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Effects of two homeopathic complexes on bovine sperm mitochondrial activity
Homeopathy (2012) 101, 99e102 Ó 2012 The Faculty of Homeopathy doi:10.1016/j.homp.2011.12.003, available online at http://www.sciencedirect.com
ORIGINAL PAPER
Effects of two homeopathic complexes on bovine sperm mitochondrial activity Dhafer M Aziz1,*, Ute Schnurrbusch2 and Heinrich Enbergs1 1
Institute of Physiology, Biochemistry and Hygiene of Animals, Bonn University, Bonn, Germany Faculty of Veterinary Medicine, Large Animal Clinic for Theriogenology and Ambulatory Services, Leipzig University, Leipzig, Germany 2
Objectives: This study was conducted to evaluate the effect of two homeopathic complexes Ubichinon compositumÒ (Ubi comp) and Coenzyme compositum ad us. vet.Ò (CoQ10 comp) on bovine sperm mitochondrial activity. Methods: Sperm viability, acrosomal integrity and sperm chromatin structure were estimated to detect the possible side effect of complexes on other sperm parameters. Results: Mitochondrial activity was significantly enhanced by both Ubi comp (P < 0.01) and CoQ10 comp (P < 0.05). No effects were detected in other tested sperm parameters. Conclusion: The tested homeopathic complex medicines stimulate the mitochondrial activity of bovine sperm without effects on their viability, acrosomal integrity or chromatin structure. The possibility that this translates into improved fertilization capacity in artificial insemination should investigated. Homeopathy (2012) 101, 99e102.
Keywords: Sperm; Mitochondria; Bovine; Ubichinon; Coenzyme; Homeopathy
Introduction Sperm motility has a strong relationship with fertility1e4 and is a useful indicator of sperm-fertilizing capacity invivo.5 There is a positive correlation between mitochondrial membrane potential and spermatozoal motility.6 Mitochondria of the sperm midpiece generate energy to support motility; therefore changes in mitochondrial membrane potential are a good indicator of functional impairment without the direct assessment of sperm motility parameters.7 Coenzyme Q10, an integral redox and proton translocation component of the mitochondrial respiratory chain, also known as ubiquinone because its wide distribution throughout mammalian tissues,8 plays a key role in energy metabolism and has potent antioxidant properties for cellular membrane integrity.9 In sperm cells, coenzyme Q10 (CoQ10) is concentrated in the mitochondria, where it is involved in energy production. It also functions as an anti-
*Correspondence: Current address: Dhafer M Aziz, Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, P.O. Box 11141, Mosul, Iraq. E-mail: [email protected] Received 17 April 2011; revised 9 November 2011; accepted 9 December 2011
oxidant, preventing lipid peroxidation of sperm membranes.10 CoQ10 levels in human seminal fluid show a significant correlation with sperm count and motility.11 Significant increases in motility were observed when sperm samples were incubated in vitro with CoQ10.12 In another study coenzyme Q7 (an analogue of CoQ10) was given to infertile men resulting in increased sperm count and motility.13 The present study was designed to evaluate the effect of two commercial homeopathic combination complexes (approved in human and veterinary medicine as intermediate catalysts to stimulate or regulate disturbed metabolic processes) on bovine sperm mitochondrial activity.
Materials and methods Fresh semen samples from six bulls, maintained at Rinder Union West e.G. (Borken, Germany) under uniform feeding and housing conditions, were used in this study. The age of the bulls ranged between 1.2 and 6 years. One ejaculate was collected from each bull with pre-warmed artificial vagina. The two homeopathic complexes Ubichinon compositumÒ (Ubi comp) and Coenzyme compositum ad us. vet.Ò (CoQ10 comp) (Heel, Baden-Baden, Germany), were tested in this study.
Homeopathic complexes and sperm activity DM Aziz et al
100
The composition of Ubichinon compositumÓ (per 2.2 ml ampoule) is 22 ml each of Coenzyme Q10 D10, Acidum ascorbicum D6; Thiaminum hydrochloricum D6, Natrium ribroflavinum phosphoricum D6, Pyridoxinum hydrochloricum D6; Nicotinamidum D6, Vaccininum myrtillus D4, Colchicum D4, Podophyllum D4, Conium D4, Hydrastis D4, Galium aparine D6, Acidum sarcolacticum D6, Hydrochinonum D8, Acidum alpha-lipoicum D8, Sulfur D8, Manganum phosphoricum D8, Natrium diethyloxalaceticum D8, Trichinoylum D10, Anthrachinonum D10, Napthochinonum D10, para-Benzochinonum D10, Adenosinum triphosphoricum D10, Coenzymum A D10, Acidum acetylosalicylicum D10, Histaminum D10, Nadidum D10, Magnesium gluconicum D10. The composition of Coenzyme compositum ad us. vet.Ó (5 ml ampoule) is 0.05 ml each of Acidum ascorbicum D6, Natrium ribroflavinum phosphoricum D6, Thiaminum hydrochloricum D6, Pyridoxinum hydrochloricum D6, Nicotinamidum D6, Acidum cis-aconiticum D8, Acidum citricum D8, Acidum fumaricum D8, Acidum a-ketoglutaricum D8, Acidum malicum D8, Acidum succinicum D8, Barium oxalsuccinicum D10, Natrium pyruvicum D8, Cysteinum D6, Pulsatilla pratensis D6, Hepar sulfuris D10, Sulfur D10, Adenosinum triphosphoricum D10, Nadidum D8, Coenzymum A D8, Beta vulgaris var. conditiva D4, Natrium diethyloxalaceticum D6, Manganum phosphoricum D6, Magnesium oroticum D6, Cerium oxalicum D8, Acidum alpha-liponicum D6. Semen samples were evaluated immediate after collection, the sample from each bull was divided to seven aliquots and diluted as below to a final concentration approximately 30 106 sperm/ml. The diluted semen samples were incubated for 30 min at water path 37 C before the analyses. Hepes-0.1% BSA solution14 was used to dilute the semen samples. For each of the tested complexes two dilutions were prepared; 1:9 and 1:4 drug:Hepes-BSA (Vol:Vol). Hepes-BSA alone, 1:9 and 1:4 normal saline:Hepes-BSA (Vol:Vol) served as control groups. Flow cytometry analysis with an argon laser at 488 nm was performed to detect excitation of the fluorescent probes (FACScan, Becton Dickinson, Heidelberg, Germany). To estimate the mitochondrial activity, sperm viability and acrosomal integrity, the cell flow rate of the flow cytometer was placed on the low reading and for testing the sperm chromatin structure on the high reading. A total of 10,000 sperm were analyzed for each sample. To estimate the sperm mitochondrial activity, stock solutions of 0.53 mM of Rhodamine 123 (R-123) (R-302, Molecular Probes, Eugene, OR, USA) were prepared in DMSO (D-8779, Sigma, Deisenhofen, Germany) and 2.99 mM of Propidium iodide (PI) (P-4170, Sigma, Deisenhofen, Germany) in Tyrode’s Salt Solution (T-2397 PI, Sigma, Deisenhofen, Germany). The final staining solution contained 3 ml of R-123 stock solution and 12 ml of PI stock solution/ml of Hepes-BSA. The diluted semen (150 ml) was stained with 300 ml of the final staining solution. The samples were incubated at 37 C for 30 min before flow cytometric examination.15
Homeopathy
To evaluate the sperm viability, Live/Dead sperm viability kit (L-7011, Molecular Probes, Eugene, OR, USA) was used. A staining solution was prepared that contained 0.8 ml of SYBR-14 and 9 ml of PI/ml of Hepes-0.1% BSA. The diluted semen (150 ml) was stained with 300 ml of the staining solution and incubated at 37 C for 15 min prior to flow cytometric examination.16 The acrosomal integrity of spermatozoa was analyzed using a stock solution of 1 mM of Lyso Tracker Green DNA-20 (L-7526, Molecular Probes, Eugene, OR, USA), which was prepared in DMSO, and 2.99 mM PI in Tyrode’s Salt Solution. Final staining solution contained 5 ml of Lyso Tracker Green DNA-20 stock solution and 12 ml of PI stock solution/ml of Hepes-BSA. The diluted semen (150 ml) was stained with 300 ml of the staining solution and incubated at 37 C for 30 min before flow cytometric examination.17 To estimate the sperm chromatin structure, 200 ml of diluted semen was mixed with 400 ml of acid detergent solution. After 30 s, 1.2 ml of the Acridine orange solution was added. The sample was covered with aluminium foil and placed in the flow cytometer.18 Samples from each bull were repeated three times. Data were normalized by square root transformation, then the data were analyzed for normal distribution using Normality test (KolmogoroveSmirnov). Normal distributed data were compared by ANOVA test and significant differences were determined by the Duncan’s Multiple Range test. The data which were not normally distribution were analyzed using KruskaleWallis test and the significant differences were determined by Dunn’s test. All statistical procedures were performed using SigmaStat (Jandel scientific software V3.1). P < 0.05 was considered as statistically significant.
Results The volume of ejaculate ranged between 5 and 8 ml with an initial sperm motility of 65e80% and a total concentration of 0.81e1.10 109 sperms/ml. The results of the effects of the tested complexes on mitochondrial activity are shown graphically in Figure 2. There was no significant variation between the sperm mitochondrial activity of semen samples in the control group (Hepes alone) and those which were treated with both concentrations of normal saline. However the incubation of the bovine spermatozoa with both concentrations of the two tested homeopathic complexes increased mitochondrial activity significantly and that in the samples of high (1:4) concentration of Ubi comp (P < 0.01) and in those of low (1:9) concentration of CoQ10 comp (P < 0.05) (Figure 1). The other concentrations of the complexes were also associated with increased mitochondrial activity, but differences were not statistically significant. There was no significant variation between the effect of the low and the high concentration for either complex. The results concerning other sperm parameters, showed no significant differences between the results of the tested sperm parameters in the homeopathic treated and control samples (Table 1).
Homeopathic complexes and sperm activity DM Aziz et al
101
Figure 1 (A) Dot plots of spermatozoa stained with R-123 and PI, (1) sperms with active mitochondria and (2) Dead sperm. (B) Dot plots of sperms stained with SYBR-14 and PI, (1) Live sperms and (2) Dead sperms. (C) Dot plots of sperms stained with Lyso Tracker Green-20 and PI, (1) sperms with intact acrosome and (2) Dead sperms. (D) Dot plots of sperms stained with Acridine orange, COMP aT show the percentage of spermatozoa with denatured DNA.
Discussion The results show that sperm mitochondrial activity is enhanced by both homeopathic complexes. These effects could be due to an increase in the transport of electrons and protons in the process of energy production, leading to increase ATP synthesis in the mitochondrial membrane. With regard to their different components the action point
Figure 2 Mitochondrial activity (mean SD) of bovine semen samples after the incubation at 37 C for 30 min with two different dilutions of the tested homeopathic complexes, n = 6, *P < 0.05, **P < 0.01.
of CoQ10 comp. is likely to be the citric acid cycle and that of Ubi comp. in the respiration chain.19 Similar results have been reported in normal and pathological human spermatozoa when incubated with Coenzyme Q10.12,20 Antioxidant treatment can produce such an improvement in seminal parameters and sperm function in vitro and represents a possible treatment for certain forms of infertility,21 for example in preventing loss of motility and inhibiting lipid peroxidation.22 A negative correlation between reactive oxygen species and mitochondrial activity was reported.23 So an antioxidant respective radical protecting or detoxification effect offers a possible explanation for our results, especially because oxidative stress on sperm due to different reactive oxygen species may occur during handling procedures in artificial insemination. Mammalian spermatozoa are rich in polyunsaturated fatty acids and, thus, are very susceptible to reactive oxygen species attack.24 The absence of negative side effects is an additional recommendation for the application of these complexes. This result agrees with the meta-analysis or systematic reviews of controlled clinical trials which showed that, homeopathic therapy is more efficacious than placebo but do not report on adverse effects of homeopathic medicines.25 Homeopathy
Homeopathic complexes and sperm activity DM Aziz et al
102 Table 1 Results (mean SD) concerning the percentage of active mitochondria, sperm viability, acrosomal integrity and sperm chromatin structure of bovine semen samples after the incubation at 37 C for 30 min with the two diluents of the tested homeopathic complexes, n = 6 Groups
Concent.
% of Active mitochondria (R-123)
% of Sperm viability (SYBR-14)
% of Acrosomal integrity (Lyso-T-G)
SCSA (Acridine orange) aT
COMP aT
Hepes alone
e
72.4 6.1
71.2 3.0
69.6 5.8
235.5 17.2
16.4 6.6
Normal saline
1:9 1:4
70.6 6.1 71.2 5.7
70.0 2.9 70.6 2.8
67.7 5.7 68.3 5.4
237.9 26.3 237.8 24.8
18.8 5.6 18.6 6.1
Ubi comp.
1:9 1:4
71.8 5.6 70.4 5.7
70.6 3.4 70.2 2.8
68.9 5.3 67.6 5.4
235.5 21.5 231.4 25.4
17.6 5.9 16.9 6.3
CoQ10 comp
1:9 1:4
71.6 5.4 70.1 5.5
71.1 2.8 70.0 2.7
68.2 2.7 67.1 5.3
235.9 22.9 239.9 25.7
18.6 6.2 19.7 6.4
Conclusion The results indicate that the tested homeopathic complex medicines stimulate the mitochondrial activity of the semen samples without effects on the viability, acrosomal integrity and chromatin structure of the sperms. Whether this positive effect translates into improved fertilization capacity in artificial insemination should be the subject of further investigations.
Conflictsof interest The homeopathic complexes tested were donated by Biologische Heilmittel HEEL GmbH, 76532 Baden-Baden, Germany. No other conflicts of interest arise.
Acknowledgments We thank Dr E. Reinhart, of Biologische Heilmittel HEEL GmbH, 76532 Baden-Baden, Germany for his support.
References 1 Budworth PR, Amann RP, Chapman PL. Relationship between computerized measurements of motion of frozen-thawed bull sperm and fertility. J Androl 1988; 9: 41e54. 2 Liu DY, Clarke GN, Baker HWG. Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rate in vitro. J Androl 1991; 12: 231e239. 3 Kjaestad H, Ropstad E, Andersen Berg K. Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta Vet Scand 1993; 34: 299e303. 4 Correa JR, Pace MM, Zavos PM. Relationships among frozenthawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 1997; 48: 721e731. 5 Tardif S, Laforest JP, Comier N, Bailey JL. The importance of porcine sperm parameter on fertility in vivo. Theriogenology 1999; 52: 447e459. 6 Troiano L, Granata AR, Cossarizza A, et al. Mitochondrial membrane potential and DNA stainability in human sperm cells, a flow cytometry analysis with implications for male infertility. Exp Cell Res 1998; 241: 384e393. 7 Ericsson SA, Garner DL, Thomas CA, Downing TW, Marshall CE. Interrelationship among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozenthawed bovine spermatozoa. Theriogenology 1993; 39: 1009e1024.
Homeopathy
8 Ernster L, Dallner G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta 1995; 1271: 195e204. 9 Ernster L, Forsmark-Andree P. Ubiquinol: an endogenous antioxidant in aerobic organisms. Clin Investig 1993; 71: 60e65. 10 Sinclair S. Male infertility: nutritional and environmental consideration. Altern Med Rev 2000; 5: 28e38. 11 Mancini A, De Marinis L, Oradei A, et al. Coenzyme Q10 concentrations in normal and pharmacological human seminal fluid. J Androl 1994; 15: 591e594. 12 Lewin A, Lavon H. The effect of coenzyme Q10 on sperm motility and function. Molec Aspects Med 1997; 18: 213e219. 13 Tanimura J. Studies on arginine in human semen. 3. The influence of several drugs on male infertility. Bull Osaka Med Sch 1967; 13: 90e100. 14 Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995; 53: 276e284. 15 Garner DL, Thomas CA, Joerg HW, DeJarnette JM, Marshall CE. Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. Biol Reprod 1997; 57: 1401e1406. 16 Graham JK, Kunze E, Hammerstedt RH. Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 1990; 43: 55e64. 17 Thomas CA, Garner DL, DeJarnette JM, Marshall CE. Fluorometric assessment of acrosomal integrity and viability in cryopreserved bovine spermatozoa. Biol Reprod 1997; 56: 991e998. 18 Ballachey BE, Hohenboken WD, Evenson DP. Heterogeneity of spermatozoa nuclear chromatin structure and its relationship to bull fertility. Biol Reprod 1987; 36: 915e925. 19 Heine H, Herzberger G, Bauer Gl. Die Therapie mit Intermedi€ aren Katalysatoren in der Praxis. Baden-Baden: Aurelia-Verlag, 1998. 20 Mazzilli F, Bisanti A, Rossi T, De Santis L, Dondero F. Seminal and biological parameters in dyspermic patients with sperm hypomotility before and after treatment with ubiquinone. J Endocrinol Invest 1990; 13: 88. 21 Conte G, Milardi D, De Marinis L, Mancini A. Reactive oxygen species in male infertility. Review of literature and personal observations. Panminerva Med 1999; 41: 45e53. 22 Kim JG, Parthasarathy S. Oxidation and the spermatozoa. Semin Reprod Endocrinol 1998; 16: 235e239. 23 Wang X, Sharma RK, Gupta A, et al. Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study. Fertil Steril 2003; 80: 844e850. 24 Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl 1992; 13: 368e386. 25 Linde K, Clausius N, Ramirez G, et al. Are the clinical effects of homeopathy placebo effects. A meta-analysis of placebocontrolled trials. Lancet 1997; 350: 834e843.
ORIGINAL PAPER
Effects of two homeopathic complexes on bovine sperm mitochondrial activity Dhafer M Aziz1,*, Ute Schnurrbusch2 and Heinrich Enbergs1 1
Institute of Physiology, Biochemistry and Hygiene of Animals, Bonn University, Bonn, Germany Faculty of Veterinary Medicine, Large Animal Clinic for Theriogenology and Ambulatory Services, Leipzig University, Leipzig, Germany 2
Objectives: This study was conducted to evaluate the effect of two homeopathic complexes Ubichinon compositumÒ (Ubi comp) and Coenzyme compositum ad us. vet.Ò (CoQ10 comp) on bovine sperm mitochondrial activity. Methods: Sperm viability, acrosomal integrity and sperm chromatin structure were estimated to detect the possible side effect of complexes on other sperm parameters. Results: Mitochondrial activity was significantly enhanced by both Ubi comp (P < 0.01) and CoQ10 comp (P < 0.05). No effects were detected in other tested sperm parameters. Conclusion: The tested homeopathic complex medicines stimulate the mitochondrial activity of bovine sperm without effects on their viability, acrosomal integrity or chromatin structure. The possibility that this translates into improved fertilization capacity in artificial insemination should investigated. Homeopathy (2012) 101, 99e102.
Keywords: Sperm; Mitochondria; Bovine; Ubichinon; Coenzyme; Homeopathy
Introduction Sperm motility has a strong relationship with fertility1e4 and is a useful indicator of sperm-fertilizing capacity invivo.5 There is a positive correlation between mitochondrial membrane potential and spermatozoal motility.6 Mitochondria of the sperm midpiece generate energy to support motility; therefore changes in mitochondrial membrane potential are a good indicator of functional impairment without the direct assessment of sperm motility parameters.7 Coenzyme Q10, an integral redox and proton translocation component of the mitochondrial respiratory chain, also known as ubiquinone because its wide distribution throughout mammalian tissues,8 plays a key role in energy metabolism and has potent antioxidant properties for cellular membrane integrity.9 In sperm cells, coenzyme Q10 (CoQ10) is concentrated in the mitochondria, where it is involved in energy production. It also functions as an anti-
*Correspondence: Current address: Dhafer M Aziz, Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, P.O. Box 11141, Mosul, Iraq. E-mail: [email protected] Received 17 April 2011; revised 9 November 2011; accepted 9 December 2011
oxidant, preventing lipid peroxidation of sperm membranes.10 CoQ10 levels in human seminal fluid show a significant correlation with sperm count and motility.11 Significant increases in motility were observed when sperm samples were incubated in vitro with CoQ10.12 In another study coenzyme Q7 (an analogue of CoQ10) was given to infertile men resulting in increased sperm count and motility.13 The present study was designed to evaluate the effect of two commercial homeopathic combination complexes (approved in human and veterinary medicine as intermediate catalysts to stimulate or regulate disturbed metabolic processes) on bovine sperm mitochondrial activity.
Materials and methods Fresh semen samples from six bulls, maintained at Rinder Union West e.G. (Borken, Germany) under uniform feeding and housing conditions, were used in this study. The age of the bulls ranged between 1.2 and 6 years. One ejaculate was collected from each bull with pre-warmed artificial vagina. The two homeopathic complexes Ubichinon compositumÒ (Ubi comp) and Coenzyme compositum ad us. vet.Ò (CoQ10 comp) (Heel, Baden-Baden, Germany), were tested in this study.
Homeopathic complexes and sperm activity DM Aziz et al
100
The composition of Ubichinon compositumÓ (per 2.2 ml ampoule) is 22 ml each of Coenzyme Q10 D10, Acidum ascorbicum D6; Thiaminum hydrochloricum D6, Natrium ribroflavinum phosphoricum D6, Pyridoxinum hydrochloricum D6; Nicotinamidum D6, Vaccininum myrtillus D4, Colchicum D4, Podophyllum D4, Conium D4, Hydrastis D4, Galium aparine D6, Acidum sarcolacticum D6, Hydrochinonum D8, Acidum alpha-lipoicum D8, Sulfur D8, Manganum phosphoricum D8, Natrium diethyloxalaceticum D8, Trichinoylum D10, Anthrachinonum D10, Napthochinonum D10, para-Benzochinonum D10, Adenosinum triphosphoricum D10, Coenzymum A D10, Acidum acetylosalicylicum D10, Histaminum D10, Nadidum D10, Magnesium gluconicum D10. The composition of Coenzyme compositum ad us. vet.Ó (5 ml ampoule) is 0.05 ml each of Acidum ascorbicum D6, Natrium ribroflavinum phosphoricum D6, Thiaminum hydrochloricum D6, Pyridoxinum hydrochloricum D6, Nicotinamidum D6, Acidum cis-aconiticum D8, Acidum citricum D8, Acidum fumaricum D8, Acidum a-ketoglutaricum D8, Acidum malicum D8, Acidum succinicum D8, Barium oxalsuccinicum D10, Natrium pyruvicum D8, Cysteinum D6, Pulsatilla pratensis D6, Hepar sulfuris D10, Sulfur D10, Adenosinum triphosphoricum D10, Nadidum D8, Coenzymum A D8, Beta vulgaris var. conditiva D4, Natrium diethyloxalaceticum D6, Manganum phosphoricum D6, Magnesium oroticum D6, Cerium oxalicum D8, Acidum alpha-liponicum D6. Semen samples were evaluated immediate after collection, the sample from each bull was divided to seven aliquots and diluted as below to a final concentration approximately 30 106 sperm/ml. The diluted semen samples were incubated for 30 min at water path 37 C before the analyses. Hepes-0.1% BSA solution14 was used to dilute the semen samples. For each of the tested complexes two dilutions were prepared; 1:9 and 1:4 drug:Hepes-BSA (Vol:Vol). Hepes-BSA alone, 1:9 and 1:4 normal saline:Hepes-BSA (Vol:Vol) served as control groups. Flow cytometry analysis with an argon laser at 488 nm was performed to detect excitation of the fluorescent probes (FACScan, Becton Dickinson, Heidelberg, Germany). To estimate the mitochondrial activity, sperm viability and acrosomal integrity, the cell flow rate of the flow cytometer was placed on the low reading and for testing the sperm chromatin structure on the high reading. A total of 10,000 sperm were analyzed for each sample. To estimate the sperm mitochondrial activity, stock solutions of 0.53 mM of Rhodamine 123 (R-123) (R-302, Molecular Probes, Eugene, OR, USA) were prepared in DMSO (D-8779, Sigma, Deisenhofen, Germany) and 2.99 mM of Propidium iodide (PI) (P-4170, Sigma, Deisenhofen, Germany) in Tyrode’s Salt Solution (T-2397 PI, Sigma, Deisenhofen, Germany). The final staining solution contained 3 ml of R-123 stock solution and 12 ml of PI stock solution/ml of Hepes-BSA. The diluted semen (150 ml) was stained with 300 ml of the final staining solution. The samples were incubated at 37 C for 30 min before flow cytometric examination.15
Homeopathy
To evaluate the sperm viability, Live/Dead sperm viability kit (L-7011, Molecular Probes, Eugene, OR, USA) was used. A staining solution was prepared that contained 0.8 ml of SYBR-14 and 9 ml of PI/ml of Hepes-0.1% BSA. The diluted semen (150 ml) was stained with 300 ml of the staining solution and incubated at 37 C for 15 min prior to flow cytometric examination.16 The acrosomal integrity of spermatozoa was analyzed using a stock solution of 1 mM of Lyso Tracker Green DNA-20 (L-7526, Molecular Probes, Eugene, OR, USA), which was prepared in DMSO, and 2.99 mM PI in Tyrode’s Salt Solution. Final staining solution contained 5 ml of Lyso Tracker Green DNA-20 stock solution and 12 ml of PI stock solution/ml of Hepes-BSA. The diluted semen (150 ml) was stained with 300 ml of the staining solution and incubated at 37 C for 30 min before flow cytometric examination.17 To estimate the sperm chromatin structure, 200 ml of diluted semen was mixed with 400 ml of acid detergent solution. After 30 s, 1.2 ml of the Acridine orange solution was added. The sample was covered with aluminium foil and placed in the flow cytometer.18 Samples from each bull were repeated three times. Data were normalized by square root transformation, then the data were analyzed for normal distribution using Normality test (KolmogoroveSmirnov). Normal distributed data were compared by ANOVA test and significant differences were determined by the Duncan’s Multiple Range test. The data which were not normally distribution were analyzed using KruskaleWallis test and the significant differences were determined by Dunn’s test. All statistical procedures were performed using SigmaStat (Jandel scientific software V3.1). P < 0.05 was considered as statistically significant.
Results The volume of ejaculate ranged between 5 and 8 ml with an initial sperm motility of 65e80% and a total concentration of 0.81e1.10 109 sperms/ml. The results of the effects of the tested complexes on mitochondrial activity are shown graphically in Figure 2. There was no significant variation between the sperm mitochondrial activity of semen samples in the control group (Hepes alone) and those which were treated with both concentrations of normal saline. However the incubation of the bovine spermatozoa with both concentrations of the two tested homeopathic complexes increased mitochondrial activity significantly and that in the samples of high (1:4) concentration of Ubi comp (P < 0.01) and in those of low (1:9) concentration of CoQ10 comp (P < 0.05) (Figure 1). The other concentrations of the complexes were also associated with increased mitochondrial activity, but differences were not statistically significant. There was no significant variation between the effect of the low and the high concentration for either complex. The results concerning other sperm parameters, showed no significant differences between the results of the tested sperm parameters in the homeopathic treated and control samples (Table 1).
Homeopathic complexes and sperm activity DM Aziz et al
101
Figure 1 (A) Dot plots of spermatozoa stained with R-123 and PI, (1) sperms with active mitochondria and (2) Dead sperm. (B) Dot plots of sperms stained with SYBR-14 and PI, (1) Live sperms and (2) Dead sperms. (C) Dot plots of sperms stained with Lyso Tracker Green-20 and PI, (1) sperms with intact acrosome and (2) Dead sperms. (D) Dot plots of sperms stained with Acridine orange, COMP aT show the percentage of spermatozoa with denatured DNA.
Discussion The results show that sperm mitochondrial activity is enhanced by both homeopathic complexes. These effects could be due to an increase in the transport of electrons and protons in the process of energy production, leading to increase ATP synthesis in the mitochondrial membrane. With regard to their different components the action point
Figure 2 Mitochondrial activity (mean SD) of bovine semen samples after the incubation at 37 C for 30 min with two different dilutions of the tested homeopathic complexes, n = 6, *P < 0.05, **P < 0.01.
of CoQ10 comp. is likely to be the citric acid cycle and that of Ubi comp. in the respiration chain.19 Similar results have been reported in normal and pathological human spermatozoa when incubated with Coenzyme Q10.12,20 Antioxidant treatment can produce such an improvement in seminal parameters and sperm function in vitro and represents a possible treatment for certain forms of infertility,21 for example in preventing loss of motility and inhibiting lipid peroxidation.22 A negative correlation between reactive oxygen species and mitochondrial activity was reported.23 So an antioxidant respective radical protecting or detoxification effect offers a possible explanation for our results, especially because oxidative stress on sperm due to different reactive oxygen species may occur during handling procedures in artificial insemination. Mammalian spermatozoa are rich in polyunsaturated fatty acids and, thus, are very susceptible to reactive oxygen species attack.24 The absence of negative side effects is an additional recommendation for the application of these complexes. This result agrees with the meta-analysis or systematic reviews of controlled clinical trials which showed that, homeopathic therapy is more efficacious than placebo but do not report on adverse effects of homeopathic medicines.25 Homeopathy
Homeopathic complexes and sperm activity DM Aziz et al
102 Table 1 Results (mean SD) concerning the percentage of active mitochondria, sperm viability, acrosomal integrity and sperm chromatin structure of bovine semen samples after the incubation at 37 C for 30 min with the two diluents of the tested homeopathic complexes, n = 6 Groups
Concent.
% of Active mitochondria (R-123)
% of Sperm viability (SYBR-14)
% of Acrosomal integrity (Lyso-T-G)
SCSA (Acridine orange) aT
COMP aT
Hepes alone
e
72.4 6.1
71.2 3.0
69.6 5.8
235.5 17.2
16.4 6.6
Normal saline
1:9 1:4
70.6 6.1 71.2 5.7
70.0 2.9 70.6 2.8
67.7 5.7 68.3 5.4
237.9 26.3 237.8 24.8
18.8 5.6 18.6 6.1
Ubi comp.
1:9 1:4
71.8 5.6 70.4 5.7
70.6 3.4 70.2 2.8
68.9 5.3 67.6 5.4
235.5 21.5 231.4 25.4
17.6 5.9 16.9 6.3
CoQ10 comp
1:9 1:4
71.6 5.4 70.1 5.5
71.1 2.8 70.0 2.7
68.2 2.7 67.1 5.3
235.9 22.9 239.9 25.7
18.6 6.2 19.7 6.4
Conclusion The results indicate that the tested homeopathic complex medicines stimulate the mitochondrial activity of the semen samples without effects on the viability, acrosomal integrity and chromatin structure of the sperms. Whether this positive effect translates into improved fertilization capacity in artificial insemination should be the subject of further investigations.
Conflictsof interest The homeopathic complexes tested were donated by Biologische Heilmittel HEEL GmbH, 76532 Baden-Baden, Germany. No other conflicts of interest arise.
Acknowledgments We thank Dr E. Reinhart, of Biologische Heilmittel HEEL GmbH, 76532 Baden-Baden, Germany for his support.
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