The effect of different concentrations of caffeine, pentoxifylline and 2’-deoxyadenosine on the biological properties of frozen-thawed canine semen

Open access


Artificial insemination (AI) and semen cryopreservation are the most accessible and commonly used techniques for breeding domestic animals. Among many parameters, such as plasma membrane integrity and acrosome structure, one of key factor that determine the quality of frozen-thawed samples for artificial insemination is sperm motility. Sperm motility is one of the key parameters that determine the quality of frozen-thawed samples for AI. The total number of progressively motile spermatozoa in thawed canine semen is correlated with fertility. A variety of substances were used to compare sperm motility with the control. The aim of this study was to determine the effect of semen extender supplementation with motility stimulants, pentoxifylline (PTX), caffeine (CAF) and 2’-deoxyadenosine (DX), after different post-thaw incubation times (30, 60, 120 min) on the motility, selected kinematic parameters, plasma membrane integrity and mitochondrial membrane potential of cryopreserved canine spermatozoa. During attempts to improve the quality of cryopreserved semen, the applied substances exerted beneficial effects at a concentration of 10 mM. We demonstrated that both phosphodiesterase inhibitors, caffeine and pentoxifylline, as well as 2’-deoxyadenosine increased the motility and selected kinematic parameters of thawed canine spermatozoa.

Antończyk A., Niżański W., Twardoń, J., Kozdrowski R., Ochota M., Błasiak K., Mikołajewska N., Stańczyk E. (2010). Current views on computer assisted sperm analysis. Med. Wet., 66: 663-667.

Bailey J.L., Bilodeau J.F., Cormier N. (2000). Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl., 21: 1-7.

Bani-Hani S.A. (2011). Semen quality and chemical oxidative stress; quantification and remediation. ETD Archive, 24,

Banihani S.A., Abu-Alhayjaa R.F. (2016). The activity of seminal creatine kinase is increased in the presence of pentoxifylline. Andrologia, 48: 603-604.

Barakat I.A., Danfour M.A., Galewan F.A., Dkhil M.A. (2015). Effect of various concentrations of caffeine, pentoxifylline, and kallikrein on hyperactivation of frozen bovine semen. Biomed. Res. Int., 2015: 1-7.

Belala R., Fatmi S., Kaidi R., Iguer-Ouada M. (2016). Benefits of cholesterol and α-tocopherol loaded cyclodextrins in dog semen cryopreservation. Revue Méd. Vét., 167: 22-27.

Brie D., Sahebkar A., Penson P.E., Dinca M., Ursoniu S., Serban M.C., Zanchetti A., Howard G., Ahmed A., Aronow W.S., Muntner P., Lip G.Y., Wong N.D., Rysz J., Banach M. (2016). Effects of pentoxifylline on inflammatory markers and blood pressure: a systematic review and meta-analysis of randomized controlled trials. J Hypertens., 34: 2318-2329.

Carrel D.T., Aston K.I. (2013). Spermatogenesis: Methods and Protocols. New York, USA, Humana Press.

Centola G.M., Cartie R.J., Cox C. (1995). Differential responses of human sperm to varying concentrations of pentoxifylline with demonstration of toxicity. J. Androl., 16: 136-142.

Comizzoli P., Crosier A.E., Songsasen N., Gunther M.S., Howard J.G., Wildt D.E. (2009). Advances in reproductive science for wild carnivore conservation. Reprod. Domest. Anim., 44: 47-52.

Dziekońska A., Fraser L., Strzeżek J. (2009). Effect of different storage temperatures on the metabolic activity of spermatozoa following liquid storage. J. Anim. Feed Sci., 18: 638-649.

Esteves S.C., Spaine D.M., Cedenho A.P. (2007). Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method. Braz. J. Med. Biol. Res., 40: 985-992.

Fraser L., Lecewicz M., Strzeżek J. (2002). Fluorometric assessments of viability and mitochondrial status of boar spermatozoa following liquid storage. Pol. J. Vet. Sci., 5: 85-92.

Garner D.L., Johnson L.A. (1995). Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol. Reprod., 553: 276-284.

Gradil C.M., Ball B.A. (2000). The use of pentoxifylline to improve motility of cryopreserved equine spermatozoa. Theriogenology, 54: 1041-1047.

Hammitt D.G., Bedia E., Rogers P.R., Syrop C.H., Donovan J.F., Williamson R.A. (1989). Comparison of motility stimulants for cryopreserved human semen. Fertil. Steril., 52: 495-502.

Imoedemhe D.A., Sigue A.B., Pacpaco E.A., Olazo A.B. (1992). Successful use of the sperm motility enhancer 2-deoxyadenosine in previously failed human in vitro fertilization. J. Assist. Reprod. Genet., 9: 53-56.

Inanc M.E., Tekin K., Olgac K.T., Yilmaz B., Cil B., Tasdemir U., Tuncer P.B., Buyukleblebici S., Durmaz E., Uysal O. (2018). Effect of cholesterol loaded cyclodextrin on semen cryopreservation of Aksaray Malakli shepherd dogs of different ages. Anim. Reprod. Sci., 193:191-200.

Jewgenow K., Braun B.C., Dehnhard M., Zahmel J., Goeritz F. (2017). Research on reproduction is essential for captive breeding of endangered carnivore species. Reprod. Domest. Anim., 52: 18-23.

Johnson A.E., Freeman E.W., Wildt D.E., Songsasen N. (2014). Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide. Cryobiology, 68: 361-370.

Karger S., Geiser B., Grau M., Heuwieser W., Arlt S.P. (2017). Short communication: Progressive motility of frozen-thawed canine semen is highest five minutes after thawing. Reprod. Domest. Anim., 52: 350-352.

Kay V.J., Coutts J.R.T., Robertson L. (1993). Pentoxifylline stimulates hyperactivation in human spermatozoa. Hum. Reprod., 8: 727-731.

Kim S.H., Yu D.H., Kim Y.J. (2010). Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm. Theriogenology, 73: 282-292.

Kordan W., Lecewicz M., Strzezek R., Dziekońska A., Fraser L. (2010). Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa. Pol. J. Vet. Sci., 13: 571-579.

Koutsarova N., Todorov P., Koutsarov G. (1997). Effect of pentoxifylline on motility and longevity of fresh and thawed dog spermatozoa. J. Reprod. Fertil., 51: 117-121.

Lanzafame F., Chapman M.G. Guglielmino A., Gearon C.M., Forman R.G. (1994). Pharmacological stimulation of sperm motility Hum. Reprod., 9:, 192-199.

Laokirkkiat P., Kunathikom S., Choavaratana R., Petyim S., Prechapanich J. (2007). Comparison between sperm treated with pentoxifylline and 2-deoxyadenosine using hypo-osmotic swelling test. J. Med. Assoc. Thai., 90: 211-215.

Lewis S.E., Moohan J.M., Thompson W. (1993). Effects of pentoxifylline on human sperm motility in normozoospermic individuals using computer assisted analysis. Fertil. Steril., 59: 418-423.

López F.J., Alvariño J.M. (2000). Effects of added caffeine on results following artificial insemination with fresh and refrigerated rabbit semen. Anim. Reprod. Sci., 58: 147-154.

Lucio C.F., Angrimani D.S.R., Brito M.M., Vannucchi C.I. (2017). Oxidative stress challenges during the sperm cryopreservation in dogs. J. Vet. Androl., 2: 1-7.

Mason S.J. (2018). A retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozen-thawed dog semen. Reprod. Domest. Anim., 52: 275-280.

Maxwell W. M., Robinson S. J., Roca J., Molinia F. C., Sanchez-Partida L. G., Evans, G. (1995). Motility, acrosome integrity and fertility of frozen ram spermatozoa treated with caffeine, pentoxifylline, cAMP, 2-deoxyadenosine and kallikrein. Reprod. Fertil. Dev., 7: 1081−1087.

Mbizvo M.T., Johnston R.C., Baker, G.H.W. (1993). The effect of the motility stimulants, caffeine, pentoxifylline, and 2-deoxyadenosine on hyperactivation of cryopreserved human sperm. Fertil. Steril., 59: 1112-1117.

Milani C., Fontbonne A., Sellem E., Stelletta C., Gérard O., Romagnoli S. (2010). Effect of post-thaw dilution with caffeine, pentoxifylline, 2’-deoxyadenosine and prostatic fluid on motility of frozen-thawed dog semen. Theriogenology, 74: 153-164.

Mundle S.D., Reza S., Ali A., Mativi Y., Shetty V., Venugopal P., Gregory S.A., Raza A. (1999). Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes. Cancer Lett., 140: 201-207.

Nabavi, N., Todehdehghan, F., Shiravi, A. (2013). Effect of caffeine on motility and vitality of sperm and in vitro fertilization of outbreed mouse in T6 and M16 media. Iran. J. Reprod. Med., 11: 741-746.

Nizanski W., Dubiel A., Bielas W., Dejneka G.J. (2001). Effects of three cryopreservation methods and two semen extenders on the quality of dog semen after thawing. J. Reprod. Fertil., 57: 365-369.

Nöthling J.O., Shuttleworth R. (2005). The effect of straw size, freezing rate, and thawing rate upon post-thaw quality of dog semen. Theriogenology, 63: 1469-1480.

Peeker R., Abramsson L., Marklund S.L. (1997). Superoxide dismutase isoenzymes in human seminal plasma and spermatozoa. Mol. Hum. Reprod., 3: 1061-1066.

Pena F.J. (2007). Detecting subtle changes in sperm membranes in veterinary andrology. Asian J. Androl., 9: 731-737.

Ponce A.A., Fiol de Cuneo M., Ruiz R.D., Vincenti L.M., Santillá M.E., Stutz G., Lacuara J.L. (1999). Influence of pentoxifylline on sperm membrane functional integrity. Arch. Androl., 43: 77-84.

Prapaiwan N., Tharasanit T., Punjachaipornpol S., Yamtang D., Roongsitthichai A., Moonarmart W., Kaeoket K., Manee-In S. (2016). Low-density lipoprotein improves motility and plasma membrane integrity of cryopreserved canine epididymal spermatozoa. Asian-Australas. J. Anim. Sci., 29: 646-651.

Rasul Z., Ahmad N., Anzar M. (2001). Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa. J. Androl., 22: 278-283.

Rijsselaere T., Van Soom A., Maes D., Niżanski W. (2012). Computer-assisted sperm analysis in dogs and cats: an update after 20 years. Reprod. Domest. Anim., 47: 204-207.

Rodenas C., Parrilla I., Roca J., Martinez E.A., Lucas X. (2014). Effects of rapid cooling prior to freezing on the quality of canine cryopreserved spermatozoa. J. Reprod. Dev., 60: 355-361.

Rota A., Ström B., Linde-Forsberg C. (1995). Effects of seminal plasma and three extenders on canine semen stored at 4 degrees C. Theriogenology, 44: 885-900.

Setyawan E.M., Kim M.J., Oh H.J., Kim G.A., Jo Y.K., Lee S.H., Choi Y.B., Lee B.C. (2015). Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective agents. Cryobiology, 71: 344-349.

Sharma R.K., Tolentino M.V. Jr, Thomas A.J. Jr, Agarwal A. (1996). Optimal dose and duration of exposure to artificial stimulants in cryopreserved human spermatozoa. J. Urol., 155: 568-573.

Silva A.R., Cardoso R.C., Silva L.D. (2006). Influence of temperature during glycerol addition and post-thaw dilution on the quality of canine frozen semen. Reprod. Domest. Anim., 41: 74-78.

Slanina T., Miškeje M., Tirpák F., Błaszczyk M., Formicki G., Massányi P. (2018). Caffeine strongly improves motility parameters of turkey spermatozoa with no effect on cell viability. Acta Vet. Hung., 66: 137-150.

Stachecki J.J., Dresser B.L., Pope C.E., Armant D.R. (1995). Stimulation of ejaculated domestic cat spermatozoa motility with caffeine, pentoxifylline, and 2’-deoxyadenosine. Arch. Androl., 34: 63-68.

Stănescu (Pascal) M., Bîrţoiu A.I. (2012). Freezing of dog’s sperm: a review. Rom. Biotechnol. Lett., 17: 7709-7716.

Stephens T.D., Brooks R.M., Carrington J.L., Cheng L., Carrington A.C., Porr C.A., Splan R.K. (2013). Effects of pentoxifylline, caffeine, and taurine on post-thaw motility and longevity of equine frozen semen. J. Equine Vet. Sci., 33: 615-621.

Strzeżek R., Polakiewicz W., Kordan W. (2015). The effect of two packaging systems on the post-thaw characteristics of canine sperm. Pol. J. Vet. Sci.,18: 249-253.

Tesarik, J., Thèbault, A. and Testart, J. (1992) Effect of pentoxifylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens. Hum. Reprod., 7, 1257-1263.

Uchoa D.C., Silva T.F., Mota Filho A.C., Silva L.D. (2012). Intravaginal artificial insemination in bitches using frozen/thawed semen after dilution in powdered coconut water (ACP-106c), Reprod. Domest. Anim., 47: 289-292.

Van den Berghe F., Paris M.C.J., Briggs M.B., Farstad W.K., Paris D.B.B.P. (2018). A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus). Cryobiology, 80: 18-25.

Yamaguchi, S., Suzuki, C., Noguchi, M., Kasa, S., Mori, M., Isozaki, Y., Ueda, S., Funahashi, H., Kikuchi, K., Nagai, T. and Yoshioka, K. (2013). Effects of caffeine on sperm characteristics after thawing and inflammatory response in the uterus after artificial insemination with frozen-thawed boar semen. Theriogenology, 79: 87-93.

Yovich J.M., Edirisinghe W.R., Cummins J.M., Yovich J.L. (1990). Influence of pentoxifylline in severe male factor infertility. Fertil. Steril., 53: 715-722.

Yu I., Songsasen N., Godke R.A., Leibo S.P. (2002). Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates. Cryobiology, 44: 62-78.

Yunes R., Fernández P., Doncel G.F., Acosta A.A. (2005). Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa. Biocell., 29: 287-293.

Zhang J. Guo H. Su J., Zhao L., Li Y., Sun W., Han H., Hu S., Zhao G., Li Y., Dai Y., Li X. (2014). A comparison of the effects of pentoxifylline on quality of fresh and frozen - thawed bull spermatozoa. Agriculture Biotechnology, 3: 20-29.

Zhang X., Sharma R.K., AgarwalA., Falcone T. (2005). Effect of pentoxifylline in reducing oxidative stress-induced embryotoxicity. J. Assist. Reprod. Genet., 22: 415-417.

Annals of Animal Science

The Journal of National Research Institute of Animal Production

Journal Information

IMPACT FACTOR 2017: 1.018
5-year IMPACT FACTOR: 0.959

CiteScore 2017: 1.01

SCImago Journal Rank (SJR) 2017: 0.413
Source Normalized Impact per Paper (SNIP) 2017: 0.822


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 58 58 40
PDF Downloads 52 52 29