João Diego de Agostini Losano, Daniel de Souza Ramos Angrimani, Raquel Fernandes Redondo, Maíra Morales Brito, Bruno Rogério Rui, Giulia Kiyomi Vechiato Kawai, Bárbara do Carmo Simões da Silva, Camilla Mota Mendes, Mayra Elena Ortiz D’Avila Assumpção, Marcilio Nichi


Cryopreservation is a key process among the canine reproductive biotechnologies. However, during sperm cryopreservation an excessive reactive oxygen species (ROS) generation occurs, leading to decrease in sperm quality. Therefore, several antioxidants were tested during sperm cryopreservation to prevent such effects, however the carnosine it has not used. Carnosine is a protein present in the seminal plasma, and unlike other antioxidants has the ability to remove products of lipid peroxidation (malondialdehyde), which are as harmful as ROS. Thus, the aim of this study was to evaluate the effects of different carnosine concentrations, during sperm cryopreservation in dogs. For this purpose, six dogs in reproductive age were used, and after sperm collection the samples were cryopreserved in Control (tris-citrate egg yolk extender), Carnosine 1mM, 50mM and 100mM groups. After thawing samples were analyzed by computer-assisted analysis of sperm motility, plasma membrane (eosin/nigrosin), acrosome integrity (fast green/rose Bengal), mitochondrial activity, DNA integrity and sperm resistance to oxidative stress (by TBARS). Decrease was observed in motility sperm kinetics (total and progressive motility) and reduced lipid peroxidation products in the group treated with 50mM and 100mM. On the other hand, 1mM was similar to control group. In conclusion, higher carnosine concentration (50 and 100mM) apparently promoted impairment in energy production and consequently was harmful to sperm kinetics. Thus, future studies must be performed using different carnosine concentrations and in association with substrates for glycolysis and oxidative phosphorylation.


La criopreservación es un proceso clave entre las biotecnologías reproductivas en caninos. Sin embargo, durante la criopreservación espermática se da una generación excesiva de especies reactivas de oxígeno (ROS), lo que lleva a una disminución en la calidad espermática. Por lo tanto, varios medios de congelación utilizando antioxidantes para evitar tales efectos han sido evaluados, aunque la carnosina todavía no se ha utilizada. La carnosina es una proteína presente en el plasma seminal que a diferencia de otros antioxidantes tiene la habilidad de remover productos de la peroxidación lipídica (malondialdehído), que son tan dañinos como los ROS. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de diferentes concentraciones de carnosina durante la congelación espermática en perros. Para este propósito se utilizaron seis perros en edad reproductiva y después de la colectar los eyaculados, las muestras fueron criopreservadas en un diluyente Control (tris, citrato, yema de huevo), Carnosina 1mM, 50mM y 100 mM. Después del descongelado, las muestras fueron evaluadas mediante el análisis computerizado de la motilidad, integridad de membrana plasmática (eosina / nigrosina), integridad del acrosoma (Fast - green / rosa de Bengala), la actividad mitocondrial (3’3 Diaminobenzidina), la integridad del ADN (SCSA) y la evaluación de la resistencia al estrés oxidativo (TBARS). Se observó una disminución en la cinética de los espermatozoides (motilidad total y progresiva) y una reducción de los productos de la peroxidación lipídica en los grupos tratados con 50 mM y 100mM de carnosina. Por otro lado, el grupo con 1 mM de carnosina fue similar al control. En conclusión, una alta concentración de carnosina (50 y 100mM) parece afectar la producción de energía del espermatozoide y por lo tanto es perjudicial para la cinética del espermatozoide. Por lo tanto, futuros estudios deben realizarse utilizando diferentes concentraciones de carnosina y en asociación con sustratos para la glucólisis y la fosforilación oxidativa.

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Angrimani DSR., Losano JD., Lucio CF., Veiga GA., Pereda MC. Nichi M., Vannucchi CI. 2014. Role of residual cytoplasm on oxidative status during sperm maturation in dogs. Animal Reproduction Science 151:256-261.

Brito MM., Lucio CF., Angrimani DSR., Losano JD., Dalmazzo A., Nichi M., Vannucchi CI. 2017. Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm. Animal Biotechnology 28:67-73.

Buege JA., Aust SD. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52:302-310.

Burcham PC., Kaminskas LM., Fontaine FR., Petersen DR., Pyke S.M. 2002. Aldehyde-sequestering drugs: tools for studying protein damage by lipid peroxidation products. Toxicology 181-182:229-36.

De Lamirande E, Gagnon C. 1992. Reactive oxygen species and human spermatozoa II: Effects on the motility of intact spermatozoa and on sperm axonemes. Journal of Andrology 13:379-386.

De Lamirande E., Jiang H., Zini A., Kodama H., Gagnon C. 1997. Reactive oxygen species and sperm physiology. Reviews of Reproduction 2:48-54.

Ducci M., Pacchini S., Niccolini A., Gazzano A., Cerri D., Gadea J. 2006. Concentrations of carnosine, anserine, L-histidine and 3-methyl histidine in boar spermatozoa and sheep milk by a modified HPLC method. Polish Journal of Veterinary Sciences 9:159-163.

Dunnett M., Harris R., Dunnett C., Harris P. 2002. Plasma carnosine concentration: diurnal variation and effects of age, exercise and muscle damage. Equine Veterinary Journal 34:283-287.

Evenson D., Jost L. Sperm chromatin structure assay is useful for fertility assessment. 2000. Methods in Cell Science 22:169-189.

Farstad W. 2009. Cryopreservation of canine semen - new challenges. Reproduction in Domestic Animals 44 Suppl 2:336-41.

Gomez E,. Irvine DS., Aitken RJ. 1998. Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. International Journal of Andrology 21:81-94.

Griveau JF., Le Lannou D. 1997a. Influence of oxygen tension on reactive oxygen species production and human sperm function. International Journal of Andrology 20:195-200.

Griveau JF., Le Lannou D. 1997b. Reactive oxygen species and human spermatozoa: physiology and pathology. International Journal of Andrology. 20:61-69.

Guiotto A., Ruzza P., Babizhayev M.A., Calderan A. 2007. Malondialdehyde scavenging and aldose-derived Schiff bases’ transglycation properties of synthetic histidyl-hydrazide carnosine analogs. Bioorganic & Medicinal Chemistry 15:6158-6163.

Hipkiss A. R,. Baye E., de Courten B. 2016. Carnosine and the processes of ageing. Maturitas. 93:28-33.

Holliday R., McFarland G.1996. Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine. British Journal of Cancer 73:966.

Iguer-ouada M., Verstegen JP. 2001. Evaluation of the "Hamilton Thorn computer-based automated system" for dog semen analysis. Theriogenology 55:733-749.

Jarosz L., Gradzki Z., Kalinowski M., Laskowska E. 2016. Quality of fresh and chilled-stored raccoon dog semen and its impact on artificial insemination efficiency. BMC Veterinary Research 12:224.

Kashou AH., Sharma R., Agarwal A. 2013. Assessment of oxidative stress in sperm and semen. Methods Molecular Biology 927:351-361.

Ko EY., Sabanegh ESJr., Agarwal A. 2014. Male infertility testing: reactive oxygen species and antioxidant capacity. 2014. Fertility and Sterility. 102:1518-1527.

Kohen R., Yamamoto Y., Cundy KC., Ames BN. 1988. Antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain. Proceedings of the National Academy of Sciences of the United States of America 85:3175-3179.

Lagergren CG. 1993. On the eosin-nigrosin stain and some other methods for the appraisal of sperm vitality with special reference to practical application. Annali di Ostetricia e Ginecologia 75:998-1005.

Lucio CF., Regazzi FM., Silva LC., Angrimani DSR., Nichi M., Vannucchi CI. 2016a. Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs. Theriogenology 85:1568-1575.

Lucio CF., Silva LC, Regazzi FM, Angrimani DSR, Nichi M., Assumpção MEAV., annucchi CI. 2016b. Effect of reduced glutathione (GSH) in canine sperm cryopreservation: In vitro and in vivo evaluation. Cryobiology 72:135-140.

Minervini F., Guastamacchia R., Pizzi F., Dell'Aquila ME., Barile VL. 2013. Assessment of different functional parameters of frozen-thawed buffalo spermatozoa by using cytofluorimetric determinations. Reproduction in Domestic Animals 48:317-24.

Moazamian R., Polhemus A., Connaughton H., Fraser B., Whiting S., Gharagozloo P. 2015. Oxidative stress and human spermatozoa: diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation. Molecular Human Reproduction 21:502-515.

Mukai C., Okuno M. 2004. Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement. Biology of Reproduction 71:540-547.

Nichi M., Goovaerts IG,. Cortada CN., Barnabe VH., De Clercq JB., Bols PE. 2007. Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C. Theriogenology 67:334-340.

Ogata K., Sasaki A., Kato Y., Takeda A., Wakabayashi M., Sarentonglaga B. 2015. Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination. The Journal of Reproduction and Development. 61:116-122.

Pagano G., Bonassi S., De Biase A., Degan P., Deeva I.B., Doronin Y.K. 1997. L-methionine induces stage-dependent changes of differentiation and oxidative activity in sea urchin embryogenesis. Pharmacology & Toxicology 81:134-143.

Pope CE., Zhang YZ., Dresser BL. 1991. A simple staining method for evaluating acrosomal status of cat spermatozoa. Journal of Zoo Wildlife Medicine 22:87-95.

Sanchez-Partida LG., Setchell BP., Maxwell WM. 1997. Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa. Reproduction, Fertility, and Development. 9:689-696.

Sharma RK., Agarwal A. 1996. Role of reactive oxygen species in male infertility. Urology 48:835-850.

Turner RM. 2003. Tales from the tail: what do we really know about sperm motility? Journal of Andrology 24:790-803.

Watson PF. 2000. The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science 60-61:481-492.

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