UPID PEROXIDATION IN SEMEN OF THE BOAR

Smutna M., o. Synek: Lipid Peroxidation in Semen of the Boar. Acta vet. Bmo, 48, 1979: 35-43. During the catalyzed aerobic incubation of the complete boar semen malonyldialdehyde (MDA) is produced. MDA is a good indicator of peroxidative decomposition of polyunsaturated fatty acids. Detection of peroxidation products was conducted both by colour reaction with 2-thiobarbituric acid (evidence of MDA), and spectrofotometrically (evidence of the conjugated dienes, oxidation of the coenzyme NADPH + H+). It was found that the progressive formation of MDA surpasses the formation of conjugated dienes especially in the early stages of peroxidation. Significant correlation for the first 30 minutes of the aerobic incubation was found between the number of spermatozoa and the amount of produced malonyldialdehyde. Enzymatic lipid peroxidation studied in isolated tails of spermatozoa was NADPH-dependent with a small dependence on pyrophosphate. Production of MDA in all studied systems was infiuenced by pH of the environment. The boar seminal plasma showed high anti-peroxidative activity limited by its dilution. Native semen, malonyldialdehyde, conjugated dienes, inhibitory effect. Properties ofthe membranes determine largely the properties of the cells and tissues. Viability of each animal cell depends on structural and functional integrity of its membrane and subcellular structures. The quality of the membrane is also important for viability of spermatozoa and for metabolic processes ensuring their contact to the environment. Changes in the properties of these membranes result in functional and morphological aberations the character of which is given by the degree of damage to the membrane. The labile compound of the membranes are unsaturated fatty acids esterified in phospholipids which together with globular proteins represent the basic structural components of all biological membrane complexes. Non-enzymatic peroxidative breakdown of unsaturated fatty acids of the membrane to small fragments impairs their function in _the lipoprotein membrane and injurs seriously their structural and functional integrity. Destruction of specific membrane phospholipids during the NADPH-induced lipid peroxidation of the microsomal fraction from rat liver was demonstrated by May and Mc Cay (1968). Decomposition by peroxidation of the chain of polyunsaturated fatty acids esterified in the beta position of phosphatidylcholine and phosphatidylethanolamine of the microsomal membrane resulted in a 40 % decrease in the amount of arachidonic acid. The harmful effect of HaO. and dialuric acid on the erythrocyte membranes in rats suffering from E hypovitaminosis lead to partial damage to phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine (Heikkila et al. 1971). Changes in the concentration of phospholipids in the boar semen were found to be caused by its aerobic incubation with Cu++ ions (Smutna 1974). Loss of plasmalogen and palmitaldehyde from the ram sperm and loss of motility of spermatozoa with their spontaneous agglutination was found by Jones and Mann (1976). Damage to ram spermatozoa due to peroxidation of endogenous phospholipids was observed by the same authors (J ones and Mann 1977). Impaired functional integrity manifesting itself by changed permeability to K + and Na+ in irradiated erythrocytes was demonstrated by Gerber and Altman (1970), glucose eftlux through an artificially prepared membrane (Hicks and Gebicki 1977) and impaired permeability of E. coli membrane for the K+ ions were observed (Suzuki and Akamatsu 1978).

Properties ofthe membranes determine largely the properties of the cells and tissues.Viability of each animal cell depends on structural and functional integrity of its membrane and subcellular structures.The quality of the membrane is also important for viability of spermatozoa and for metabolic processes ensuring their contact to the environment.Changes in the properties of these membranes result in functional and morphological aberations the character of which is given by the degree of damage to the membrane.
The labile compound of the membranes are unsaturated fatty acids esterified in phospholipids which together with globular proteins represent the basic structural components of all biological membrane complexes.Non-enzymatic peroxidative breakdown of unsaturated fatty acids of the membrane to small fragments impairs their function in _the lipoprotein membrane and injurs seriously their structural and functional integrity.Destruction of specific membrane phospholipids during the NADPH-induced lipid peroxidation of the microsomal fraction from rat liver was demonstrated by May and Mc Cay (1968).Decomposition by peroxidation of the chain of polyunsaturated fatty acids esterified in the beta position of phosphatidylcholine and phosphatidylethanolamine of the microsomal membrane resulted in a 40 % decrease in the amount of arachidonic acid.The harmful effect of HaO. and dialuric acid on the erythrocyte membranes in rats suffering from E hypovitaminosis lead to partial damage to phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine (Heikkila et al. 1971).Changes in the concentration of phospholipids in the boar semen were found to be caused by its aerobic incubation with Cu++ ions (Smutna 1974).Loss of plasmalogen and palmitaldehyde from the ram sperm and loss of motility of spermatozoa with their spontaneous agglutination was found by Jones and Mann (1976).Damage to ram spermatozoa due to peroxidation of endogenous phospholipids was observed by the same authors (J ones and Mann 1977).Impaired functional integrity manifesting itself by changed permeability to K + and Na+ in irradiated erythrocytes was demonstrated by Gerber and Altman (1970), glucose eftlux through an artificially prepared membrane (Hicks and Gebicki 1977) and impaired permeability of E. coli membrane for the K+ ions were observed (Suzuki and Akamatsu 1978).
The boar sperm with the high content of unsaturated fatty acids in phospholipids of spermatozoa is a suitable substrate for the study of non-enzymatic and enzymatic lipid peroxidation.Particularly high unsaturated fatty acid content, namely 73 %, is in phosphatidylcholine, followed by phosphatidylethanolamine containing 64% of unsaturated fatty acids.Among these predominate the docosapentaenoic acid (22: 5w6) and docosahexaenoic acid (22: 6).Smaller proportions are of the oleic, linoleic, linolenic and arachidonic acids (Johnson et al. 1969a(Johnson et al. , 1969b(Johnson et al. , 1972)).
Lipid peroxidation in the sperm of farm animals has been hitherto studied only scarcely although the deteriorating effect of air oxygen and H,O, (both exogenous and endogenous) on viability and metabolic activity of spermatozoa of various animals species has betn known for more than 30, years (Mc Leod 1943;Prince and Almquist 1948).The influence of ageing of sperm on lipid peroxidation of the boar spermatozoa was studied by Mrotek et al. (1966), lipid peroxidation in the boar sperm was described by Hruska et al. (1970), and in the ram sperm by Jones and Mann (1973).
These data indicate that the phospholipids form a dynamic component in the membrane of spermatozoa which reacts rapidly and sensitively to the injurious factors of the environment (undue manipulation, dilution, cold shock, freezing and thawing of the sperm, method of sperm conservation) inducing changes of the fatty acids.Oxidized or decomposed fatty acids do not fulfill their' functions, and consequently, impaired integrity of cell membranes and fertilizing ability of spermatozoa follow.
The aim of the present work is to study the changes of fatty acids from spermatozoa phospho-lipids during a catalyzed aerobic incubation of native boar semen.

Material and Methods
For experiments, semen from 4 White Purebred and one Pietrain boars of the Veterinary Re-search Institute, Brno, was used.Housing and feeding of the animals was in agreement with the Czechoslovak norm standard.Semen was collected by artificial vagina and a dummy.The laboratory control was provided at the above-mentioned Institute.The semen separated from the geL was carefully transported to our laboratory and processed immediately.
For the study of lipid peroxidation in the boar sperm the following methods were elaborated and successfully employed: 1) Assay of malonyldialdehyde (MDA), the secondary product of lipid peroxi-• dation Native semen (2.7 ml) was incubated in the vessel-9f the Warburg apparatus at a temperature of 310 K and continuous shaking (80/min.)which ensured the aerobic conditions.Activator (0.3 ml) was added through the side arm of the vessel after a lO-minute long preincubation.In the control experiment, 0.3 ml of 0.145 M NaCI was added instead of the activator.
Within given time intervals (60 or 90 minutes) the reaction was finished after the aerobic incubation both with and without the activator by addition of 3 ml 20 % trichloracetic acid.The_ aliquot of non-protein filtrate was mixed with equal volume of 0.5 % 2-thiobarbituric acid in 10 %.
trichloracetic acid.Mter a 10-minute long heating to 373 K in boiling water bath, the sample was: quickly cooled to room temperature.The colour intensity of the mixture dependent on the amountof malonyldialdehyde was measured on a spectrophotometer UV -VIS Model 635 by Varian Techtron at 532nm.Calibration: reproducibility of the method was tested by measuring the extinction molar coefficient of the MDA for 532 nm.Hydrolysis of 10-4 ethanol solution of 1,1,3,3-tetraethoxypropane-(Fluka AG) with 0.7 M hydrochloric acid (Schmidt 1959).From the calibration graph the extinction molar coefficient for MDA (15.4 m'moJ-l) was determined and employed for quantitati-ve evaluation of the results.
2) Determination of conjugated dienes in aerobically incubated boar semen with or without an activator was made using the method' by Placer (1968) originally developed for measurement of conjugated dienes isolated from liver tissue.For reproducible results it was necessary to extract only 0.5 ml of semen after the finished aerobic incubation with or without the activator.The final heptane layer was measured in spectrophotometer (Specord UV-VIS, Zeiss Jena) at 200-350nm.The results were expressed in mmoI of conjugated dienes per 10' spermatozoa.The extinction molar coefficient for calculation of concentration of the conjugated dienes was used according to Placer et al. (1970).
Purification and control of the solvents employed was made according to Pestemer (1951)_ 3) Oxidation of the coenzyme NADPH + H+ resulting in its disappearence is also a measure of changes in the quality of unsaturated fatty acids during the lipid peroxidation (Hochstein and Ernster 1963;May and McCay 1968).The -oxidation ofNADPH + H+ was followed in the system containing 2.7 ml 0.1 M phosphate buffer .at pH 7.20 or 0.1 M KCI-Tris-HCI buffer at pH 7.40 and 0.1 ml of the native boar semen, 3 mM •ofpyrophosphate, 3 mM NADPH + H+ (final concentration in 3.0 ml of total volume).The mix-"ture was incubated at a temperature of 310 K. Decrease in the amount of NADPH + H+ was _followed at 336 nm with help of a registration equipment (VIT ATRON) and was expressed in }tmol of decreased coenzyme amount per minute and ml of semen.
The intracellular lipids of spermatozoa with high content of unsaturated fatty acids are concentrated besides the head also in the mitochondrial sheath of the middle piece of the tail; we there-:fore isolated the tails according to Ahluwalia and Holman (1969).For disintegration we used -a fine sterile ball-balotine (No. 12) on a high-rotation laboratory shaker.The efectiveness of .disintegrationwas controlled in a micrpscope.Finally, the centrifuged tails were stirred in cooled O().154 M NaCI solution to obtain tails from 1 g of spermatozoa in 1 ml of the suspension.This :suspension covered with ice was processed immediately.
The course of enzymatic lipid peroxidation was followed in the system composed of 0.1 ml :sperm tail suspension, 4 mM ADP, 3 mM NADPH + H+ (final concentration in total volume -of 2.0 ml) in a phosphate buffer at pH 7.50.The second system contained 0.1 M Tris-HCI buffer .atpH 7.52.

4) Anti-peroxidative activity of the boar seminal plasma
The boar sperm was centrifuged (3000 g/10 min.), the seminal plasma separated.A portion of .heplasma was diluted in the phosphate buffer (pH 5.9) 1 : 10 to 1 : 100.Another portion of the plasma was left undiluted.For an active system,10 % homogenate of mouse liver was used.Imme-<Hately before use it was shortly centrifuged at 400 g and stored in ice (Placer 1968).The diluted .andintact seminal plasma (both 0.5 ml) were added to 0.2 ml homogenate in 4.0 ml of buffer.The total volume (4.7 ml) was incubated for 90 minutes.The MDA concentration in deproteined :filtrate was measured.The inhibition percentage was calculated according to VeseU"" 'va et al. (1967).
Statistical evaluation of the results was made according to Roth et al. (1962) . .

Results
The aerobic incubation alone of the boar semen does not result in measurable amount of MDA.Added inorganic Cu + + ions increase considerably the production of MDA which is highest after 90 minutes.The spectral characteristics of the product of semen peroxidation (Cu ++, 60 min.)with the MDA standard prepared by acid hydrolysis of 1,1,3,3-tetraethoxypropane are shown in Fig. 1.The typical absorption curve of the MDA standard shows •a sharp absorbance maximum at 532 nm corresponding to the MDA from the catalyzed peroxidation of the unsaturated fatty acids from phospholipids of the boar spermatozoa.Relationship between the MDA and conjugated diene concentration in mmol 10 9 spermatozoa Peroxidation induced by the eu + + ions is dependent upon concentration of the activator and the reaction time.With decreasing activator concentration the reaction velocity slows down and the lowest CuSO, concentration at which still measurable MDA production was found to be 2 x 10-5 M(Hruskaeta1.1970) .The optimum CuSO, concentration inducing peroxidation of lipids in boar semen was 100 x x 10 -'M (final concentration in the incubated system 100 x x 10 -5M).This concentration has been.used since in our laboratory.
The total yield of MDA produced in boar semen (20 specimens under identical experimental conditions) were examined depending on time during a catalyzed and non-catalyzed aerobic incubation is given in Table 1.
The amount of cellular elements (protein) present in the semen does not influence substantially the yield of MDA produced in the catalyzed aerobic incubation except for the first 30 minutes of incubation.The relationship between the final yield of MDA and the concentration of spermatozoa in boar semen expressed by a correlation coefficient r = 0.485 was significant (P < 0.05); see The lipid peroxidation activity was also evaluated by a simultaneous determination of conjugated dienes in the lipid extract from the boar semen.Production of conjugated dienes was ob- served on induction of the lipid peroxidation by inorganic ions of Fe++ and Cu + +.The absorbance at 233 run increased with the incubation time.Relationship between the concentration of MDA and conjugated dienes after Fe ++ and Cu ++ activation is shown in Fig. 3.The Fe++ ions are clearly more potent activator of the lipid peroxidation in the boar semen.In the early phase of lipid peroxidation with a low level of conjugated dienes (up to 1 mmol/l0 9 spermatozoa) a rapid MDA production occurs.
The oxidation of NADPH + H + is significantly influenced by the kind of the employed buffer.In the KCI-Tris-HCI buffer at pH 7.40 it was 3.5 times more rapid as compared to the reaction in phosphate buffer at the same pH.The influence of pyrophosphate made itself felt in both cases by a mild slow-down of the NADPH + H + oxidation (Table 2).
The tails of spermatozoa carrying the mitochondrial sheath rich in lipids in their middle piece were subjected to the enzymatic NADPH-dependent lipid peroxidation.The results presented in Table 3 show that the Cu + + ions enhance the peroxidation effect of NADPH 2.3 times and the Fe++ ions 2.6 times.The stimulatory effect of ADP in concentration of 4mM was not so obvious (21 %) as was the case with mitochondrial and microsomal suspensions (Placer 1967;Hochstein and Ernster 1963).Also the type of the employed buffer made itself felt to a certain degree.
In connection with the study of lipid peroxidation in the boar semen it was  of interest to explore if and to what extent the seminal plasma is capable to inhibit the lipid peroxidation activity of some other biological system.Table 4 shows that the seminal plasma diluted 1: 10 to 1: 30 has a high anti-peroxidative activity which tends to-decrease with its increasing dilution.No anti-peroxidative activity of undiluted seminal plasma of the boar is given in the Table as we failed to eliminate turbidity of the parallel samples after deproteination.In the repeated experiments, however, this problem was solved and we detected a 92 % anti--peroxidative activity of the undiluted seminal plasma.This inhibitory activity is connected with the macromolecular substances of the seminal plasma and it does not disappear during dialysis.
Table 4 Inhibitory activity of the seminal plasma of the boar Activity of the lipidperoxidative system (pmoIMDA) Inhibition of lipid peroxidation (%) The vast knowledge on lipid peroxidation and changes of the polyunsaturated fatty acids of phospholipids contained in mitochondrial membranes, in microsomes, lysosomes, chloroplasts and in other natural and artificial systems indicate a connection of these changes with impairment of membranes.They provide a broad basis for the study of these questions in spermatozoa.Recent results show that lipid peroxidation of the fatty acids of sperm lipids of the boar in vitro can proceed under certain conditions, and that it mirrors some influences upon the spermatozoa.Thorough analysis of these facts can lead to practical conclusions necessary for field insemination.
Demonstration of the lipid peroxidative activity in the native boar sperm can be regarded as a contribution to the general physiology and cell metabolism because up to now the in vivo lipid peroxidation had been connected with the toxic effect of oxygen on cellular level (Hatigaard 1968), with the cellular mechanisms of senescence (Barber and Bernheim 1967;Chio et al. 1969;Okuma et a1. 1969), with damage to liver parenchyma in CCl4 intoxication (Reck-nage11967) and especially with insufficient antioxidant ability of tissues connected with lack of vitamin E and selenium (Kitabchi et a1. 1960(Kitabchi et a1. , 1968;;Zalkin and Tappel 1960).
At the aerobic incubation of boar sperm with Cu + + ions a significant dependence between the concentration of spermatozoa in the semen and MDA occurrence during the first 30 minutes of incubation was demonstrated.The correlation coefficient r = 0.485 (P < 0.05) indicates that the number of spermatozoa is not the only and decisive factor of lipid peroxidation.The changing amount of seminal plasma can make itself felt by varying lipid antiperoxidation activity.
MDA is produced progressively in the early stage of peroxidation.Therefore we consider its determination in these stages to be a more valuable and reproducible indicator of peroxidative activity than determination of conjugated dienes which in turn is being used for evaluation of long-time catalyzed aerobic incubation (Placer et al. 1970;Goldstein and Harber 1972;Tsai 1975).
The Fe + + ions are highly aggressive in the peroxidative reaction which is in agreement with their oxidoreduction properties.In the reduced state they are probably maintained in the reaction by the ascorbic acid which is a natural component of boar semen.
Connection of NADPH + H + with pyrophosphate had no stimulatory effect as described with mitochondrial and microsomal suspensions from rat liver (placer 1967;Tam and McCay 1970).One-minute long exposure to a temperature of 343 K inactivated the system, the oxidation of coenzyme did not proceed but the final reaction with thiobarbituric acid was weakly positive.This finding is different from that of the course of peroxidation of microsomal fraction frorn.rat liver which is also NADPH-dependent but highly labile to the effect of temperature (May and McCay 1968).
From the viewpoint of profound study of lipid peroxidation we consider important to follow the interaction of organic peroxides with-SH groups of proteins and enzymes in the semen.

Table 1
InflueDce of Cu + + on production of MDA in the semen of the boar at aerobic incubation

Table 2 Table 3
Oxidation of NADPH + H +