Mechanism of Action of Low Dose Preparations from Coffea arabica, Gelsemium and Veratrum Based on in Vivo and in Vitro Neurophysiological Findings
ABSTRACT
Low dose remedies are widely administered in medicine. We used Tele-Stereo-EEG and the hippocampal slice preparation to measure physiological effects of orally given Coffea D6 (40 mg/kg), Gelsemium D4 (10 mg/kg) and Veratrum D6 (30 mg/kg) in rats. Adult rats were implanted with electrodes positioned stereotactically into four brain regions. Changes in field potentials were transmitted wirelessly. After frequency analysis data from 6 - 8 animals were averaged. For in vitro testing, preparations were superfused directly on hippocampal slices. Stimulation of Schaffer Collaterals by single stimuli (SS) or theta burst stimulation (TBS) resulted in stable population spike amplitudes. All three low dose preparations produced decreases of spectral power. Statistically significant changes were observed in delta, theta and alpha2 spectral power. In the hippocampal slice preparation Coffea facilitated signal transfer presumably by enhancing glutamate AMPA receptor transmission. Gelsemium showed a similar effect, but only after single shock stimulation. Opposite to this, attenuation of the electric pathway was recognized after theta burst stimulation due to AMPA receptor and glutamate metabotropic II receptor mediated transmission. Veratrum was able to attenuate glutamatergic due to receptor-mediated signalling sensitive to AMPA and NMDA. The results strongly speak in favour of the existence of biologically active molecules in these low dose preparations.
Low dose remedies are widely administered in medicine. We used Tele-Stereo-EEG and the hippocampal slice preparation to measure physiological effects of orally given Coffea D6 (40 mg/kg), Gelsemium D4 (10 mg/kg) and Veratrum D6 (30 mg/kg) in rats. Adult rats were implanted with electrodes positioned stereotactically into four brain regions. Changes in field potentials were transmitted wirelessly. After frequency analysis data from 6 - 8 animals were averaged. For in vitro testing, preparations were superfused directly on hippocampal slices. Stimulation of Schaffer Collaterals by single stimuli (SS) or theta burst stimulation (TBS) resulted in stable population spike amplitudes. All three low dose preparations produced decreases of spectral power. Statistically significant changes were observed in delta, theta and alpha2 spectral power. In the hippocampal slice preparation Coffea facilitated signal transfer presumably by enhancing glutamate AMPA receptor transmission. Gelsemium showed a similar effect, but only after single shock stimulation. Opposite to this, attenuation of the electric pathway was recognized after theta burst stimulation due to AMPA receptor and glutamate metabotropic II receptor mediated transmission. Veratrum was able to attenuate glutamatergic due to receptor-mediated signalling sensitive to AMPA and NMDA. The results strongly speak in favour of the existence of biologically active molecules in these low dose preparations.
KEYWORDS
Neurophysiology, Rat, Gelsemium sempervirens, Veratrum album, Coffea arabica, Electropharmacogram, Hippocampus Slice
Neurophysiology, Rat, Gelsemium sempervirens, Veratrum album, Coffea arabica, Electropharmacogram, Hippocampus Slice
Cite this paper
Dimpfel, W. and Biller, A. (2015) Mechanism of Action of Low Dose Preparations from Coffea arabica, Gelsemium and Veratrum Based on in Vivo and in Vitro Neurophysiological Findings. Journal of Behavioral and Brain Science, 5, 368-380. doi: 10.4236/jbbs.2015.59036.
Dimpfel, W. and Biller, A. (2015) Mechanism of Action of Low Dose Preparations from Coffea arabica, Gelsemium and Veratrum Based on in Vivo and in Vitro Neurophysiological Findings. Journal of Behavioral and Brain Science, 5, 368-380. doi: 10.4236/jbbs.2015.59036.
References
[1] Dimpfel, W. and Hoffmann, J.A. (2010) Electropharmacograms of Rasagiline, Its Metabolite Aminoindan and Selegiline in the Freely Moving Rat. Neuropsychobiology, 62, 213-220.
http://dx.doi.org/10.1159/000319947 [2] Dimpfel, W. (1995) Effects of Memantine on Synaptic Transmission in the Hippocampus in Vitro. Arzneimittelforschung, 45, 1-5. [3] Dimpfel, W. and Hoffmann, J.A. (2011) Effects of Rasagiline, Its Metabolite Aminoindan and Selegiline on Glutamate Receptor Mediated Signalling in the Rat Hippocampus Slice in Vitro. BMC Pharmacology, 11, 2.
http://dx.doi.org/10.1186/1471-2210-11-2 [4] Paxinos, G. and Watson, C. (1982) The Rat Brain in Stereotactic Coordinates. Academic Press, New York. [5] Dimpfel, W. (2013) Pharmacological Classification of Herbal Extracts by Means of Comparison to Spectral EEG Signatures Induced by Synthetic Drugs in the Freely Moving Rat. Journal of Ethnopharmacology, 149, 583-589.
http://dx.doi.org/10.1016/j.jep.2013.07.029 [6] Dimpfel, W. (2003) Preclinical Data Base of Pharmaco-Specific Rat EEG Fingerprints (Tele-Stereo-EEG). European Journal of Medical Research, 8, 199-207. [7] Dingledine, R. (1984) Brain Slices. Plenum Press, New York.
http://dx.doi.org/10.1007/978-1-4684-4583-1 [8] Dimpfel, W., Dalhoff, B., Hofmann, W. and Schlüter, G. (1994) Electrically Evoked Potentials in the Rat Hippocampus Slice in the Presence of Aminophylline Alone and in Combination with Quinolones. European Neuropsychopharmacology, 4, 151-156.
http://dx.doi.org/10.1016/0924-977X(94)90009-4 [9] Dimpfel, W., Spüler, M., Dalhoff, A., Hoffmann, W. and Schlüter, G. (1991) Hippocampal Activity in the Presence of Quinolones and Fenbufen In-Vitro. Antimicrobial Agents and Chemotherapy, 35, 1142-1146.
http://dx.doi.org/10.1128/AAC.35.6.1142 [10] Haas, H.L., Schaerer, B. and Vosmansky, M. (1979) A Simple Perfusion Chamber for the Study of Nervous Tissue Slices in Vitro. Journal of Neuroscience Methods, 1, 323-325.
http://dx.doi.org/10.1016/0165-0270(79)90021-9 [11] Schiff, S.J. and Somjen, G.G. (1985) The Effects of Temperature on Synaptic Transmission in Hippocampal Tissue Slices. Brain Research, 345, 279-284.
http://dx.doi.org/10.1016/0006-8993(85)91004-2 [12] Lynch, G. and Schubert, P. (1980) The Use of in Vitro Brain Slices for Multidisciplinary Studies of Synaptic Function. Annual Review of Neuroscience, 3, 1-22.
http://dx.doi.org/10.1146/annurev.ne.03.030180.000245 [13] Allan, R.D., Hanrahan, J.R., Hambley, T.W., Johnston, G.A., Mewett, K.N. and Mitrovic, A.D. (1990) Synthesis and Activity of a Potent N-Methyl-D-Aspartic Acid Agonist, Trans-1-Aminocyclobutane-1,3-Dicarboxylic acid, and Related Phosphonic and Carboxylic Acids. Journal of Medicinal Chemistry, 33, 2905-2915.
http://dx.doi.org/10.1021/jm00172a036 [14] Wong, L.A., Mayer, M.L., Jane, D.E. and Watkins, J.C. (1994) Willardiines Differentiate Agonist Binding Sites for Kainate-Versus AMPA-Preferring Glutamate Receptors in DRG and Hippocampal Neurons. The Journal of Neuroscience, 14, 3881-3897. [15] Hawkins, L.M., Beaver, K.M., Jane, D.E., Taylor, P.M., Sunter, D.C. and Roberts, P.J. (1995) Binding of the New Radioligand (S)-[3H]AMPA to Rat Brain Synaptic Membranes: Effects of a Series of Structural Analogues of the Non-NMDA Receptor Agonist Willardiine. Neuropharmacology, 34, 405-410.
http://dx.doi.org/10.1016/0028-3908(94)00157-N [16] Jane, D.E., Hoo, K., Kamboj, R., Deverill, M., Bleakman, D. and Mandelzys, A. (1997) Synthesis of Willardiine and 6-Azawillardiine Analogs: Pharmacological Characterization on Cloned Homomeric Human AMPA and Kainate Receptor Subtypes. Journal of Medicinal Chemistry, 40, 3645-3450.
http://dx.doi.org/10.1021/jm9702387 [17] Matzen, L., Engesgaard, A., Ebert, B., Didriksen, M., Frolund, B., Krogsgaard-Larsen, P. and Jaroszewski, J.W. (1997) AMPA Receptor Agonists: Synthesis, Protolytic Properties, and Pharmacology of 3-Isothiazolol Bioisosteres of Glutamic Acid. Journal of Medicinal Chemistry, 40, 520-527.
http://dx.doi.org/10.1021/jm9607212 [18] Clarke, V.R., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D. and Bleakman, D. (1997) A Hippocampal GluR5 Kainate Receptor Regulating Inhibitory Synaptic Transmission. Nature, 389, 599-603.
http://dx.doi.org/10.1038/39315 [19] Moldrich, R.X., Cheung, N.S., Pascoe, C.J. and Beart, P.M. (1999) Excitotoxic Injury Profiles of Low-Affinity Kainate Receptor Agonists in Cortical Neuronal Cultures. European Journal of Pharmacology, 378, R1-R3.
http://dx.doi.org/10.1016/S0014-2999(99)00456-2 [20] Kaminski, R.M., Banerjee, M. and Rogawski, M.A. (2004) Topiramate Selectively Protects Against Seizures Induced by ATPA, a GluR5 Kainate Receptor Agonist. Neuropharmacology, 46, 1097-1104.
http://dx.doi.org/10.1016/j.neuropharm.2004.02.010 [21] Irving, A.J., Schofield, J.G., Watkins, J.C., Sunter, D.C. and Collingridge, G.L. (1990) 1S,3R-ACPD Stimulates and L-AP3 Blocks Ca2+Mobilization in Rat Cerebellar Neurons. European Journal of Pharmacology, 186, 363-365.
http://dx.doi.org/10.1016/0014-2999(90)90462-F [22] Pin, J.P. and Duvoisin, R. (1995) The Metabo-tropic Glutamate Receptors: Structure and Functions. Neuropharmacology, 34, 1-26
http://dx.doi.org/10.1016/0028-3908(94)00129-G [23] Knopfel, T., Kuhn, R. and Allgeier, H. (1995) Metabo-tropic Glutamate Receptors: Novel Targets for Drug Development. Journal of Medicinal Chemistry, 38, 1417-1426.
http://dx.doi.org/10.1021/jm00009a001 [24] Bennett, D.A., Bernard, P.S., Amrick, C.L., Wilson, D.E., Liebman, J.M. and Hutchison, A.J. (1989) Behavioral Pharmacological Profile of CGS 19755, a Competitive Antagonist at N-Methyl-D-Aspartate Receptors. Journal of Pharmacology and Experimental Therapeutics, 250, 454-460. [25] Gill, R., Nordholm, L. and Lodge, D. (1992) The Neuroprotective Actions of 2,3-Dihydroxy-6-Nitro-7-Sulfamoyl-Ben-zo(F)Quinoxaline (NBQX) in a Rat Focal Ischaemia Model. Brain Research, 580, 35-43.
http://dx.doi.org/10.1016/0006-8993(92)90924-X [26] More, J.C., Troop, H.M., Dolman, N.P. and Jane, D.E. (2003) Structural Requirements for Novel Willardiine Derivatives Acting as AMPA and Kainate Receptor Antagonists. British Journal of Pharmacology, 138, 1093-1100.
http://dx.doi.org/10.1038/sj.bjp.0705148 [27] Xi, Z.X., Baker, D.A., Shen, H., Carson, D.S. and Kalivas, P.W. (2002) Group II Metabotropic Glutamate Receptors Modulate Extracellular Glutamate in the Nucleus Accumbens. Journal of Pharmacology and Experimental Therapeutics, 300, 162-171.
http://dx.doi.org/10.1124/jpet.300.1.162 [28] Dutt, V., Dhar, V.J. and Sharma, A. (2010) Antianxiety Activity of Gelsemium Sempervirens. Pharmaceutical Biology, 48, 1091-1096.
http://dx.doi.org/10.3109/13880200903490521 [29] Bellavite, P., Magnani, P., Zanolin, E. and Conforti, A. (2009) Homeopathic Doses of Gelsemium Sempervirens Improve the Behavior of Mice in Response to Novel Environments. Evidence-Based Complementary and Alternative Medicine, 2011, Article ID: 362517. [30] Paris, A., Schmidlin, S., Mouret, S., Hodaj, E., Marijinen, P., Boujedaini, N., Polosan, M. and Crasowski, J.L. (2011) Effect of Gelsemium 5CH and 15CH on Anticipatory Anxiety: A Phase III, Single Centre, Randomized, Placebo-Controlled Study. Fundamental & Clinical Pharmacology, 26, 751-760. [31] Holscher, C. (1999) Synaptic Plasticity and Learning and Memory: LTP and Beyond. Journal of Neuroscience Research, 58, 62-75.
http://dx.doi.org/10.1002/(SICI)1097-4547(19991001)58:1<62::AID-JNR7>3.0.CO;2-G [32] Danysz, W., Parsons, C.G., Mobius, H.J., Stoffler, A. and Quack, G. (2000) Neuroprotective and Symptomatological Action of Memantine Relevant for Alzheimers’s Disease—A Unified Glutamatergic Hypothesis on the Mechanism of Action. Neurotoxicity Research, 2, 85-97.
http://dx.doi.org/10.1007/BF03033787 [33] Planells-Cases, R., Lerma, J. and Ferrer-Montiel, A. (2006) Pharmacological Interventions at Ionotropic Glutamate Receptor Complexes. Current Pharmaceutical Design, 12, 4583-4596.
http://dx.doi.org/10.2174/138161206778522092 [34] Dimpfel, W. (2015) Drug Discovery and Translational Medicine. Neurophysiological Techniques Provide a Holistic Approach to Saving Animals. BoD Verlag, Norderstetten.
[1] Dimpfel, W. and Hoffmann, J.A. (2010) Electropharmacograms of Rasagiline, Its Metabolite Aminoindan and Selegiline in the Freely Moving Rat. Neuropsychobiology, 62, 213-220.
http://dx.doi.org/10.1159/000319947 [2] Dimpfel, W. (1995) Effects of Memantine on Synaptic Transmission in the Hippocampus in Vitro. Arzneimittelforschung, 45, 1-5. [3] Dimpfel, W. and Hoffmann, J.A. (2011) Effects of Rasagiline, Its Metabolite Aminoindan and Selegiline on Glutamate Receptor Mediated Signalling in the Rat Hippocampus Slice in Vitro. BMC Pharmacology, 11, 2.
http://dx.doi.org/10.1186/1471-2210-11-2 [4] Paxinos, G. and Watson, C. (1982) The Rat Brain in Stereotactic Coordinates. Academic Press, New York. [5] Dimpfel, W. (2013) Pharmacological Classification of Herbal Extracts by Means of Comparison to Spectral EEG Signatures Induced by Synthetic Drugs in the Freely Moving Rat. Journal of Ethnopharmacology, 149, 583-589.
http://dx.doi.org/10.1016/j.jep.2013.07.029 [6] Dimpfel, W. (2003) Preclinical Data Base of Pharmaco-Specific Rat EEG Fingerprints (Tele-Stereo-EEG). European Journal of Medical Research, 8, 199-207. [7] Dingledine, R. (1984) Brain Slices. Plenum Press, New York.
http://dx.doi.org/10.1007/978-1-4684-4583-1 [8] Dimpfel, W., Dalhoff, B., Hofmann, W. and Schlüter, G. (1994) Electrically Evoked Potentials in the Rat Hippocampus Slice in the Presence of Aminophylline Alone and in Combination with Quinolones. European Neuropsychopharmacology, 4, 151-156.
http://dx.doi.org/10.1016/0924-977X(94)90009-4 [9] Dimpfel, W., Spüler, M., Dalhoff, A., Hoffmann, W. and Schlüter, G. (1991) Hippocampal Activity in the Presence of Quinolones and Fenbufen In-Vitro. Antimicrobial Agents and Chemotherapy, 35, 1142-1146.
http://dx.doi.org/10.1128/AAC.35.6.1142 [10] Haas, H.L., Schaerer, B. and Vosmansky, M. (1979) A Simple Perfusion Chamber for the Study of Nervous Tissue Slices in Vitro. Journal of Neuroscience Methods, 1, 323-325.
http://dx.doi.org/10.1016/0165-0270(79)90021-9 [11] Schiff, S.J. and Somjen, G.G. (1985) The Effects of Temperature on Synaptic Transmission in Hippocampal Tissue Slices. Brain Research, 345, 279-284.
http://dx.doi.org/10.1016/0006-8993(85)91004-2 [12] Lynch, G. and Schubert, P. (1980) The Use of in Vitro Brain Slices for Multidisciplinary Studies of Synaptic Function. Annual Review of Neuroscience, 3, 1-22.
http://dx.doi.org/10.1146/annurev.ne.03.030180.000245 [13] Allan, R.D., Hanrahan, J.R., Hambley, T.W., Johnston, G.A., Mewett, K.N. and Mitrovic, A.D. (1990) Synthesis and Activity of a Potent N-Methyl-D-Aspartic Acid Agonist, Trans-1-Aminocyclobutane-1,3-Dicarboxylic acid, and Related Phosphonic and Carboxylic Acids. Journal of Medicinal Chemistry, 33, 2905-2915.
http://dx.doi.org/10.1021/jm00172a036 [14] Wong, L.A., Mayer, M.L., Jane, D.E. and Watkins, J.C. (1994) Willardiines Differentiate Agonist Binding Sites for Kainate-Versus AMPA-Preferring Glutamate Receptors in DRG and Hippocampal Neurons. The Journal of Neuroscience, 14, 3881-3897. [15] Hawkins, L.M., Beaver, K.M., Jane, D.E., Taylor, P.M., Sunter, D.C. and Roberts, P.J. (1995) Binding of the New Radioligand (S)-[3H]AMPA to Rat Brain Synaptic Membranes: Effects of a Series of Structural Analogues of the Non-NMDA Receptor Agonist Willardiine. Neuropharmacology, 34, 405-410.
http://dx.doi.org/10.1016/0028-3908(94)00157-N [16] Jane, D.E., Hoo, K., Kamboj, R., Deverill, M., Bleakman, D. and Mandelzys, A. (1997) Synthesis of Willardiine and 6-Azawillardiine Analogs: Pharmacological Characterization on Cloned Homomeric Human AMPA and Kainate Receptor Subtypes. Journal of Medicinal Chemistry, 40, 3645-3450.
http://dx.doi.org/10.1021/jm9702387 [17] Matzen, L., Engesgaard, A., Ebert, B., Didriksen, M., Frolund, B., Krogsgaard-Larsen, P. and Jaroszewski, J.W. (1997) AMPA Receptor Agonists: Synthesis, Protolytic Properties, and Pharmacology of 3-Isothiazolol Bioisosteres of Glutamic Acid. Journal of Medicinal Chemistry, 40, 520-527.
http://dx.doi.org/10.1021/jm9607212 [18] Clarke, V.R., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A., Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D. and Bleakman, D. (1997) A Hippocampal GluR5 Kainate Receptor Regulating Inhibitory Synaptic Transmission. Nature, 389, 599-603.
http://dx.doi.org/10.1038/39315 [19] Moldrich, R.X., Cheung, N.S., Pascoe, C.J. and Beart, P.M. (1999) Excitotoxic Injury Profiles of Low-Affinity Kainate Receptor Agonists in Cortical Neuronal Cultures. European Journal of Pharmacology, 378, R1-R3.
http://dx.doi.org/10.1016/S0014-2999(99)00456-2 [20] Kaminski, R.M., Banerjee, M. and Rogawski, M.A. (2004) Topiramate Selectively Protects Against Seizures Induced by ATPA, a GluR5 Kainate Receptor Agonist. Neuropharmacology, 46, 1097-1104.
http://dx.doi.org/10.1016/j.neuropharm.2004.02.010 [21] Irving, A.J., Schofield, J.G., Watkins, J.C., Sunter, D.C. and Collingridge, G.L. (1990) 1S,3R-ACPD Stimulates and L-AP3 Blocks Ca2+Mobilization in Rat Cerebellar Neurons. European Journal of Pharmacology, 186, 363-365.
http://dx.doi.org/10.1016/0014-2999(90)90462-F [22] Pin, J.P. and Duvoisin, R. (1995) The Metabo-tropic Glutamate Receptors: Structure and Functions. Neuropharmacology, 34, 1-26
http://dx.doi.org/10.1016/0028-3908(94)00129-G [23] Knopfel, T., Kuhn, R. and Allgeier, H. (1995) Metabo-tropic Glutamate Receptors: Novel Targets for Drug Development. Journal of Medicinal Chemistry, 38, 1417-1426.
http://dx.doi.org/10.1021/jm00009a001 [24] Bennett, D.A., Bernard, P.S., Amrick, C.L., Wilson, D.E., Liebman, J.M. and Hutchison, A.J. (1989) Behavioral Pharmacological Profile of CGS 19755, a Competitive Antagonist at N-Methyl-D-Aspartate Receptors. Journal of Pharmacology and Experimental Therapeutics, 250, 454-460. [25] Gill, R., Nordholm, L. and Lodge, D. (1992) The Neuroprotective Actions of 2,3-Dihydroxy-6-Nitro-7-Sulfamoyl-Ben-zo(F)Quinoxaline (NBQX) in a Rat Focal Ischaemia Model. Brain Research, 580, 35-43.
http://dx.doi.org/10.1016/0006-8993(92)90924-X [26] More, J.C., Troop, H.M., Dolman, N.P. and Jane, D.E. (2003) Structural Requirements for Novel Willardiine Derivatives Acting as AMPA and Kainate Receptor Antagonists. British Journal of Pharmacology, 138, 1093-1100.
http://dx.doi.org/10.1038/sj.bjp.0705148 [27] Xi, Z.X., Baker, D.A., Shen, H., Carson, D.S. and Kalivas, P.W. (2002) Group II Metabotropic Glutamate Receptors Modulate Extracellular Glutamate in the Nucleus Accumbens. Journal of Pharmacology and Experimental Therapeutics, 300, 162-171.
http://dx.doi.org/10.1124/jpet.300.1.162 [28] Dutt, V., Dhar, V.J. and Sharma, A. (2010) Antianxiety Activity of Gelsemium Sempervirens. Pharmaceutical Biology, 48, 1091-1096.
http://dx.doi.org/10.3109/13880200903490521 [29] Bellavite, P., Magnani, P., Zanolin, E. and Conforti, A. (2009) Homeopathic Doses of Gelsemium Sempervirens Improve the Behavior of Mice in Response to Novel Environments. Evidence-Based Complementary and Alternative Medicine, 2011, Article ID: 362517. [30] Paris, A., Schmidlin, S., Mouret, S., Hodaj, E., Marijinen, P., Boujedaini, N., Polosan, M. and Crasowski, J.L. (2011) Effect of Gelsemium 5CH and 15CH on Anticipatory Anxiety: A Phase III, Single Centre, Randomized, Placebo-Controlled Study. Fundamental & Clinical Pharmacology, 26, 751-760. [31] Holscher, C. (1999) Synaptic Plasticity and Learning and Memory: LTP and Beyond. Journal of Neuroscience Research, 58, 62-75.
http://dx.doi.org/10.1002/(SICI)1097-4547(19991001)58:1<62::AID-JNR7>3.0.CO;2-G [32] Danysz, W., Parsons, C.G., Mobius, H.J., Stoffler, A. and Quack, G. (2000) Neuroprotective and Symptomatological Action of Memantine Relevant for Alzheimers’s Disease—A Unified Glutamatergic Hypothesis on the Mechanism of Action. Neurotoxicity Research, 2, 85-97.
http://dx.doi.org/10.1007/BF03033787 [33] Planells-Cases, R., Lerma, J. and Ferrer-Montiel, A. (2006) Pharmacological Interventions at Ionotropic Glutamate Receptor Complexes. Current Pharmaceutical Design, 12, 4583-4596.
http://dx.doi.org/10.2174/138161206778522092 [34] Dimpfel, W. (2015) Drug Discovery and Translational Medicine. Neurophysiological Techniques Provide a Holistic Approach to Saving Animals. BoD Verlag, Norderstetten.