The Effect of Tarantula cubensis (Theranekron) on Some Reproductive Parameters in Male Rats

Document Type : Original Articles

Authors

1 Department of Artificial Insemination, Faculty of Veterinary, Van Yuzuncu Yil University, Van, Turkey.

2 Department of Pathology, Faculty of Veterinary, Van Yuzuncu Yil University, Van, Turkey.

10.32598/ijvm.17.2.1005343

Abstract

Background: Tarantula cubensis (theranekron), obtained from the poison of Cuban Tarantula, is a homeopathic drug frequently used in animal health. 
Objectives: The effect of theranekron on reproductive parameters in male animals is unknown. In this study, the effect of theranekron on male reproductive parameters was investigated. 
Methods: Three different groups of rats were created: control (n=8) (no application), theranekron I (n=8) (subcuteneous injection of 0.3 mg/kg on days 0, 5, and 10), and theranekron II (n=8) (subcuteneous injection of 0.3 mg/kg on days 0, 5, and 10). Control and theranekron I groups were sacrificed at the end of the 4 weeks, and the theranekron II group was sacrificed at the end of the 6 weeks. Sperm motility, density, abnormal rate, DNA damage, and testicular histopathology were examined. 
Results: No statistical difference was observed in the parameters examined between the study groups (P>0.05). It was determined that the theranekron did not change sperm motility, density and spermatozoa ratio, also no histopathological changes, and sperm DNA damage.
Conclusion: There is no harm in using theranekron in male animals during the breeding and nonbreeding seasons.

Keywords


Article Title [Persian]

اثرترانتولا کوبنسیس(ترانکرون) بر برخی پارامترهای تولید مثلی در موش های صحرایی نر

Authors [Persian]

  • ولکان کوشال 1
  • اومل مر فاروغ کلش 2
1 گروه تلقیح مصنوعی،دانشکده دامپزشکی یوزونجو ایل،وان،ترکیه
2 گروپاتولوژی،دانشکده دامپزشکی یوزونجو ایل،وان،ترکیه

Keywords [Persian]

  • ترانتولا کوبنسیس:ترانکرون:اسپرم:بیضه:موش صحرایی

1. Introduction
Tarantula cubensis extract, a homeopathic medicine, is obtained from the poison of the Cuban tarantula (Sencar et al., 2021). This drug is commercially available under the trade name theranekron® (Richter Pharma, Wels, Austria). Although the mechanism of action of T. cubensis venom is not clear, it is believed that it stimulates the immune system, activates vitality, and suppresses inflammation spontaneously in proliferative lesions (Iacopetti et al., 2020; Gültekin and Vural, 2007; Sencar et al., 2021). Theranekron helps in the treatment of inflammation and necrosis. It has been reported that it provides healing with demarcation, regeneration, resorption, and antiphlogistic action (Iacopetti et al., 2020; Gültekin & Vural, 2007; Sencar et al., 2021; Song et al., 2017; Karabacak et al., 2015; Adib-Hashemi et al., 2015; Güven et al., 2022).
Theranekronis used in the treatment of many genital diseases such as mastitis, breast edema, retention of secundinarium, papillomatosis, breast tumor, genital organ tumors, metritis (Kaçar et al., 2007; Oryan et al., 2012; Swamy et al., 2020; Sardari et al., 2007; Ghasemi et al., 2016; Lotfollahzadeh, 2020; Gürbulak et al., 2014; Stampa, 1986; Icen et al., 2011).
In the literature review, no study examined the effect of theranekron application on male reproductive parameters. This study aims to investigate the changes in the testis and spermatological parameters resulting from the theranekron application. It is the first study carried out in this context and has a pioneering character.

 

2. Materials and Methods
Study animals
Adult, pathogen-free, male Albino Wistar rats were obtained from Van Yüzüncü Yıl University. The rats aged 3-4 months on average and weighing 200-250 g were used in the study. Animals were fed ad libitum and kept under 12 hours of light and 12 hours of dark per day. The living areas had an average temperature of 26°C and 60% relative humidity. 
Study groups
Twenty-four rats were randomly divided into 3 groups.
Control (n=8): No medication was administered. The rats were sacrificed after anesthesia (using ketamine+xylazine) at the end of the fourth week.
Theranekron I (n=8): theranekron (Richter-Pharma AG, Wels, Austria) was administered subcutaneously at a dose of 0.3 mg/kg on days 0, 5, and 10. The rats were sacrificed after anesthesia (using ketamine+xylazine) at the end of the fourth week (Dolapcioglu et al., 2013; Kozlu et al., 2021).
Theranekron II (n=8): theranekron (Richter-Pharma AG, Wels, Austria) was administered subcutaneously at a dose of 0.3 mg/kg on days 0, 5, and 10. The rats were sacrificed after anesthesia (using ketamine+xylazine) at the end of the sixth week (Dolapcioglu et al., 2013; Kozlu et al., 2021).
Anesthesia protocol
Xylazine hydrochloride (5 mg/kg) (Basilazine® 2%, Bavet, Turkey)+ketamine hydrochloride (80 mg/kg) (Ketasol®, Interhas, Turkey) were drawn into the same injector, administered intraperitoneally (Smith, 1993; Korkmaz & Sancak, 2022).
Sperm examination
Motility examination (progressive motility)
The sperm sample was obtained by epididymis puncture immediately after sacrifice and was placed on a glass slide on the heating table set to 38°C. The coverglass was closed at an angle of 45°, and motility (in %) was detected under microscope at 40x magnification. Uniform, linear, forward-moving spermatozoa were compared to immobile, swirling, and quivering spermatozoa (Hafez & Hafez, 2013).
Density analysis
After an epididymal puncture, 0.1 mL of sperm sample was added to eppendorf tubes with 0.5 mL Hayem solution (Norateks, Germany). Sperm count per mL was calculated on a thoma cell counting chamber (Hafez & Hafez 2013).
Abnormal sperm ratio
The sperm obtained by epididymis puncture was transferred to eppendorf tubes with 0.5 mL Hancock solution (Norateks, Germany). At least 400 sperm samples were examined at 40x magnification to determine the ratio. The proportion of spermatozoa with anomalies in the head, tail, and neck was determined (Hafez & Hafez, 2013).
Sperm DNA damage
DNA fragmentation index was assessed using the Halomax® (Spain) kit. Sperm DNA damage was calculated according to the protocol of the Halotech, Halomax HT-RN40 kit. A total of 600 sperms were counted for each group. Spermatozoa were evaluated according to Figure 1 (normal, fragmented, degraded).

 

The damage ratio was calculated according to the Equation 1.
1) Sperm DNA fragmentation (SDF)=(fragmented+degraded)/(total sperm counted)
Histopathological examination
At the end of the experiment, necropsies of the rats were performed, and testis tissue samples were taken. After the tissue pieces were fixed in 10% buffered formaldehyde solution, routine tissue follow-up was performed and embedded in paraffin blocks, and 4 μm sections were taken with a microtome. They were stained with hematoxylin-eosin (H&E) and examined under a light microscope, and morphological findings were photographed and evaluated.

Statistical analysis
    SPSS software, version 20 (Chicago, IL, USA) was used for statistical analysis. All data were expressed as Mean±SD. Statistical studies of the groups were conducted using the one-way ANOVA followed by post hoc multiple comparisons (Tukey test) for comparative analysis between the study groups. P<0.05 was regarded as statistically significant.

 

3. Results
Figure 2 shows the sperm DNA damages and DNA fragmentation ratio is mentioned in Table 1.

 

 

 

In the histopathological examinations, normal histological structures of the testis were observed in all groups (A-B Control; C-D theranekron I; theranekron II E-F) (Figure 3).

 

The ratio of motility, density, abnormal sperm ratios ratio are mentioned in Table 2.

 

4. Discussion
The effect of theranekron depends on spider venom in its structure, and this component is effective for a long time. Although theranekron is frequently used in treating many diseases in veterinary medicine, no research has investigated its effects on male reproductive parameters. In this study, the impact of theranekron on reproductive parameters in male animals was investigated.
In the study, sperm motility, density, and abnormal sperm rates were examined. There was no statistical difference between the study groups (P>0.05). As a result of histopathological examination, no difference was observed between the groups in seminiferous tubules contours, Sertoli cells, Leydig cells, and spermatogonia. These results show that theranekron does not have a negative effect on spermatogenesis.
In examining sperm DNA damage, the ratios of breaks in the DNA chain, the proportion of DNA-damaged spermatozoa, and fragmented and degraded sperm do not differ statistically between groups (P>0.05).

 

5. Conclusion
Based on this study, theranekron did not have a negative effect on sperm motility, density, and abnormal spermatozoa rates; it did not cause a histopathological change, or affect the sperm DNA damage rate. For these reasons, theranekron does not have a negative effect on male reproductive parameters, and there is no harm in using it during breeding and non-breeding seasons.

 

Ethical Considerations

 

Compliance with ethical guidelines
The study was undertaken under agreement No. 2022/11-11 of Van Yüzüncü Yıl University Animal Experiments Local Ethics Committee, dated 03/11/2022.

 

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

 

Authors' contributions
Conceptualization, supervision, investigation and writing-original draft: Volkan Koşal, and Ömer Faruk Keleş; Writing–review & editing:Volkan Koşal; Data collection: Ömer Faruk Keleş. 

 

Conflict of interest
All authors declared no conflict of interest.

 

Acknowledgments
The authors would like to thank the Van Yüzüncü Yıl University.

 

References

 

Adib-Hashemi, F., Farahmand, F., Hesari, S. F., Rezakhaniha, B., Fallah, E., & Fayyaz, A. F., et al. (2015). Anti-inflammatory and protective investigations on the effects of theranekron "an alcoholic extract of the tarantula cubensis" on wound healing of peritoneal in the rat: An in vivo comparative study. Diagnostic pathology, 10, 19. [DOI:10.1186/s13000-015-0252-x] [PMID] [PMCID]

Dolapcioglu, K., Dogruer, G., Ozsoy, S., Ergun, Y., Ciftci, S., & Soylu Karapinar, O., et al. (2013). Theranekron for treatment of endometriosis in a rat model compared with medroxyprogesterone acetate and leuprolide acetate. European Journal Of Obstetrics, Gynecology, and Reproductive Biology, 170(1), 206–210. [DOI:10.1016/j.ejogrb.2013.05.026] [PMID]

Ghasemi-Dizgahl, A., Nami, B., & Amirmozafari, N. (2016). Tarantula cubensis venom (theranekron) selectively destroys human cancer cells via activating caspase-3-mediated apoptosis. Acta Medica International, 4(1), 73-79. [DOI:10.5530/ami.2017.4.14]

Gültiken, N. & Vural, M. R. (2007). The effect of Tarantula Cubensis extract applied in pre and postoperative period of canine mammary tumours . Journal of Istanbul Veterinary Sciences , (2) , 13-23. [Link]

Gürbulak, K., Akcay, A., Gümüşsoy, K. S., Sist, B., Steiner, S., & Abay, M., et al. (2014). Investigation of the efficacy of tarantula cubensis extract (theranekron D6) in the treatment of subclinical and clinical mastitis in dairy cows. Turkish Journal of Veterinary & Animal Sciences. 38(6), 712-718. [DOI:10.3906/vet-1405-101].

Güven, N., Özkan, S., Türközü, T., Koç, S., Keleş, Ö. F., Yener, Z., & Karasu, A. (2022). The effect of theranekron on femur fracture healing in an experimental rat model. Joint diseases and related surgery, 33(2), 374–384. [DOI: 10.52312/jdrs.2022.640] [PMID]

Hafez, E. S. E., & Hafez, B. (2013). Reproduction in farm animals. 7th ed. Hoboken: WileyBlackwell. [Link]

Iacopetti, I., Patruno, M., Melotti, L., Martinello, T., Bedin, S., Badon, T., et al. (2020). Autologous platelet-rich plasma enhances the healing of large cutaneous wounds in dogs. Frontiers in Veterinary Science, 7, 575449. [DOI:10.3389/fvets.2020.575449] [PMID] [PMCID] 

Icen, H., Sekin, S., Simsek, A., Kochan, A., & Tunik, S. (2011). The efficacy of tarantula cubensis extract (theranekron) in treatment of canine oral papillomatosis. Asian Journal of Animal and Veterinary Advances. 6(7), 744-749. [DOI:10.3923/ajava.2011.744.749]

Kaçar C., Zonturlu, A. K., Oral, H., Yıldız, S., & Arı, U. Ç. (2007). [The effects of theranekron application on uterus involution and vaginal efluence in cows early puerperal period (Turkish)]. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 13 (1), 11-15. [DOI:10.9775/kvfd.2006.36-A]

Karabacak, M., Eraslan, G., Kanbur, M., & Sarıca, Z. S. (2015). Effects of tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats. Homeopathy : The Journal of the Faculty of Homeopathy, 104(3), 205–210. [DOI:10.1016/j.homp.2015.02.005] [PMID]

Korkmaz, M., & Sancak, T. (2022). Clinical and histopathological comparison of the efficacy of ointments containing centella asiatica extract, zinc oxide and alaptidum in wound healing. Van Veterinary Journal, 33(3), 141-148. [DOI:10.36483/vanvetj.1187715]

Kozlu, T., Güler, F., Peker Akalın, P., Kazak, F., Ergün, Y., & Aslan E. (2021). The influence of theranekron on glucose6-phosphate dehydrogenase activity in rat ovaries, intact and with ischemiareperfusion injury. Veterinarski Arhiv, 91(1), 81-88. [DOI:10.24099/vet.arhiv.0828] 

Lotfollahzadeh, S. (2020). Comparative therapeutic effects of Theranekron D2 and Theranekron D6 in the treatment of foot- and- mouth disease (FMD) in cattle. Journal of Istanbul Veterinary Sciences, Vetexpo-2019, KHVD-2019 , 53-53. [Link]

Oryan, A., Moshiri, A., & Raayat, A. R. (2012). Novel application of Theranekron® enhanced the structural and functional performance of the tenotomized tendon in rabbits. Cells, Tissues, Organs, 196(5), 442–455. [DOI:10.1159/000337860] [PMID]

Sardari, K., Kakhki, E., & Mohri, M. (2007). Evaluation of wound contraction and epithelialization after subcutaneous administration of theranekron in cows. Comparative Clinical Pathology, 16, 197-200. [DOI:10.1007/s00580-006-0657-8]

Sencar, L., Coşkun, G., Şaker, D., Sapmaz, T., Kara, S., & Çelenk, A., et al. (2021). Effects of theranekron and alpha-lipoic acid combined treatment on GAP-43 and Krox-20 gene expressions and inflammation markers in peripheral nerve injury. Ultrastructural Pathology, 45(3), 167–181. [DOI:10.1080/01913123.2021.1923600] [PMID: 34184615].] [PMID]

Smith W. (1993). Responses of laboratory animals to some injectable anaesthetics. Laboratory Animals, 27(1), 30–39. [DOI:10.1258/002367793781082377] [PMID]

Song, P. H., Chun, S. Y., Chung, J. W., Kim, Y. Y., Lee, H. J., & Lee, J. N., et al. (2017). Comparison of 5 different rat models to establish a standard animal model for research into interstitial cystitis. International Neurourology Journal, 21(3), 163–170. [DOI:10.5213/inj.1734898.449] [PMID] [PMCID]

Stampa S. (1986). A field trial comparing the efficacy of sulphamonomethoxine, penicillin, and tarantula poison in the treatment of pododermatitis circumspecta of cattle. Journal of the South African Veterinary Association, 57(2), 91–93. [PMID]

Swamy Babu, M. V., Veena, P., Suresh Kumar, R. V., Amaravathi, P., & Vaniet, G., (2020). Effectiveness of tarantula cubensis extract in the treatment of bovine papillomatosis. International Journal of Current Microbiology and Applied Sciences. 9(5), 119-122. [DOI:10.20546/ijcmas.2020.905.012]

Adib-Hashemi, F., Farahmand, F., Hesari, S. F., Rezakhaniha, B., Fallah, E., & Fayyaz, A. F., et al. (2015). Anti-inflammatory and protective investigations on the effects of theranekron "an alcoholic extract of the tarantula cubensis" on wound healing of peritoneal in the rat: An in vivo comparative study. Diagnostic pathology, 10, 19. [DOI:10.1186/s13000-015-0252-x] [PMID] [PMCID]
Dolapcioglu, K., Dogruer, G., Ozsoy, S., Ergun, Y., Ciftci, S., & Soylu Karapinar, O., et al. (2013). Theranekron for treatment of endometriosis in a rat model compared with medroxyprogesterone acetate and leuprolide acetate. European Journal Of Obstetrics, Gynecology, and Reproductive Biology, 170(1), 206–210. [DOI:10.1016/j.ejogrb.2013.05.026] [PMID]
Ghasemi-Dizgahl, A., Nami, B., & Amirmozafari, N. (2016). Tarantula cubensis venom (theranekron) selectively destroys human cancer cells via activating caspase-3-mediated apoptosis. Acta Medica International, 4(1), 73-79. [DOI:10.5530/ami.2017.4.14]
Gültiken, N. & Vural, M. R. (2007). The effect of Tarantula Cubensis extract applied in pre and postoperative period of canine mammary tumours . Journal of Istanbul Veterinary Sciences , (2) , 13-23. [Link]
Gürbulak, K., Akcay, A., Gümüşsoy, K. S., Sist, B., Steiner, S., & Abay, M., et al. (2014). Investigation of the efficacy of tarantula cubensis extract (theranekron D6) in the treatment of subclinical and clinical mastitis in dairy cows. Turkish Journal of Veterinary & Animal Sciences. 38(6), 712-718. [DOI:10.3906/vet-1405-101].
Güven, N., Özkan, S., Türközü, T., Koç, S., Keleş, Ö. F., Yener, Z., & Karasu, A. (2022). The effect of theranekron on femur fracture healing in an experimental rat model. Joint diseases and related surgery, 33(2), 374–384. [DOI: 10.52312/jdrs.2022.640] [PMID]
Hafez, E. S. E., & Hafez, B. (2013). Reproduction in farm animals. 7th ed. Hoboken: WileyBlackwell. [Link]
Iacopetti, I., Patruno, M., Melotti, L., Martinello, T., Bedin, S., Badon, T., et al. (2020). Autologous platelet-rich plasma enhances the healing of large cutaneous wounds in dogs. Frontiers in Veterinary Science, 7, 575449. [DOI:10.3389/fvets.2020.575449] [PMID] [PMCID] 
Icen, H., Sekin, S., Simsek, A., Kochan, A., & Tunik, S. (2011). The efficacy of tarantula cubensis extract (theranekron) in treatment of canine oral papillomatosis. Asian Journal of Animal and Veterinary Advances. 6(7), 744-749. [DOI:10.3923/ajava.2011.744.749]
Kaçar C., Zonturlu, A. K., Oral, H., Yıldız, S., & Arı, U. Ç. (2007). [The effects of theranekron application on uterus involution and vaginal efluence in cows early puerperal period (Turkish)]. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 13 (1), 11-15. [DOI:10.9775/kvfd.2006.36-A]
Karabacak, M., Eraslan, G., Kanbur, M., & Sarıca, Z. S. (2015). Effects of tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats. Homeopathy : The Journal of the Faculty of Homeopathy, 104(3), 205–210. [DOI:10.1016/j.homp.2015.02.005] [PMID]
Korkmaz, M., & Sancak, T. (2022). Clinical and histopathological comparison of the efficacy of ointments containing centella asiatica extract, zinc oxide and alaptidum in wound healing. Van Veterinary Journal, 33(3), 141-148. [DOI:10.36483/vanvetj.1187715]
Kozlu, T., Güler, F., Peker Akalın, P., Kazak, F., Ergün, Y., & Aslan E. (2021). The influence of theranekron on glucose6-phosphate dehydrogenase activity in rat ovaries, intact and with ischemiareperfusion injury. Veterinarski Arhiv, 91(1), 81-88. [DOI:10.24099/vet.arhiv.0828] 
Lotfollahzadeh, S. (2020). Comparative therapeutic effects of Theranekron D2 and Theranekron D6 in the treatment of foot- and- mouth disease (FMD) in cattle. Journal of Istanbul Veterinary Sciences, Vetexpo-2019, KHVD-2019 , 53-53. [Link]
Oryan, A., Moshiri, A., & Raayat, A. R. (2012). Novel application of Theranekron® enhanced the structural and functional performance of the tenotomized tendon in rabbits. Cells, Tissues, Organs, 196(5), 442–455. [DOI:10.1159/000337860] [PMID]
Sardari, K., Kakhki, E., & Mohri, M. (2007). Evaluation of wound contraction and epithelialization after subcutaneous administration of theranekron in cows. Comparative Clinical Pathology, 16, 197-200. [DOI:10.1007/s00580-006-0657-8]
Sencar, L., Coşkun, G., Şaker, D., Sapmaz, T., Kara, S., & Çelenk, A., et al. (2021). Effects of theranekron and alpha-lipoic acid combined treatment on GAP-43 and Krox-20 gene expressions and inflammation markers in peripheral nerve injury. Ultrastructural Pathology, 45(3), 167–181. [DOI:10.1080/01913123.2021.1923600] [PMID: 34184615].] [PMID]
Smith W. (1993). Responses of laboratory animals to some injectable anaesthetics. Laboratory Animals, 27(1), 30–39. [DOI:10.1258/002367793781082377] [PMID]
Song, P. H., Chun, S. Y., Chung, J. W., Kim, Y. Y., Lee, H. J., & Lee, J. N., et al. (2017). Comparison of 5 different rat models to establish a standard animal model for research into interstitial cystitis. International Neurourology Journal, 21(3), 163–170. [DOI:10.5213/inj.1734898.449] [PMID] [PMCID]
Stampa S. (1986). A field trial comparing the efficacy of sulphamonomethoxine, penicillin, and tarantula poison in the treatment of pododermatitis circumspecta of cattle. Journal of the South African Veterinary Association, 57(2), 91–93. [PMID]
Swamy Babu, M. V., Veena, P., Suresh Kumar, R. V., Amaravathi, P., & Vaniet, G., (2020). Effectiveness of tarantula cubensis extract in the treatment of bovine papillomatosis. International Journal of Current Microbiology and Applied Sciences. 9(5), 119-122. [DOI:10.20546/ijcmas.2020.905.012]