Phospholipid fatty acid composition, vitamin e content and susceptibility to lipid peroxidation of duck spermatozoa

ELSEVIER

PHOSPHOLIPID FATTY ACID COMPOSITION, SUSCEPTIBILITY TO LIPID PEROXIDATION P.F. Surai,” J-P. Brillard,’

VITAMIN E CONTENT AND OF DUCK SPERMATOZOA

B.K. Speake,’ E. Blesbois,* F. Seigneurin N.H.C. Sparks’

* and

‘Department

of Biochemistry and Nutrition, Scottish Agricultural College Auchincruive, Ayr KA6 5HW, Scotland UK ‘Station De Recherches Avicoles, INRA, F-7380 Nouzilly, France Received for publication: Accepted:

May 20, I999 September 9, 1999

ABSTRACT Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using CC-MS and HPLC based methods, The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction, Phosphatidylethanolamine (PE) also contained high proportions of these four C20-22polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe*‘-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress. 0 2000 by Elsevier Science Inc. Key words: duck spermatozoa, fatty acids, docosahexaenoic acid, peroxidation, vitamin E Acknowledgments The authors thank the Scottish Executive Rural AfIairs Department for support and to MS. R. McCartney and Mr. F. Thacker for technical assistance. ‘Correspondence and reprint requests: Department of Biochemistry and Nutrition, SAC, Auchincruive, Ayr, KA6 XIW, Scotland, UK Tel. 01292-525-160; Fax: 01292-525-177; e-mail: [email protected] Theriogenology 0 2000 Elsevier

63:1026-1039, Science Inc.

2000

0093-691WOO/$-see PII SOO93-691

front matter X(00)00249-1

Theriogenology

1026 INTRODUCTION

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility (4,6,19). The phospholipids of avian spermatozoa are characterized by very high proportions of C20-22 n-6 polyunsaturated fatty acids (PUFAs), mainly docosatetraenoic (22:4n-6) and arachidonic (20:4n-6) acids (4,6,19,34). This contrasts with the spermatozoa of many mammalian species, in which docosahexaenoic acid (22:6n-3) is the main PUFA, comprising as much as 80% by weight of the phospholipid fatty acid groups (9,14,20,24). Thus there is, to some extent, a dichotomy between birds and mammals regarding the type of PUFA present in spermatozoa: the n-6 fatty acids predominate in avian sperm phospholipids, whereas n-3 fatty acids are more typical of mammalian spermatozoa (I 8). However, in contrast to the other avian species that have been studied, duck spermatozoa contain significant proportions of 22:6n-3 (33). Moreover, this distinction is maintained when ducks and other avian species are maintained on diets with very similar fatty acid profiles (33) suggesting the possibility of species-specific differences in the synthesis of C20.Zz PUFAs from their CrR dietary precursors and/or the incorporation of these PUFAs into the sperm phospholipids. The protection superoxide activities in lipid-soluble

highly polyunsaturated lipids of spermatozoa clearly require adequate antioxidant to prevent peroxidative damage to the cells. Although the antioxidant enzymes dismutase (SOD) and glutathione peroxidase (GSH-Px) are expressed at high duck spermatozoa (33) there is no information on the content of vitamin E, the main antioxidant in duck semen.

As a step towards elucidating the relationship between the PUFA profile, antioxidant content, viability and fertilizing ability of avian semen, the aims of the present study were 1) to provide t?nther compositional information on duck spermatozoan phospholipids by determining the fatty acid profiles of the phospholipid classes; 2) to measure the vitamin E concentration in duck semen and the distribution of this antioxidant between spermatozoa and seminal plasma; 3) to determine the in vitro peroxidative susceptibility of duck spermatozoa; and 4) to investigate the potential ability of the seminal plasma of the duck to protect spermatozoa against lipid peroxidation. MATERIALS Semen Collection

and Preparation

AND METHODS

for Analysis

Male Muscovy ducks from a commercial line (Grimaudfreres, 49450 Roussay, France) were housed in individual battery cages and fed a standard commercial diet designed for this species. Upon sexual maturity (26 to 29 wk), semen was routinely collected twice a week using female teasers. Semen was also collected from roosters (Ross 308 Broiler Breeder strain, 28 to 30 wk) 3 times a week by massage as described previously (33). Fifteen individual duck ejaculates from 15 males, collected on the same day, were prepared for analysis. Sperm concentrations were estimated using a calibrated photometer (wavelength 535 nm). Spermatozoa were separated from seminal plasma by centrihrgation (500 g, 10 min), and the resulting pellets were frozen in liquid nitrogen for further analyses. Seminal plasma was prepared

1027

Theriogenology by successive centrifugations, t%rther analyses.

as previously

described (2) and then frozen in liquid nitrogen

for

Lipid Analysis Spermatozoa were extracted for total lipid by standard procedures following homogenization in chloroform/methanol (2:1, v:v; 7). The extracts were subjected to thin-layer chromatography on silica gel G, using a solvent system of hexane : diethyl ether : formic acid (80:20: 1, v:v), after which the bands corresponding to phospholipids, triacylglycerol, free fatty acids and cholesteryl esters were eluted from the silica. Phospholipids were eluted from the silica by methanol, and the other lipid classes by diethyl ether. The isolated phospholipid fraction of the spermatozoa was further fractionated by thin-layer chromatography on silica gel D using a solvent system of chloroform-methanol-acetic acid-water (25: 15:4:2, v:v). The major phospholipid classes present (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylserine, PS; and sphingomyelin, Sp) were visualized and eluted from the silica (25). The isolated acyl-containing lipid and phospholipid classes were subjected to transmethylation by refluxing with methanol:toluene:sulphuric acid (20: 10: 1, v:v) in the presence of pentadecaenoic acid standard (8) and the fatty acid composition was determined by gas-liquid chromatography/mass spectrometry (GC-MS), using a capillary column system (SGE, BPX70, 60 m x 0.22 mm, film thickness 0.25 urn; Burke Analytical, Glasgow, UK) in a Fisons Instrument, MD 800 GC-MS fitted with AS 800 autosampler. Integration of the peaks and subsequent data handling was performed using the built-in data handling software The identities of the peaks were verified by comparison with the retention times of standard fatty acid methyl esters (Sigma. Poole, UK) and GC-MS library comparison. Lipid analysis of the feed was performed by the same method. Individual phospholipid classes were also separated by high-performance thin-layer chromatography (HPTLC) using a solvent system of methyl acetateisopropanol:chloroform:methanol:0.25% (w:v in H20) KC1 (25:25:25:10:9, v:v:v:v), as described by Olsen and Henderson (27). After charring, quantification was performed by densitometry using a Shimadzu CS-9001 PC dual wavelength flying spot thin-layer scanner (Shimadzu Corporation, Tokyo, Japan). Vitamin E Determinations Vitamin E (cc-tocopherol) was measured by the HPLC method of McMurray et al. (23) as previously described (37). In brief, under subdued light, the samples were saponified with alcoholic potassium hydroxide in the presence of pyrogallol for 30 min at 70” C, then cooled and extracted with light petroleum spirit (b.p. 40 to 60” C). The tocopherol content of the petroleum spirit extract was measured after evaporation of solvent under nitrogen and solubilization of the sample in methanol, and then injected into an HPLC system (Shimadzu Liquid Chromatograph, LC-lOAD, Japan Spectroscopic Co. LTD with JASCO Intelligent Spectrofluorometer 821-FP) fitted with a Spherisorb HPLC column, type S30DS2, 15 cm x 4.6 mm, 3 micron Cl 8 reverse phase (phase separation limited). The mobile phase was a solution of methanol-water (97:3, v:v) and the flow rate was 1.1 mL/min. Fluorescence detection for vitamin E was used with an excitation wavelength of 295 nm and emission wavelength of 330 nm. Calibration was performed using standard solutions of cc-tocopherol in methanol. Tocol was used as the internal standard.

1028 Susceptibility

Theriogenology to Lipid Peroxidation

Susceptibility of spermatozoa to lipid pet-oxidation was studied as previously described (37). In brief, spermatozoa were washed twice with sodium-phosphate buffer (pH 7.4, 0.01 M) containing 1.15% (w/v) KC1 and suspended in the same buffer to give a final concentration of 1.5 x lo9 sperm/ml. The sperm suspension was incubated for 60 min at 37°C in the presence of FeS04 (0.1 or 1.0 mmol 1-r) with vigorous shaking. At the end of incubation, butylated hydroxytoluene was added for a final concentration of 0.01% (v/v), and the concentration of thiobarbituric acid-reactive substances (TBARS) was determined by the method of Ohkawa et al. (26) with 1,1,3,3-tetramethoxypropane as the standard. Results were expressed as ug malondialdehyde (MDA) generated/lo’ spermatozoa during the 60-min incubation. Antioxidant

Effect of Seminal Plasma

The antioxidant protective effect of seminal plasma was studied in an in vitro model system (40). The model system consisted of 0.5 mL of homogenate (IO%, w:v in phosphate buffer, 0.05 M containing 1.15% KCI, w/v) of turkey embryo brain mixed with different volumes of seminal plasma, and the volume was made up to 1 mL with the same buffer. A control sample consisted only of brain homogenate and phosphate buffer without addition of seminal plasma. The avian embryonic brain homogenate was present as a substrate for peroxidation because it is rich in polyunsaturated lipids and is highly susceptible to in vitro peroxidation (38). After 1 h of incubation the accumulation of MDA was determined, as described above, and the inhibition of spontaneous lipid peroxidation due to the presence of seminal plasma was calculated. Statistical Analysis Results are presented as mean f SEM of measurements on samples from 6 to 10 replicate determinations. Statistical analysis was performed using single factor ANOVA. Differences between means were evaluated at 5% level of significance using Student’s t-test. RESULTS The lipids of the feed provided to the ducks consisted almost entirely of C16 and CIS fatty acids, with a 13-fold preponderance of 18:2n-6 over 18:3n-3; a similar fatty acid profile is found in diets formulated for male breeder chicken (Table 1) The total phospholipid fraction of duck spermatozoa (Table 1) was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6) docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids, but also present was a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid. Palmitic (16:0) and stearic (18:O) fatty acids were the major saturates in sperm phospholipids. The proportion of monounsaturated fatty acids was low by comparison with the saturated and polyunsaturated fatty acids

Theriogenology

1029

Table 1. Fatty acid composition of the diet and of the spermatozoan phospholipids of the duck Fatty acids

Palmitic (16:0) Stearic (18:0) Oleic (18: ln-9) Vaccenic ( 18: 1n-7) Linoleic (18:2n-6) Linolenic (18:3n-3) Gadoleic (20: ln-9) Arachidonic (20:4n-6) Adrenic (22:4n-6) Docosapentaenoic (22:5n-6) Tetracosanoic (24:0) Nervonic (24: ln-9) Docosahexaenoic (22:6n-3)

Feed

Spermatozoan Phospholipids

15.H 1.1 1.8 +O.l 17.0 + 0.8 1.3+ 0.1

17.6 * 2.2 10.8 * 0.3

58.3 f3.2 4.4 kO.3

kO.1 nd 0.7 kO.1 14.0 + 0.5 20.1 * 1.9 11.2+0.7 0.9 kO.1

0.5 f 0.0 nd nd nd nd nd nd

4.7 kO.3 2.5 +0.1 1.8

2.6 f0.2

10.5 k 0.6

Results are mean f SEM of the percentage (w:w of fatty acid) composition of the total lipid of the feed (n=3 replicate analyses) and of the phospholipid fraction of the spermatozoa (n=7 semen samples). Fatty acid composition of chicken feed includes 4.4% 18:3n-3, 46.1% 18:2n-6, 1.8% 18:ln-7, 31.1% 18:ln-9, 1.8% 18:0 and 11.5% 16:0. nd = not detected. In the triglyceride fraction of spermatozoan lipids, oleic acid (18: ln-9) comprised more than 50% of all fatty acids with pahnitic (18.0%) and linoleic (15.7%) acids as the major saturates and polyunsaturates respectively. The proportions of C&Z polyunsaturates in triglyceride were very low. The cholesteryl ester fraction of spermatozoan lipids was more unsaturated than the triglyceride fraction, with arachidonic (8. lo/,) and linoleic (16.2%) acids being the major polyunsaturates and oleic (33.2%) palmitic (21.8%) and stearic (15.2%) acids being the major monounsaturates and saturates of this fraction (data are not shown). In duck semen PC and PE were the major phospholipid fractions (Table 2). Among the phospholipid classes, PS had the highest level of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in the fraction (Table 2). Phosphatidylethanolamine also contained high proportions of these four Czo-2~polyunsaturates, which together formed 60% of total PE fatty acids. In contrast, PC and Sp were much more saturated than the other phospholipid classes studied and contained pahnitic acid as a major saturate, comprising about half of all fatty acid present.

Theriogenology

1030 Table 2. Fatty acid composition

Percentage of total phospholipids

of the spermatozoan

phospholipid

classes of the duck

PE

PC

PS

SP

28.0 * 1.1

40.6 i 0.3

11.2 rtO.8

9 1 f 0.7

7.2 f 1.3 13.3 5 0.3 4.4 f 0.4 2.2 f 0.2 1.4fO.l 1.1 rto.2 18.4 + 0.5 19.5 i 1.4 12.3 + 1.0 1.9 * 0.5 4.8 ix 0.3 10.3 f 0.5

48.3 3~ 1.6 13.8 f 0.5 4.9 + 0.4 1.5 10.1 0.8 f 0.2 0.7 fO.l 10.4 f 0.7 5.5 f 0.4 6.4 3~0.7 0.1 +o.o 0.6 rt 0.1 6.7 k 0.6

6.9 I!Z1.2 9.6 f 0.5 3.4 l!z 0.2 0.4 * 0.1 0.5 fO.1 0.6 + 0.2 17.1 f0.3 17.8 f 1.1 18.6 * 1.2 0.5 io.l 1.OfO.l 23.8 310.9

45.0 * 1 7 8.4 I& 0.3 3.1 +- 0.3 nd nd nd 10.9 t 0.4 4.8 f 0.4 5.5 f 0.6 3.2 f 0.2 9.9 i 0.8 8.2 3~0.4

Fatty acids Palmitic (16:O) Stearic (18:0) Oleic (18:ln-9) Vaccenic ( 18 : 1n-7) Linoleic (18:2n-6) Gondoic (20: 1n-9) Arachidonic (20:4n-6) Adrenic (22:4n-6) Docosapentaenoic (22:5n-6) Lignoceric (24:0) Nervonic (24: 1n-9) Docosahexaenoic (22:6n-3)

Results are mean ?I SEM (“XI w:w of fatty acid) in individual phospholipid classes (n=7). PE, phosphatidylethanolamine; PC, phosphatidylcholine, PS, phosphatidylserine, Sp, sphingomyelin.

The spermatozoa and seminal plasma of duck semen contained unexpectedly low levels of vitamin E, which were more than 4-fold lower than in chicken semen (Table 3). In duck, as in chicken semen, the highest proportion of vitamin E is found in spermatozoa. Table 3. Vitamin E levels in duck and chicken spermatozoa (ng/mL)

(rig/l O’cells) and seminal plasma

Spermatozoa

Duck 48.1 z!z2.4

Chicken 182.48 f 8 S”

Seminal plasma

32.4 + 2.6

163.7 i 6.5”

Results are mean f SEM. of 6 semen samples *The concentration of cc-tocopherol in duck and chicken feeds were 20.63 and 19.88 (mgkg wt of feed) respectively. Significance of difference between duck and chicken “P
fresh

The susceptibilities to Fe*’ -stimulated lipid peroxidation of duck and chicken spermatozoa were very similar (Figure 1). Inclusion of 1 mM Fe*‘ In the incubation medium had a higher

Theriogenology

ImM

Fe

O.lmM

Fe

Figure 1. Malondialdehydeformationin duck andchickensemenasa result of Fe- stimulatedlipid peroxidation. Valuesaremeansof measurements of 5 replicatesemensamples with SEM indicatedby the error bars.

Percentage of Inhibition of MDA Formation

Theriogenology

80

B

A

Duck

A

B

Chicken

Figure 3 Effect of fresh(1) andboiled(2) seminalplasmaon lipid peroxidation.Values aremeansof measurements of 5 replicatesemensamples.Significanceof differencebetweenfreshandboiledseminalplasma** *P
1034

Theriogenology

stimulating effect on lipid peroxidation in the spermatozoa than 0.1 mM Fe”. In an in vitro peroxidation system based on embryo brain homogenate, seminal plasma from ducks showed much higher (2-fold) antioxidant activity compared with that of the chicken (Figure 2). For example, 50% inhibition of lipid peroxidation in this model was achieved by inclusion in the incubation medium of duck seminal plasma at 5% (v:v of incubation mixture) or the chicken seminal plasma at 10% (v:v of incubation medium). To estimate the antioxidant effect of thermolabile compounds in the seminal plasma (proteins, including antioxidant enzymes) it was boiled for 20 min. As can be seen from the data (Figure 3), thermolabile compounds in the seminal plasma are responsible for about half of its antioxidant activity. DISCUSSION Chicken spermatozoa have been shown to contain high proportions of n-6 polyunsaturated fatty acids, mainly 20:4n-6 and 22:4n-6 (6,34). However, duck spermatozoa are unique in also containing a much higher proportion of 22:6n-3 in the sperm lipids compared with that of other avian species that have been studied (33). In this study, fatty acid profiles of duck and chicken diets were very similar to each other and to those reported for the chicken earlier (33). Therefore, the results of the present study show that a very high proportion of 22:6n-3 in the PS fraction is a distinctive feature of duck spermatozoa. Phosphatidylserine may be an important phospholipid fraction in avian spermatozoa since it has the highest degree of unsaturation, its level decreases in chicken spermatozoa during ageing (19), and it shows a significant positive correlation with the fertilizng ability of spermatozoa during the reproductive cycle (6) Commercial diets formulated for the duck and for other poultry species usually provide polyunsaturates, mainly as linoleic acid (18:2n-6), with a much smaller proportion of cr-linolenic acid (18:3n-3). Consequently the difference in 18:3n-3 levels of these diets of the various avian species is minimal (33). It may be suggested that the efficiency of conversion of 18:3n-3 to 22:6n-3 by desaturation/elongation, the transport of 22:6n-3 to the testis, and/or the incorporation of 22:6n-3 into spermatozoan phospholipid is higher in the duck than in other avian species, including the chicken. Similar levels of 22:6n-3 (9.1-9.8%) in the phospholipid fraction of chicken spermatozoa were only achieved by inclusion of high levels of 22:6n-3-rich fish oil in the diet (4, 18). There is a lack of information on the mechanisms regulating the delivery of fatty acids from the circulation to the testis and their incorporation into the phospholipids of the developing spermatozoa. Confirmation of the low efficiency of 22 6n-3 synthesis and/or transport to and incorporation by chicken spermatozoa comes from the results of Kelso et al (19). where 49..wk dietary supplementation of the cockerel’s diet with linseed oil. rich in 18:3n-3, failed to significantly increase the proportion of 22:6n-3 in the spermatozoan phosphoiipid. It was suggested (19) that the microsomal elongation of 22:5n-3 and the subsequent peroximal retroconversion pathway may not occur in cockerel spermatozoa. The biological reason for such differences in fatty acid composition between duck and chicken spermatozoa is not clear. There is a growing body of evidence that the fatty acid composition of sperm membranes, especially their unsaturated components, determine their biophysical characteristics such as fluidity and flexibility as appropriate for their specific functions, including sperm motility and fertilizing capacity (2 1)

Theriogenology Phospholipids which contain 22:6n-3 are not equally distributed throughout the membranes of mammalian spermatozoa, being located mainly in the tail (9). It remains to be determined if the same is true for avian spermatozoa, and, in particular, to assess whether the relative proportion of tail to whole spermatozoa accounts for the differences in levels of 22:6n-3 between the duck and chicken sperm lipids. In contrast to the male, it seems likely that a completely different strategy with regard to PUFA metabolism takes place in the female duck. Our previous observations (39) indicate that despite similar fatty acid profiles of the diets, the phospholipid fraction of duck egg yolk is characterized by significantly lower proportions 22:6n-3 compared with those in chicken egg yolk. Thus, our data suggest sex-specific differences in the strategy of 22:6n-3 synthesis and delivery to specific tissues, including spermatozoa and egg yolk. Duck spermatozoa are characterized by significantly lower levels of a-tocopherol than chicken spermatozoa. This was an unexpected finding, because an increased level of lipid unsaturation needs an effective system of antioxidant defense, in which vitamin E is a major component (32). Because the vitamin E level in the diets did not differ significantly, speciesspecific differences in vitamin E assimilation and delivery to specific tissues are indicated. However, data about such differences between chickens and ducks are not currently available, but our previous observations with egg yolk (35) indicate that the efficiency of vitamin E assimilation and accumulation in the chicken egg yolk is much higher than that in the duck yolk. It is not clear at present how vitamin E is delivered to the testis and to the developing spermatozoa. Mammalian testes contain less a-tocopherol per gram of tissue than most other organs (12). Previous studies (36) have shown that there is a direct relationship between the vitamin E level in the cockerel’s feed and that in the liver, testes and semen. The question arises as to whether it is possible to enrich duck spermatozoa with vitamin E by dietary means, as has been demonstrated in cockerels (36, 37) and turkeys (3 1). The lack of cytoplasm in the spermatozoa would probably preclude the accumulation of tocopherol in cellular lipid droplets, and probably all the tocopherol in the spermatozoa is distributed in the cell membranes, It is generally accepted that the physiological level of vitamin E in biological membranes is less than 1 mol per 1000 mol of phospholipids (28). Thus spermatozoa have a limited ability to incorporate a-tocopherol into their membrane, and this ability would depend on many factors, among which fatty acid composition could be the major one. The inclusion of vitamin E directly into sperm diluent can be an alternative way to stabilize sperm membrane (11). The levels of vitamin E found in chicken spermatozoa were consistent with that in our previous observations (3, 36, 37). Vitamin E concentration and distribution in duck semen are reported here for the first time. The distribution of vitamin E in duck and chicken spermatozoa was also similar to that in the above mentioned publications. Data on vitamin E concentrations in spermatozoa are quite limited and sometimes inconsistent, although there is evidence that in mammalian spermatozoa the vitamin E level is higher than that in avian species. For example, in boar semen, the a-tocopherol level was found to be 365 to 497 ng/mL, which corresponds to about 1600 ng/109 spermatozoa (22). A wide range of a-tocopherol concentrations has been detected in human spermatozoa (100 to 2450 ng/109 cells) with the percentage of motile spermatozoa being significantly related to sperm a-tocopherol content (r=O.84, P
1036

Theriogenology

Because of the high degree of sperm lipid unsaturation, lipid peroxidation can take place during storage of chicken (42) and turkey (5) semen. and is responsible for a significant decrease of sperm fertilizing ability (42). Although a highly significant negative correlation was found in chicken semen between vitamin E content and the susceptibility of the sperm to lipid peroxidation (37), a comparison of the susceptibility of duck and chicken semen to peroxidation showed that there was no significant difference. Taking into account the higher level of unsaturation and the lower concentration of vitamin E in duck spermatozoa compared with that in chicken spermatozoa, an increase in susceptibility to peroxidation could be expected However. the susceptibility to peroxidation depends on the balance between prooxidants and antioxidants in the system (17), and the complete antioxidant system of spermatozoa has not been fully characterized Our previous results (33) showed that compared with chicken spermatozoa, duck spermatozoa are characterized by significantly higher SOD and GSH-Px activities. Chicken spermatozoa also contain the water-soluble antioxidants ascorbic acid and reduced glutathione (34), and these antioxidants presumably also contribute to the protection of duck spermatozoa against lipid peroxidation. Sperm storage within oviductal sperm storage tubules (1) at a body temperature of 41°C can be considered a risk factor for lipid peroxidation. and an antioxidant role of the SST has been proposed (33) In the chicken and turkey, natural mating consists of semen deposition in the lower (abovarian) vagina due to the presence of a vestigial penis, which, in these species, prevents any vaginal penetration of the female at copulation. Spermatozoa in these species must therefore migrate through most of the luminal portion of the vagina before reaching the storage sites, a migration performed in the somewhat oxygenated environment of the vaginal mucosa By contrast, in ducks, the existence in the female of a nontubular vagina (presence of a double “Slike” portion) located approximately at mid-vagina (29) and in the male of a well-developed penis suggest that ejaculated semen is deposited in the upper vagina, which, due to its anatomical specificities, is likely to be isolated from a direct contact with ambient air Therefore. it is reasonable to suggest that the antioxidant defense of duck spermatozoa may not. in the natural situation, be as crucial as it is in the chicken and the turkey The observation that seminal plasma can protect spermatozoa against lipid peroxidation was first reported for mammalian semen (IO, 14, 15, 16. 30). In avian species, there is also evidence that seminal plasma can protect spermatozoa against peroxidation (5, 13) Nevertheless, the precise protective mechanisms of avian seminal plasma, as well as the antioxidants involved. have not been characterized. To investigate the antioxidant protective activity of seminal plasma an in vitro model system was designed (40) and it was shown that Muscocy duck seminal plasma possesses higher antioxidant activity than chicken seminal plasma. These data are similar to our observation (33). indicating a significantly higher free-radical trapping activity of duck seminal plasma compared with that in chickens. Exposure of seminal plasma to high temperature decreased its protective effect by 50%. which reflects the importance of thermolabile compounds, including the antioxidant enzymes SOD and GSH-Px. in the antioxidant activity of the plasma, After similar treatment of the plasma its free-radical trapping activity did not change (34) Based on the very low level of vitamin E in avian seminal plasma (34). we suggested that vitamin E plays a minor role as a protective element of seminal plasma but is more important as a stabilizer of the sperm membranes as a result of its incorporation into the sperm membrane structure (32)

Theriogenology

1037

In conclusion, the main finding of our study was the very high proportion of 22:6n-3 in PS and PE fractions of duck spermatozoa lipids and the comparatively low levels of vitamin E. These factors could predispose semen to lipid peroxidation. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation, from oxidative stress. Whether dietary supplementation of natural antioxidants could further increase such protection awaits investigation. REFERENCES

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