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

Can we use LDL instead of egg yolk in BotuCrio® extender to cryopreserve sperm from the Mangalarga Marchador stallion?

Paola Pereira das Neves Snoeck, Tainá Hortência Oliveira Pessoa, Marcelo Gonçalves Sousa Pereira, Inara Cristina Leal Bastos, Maria Isabel Vaz de Melo

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The objective of this study was to compare the BotuCrio® extender with the Merk - egg yolk and the INRA 82 modified by the inclusion of acetamide, methyl cellulose and trehalose in substitution of glycerol for freezing equine semen. The semen was diluted after centrifugation to obtain 100 x 106 of sperm/ml in: BotuCrio® (control); Merk - egg yolk or INRA 82 modified (Experiment 1). The extended semen was packaged in 0.5 ml straws, cooled and frozen in a freezing machine. The control extender was superior in preserving the motility, VCL, VSL, VAP, LIN, STR and the BCF when compared to the Merk - egg yolk and INRA 82 modified (P < 0.05). The BotuCrio® preserved more effectively the equine sperm viability characteristics evaluated in Experiment 1 and was used as a control extender in Experiment 2 to test the effectiveness of using LDL in replacement of egg yolk. BotuCrio® was superior to preserve progressive motility, VCL, VSL, VAP, LIN, STR and the percentage of functional integrity of sperm membranes compared to BotuCrio LDL (P < 0.05). However, both extenders preserved similarly the total motility, ALH, BCF and the structural integrity of the membranes (P > 0.05). The fertility rate after AI with frozen semen in BotuCrio LDL was 37.5%.


sperm, lipoproteins, amide, cryoprotection. equine.


Alvarenga MA, Graham JK, Keith SL, Landim-Alvarenga FC, Squires EL. 2000. Alternative cryoprotectors for freezing stallion spermatozoa. In Abstracts of the 14th International Congress on Animal Reproduction, 2000, Stockholm, Sweden. Stockholm: ICAR. pp.157.

Alvarenga MA, Leão KM, Papa FO, Graham Jk. 2002. Improvement of stallion semen post-thaw motility with the utilization of dimethyl-formamide as cryoprotector. Theriogenology, 57:459.

Alvarenga MA, Leão KM, Papa FO, Landim-Alvarenga FC, Medeiros ASL, Gomes GM. 2003. The use of alternative cryoprotectors for freezing stallion semen. In Proceedings of the Workshop on Transporting Gametes and Embryos, 2003, Massachusetts, USA. USA: Havemeyer Foundation. pp.74-76.

Alvarenga MA, Papa FO, Landim-Alvarenga FC, Medeiros ASL. 2005. Amides as cryoprotectans for freezing stallion semen: a review. Anim Reprod Sci, 89:105-113.

Álvarez C, Gil L, González N, Olaciregui M, Luño V. 2014. Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96® extender. Cryobiology, 69:144-148.

Amirat L, Tainturier D, Jeanneau L, Thorin C, Gérard O, Courtens JL, Anton M. 2004. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender. Theriogenology, 61:895-907.

Avanzi BR, Ramos RS, Araujo GHM, Fioratii EG, Trinca LA, Dell´Aqua Jr JA, Melo E, Oña CM, Zahn FS, Martin I, Alvarenga MA, Papa FO. 2015. Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions? Theriogenology, 83:1389-1393.

Bergeron A, Crête MH, Brindle Y, Manjunath P. 2004. Low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod, 70:708-717.

Bencharif D, Amirat-Briand L, Garand A, Anton M, Schmitt E, Desherces S, Delhomme G, Langlois ML, Barrière P, Destrumelle S, Vera-Munoz O, Tainturier D. 2010. Freezing canine sperm: Comparison of semen extenders containing Equex® and LDL (Low Density Lipoproteins). Anim Reprod Sci, 119:305-313.

Candeias ML, Alvarenga MA, Carmo MT, Ferreira HN, Maior MRS, Torres Filho RA, Rodrigues ALR, Brandão FZ. 2012. Semen cryopreservation protocols of Mangalarga Marchador stallions. R Bras Zootec, 41:1989-1995.

CBRA (Colégio Brasileiro de Reprodução Animal). 2013. Manual for Andrological Examination and Evaluation of Animal Semen. 3rd ed. Belo Horizonte: CBRA, 104p, 2013.

Costa DNM, Silva DAM, Boakari YL, Ferreira SB, Branco MAC, Souza JAT. 2014. Efficiency of tris extenders and Botu-crio® on seminal parameters in

stallion breeds Quarter Horse and Mangalarga Marchador. Cienc Anim Bras, 15:322-329.

Dalimata AM, Graham J K. 1997. Cryopresevation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose. Theriogenology, 48:831-841.

Gibb Z, Morris LHA, Maxwell WMC, Grupen CG. 2013. Dimethyl formamide improves the postthaw characteristics of sex-sorted and nonsorted stallion sperm. Theriogenology, 79:1027-1033.

Gomes GM, Papa F O, Jacob JCF, Macedo LP, Leão KM, Machado MS, Alvarenga MA. 2002. Improvement of frozen-thawed spermatic parameters with utilization of MP-50 extender to Mangalarga Machador stallions. Rev Bras Reprod Anim, 26:187-189.

Graham JK. 2000. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. In Abstracts of the 14th International Congress on Animal Reproduction, 2000, Stockholm, Sweden. Stockholm: ICAR. pp.307.

Hanada A, Nagase H. 1980. Cryoprotective effects of some amides on rabbit spermatozoa. J Reprod Fert, 60:247-252.

Harrison RAP, Vickers SE. 1990.Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil, 88: 343-352.

Henry M, Snoeck PPN, Cottorello ACP. 2002. Post-thaw spermatozoa plasma membrane integrity and motility of stallion semen frozen with different cryoprotectants. Theriogenology, 58:245-248.

Hoffmann N, Oldenhof H, Morandini C, Rohn K, Sieme H. 2011. Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing. Anim Reprod Sci, 125:112-118.

Jiang ZL, Li QW, Li WY, Hu JH, Zhao HW, Zhang SS. 2007. Effect of low density lipoprotein on DNA integrity of freezing-thawing boar sperm by neutral comet assay. Anim Reprod Sci, 99:401-407.

Juliani G, Henry M, Melo MIV. 2004. Freezing of equine semen in extenders with low density lipoproteins. In Abstracts of the 15th International Congress on Animal Reproduction, 2004, Bahia, Brazil. Brazil: ICAR . pp.498.

Magistrini M, Couty I, Palmer E. 1992. Factors influencing stallion sperm survival. Acta Vet Scand Suppl, 88:97-110.

Manjunath P, Nauc V, Bergeron A, Ménard M. 2002. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen’s egg yolk. Biol Reprod, 67:1250-1258.

Martin JC, Klug E, Günzel AR. 1979. Centrifugation of stallion semen and its storage in large volumes straws. J Reprod Fertil Suppl, 27:47-51.

Medeiros ASL, Gomes GM, Carmo MT, Papa FO, Alvarenga MA. 2002. Cryopreservation of stallion using different amides. Theriogenology, 58:273-276.

Melo CM, Zahn FS, Martin I, Orlandi C, Dell’Aqua Jr. JA, Alvarenga MA, Papa FO. 2007. Influence of semen storage and cryoprotectant on post-thaw viability and fertility of stallion spermatozoa. J Equine Vet Sci, 27:171-175.

Melo MIV, Henry M. 1999. Hypoosmotic test for the evaluation of equine semen. Arq Bras Med Vet Zootec, 51:71-78.

Melo MIV, Resende YF, Rezende EP, Snoeck PPN, Gomes DML, Carvalho TTC, Silva AVD, Neves MM, Bastos DG, Henry MRJM. 2012. Low density lipoproteins and egg yolk to preserve cooled donkey semen. Rev Bras Med Vet, 34:288-294.

Moreno D, Bencharif D, Amirat-Briand L, Neira A, Destrumelle S, Tainturier D. 2013. Preliminary results: the advantages of low-density lipoproteins for the cryopreservation of equine semen. J Equine Vet Sci, 33:1068-1075.

Moussa M, Martinet V, Trimeche A, Tainturier D, Anton M. 2002. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology, 57:1695-1706.

Moustacas VS, Zaffalon FG, Lagares MA, Loaiza-Eccheverri AM, Varago FC, Neves MM, Heneine LGD, Arruda RP, Henry M. 2011. Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram. Theriogenology, 75:300-307.

Neves MM, Heneine LGD, Henry M. 2014. Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa. Arq Bras Med Vet Zootec, 66:769-777.

Oliveira RA, Rubin MIB, Silva CAM. 2013. Pregnancy rates using frozen semen of Criollo stallions with glycerol or dimethylformamid as cryoprotectants. Cienc Anim Bras, 14:488-494.

Papa FO, Zahn FS, Dell´Aqua Jr JA, Alvarenga MA. 2002. Using the MP 50 extender for cryopreservation of equine semen. Rev Bras Reprod Anim, 26:184-187.

Pillet E, Batellier F, Duchamp G, Furstoss V, Vern YLE, Kerboeuf D, Vidament M, Renseigné N. 2008. Freezing stallion semen in INRA96® based extender improves fertility rates in comparison with INRA82. Dairy Sci Technol, 88:257-265.

Pillet E, Duchamp G, Batellier F, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M. 2011. Egg yolk plasma can replace egg yolk in stallion freezing extenders. Theriogenology, 75:105-114.

Ramires Neto C, Monteiro GA, Sancler-Silva YFR, Papa P, Guasti PN, Resende HL, Papa FO, Dell’Áqua Jr JA, Alvarenga MA. 2014. Comparison of different freezing extenders for semen cryopreservation from stallions with poor and good semen freezability. J Equine Vet Sci, 34:58-60.

Rossi TC, Papa FO, Santos TB, Macedo LP, Alvarenga MA, Melo CM, Dell’Aqua Junior JA. 2003. Efeito da utilização de diferentes crioprotetores e suas associações no processo de congelação de sêmen eqüino com meio MP50. Rev Bras Reprod Anim, 27:350-352.

Silva MC, Moura LCO, Melo MIV Mambrini JVM, Neves MM, Henry MRJM, Snoeck PPN. 2014. Prolonged post cooling but not pre-cooling equilibrium lengh improves the viability of ram sperm cryopreserved in an extender containing low-density lipoproteins. Small Ruminant Res, 119: 88-95.

Snoeck PPN, Cottorello ACP, Henry M. 2012.

Viability and fertility of stallion semen frozen with ethylene glycol and acetamide as a cryogenic agent. Anim Reprod, 9:33-39.

Snoeck PPN, Henry M, Melo MIV. 2007. Effect of differents freezing extenders on post thaw equine spermatozoa viability. Arq Bras Med Vet Zootec, 59: 56-64.

Snoeck PPN, Moura LCO, Silva MC, Machado-Neves M, Melo MIV, Heneine LGD, Henry M. 2017. Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation. Cryobiology, 75:88-90.

Squires EL, Keith SL, Graham JK. 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology, 62:1056-1065.

Terraciano PB, Bustamante – Filho IC, Miquelito LV, Arlas TR, Castro F, Mattos RC, Passos EP, Oberst ER, Lima EOC. 2008. Cryopreservation of equine spermatozoa comparing different freezing rates combined with commercial extenders: laboratorial analysis. Cienc Rural, 38:1972-1977.

Varela Junior AS, Corcini CD, Ulguim RR, Alvarenga MVF, Bianchi I, Corrêa MN, Lucia Jr T, Deschamps JC. 2009. Effect of low density lipoprotein on the quality of cryopreserved dog semen. Anim Reprod Sci, 115:323-327.

Vidament M, Daire C, Yvon J M. Doligez P, Bruneau B, Magistrini M, Ecot P. 2002. Motility and fertility of stallion semen frozen with glycerol and/or dimethyl formamide. Theriogenology, 58: 249-251.

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