related to maintenance of osmotic equilibria). The readily metabolizable hexoses glucose, mannose, and fructose support spermatozoal survival very well at 37°0, as also do the disaccharides lactose and sucrose. Increasing the proportion of these sugars in the sugar part of the diluents generally increases viability. The two disaccharides may be effective in supporting survival of spermatozoa only after breakdown to metabolizable monosaccharides. High metabolic requirements at this temperature may be a reason for their beneficial effect. However, an increase in the proportion of these sugars, even when already well above estimated metabolic needs, leads to an increase of spermatozoal activity at 37°0, which suggests that other factors may be involved. There is no apparent structural relationship between these beneficial sugars. At 5°0 the pentoses and galactose were superior to the other sugars, including those which are readily metabolized by spermatozoa. This was especially true of glucose which gave very poor results at this temperature. Even though White, Blackshaw, and Emmens (1954) and O'Dell, Almquist, and Flipse (1959) have shown that spermatozoa can metabolize arabinose, it is unlikely that a preferential increase in the metabolism of these sugars at lower temperatures could account for these effects, as the addition of fructose to the pentose-containing diluents for washed spermatozoa does increase survival. It appears more likely that the pentoses are protective against cold, possibly due to their being able to cross the cell membrane more readily than the hexoses which have larger molecules. Flipse (1962) has shown that the hexose, galactose-I-014, readily enters spermatozoa and at 37°0 is only slowly metabolized by bull spermatozoa; its accumulation in the cell may be protective. Observations by other workers (Salisbury and Van Demark 1946; Ohms and Willett 1958; and Foote and Bratton 1960), using bull semen, and Wilcox (1960) using cock semen, showed that incorporation of glucose into semen diluents for storage at chilling temperatures increases motility or fertility or both. In diluents for ram semen, replacement of sodium chloride with up to 185 mM sugar gave increased survival rates for spermatozoa stored at 37 and 5°0 (Martin, unpublished data), but results in this paper indicate that glucose is the least satisfactory of these sugars for use at the lower temperature. Wilcox (1959) found that the addition of glucose, mannose, fructose, and galactose to diluted cock semen after storage at 10°0 increased motility and fertility while the pentoses had no effect on motility and were not tested for their effects on fertility. The present experiments showed that the inclusion of glucose and mannose reduces survival rates of ram spermatozoa stored at 5°C, while the pentoses and galactose increase survival. Increasing or decreasing the osmotic pressure of the diluent by 10% of the theoretical isosmotic values had no effect on spermatozoal viability, except when maltose was the sugar used. In this case an increase in the maltose content of the 670 K. R. LAPWOOD AND I. C. A. MARTIN

diluent, with concurrent increase in tonicity, reduced viability at 37°0. The low viability of spermatozoa in diluents containing 185 mM maltose in experiment 3, and the drop in survival rates when the maltose replacement of fructose increased from 62 to 123 mM in experiment 1, together with the fact that no other sugars showed such a tonicity effect, would indicate that it is the maltose content of the diluent, rather than tonicity due to it, which is important in producing this effect. Other workers have found that much larger changes in tonicity are deleterious to ram, bull, and human spermatozoa (Blackshaw and Emmens 1951); fowl spermatozoa (Wales and White 1958a); and dog spermatozoa (Wales and White 1958b), hypotonicity being more harmful than hypertonicity. Addition of 17 mM fructose to the pentose-containing diluents leads to a signifi cant increase in motility and percentage of motile spermatozoa in diluents containing ribose and arabinose at 5°0 but not at 37°0. This may be the result of these sugars being harmful at the higher temperature either to the structure of the spermatozoa or by blocking their metabolism of fructose, an effect overriding any benefit from the use of fructose. Additions of fructose to xylose diluents at 5°0 was not beneficial. Xylose was generally superior at 37°0 to ribose and arabinose, and this may indicate that ram spermatozoa do in fact metabolize appreciable amounts of xylose.

V. ACKNOWLEDGMENTS We are grateful to Professor O. W. Emmens for advice and criticism of this paper. The work was carried out with the aid of grants from the Australian Wool Board. VI. REFERENCES BLACKSHAW, A. W. (1954).-A bipolar rectal electrode for the electrical production of ejaculation in sheep. AU8t. Vet. J. 30: 249. BLACKSHAW, A. W., and EMMENS, C. W. (1951).-The interaction of pH, osmotic pressure, and electrolyte concentration on the motility of ram, bull, and human spermatozoa. J. PhyBiol. 114: 16. CHOONG, C. H., and WALES, R. G. (1963).-The use of various diluents for deep-freezing bull spermatozoa. AUBt. J. Biol. Sci. 16: 896_ EMMENS, C. W. (1947).-The motility and viability of rabbit spermatozoa at different hydrogen ion concentrations. J. PhY8iol. 106: 471. EMMENS, C. W. (1948).-The effect of variations in osmotic pressure and electrolyte concentration on the motility of rabbit spermatozoa at different hydrogen-ion concentrations. J. PhyBiol. 107: 129. EMMENS, C. W., and BLACKSHAW, A. W. (1950).-The low temperature storage of ram, bull, and rabbit spermatozoa. AUBt. Vet. J. 26: 226. FLIPSE, R. J. (1962).-Metabolism of bovine sperm: Galactose as an indicator of hexose trans port by bovine spermatozoa. J. Dairy Sci. 45: 1083. FOOTE, R. H., and BRATTON, R. W. (1960).-Survival of bovine spermatozoa at 5 and 25°C in extenders containing varying levels of egg yolk, glucose, glycine, glycerol, citrate, and other salts. J. Dairy Sci. 43: 1322. JONES, R. C. (1965).-Studies of the handling and insemination of ram semen. Ph.D. Thesis, University of Sydney. JONES, R. C., and MARTIN, I. C. A. (1965).-Deep-freezing ram spermatozoa: The effects of milk, yolk-citrate, and synthetic diluents containing sugar. J. Reprod. Fertil. 10: 413. KAMPSCHMIDT, R. F., MAYER, D. T., and HERMAN, H. A. (1953).-Viability of bull spermatozoa as influenced by various sugars and electrolytes in the storage medium. Res. Bull. Univ. Mo. Agric. Exp. Stn. No. 519. DILUENTS FOR RAM SPERMATOZOA 671

LORENZ, F. W. (1958}.-Carbohydrate metabolism of cock spermatozoa. Nature, Land. 182: 397. MANN, T. (1964}.-"Biochemistry of Semen and of the Male Reproductive Tract." (Methuen & Co., Ltd.: London.) MARTIN, 1. C. A. (1965}.-Effects of rate of cooling to 5°C, storage at 5°C, glycerol concentration, sodium chloride, fructose, and glycine on the revival of deep. frozen bull spermatozoa. J. Agric. Sci. 64: 425. MARTIN, 1. C. A. (1966}.-Diluents for the preservation of ram spermotozoa. 1. Diluents used at 37°C and 5°C containing casein. Aust. J. Bioi. Sci. 19: 645. O'DELL, W. T., ALMQUIST, J. 0., and FLIPSE, R. J. (1959}.-Metabolism of bovine semen: Effect of fructose and arabinose on the uptake and metabolic utilization of glycerol-I-C14 by bovine spermatozoa. J. Dairy Sci. 42: 89. OHMS, J. 1., and WILLETT, E. L. (1958}.-Fertility of bull spermatozoa in yolk~citrate partially replaced by glucose. J. Dairy Sci. 41: 1800. SALISBURY, G. W., and VAN DEMARK, N. L. (1946}.-Stimulation of livability and glycolysis by additions of glucose to the egg yolk~citrate diluent for ejaculated bovine semen. J. Dairy Sci. 23: 692. VANTIENHOVEN, A., SALISBURY, G. W., VAN DEMARK, N. L., and HANSEN, R. G. (1952}.-The preferential utilization by bull spermatozoa of glucose as compared to fructose. J. Dairy Sci. 35: 637. WALES, R. G., and WHITE, 1. G. (1958a}.-The interaction of pH, tonicity, and electrolyte con centration on the motility of fowl spermatozoa. A ust. J. Bioi. Sci. 11: 177. WALES, R. G., and WHITE, 1. G. (1958b}.-The interaction of tonicity and electrolyte concentra tion on the motility of dog spermatozoa. J. Physiol. 141: 273. WHITE, 1. G., BLACKSHAW, A. W., and EMMENS, C. W. (1954}.-Metabolic and motility studies relating to the low temperature storage of ram and bull spermatozoa. Aust. Vet. J. 30: 85. WILCOX, F. H. (1959}.-Effect of the addition of carbohydrates after storage on the motility and fertilizing ability of chicken sperm. Poultry Sci. 38: 1162. WILCOX, F. H. (1960}.-Effect on fertility of temperature, handling methods, Lake's solution, and the addition of egg white, egg yolk, and sugars to the diluent used in storing chicken semen. Pou,ltry Sci. 39: 459. WILCOX, F. R., and SCHAFFNER, C. S. (1958}.-The effect of different handling methods and added fructose on the fertilizing ability of chicken spermatozoa after storage. Poultry Sci. 37: 1353.