Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Association of L-arginine with heparin on the sperm capacitation improves in vitro embryo production in bovine

Gester Breda Aguiar; Maria Clara Caldas-Bussiere; Valter Luiz Maciel Jr.; Carla Sobrinho Paes de Carvalho; Cláudio Luiz Melo de Souza

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Abstract: We aimed to evaluate the effects of L-arginine (L-arg) in the quality of in vitro heparin-induced capacitation of cryopreserved bovine spermatozoa and its effects on IVP. The experimental groups were: Control 0 hour without pre-capacitation, and groups of sperm capacitated for 30 min in the absence of COC with heparin (Control 30 min), with 1 mM L-arg and with 1 mM L-arg + heparin. The capacitation pattern was evaluated by chlortetracycline assay and the integrity of the plasma membrane (PM) and acrosome membrane (AM) by the association of Hoescht 33342 and propidium iodide. Further, we assess the sperm quality by the rate of in vitro blastocysts production. Treatment with 1 mM L-arg + heparin increased the percentage of capacitated sperm when compared to Control 0 hour and the treatment with heparin (61.1 vs. 18.2 and 47.0%, respectively, P<0.05). The addition of 1 mM L-arg to the medium has capacitated the spermatozoa (26.2 ± 3.8) but was less effective than heparin (47.0 ± 4.0) (P<0.05). There was no difference in the percentage of sperm with intact PM between treatments when compared to Control 0 hour (P>0.05). The group capacitated with 1 mM L-arg + heparin for 30 min increased the blastocyst rate compared to Control IVF (53.7 vs. 40.8%, P<0.05). We conclude that the addition of L-arg with heparin increases the number of capacitated spermatozoa in vitro with 30 min of pre-incubation in the absence of COC not altering the integrity of plasma and acrosomal membrane. This treatment in the absence of COC was the most effective method for blastocysts production, and the method of pre-incubation could be used to assess the role of other substances in the sperm capacitation and its effect on IVP.


in vitro fertilization, nitric oxide, blastocyst, membrane integrity, cattle


Bailey JL. Factors regulating sperm capacitation. Syst Biol Reprod Med. 2010;56(5):334-48. PMid:20849222.

Breininger E, Cetica PD, Beconi MT. Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm. Theriogenology. 2010;74(6):1036-49. PMid:20580081.

Casey PJ, Hillman RB, Robertson KR, Yudin AI, Liu IK, Drobnis EZ. Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm. J Androl. 1993;14(4):289-97. PMid:7693637.

Celeghini ECC, Arruda RP, Andrade AFC, Nascimento J, Raphael CF. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reprod Domest Anim. 2007;42(5):479-88. PMid:17845603.

Chamberland A, Fournier V, Tardif S, Sirard MA, Sullivan R, Bailey JL. The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation. Theriogenology. 2001;55(3):823-35. PMid:11245268.

Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl. 1997;18(4):461-8. PMid:9283961.

De Jonge CJ, Barratt CLR. 2017. The Sperm Cell - Production, Maturation, Fertilization, Regeneration. 2.ed. Cambridge, UK: Cambridge University Press.

de Loos F, van Vliet C, van Maurik P, Kruip TA. Morphology of immature bovine oocytes. Gamete Res. 1989;24(2):197-204. PMid:2793058.

Dixit VD, Parvizi N. Nitric oxide and the control of reproduction. Anim Reprod Sci. 2001;65(1-2):1-16. PMid:11182504.

Dubeibe DF, Caldas-Bussiere MC, Maciel VL Jr, Sampaio WV, Quirino CR, Gonçalves PBD, De Cesaro MP, Faes MR, Paes de Carvalho CS. L-arginine affects the IVM of cattle cumulus-oocyte complexes. Theriogenology. 2017;88:134-44. PMid:27743687.

Eid LN, Lorton SP, Parrish JJ. Paternal influence on S-phase in the first cell cycle of the bovine embryo. Biol Reprod. 1994;51(6):1232-7. PMid:7888500.

Fraser L. Ionic control of sperm function. Reprod Fertil Dev. 1995;7(4):905-25. PMid:8711223.

Fukui Y, Sonoyama T, Mochizuki H, Ono H. Effects of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro. Theriogenology. 1990;34(3):579-91. PMid:16726862.

Funahashi H. Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system. Reproduction. 2002;124(6):857-64. PMid:12530923.

Gangwar DK, Atreja SK. Signalling events and associated pathways related to the mammalian sperm capacitation. Reprod Domest Anim. 2015;50(5):705-11. PMid:26294224.

Graham JK, Kunze E, Hammerstedt RH. Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod. 1990;43(1):55-64. PMid:2393693.

Hung P-H, Suarez SS. Alterations to the bull sperm surface proteins that bind sperm to oviductal epithelium. Biol Reprod. 2012;87(4):88-88. PMid:22837481.

Ignarro LJ. 2000. Nitric oxide: biology and pathobiology. California: Academic Press.

Jagan Mohanarao G, Atreja SK. Identification of NO induced and capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) spermatozoa. Res Vet Sci. 2012;93(2):618-23. PMid:22035659.

Leal ACMS, Caldas-Bussiere MC, Carvalho CSP, Viana KS, Quirino CR. Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation. Anim Reprod Sci. 2009;116(1-2):38-49. PMid:19188034.

Leal ACMS, Caldas-Bussiere MC, Ohashi OM, Cordeiro MS, Quirino C. Assessment of bovine sperm capacitation pathway L-arginine/NO/GMPc through in vitro embryo production. In: Proceedings of the 17th International Congress on Animal Reproduction (ICAR). Reprod Domest Anim. 2012;47:586-586.

Lefièvre L, Chen Y, Conner SJ, Scott JL, Publicover SJ, Ford WCL, Barratt CLR. Human spermatozoa contain multiple targets for protein S-nitrosylation: an alternative mechanism of the modulation of sperm function by nitric oxide? Proteomics. 2007;7(17):3066-84. PMid:17683036.

Maciel VL Jr, Caldas-Bussiere MC, Silveira V, Reis RS, Rios AFL, Paes de Carvalho CS. L-arginine alters the proteome of frozen-thawed bovine sperm during in vitro capacitation. Theriogenology. 2018;119:1-9. PMid:29958134.

Matta SGC, Caldas-Bussiere MC, Viana KS, Faes MR, Paes de Carvalho CS, Dias BL, Quirino CR. Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle. Anim Reprod Sci. 2009;111(2-4):189-201. PMid:18439771.

O’Flaherty C, Rodriguez P, Srivastava S. L-Arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa. Biochim Biophys Acta, Gen Subj. 2004;1674(2):215-21. PMid:15374626.

Paes de Carvalho CS, Caldas-Bussiere MC, Ueno VG, Carvalho FP, Matta SGC, Quirino C. Efeito do óxido nítrico na capacitação in vitro de espermatozóides de bovinos da raça Nelore. In: Anais do Congresso de Integração em Biologia da Reprodução, Ribeirão Preto. São Paulo: TEC Art Editora Ltda; 2003. p. 216-17.

Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod. 1988;38(5):1171-80. PMid:3408784.

Parrish JJ. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology. 2014;81(1):67-73. PMid:24274411.

Pons-Rejraji H, Bailey JL, Leclerc P. Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis. Reprod Fertil Dev. 2009;21(4):525-37. PMid:19383259.

Robertson I, Nelson R. Certification and identification of the embryo. In: Stringfellow S, Seidel DA, editors. Manual of the international embryo transfer society. USA: IETS; 1998. p. 170.

Rodriguez PC, O’Flaherty CM, Beconi MT, Beorlegui NB. Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways. Anim Reprod Sci. 2005;85(3-4):231-42. PMid:15581507.

Roy SC, Atreja SK. Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway. Int J Androl. 2008;31(1):12-24. PMid:17355240.

Santana PPB, Silva BB, Silva TVG, Costa NN, Cordeiro MS, Santos SSD, Ohashi OM, Miranda MS. Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates. Theriogenology. 2016;85(6):1132-8. PMid:26733119.

Silva TVG, Silva BB, Sá ALA, Costa NN, Sampaio RV, Cordeiro MS, Santana PDPB, Adona PR, Santos SSD, Miranda MS, Ohashi OM. Influence of L-arginine during bovine in vitro fertilization. Indian J Exp Biol. 2014;52(12):1159-64. PMid:25651608.

Statistical Analysis System – SAS. SAS/STAT 9.3 User’s Guide. User’s Guid. Cary: SAS Inst. Inc.; 2011.

Viana KS, Caldas-Bussiere MC, Matta SGC, Faes MR, Carvalho CSP, Quirino CR. Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes. Anim Reprod Sci. 2007;102(3-4):217-27. PMid:17145142.

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