- Email: [email protected]
A comparative evaluation of thiobarbituric acid methods for determination of malondialdehyde in equine spermatozoa
Theriogenology 58 (2002) 347±350
Abstract
A comparative evaluation of thiobarbituric acid methods for determination of malondialdehyde in equine spermatozoa G. Stradaiolia,*, M. Magistrinib a
Dipartimento di Scienze della Produzione Animale, Via delle Scienze 208, 33100 Udine, Italy b Equipe Reproduction Equine, Unite PRC, INRA, 37380 Nouzilly, France
1. Introduction The decrease in the fertility potential, that characterizes preservation of equine sperm in the liquid state, is not systematically associated with a decrease in sperm motility. One possible cause of sperm deterioration during in vitro preservation could be linked to oxidative stress; in fact, sperm cells possess the unusual capacity to generate toxic oxygen metabolites [1±3], as recently evidenced in equine spermatozoa [4]. Spermatozoa are susceptible to oxidative stress from the relative abundance of unsaturated fatty acids which undergo lipid peroxidation (LPO); moreover, the limited availability of antioxidant defensive enzymes increases the exposure of cells to this attack. The LPO process generate malondialdehyde (MDA) as one of the breakdown products. We have recently adapted the thiobarbituric acid (TBA) reaction to measure MDA [5] for equine semen [6]. However, we found in extenders routinely employed in our lab, the presence of compounds other than MDA, which generate reaction products with similar spectral characteristics to the true TBA adduct. To partially resolve these drawbacks, the high performance liquid chromatography (HPLC) was proposed [7] to separate the TBA±MDA complex from interfering compounds and other TBA reactive substances (TBARS). The purposes of this study were: (1) to compare the direct ¯uorimetric evaluation of TBARS with the reverse phase HPLC procedure for isolation and measurement of the MDA±TBA complex in equine semen; and (2) to test the ef®cacy of the two methods in recovering MDA from some extenders commonly employed for equine semen preservation.
* Corresponding author. E-mail addresses: [email protected] (G. Stradaioli), [email protected] (M. Magistrini).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 8 5 - 3
348
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
2. Materials and methods 2.1. Experiment 1 Semen was collected from eight Welsh pony stallions and diluted in Hank's-Hepes (HH) salt solution, then centrifuged to eliminate seminal plasma and resuspended to obtain concentrations ranging from 80 to 2:5 106 cells/ml. LPO was assessed by using the TBA reaction for MDA evaluation followed by ¯uorimetric (lex 532 nm; lem 550 nm) detection with or without HPLC separation before detection. 2.2. Experiment 2 As a variety of non-lipid sources are reported to produce MDA, including proteins and carbohydrates [8]; and considering that sugars and proteins (from milk and BSA) usually go into the composition of extenders for equine sperm storage, studies were performed on the interferences generated by the extenders routinely used in our lab on MDA±TBA test. Moreover, free MDA reacts extensively with the serum albumin and other protein present in cell culture media [9]. So, we performed some recovery tests of MDA standard added to the extenders. For these studies, the extenders tested were the HH modi®ed solution, containing large amounts of glucose and lactose (HGLL), supplemented or not with 1% BSA [10], and the commercial extender INRA96 (IMV Technologies, L'Aigle Cedex, France) [11]. Levels of MDA±TBA adducts were produced by the extenders themselves, and the recovery rate of MDA standard dilutions added to the extenders were evaluated. 3. Results 3.1. Experiment 1 The TBA test, in conjunction with ¯uorimetric detection, showed the production of MDA by equine spermatozoa. This production is proportional to cell concentration; in fact, linear regression analysis of our data revealed a high correlation between MDA levels and spermatozoa concentrations for both spectro¯uorimetric methods (y 1:5834x 1:4146; R2 0:999) and HPLC (y 1:0838x 1:372; R2 0:995). The regression between the two methods for the MDA evaluation demonstrated that they are in strong agreement (R2 0:996). The analysis of chromatographic reports never evidenced signi®cant peaks other than the one corresponding to MDA standard. 3.2. Experiment 2 The level of false MDA evidenced in the four tested extenders did not differ (P > 0:05) between the two methods, whereas MDA content was elevated in both HGLL and INRA96, where sugars are more abundant. The calibration curves performed in aqueous solution and in each of the tested extenders for both methods (with or without HPLC separation) revealed that the slopes were mainly the same for curves in HH and aqueous solution,
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
349
whereas, the presence of sugars and protein in the other extenders signi®cantly reduced the slope, indicating that the recovery was not complete in this condition; in fact, recovery values ranged from 90 to 80% in HH, whereas in HGLL and INRA96, recovery was never more than 70% and frequently less than 50%. 4. Discussion The use of HPLC for MDA dosage did not give us any improvement in the speci®city or sensitivity of the methods, while it was more cumbersome and time consuming. HPLC method was proposed for isolation of genuine MDA±TBA adduct from other adducts and/ or interfering substances. However, in our conditions, no apparent interference was evidenced which can be resolved by chromatographic separation, as reported for other substrates [8]. This was evident in extender alone, particularly for HGLL extender which is only composed of sugars and salts (no lipid or lipid peroxides); in that case, HPLC was not able to separate false MDA (due to sugars) from genuine MDA. The other relevant result was that extender containing large amounts of sugars and proteins bound free MDA. So, it is necessary to wash spermatozoa from extenders before LPO determination by the MDA± TBA reaction. In conclusion, we demonstrated that: (1) ¯uorimetry is a rapid and ef®cient method for determination of TBARS, and previous chromatographic separation does not improve method speci®city; and (2) extenders could induce artefactual MDA±TBA adducts in sperm LPO determination. Acknowledgements This research was supported by INRA, Haras Nationaux. G. Stradaioli was supported, in part, by a grant from Organization for Economic Cooperation and Development (OECD). References [1] Tosic J, Walton A. Formation of hydrogen peroxide by spermatozoa and its inhibitory effect on respiration. Nature 1946;158:485. [2] Tosic J, Walton A. Metabolism of spermatozoa. Formation of hydrogen peroxide by spermatozoa and its effects on motility and survival. Biochem J 1950;47:199±212. [3] Holland MK, Storey BT. Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa. Biochem J 1981;198:273±80. [4] Ball BA, Vo AT, Baumber J. Generation of reactive oxygen species by equine spermatozoa. Am J Vet Res 2001;62:508±15. [5] Aitken RJ, Harkiss D, Buckingham D. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 1993;35:302±15. [6] Stradaioli G, Genevieve A, Magistrini M. Evaluation of lipid peroxidation by thiobarbituric acid test in equine spermatozoa. In: Proceedings of the Third International Symposium on Stallion Reproduction, 2001, Fort Collins (CO) [abstract 40]. [7] Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological samples. Free Radic Biol Med 1993;15:353±63.
350
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
[8] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515±40. [9] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421±31. [10] Magistrini M, Cuty I, Palmer E. Interaction between sperm packaging, gas environment, temperature and diluent on fresh stallion sperm survival. Acta Vet Scand 1992;(Suppl 88):97±110. [11] Batellier F, Duchamp G, Vidament M, Arnaud G, Palmer E, Magistrini M. Delayed insemination is successful with a new extender for storing fresh equine semen at 15 8C under aerobic conditions. Theriogenology 1998;50:229±36.
Abstract
A comparative evaluation of thiobarbituric acid methods for determination of malondialdehyde in equine spermatozoa G. Stradaiolia,*, M. Magistrinib a
Dipartimento di Scienze della Produzione Animale, Via delle Scienze 208, 33100 Udine, Italy b Equipe Reproduction Equine, Unite PRC, INRA, 37380 Nouzilly, France
1. Introduction The decrease in the fertility potential, that characterizes preservation of equine sperm in the liquid state, is not systematically associated with a decrease in sperm motility. One possible cause of sperm deterioration during in vitro preservation could be linked to oxidative stress; in fact, sperm cells possess the unusual capacity to generate toxic oxygen metabolites [1±3], as recently evidenced in equine spermatozoa [4]. Spermatozoa are susceptible to oxidative stress from the relative abundance of unsaturated fatty acids which undergo lipid peroxidation (LPO); moreover, the limited availability of antioxidant defensive enzymes increases the exposure of cells to this attack. The LPO process generate malondialdehyde (MDA) as one of the breakdown products. We have recently adapted the thiobarbituric acid (TBA) reaction to measure MDA [5] for equine semen [6]. However, we found in extenders routinely employed in our lab, the presence of compounds other than MDA, which generate reaction products with similar spectral characteristics to the true TBA adduct. To partially resolve these drawbacks, the high performance liquid chromatography (HPLC) was proposed [7] to separate the TBA±MDA complex from interfering compounds and other TBA reactive substances (TBARS). The purposes of this study were: (1) to compare the direct ¯uorimetric evaluation of TBARS with the reverse phase HPLC procedure for isolation and measurement of the MDA±TBA complex in equine semen; and (2) to test the ef®cacy of the two methods in recovering MDA from some extenders commonly employed for equine semen preservation.
* Corresponding author. E-mail addresses: [email protected] (G. Stradaioli), [email protected] (M. Magistrini).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 8 5 - 3
348
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
2. Materials and methods 2.1. Experiment 1 Semen was collected from eight Welsh pony stallions and diluted in Hank's-Hepes (HH) salt solution, then centrifuged to eliminate seminal plasma and resuspended to obtain concentrations ranging from 80 to 2:5 106 cells/ml. LPO was assessed by using the TBA reaction for MDA evaluation followed by ¯uorimetric (lex 532 nm; lem 550 nm) detection with or without HPLC separation before detection. 2.2. Experiment 2 As a variety of non-lipid sources are reported to produce MDA, including proteins and carbohydrates [8]; and considering that sugars and proteins (from milk and BSA) usually go into the composition of extenders for equine sperm storage, studies were performed on the interferences generated by the extenders routinely used in our lab on MDA±TBA test. Moreover, free MDA reacts extensively with the serum albumin and other protein present in cell culture media [9]. So, we performed some recovery tests of MDA standard added to the extenders. For these studies, the extenders tested were the HH modi®ed solution, containing large amounts of glucose and lactose (HGLL), supplemented or not with 1% BSA [10], and the commercial extender INRA96 (IMV Technologies, L'Aigle Cedex, France) [11]. Levels of MDA±TBA adducts were produced by the extenders themselves, and the recovery rate of MDA standard dilutions added to the extenders were evaluated. 3. Results 3.1. Experiment 1 The TBA test, in conjunction with ¯uorimetric detection, showed the production of MDA by equine spermatozoa. This production is proportional to cell concentration; in fact, linear regression analysis of our data revealed a high correlation between MDA levels and spermatozoa concentrations for both spectro¯uorimetric methods (y 1:5834x 1:4146; R2 0:999) and HPLC (y 1:0838x 1:372; R2 0:995). The regression between the two methods for the MDA evaluation demonstrated that they are in strong agreement (R2 0:996). The analysis of chromatographic reports never evidenced signi®cant peaks other than the one corresponding to MDA standard. 3.2. Experiment 2 The level of false MDA evidenced in the four tested extenders did not differ (P > 0:05) between the two methods, whereas MDA content was elevated in both HGLL and INRA96, where sugars are more abundant. The calibration curves performed in aqueous solution and in each of the tested extenders for both methods (with or without HPLC separation) revealed that the slopes were mainly the same for curves in HH and aqueous solution,
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
349
whereas, the presence of sugars and protein in the other extenders signi®cantly reduced the slope, indicating that the recovery was not complete in this condition; in fact, recovery values ranged from 90 to 80% in HH, whereas in HGLL and INRA96, recovery was never more than 70% and frequently less than 50%. 4. Discussion The use of HPLC for MDA dosage did not give us any improvement in the speci®city or sensitivity of the methods, while it was more cumbersome and time consuming. HPLC method was proposed for isolation of genuine MDA±TBA adduct from other adducts and/ or interfering substances. However, in our conditions, no apparent interference was evidenced which can be resolved by chromatographic separation, as reported for other substrates [8]. This was evident in extender alone, particularly for HGLL extender which is only composed of sugars and salts (no lipid or lipid peroxides); in that case, HPLC was not able to separate false MDA (due to sugars) from genuine MDA. The other relevant result was that extender containing large amounts of sugars and proteins bound free MDA. So, it is necessary to wash spermatozoa from extenders before LPO determination by the MDA± TBA reaction. In conclusion, we demonstrated that: (1) ¯uorimetry is a rapid and ef®cient method for determination of TBARS, and previous chromatographic separation does not improve method speci®city; and (2) extenders could induce artefactual MDA±TBA adducts in sperm LPO determination. Acknowledgements This research was supported by INRA, Haras Nationaux. G. Stradaioli was supported, in part, by a grant from Organization for Economic Cooperation and Development (OECD). References [1] Tosic J, Walton A. Formation of hydrogen peroxide by spermatozoa and its inhibitory effect on respiration. Nature 1946;158:485. [2] Tosic J, Walton A. Metabolism of spermatozoa. Formation of hydrogen peroxide by spermatozoa and its effects on motility and survival. Biochem J 1950;47:199±212. [3] Holland MK, Storey BT. Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa. Biochem J 1981;198:273±80. [4] Ball BA, Vo AT, Baumber J. Generation of reactive oxygen species by equine spermatozoa. Am J Vet Res 2001;62:508±15. [5] Aitken RJ, Harkiss D, Buckingham D. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 1993;35:302±15. [6] Stradaioli G, Genevieve A, Magistrini M. Evaluation of lipid peroxidation by thiobarbituric acid test in equine spermatozoa. In: Proceedings of the Third International Symposium on Stallion Reproduction, 2001, Fort Collins (CO) [abstract 40]. [7] Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological samples. Free Radic Biol Med 1993;15:353±63.
350
G. Stradaioli, M. Magistrini / Theriogenology 58 (2002) 347±350
[8] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515±40. [9] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421±31. [10] Magistrini M, Cuty I, Palmer E. Interaction between sperm packaging, gas environment, temperature and diluent on fresh stallion sperm survival. Acta Vet Scand 1992;(Suppl 88):97±110. [11] Batellier F, Duchamp G, Vidament M, Arnaud G, Palmer E, Magistrini M. Delayed insemination is successful with a new extender for storing fresh equine semen at 15 8C under aerobic conditions. Theriogenology 1998;50:229±36.