Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates
C. M. Kershaw A B C , G. Evans B , R. Rodney B and W. M. C. Maxwell B
+ Author Affiliations
- Author Affiliations
A Department of Animal Production, Welfare and Veterinary Sciences, Harper Adams University, Newport, Shropshire TF10 8NB, UK.
B Faculty of Veterinary Science, The University of Sydney, Camperdown, Sydney, NSW 2006 Australia.
C Corresponding author. Email: ckershaw@harper-adams.ac.uk
Reproduction, Fertility and Development 29(6) 1107-1114 https://doi.org/10.1071/RD15261
Submitted: 29 June 2015 Accepted: 5 January 2016 Published: 9 May 2016
Abstract
In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1 mg mL–1, 20 min, 37°C) eliminated alpaca semen viscosity while maintaining sperm motility, viability, acrosome integrity and DNA integrity. Furthermore E-64 (10 µM at 37°C for 5 min after 20 min papain) inhibited the papain without impairing sperm function. Cryopreserved, papain-treated alpaca spermatozoa exhibited higher total motility rates after chilling and 0 and 1 h after thawing compared with control (untreated) samples. Papain treatment, followed by inhibition of papain with E-64, is effective in reducing alpaca seminal plasma viscosity without impairing sperm integrity and improves post-thaw motility rates of cryopreserved alpaca spermatozoa. The use of the combination of papain and E-64 to eliminate the viscous component of camelid semen may aid the development of assisted reproductive technologies in camelids.
Additional keywords: acrosome, alpaca, cryopreservation.
References
Adams, G. P., Ratto, M. H., Collins, C. W., and Bergfelt, D. R. (2009). Artificial insemination in South American camelids and wild equids. Theriogenology 71, 166–175.| Artificial insemination in South American camelids and wild equids.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjntFyhtw%3D%3D&md5=598f5d198cdc8f9c7a0cf04bd0b19ac0CAS | 18922569PubMed |
Ali, H. A., Moniem, K. A., and Tingari, M. D. (1976). Some histochemical studies on the prostate, urethral and bulbourethral glands of the one-humped camel (Camelus dromedarius). Histochem. J. 8, 565–578.
| Some histochemical studies on the prostate, urethral and bulbourethral glands of the one-humped camel (Camelus dromedarius).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE2sXktVKgsg%3D%3D&md5=a3244e8e2d90cc4cf375b42f45394fd8CAS | 186443PubMed |
Barrett, A. J., Kembhavi, A. A., and Hanada, K. (1981). E-64 [l-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] and related epoxides as inhibitors of cysteine proteinases. Acta Biol. Med. Ger. 40, 1513–1517.
| 1:CAS:528:DyaL38Xhtlymsr8%3D&md5=b358b877d310d29bc18f7074e6f5ed10CAS | 7044005PubMed |
Barrett, A. J., Kembhavi, A. A., Brown, M. A., Kirschke, H., Knight, C. G., Tamai, M., and Hanada, K. (1982). l-Trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. 201, 189–198.
| l-Trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL38XhvFGksr0%3D&md5=2ce27687f2c837a7ade8f4be96c1e175CAS | 7044372PubMed |
Bravo, P. W., Pacheco, C., Quispe, G., Vilcapaza, L., and Ordonez, C. (1999). Degelification of alpaca semen and the effect of dilution rates on artificial insemination outcome. Arch. Androl. 43, 239–246.
| Degelification of alpaca semen and the effect of dilution rates on artificial insemination outcome.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c%2FovFKgtw%3D%3D&md5=6e3dcdf4a866fad6e610a45855e0a449CAS | 10624509PubMed |
Bravo, P. W., Ccallo, M., and Garnica, J. (2000a). The effect of enzymes on semen viscosity in llamas and alpacas. Small Rumin. Res. 38, 91–95.
| The effect of enzymes on semen viscosity in llamas and alpacas.Crossref | GoogleScholarGoogle Scholar | 10924884PubMed |
Bravo, P. W., Skidmore, J. A., and Zhao, X. X. (2000b). Reproductive aspects and storage of semen in Camelidae. Anim. Reprod. Sci. 62, 173–193.
| Reproductive aspects and storage of semen in Camelidae.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1Kgtr4%3D&md5=c2af83b53726bf9663d2346c53a0b06aCAS | 10924824PubMed |
Conde, P. A., Herrera, C., Trasorras, V. L., Giuliano, S. M., Director, A., Miragaya, M. H., Chaves, M. G., Sarchi, M. I., Stivale, D., Quintans, C., Aguero, A., Rutter, B., and Pasqualini, S. (2008). In vitro production of llama (Lama glama) embryos by IVF and ICSI with fresh semen. Anim. Reprod. Sci. 109, 298–308.
| In vitro production of llama (Lama glama) embryos by IVF and ICSI with fresh semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht12gu7zE&md5=6f83610b6607e8fdf4a53e73eb0606feCAS | 18054452PubMed |
Crichton, E. G., Pukazhenthi, B. S., Billah, M., and Skidmore, J. A. (2015). Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa. Theriogenology 83, 168–174.
| Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXhs1KmtLnK&md5=4c6f3327f910c6635da9ad4c81a0112bCAS | 25312816PubMed |
Deen, A., Vyas, S., and Sahani, M. S. (2003). Semen collection, cryopreservation and artificial insemination in the dromedary camel. Anim. Reprod. Sci. 77, 223–233.
| Semen collection, cryopreservation and artificial insemination in the dromedary camel.Crossref | GoogleScholarGoogle Scholar | 12695056PubMed |
Evans, G., and Maxwell, W. M. C. (1987). ‘Salamon’s Artificial Insemination of Sheep and Goats.’ (Butterworths: Sydney.)
Garnica, J., Achata, R., and Bravo, P. W. (1993). Physical and biochemical characteristics of alpaca semen. Anim. Reprod. Sci. 32, 85–90.
| Physical and biochemical characteristics of alpaca semen.Crossref | GoogleScholarGoogle Scholar |
Giuliano, S., Carretero, M., Gambarotta, M., Neild, D., Trasorras, V., Pinto, M., and Miragaya, M. (2010). Improvement of llama (Lama glama) seminal characteristics using collagenase. Anim. Reprod. Sci. 118, 98–102.
| Improvement of llama (Lama glama) seminal characteristics using collagenase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFGrt7zJ&md5=74b3e95d5064d1cba78e2de79e841b35CAS | 19616391PubMed |
Kershaw-Young, C. M., and Maxwell, W. M. C. (2011). The effect of seminal plasma on alpaca sperm function. Theriogenology 76, 1197–1206.
| The effect of seminal plasma on alpaca sperm function.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3Mfks12msQ%3D%3D&md5=34d7bdf1e3594eec73d8c8527c65c4d2CAS | 21820722PubMed |
Kershaw-Young, C. M., Evans, G., and Maxwell, W. M. (2012). Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram. Reprod. Fertil. Dev. 24, 362–369.
| Glycosaminoglycans in the accessory sex glands, testes and seminal plasma of alpaca and ram.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xht1OitL8%3D&md5=3adecaae2d73ef83756e510615163118CAS | 22281083PubMed |
Kershaw-Young, C. M., Stuart, C., Evans, G., and Maxwell, W. M. C. (2013). The effect of glycosaminoglycan enzymes and proteases on the viscosity of alpaca seminal plasma and sperm function. Anim. Reprod. Sci. 138, 261–267.
| The effect of glycosaminoglycan enzymes and proteases on the viscosity of alpaca seminal plasma and sperm function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXks1Slsb0%3D&md5=18fa1c7777b42df663d0229a28041e13CAS | 23537479PubMed |
Leahy, T., Celi, P., Bathgate, R., Evans, G., Maxwell, W. M., and Marti, J. I. (2010). Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase. Reprod. Fertil. Dev. 22, 1131–1140.
| Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtVOgu7vI&md5=9e596909736b2d1acc00be67de8d0492CAS | 20797351PubMed |
Morton, K. M., Bathgate, R., Evans, G., and Maxwell, W. M. (2007). Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws. Reprod. Fertil. Dev. 19, 792–796.
| Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVWhtrzO&md5=b653bdbdf76845ec0b9db6012e7e03a3CAS | 17897581PubMed |
Morton, K. M., Vaughan, J., and Maxwell, W. M. (2008). ‘Continued Development of Artificial Insemination Technology in Alpacas’. (Rural Industries Research and Development Corporation: Canberra.)
Morton, K. M., Evans, G., and Maxwell, W. M. (2010b). Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen–thawed epididymal alpaca (Vicugna pacos) sperm. Theriogenology 74, 311–316.
| Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen–thawed epididymal alpaca (Vicugna pacos) sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXotVahsr4%3D&md5=e97f2479958ca272c76e5ea60688d2f0CAS | 20416935PubMed |
Morton, K. M., Thomson, P. C., Bailey, K., Evans, G., and Maxwell, W. M. (2010a). Quality parameters for alpaca (Vicugna pacos) semen are affected by semen collection procedure. Reprod. Domest. Anim. 45, 637–643.
| 1:STN:280:DC%2BC3cnpsFSksw%3D%3D&md5=56c3c203b3071836808e758392174885CAS | 19144029PubMed |
Niasari-Naslaji, A., Mosaferi, S., Bahmani, N., Gharahdaghi, A. A., Abarghani, A., Ghanbari, A., and Gerami, A. (2006). Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen. Cryobiology 53, 12–21.
| Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XntV2mt7c%3D&md5=ee52129e477b77b5610f4ada6bd97313CAS | 16647050PubMed |
Niasari-Naslaji, A., Mosaferi, S., Bahmani, N., Gerami, A., Gharahdaghi, A.A., Abarghani, A., and Ghanbari, A. (2007). Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations. Theriogenology 68, 618–625.
| Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXotlWqu7g%3D&md5=9c1b8decc0c56a23202a4f20ce57792dCAS | 17588650PubMed |
Perk, K. (1962). Seasonal changes in the glandula bulbo-urethralis of the camel. Bull. Res. Counc. Isr. Sect. E Exp. Med. 10, 37–44.
| 1:STN:280:DyaF383jtVeiug%3D%3D&md5=0adf1f0ba6a9c45ea0261f4e3434caf1CAS | 14038281PubMed |
Santiani, A., Huanca, W., Sapana, R., Huanca, T., Sepulveda, N., and Sanchez, R. (2005). Effects on the quality of frozen–thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. Asian J. Androl. 7, 303–309.
| Effects on the quality of frozen–thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFSksL%2FE&md5=5836bbd471da2df6ce5a7aafbbfff9a1CAS | 16110359PubMed |
Skidmore, J. A., and Billah, M. (2006). Comparison of pregnancy rates in dromedary camels (Camelus dromedarius) after deep intra-uterine versus cervical insemination. Theriogenology 66, 292–296.
| Comparison of pregnancy rates in dromedary camels (Camelus dromedarius) after deep intra-uterine versus cervical insemination.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zotlWmtA%3D%3D&md5=b6fd1d370eb988619777128d118e7a2cCAS | 16356542PubMed |
Tamai, M., Hanada, K., Adachi, T., Oguma, K., Kashiwagi, K., Omura, S., and Ohzeki, M. (1981). Papain inhibitions by optically active E-64 analogs. J. Biochem. 90, 255–257.
| 1:CAS:528:DyaL3MXltFSrsb8%3D&md5=cfc0798a7fc65a3c120425c0c7f72c6aCAS | 7287681PubMed |
Tibary, J., and Vaughan, J. (2006). Reproductive physiology and infertility in male South American camelids: a review and clinical observations. Small Rumin. Res. 61, 283–298.
| Reproductive physiology and infertility in male South American camelids: a review and clinical observations.Crossref | GoogleScholarGoogle Scholar |
Vaughan, J., Galloway, D., and Hopkins, D. (2003). ‘Artificial Insemination in Alpacas (Lama Pacos).’ (Rural Industries Research and Development Corporation: Canberra.)
Vyas, S., Goswani, P., Rai, A. K., and Khanna, N. D. (1998). Use of Tris and lactose extenders in preservation of camel semen at refrigerated temperature. Indian Vet. J. 75, 810–812.
Wani, N. A., Billah, M., and Skidmore, J. A. (2008). Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen. Anim. Reprod. Sci. 109, 309–318.
| Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht12gu7zF&md5=5a62ae9d37ce7603492e9563da986f7cCAS | 18082979PubMed |
Zhao, X. X. (2000). Semen characteristics and artificial insemination in the Bactrian camel. In ‘Recent Advances in Camelid Reproduction’. (Eds J. A. Skidmore and G. P. Adams.) (International Veterinary Information Service: Ithaca, NY.)
