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

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

Jaci Almeida; Beatriz Parzewski Neves; Mayara Ferreira Brito; Robson Ferreira Freitas; Lílian Gabriel Lacerda; Lira Santos Grapiuna; João Paulo Haddad; Patrícia Alencar Auler; Marc Henry

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Abstract: The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.


oocyte donors, OPU, in vitro fertilization


ABS Brasil [homepage on the Internet]. Delta: ABS Brasil; 2016 [cited 2020 Mar 31]. Available from:

Akhter S, Ansari MS, Rakha BA, Ullah N, Andrabi SMH, Khalid M. In vitro evaluation of liquid-stored buffalo semen at 5oC diluted in soya-lecithin based extender (Bioxcell®), Tris-citric egg yolk, skim milk and egg yolk-citrate extenders. Reprod Domest Anim. 2011;46(1):45-9. PMid:20070582.

Almeida J, Becerra VAB, Neves BP, Auler PA, Andrade GO, Brito MF, Henry M. Refrigeração de sêmen bubalino (Bubalus bubalis) frente a diferentes extensores e avaliação da motilidade espermática no CASA. Proceedings of the 30th Annual of the Brazilian Embryo Technology Society (SBTE), Foz do Iguaçú, PR, Brazil. August 25th to 27th, 2016, and 32th Meeting of the European Embryo Transfer Association (AETE), Barcelona, Spain, Septembre 9th and 10th, 2016. Abstracts. Anim Reprod. 2016;13(3):557.

Almeida J. Sêmen refrigerado e seu potencial de uso na inseminação artificial de búfalas (Bubalus bubalis). [tese]. Belo Horizonte: Universidade Federal de Minas Gerais; 2018.

Baruselli PS, Ferreira RM, Vieira LM, Souza AH, Bó GA, Rodrigues CA. Use of embryo transfer to alleviate infertility caused by heat stress. Theriogenology. 2020;155:1-11. PMid:32562738.

Baruselli PS, Mucciolo RG, Visintin JA, Viana WG, Arruda RP, Madureira EH, Oliveira CA, Molero-Filho JR. Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis). Theriogenology. 1997;47(8):1531-47. PMid:16728096.

Becerra VAB. Efeito da adição de Aniba canelilla, blueberry e polifenol de chá verde sobre a viabilidade in vitro de espermatozoides de búfalo (Bubalus bubalis) resfriados a 5ºC [dissertação]. Belo Horizonte: Universidade Federal de Minas Gerais; 2017.

Bernardes J. Nascem bezerras geradas de embriões de animais de dois meses. J. USP. [serial on the Internet]. 2017 [cited 2020 Mar 31];02-05(17). Available from:

Bucher A, Kasimanickam R, Hall JB, DeJarnette JN, Whittier WD, Kähn W, Xu Z. Fixed-time AI pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented CO-Synch protocol in beef cows. Theriogenology. 2009;71(7):1180-5. PMid:19195696.

Campanile G, Baruselli PS, Neglia G, Vecchio D, Gasparrini B, Gimenes LU, Zicarelli L, D’Occhio MJ. Ovarian function in the buffalo and implications for embryo development and assisted reproduction. Anim Reprod Sci. 2010;121(1-2):1-11. PMid:20430540.

Colégio Brasileiro de Reprodução Animal – CBRA. Manual para exame andrológico e avaliação de sêmen animal. 3. ed. Belo Horizonte: CBRA; 2013. 104 p.

Danell B. Oestrous behaviour, ovarian morphology and cyclical variation in folicular system and endocrine pattern in water buffalo heifers [thesis]. Sweden: Swedish University of Agricultural Sciences; 1987.

Di Francesco S, Boccia L, Campanile G, Di Palo R, Vecchio D, Neglia G, Zicarelli L, Gasparrini B. The effect of season on oocyte quality and developmental competence in Italian Mediterranean buffaloes (Bubalus bubalis). Anim Reprod Sci. 2011;123(1-2):48-53. PMid:21168984.

El-Sisy GA, ElNatat WS, El-Sheshtawy RI. Buffalo semen quality, antioxidants and peroxidation during chilling and cryopreservation. J Vet Res (Pulawy). 2007;11:55-61.

Erickson BH. Development and radio-response of pre-natal bovine ovary. J Reprod Fertil. 1966;10(1):97-105.

Farrell PB, Presicce GA, Brockett CC, Foote RH. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology. 1998;49(4):871-9. PMid:10732095.

Ferraz ML, Sá MF Fo, Batista EOS, Watanabe YF, Watanabe MR, Dayan A, Joaquim DC, Accorsi MR, Gimenes LU, Vieira LM, Baruselli PS. Paradoxical effects of bovine somatotropin treatment on the ovarian follicular population and in vitro embryo production of lactating buffalo donors submitted to ovum pickup. Anim Reprod Sci. 2015;154:1-7. PMid:25623138.

Gasparrini B, Neglia G, Di Palo R, Vecchio D, Albero G, Esposito L, Campanile G, Zicarelli L. Influence of oocyte donor on in vitro embryo production in buffalo. Anim Reprod Sci. 2014;144(3-4):95-101. PMid:24374181.

Gasparrini B. Effects of reproductive season on embryo development in the buffalo. Reprod Fertil Dev. 2018;31(1):68-81. PMid:32188543.

Gillan L, Kroetsch T, Chis Maxwell WM, Evans G. Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls. Anim Reprod Sci. 2008;103(3-4):201-14. PMid:17208395.

Gimenes LU, Fantinato P No, Arango JSP, Ayres H, Baruselli PS. Follicular dynamics of Bos indicus, Bos taurus and Bubalus bubalis heifers treated with norgestomet ear implant associated or not to injectable progesterone. Anim Reprod. 2009;6:256.

Gimenes LU, Ferraz ML, Araujo A, Fantinato Neto P, Chiarati MR, Mesquita LG, Arango JSP, Raposo M, Souza DC, Calomeni GD, Gardinal R, Rodriguez CLV, Trinca LA, Meirelles FV, Baruselli PS. OPU at different times of a synchronized follicular wave did not affect IVP in Bos indicus, Bos taurus and Bubalus bubalis. Reprod Fertil Dev. 2010;22(1):293-4.

Hancock JL. The morphology of boar espermatozoa. J R Microsc Soc. 1957;76(3):84-97. PMid:13476430.

Henry M, Brito MF, Neves BP, Auler PA, Almeida J, Andrade GO, Becerra VB, Bergmann L. Peculiarities of the buffalo species for andrological evaluation-results of four years of study and weekly semen collection schedule. Anim Reprod. 2017;14(Suppl. 1):1225-33.

Le Van Ty D, Chupin D, Driancourt DA. Ovarian follicular population in buffaloes and cows. Anim Reprod Sci. 1989;19(3-4):171-8.

Liang A, Salzano A, D’Esposito M, Comin A, Montillo M, Yang L, Campanile G, Gasparrini B. Anti-Mullerian hormone (AMH) concentration in follicular fluid and mRNA expression of AMH receptor type II and LH receptor in granulosa cells as predictive markers of good buffalo (Bubalus bubalis) donors. Theriogenology. 2016;86(4):963-70. PMid:27087535.

Loaiza-Echeverri AM, Cruz BC, Snoeck PPN, Moura LCO, Neves BP, Neves MM, Heneine LGD, Henry M. Low density lipoproteins added to an extender frozen or lyophilized are evenly efficient in cryoprotecting ovine sperm cells than when 16% whole egg yolk was added. Semina: Ciênc Agrár. 2015;3(36):1315-46.

Madan ML, Singla SK, Jain GC. Ovulatory response to different superovulatory treatment amongst buffaloes (Bubalus bubalis). In: Abstract of the 11th International Congress on Animal Reproduction and AI; 1992; Dublin. Dublin: ICAR; 1992.

Mahmoud KGM, El-Sokary AAE, Abou El-Roos MEA, Abdel-Ghaffar AE, Nawito M. Sperm characteristics in cryopreserved buffalo bull semen in relation to field fertility. Iran J Appl Anim Sci. 2013;3(4):777-83.

Mahmoud KGM, El-Sokary AAE, Abdel-Ghaffar AE, Abou El-Roos MEA, Ahmed YF. Analysis of chromatin integrity and DNA damage in cryopreserved buffalo semen. Iran J Vet Res. 2015a;16(2):161-6.

Mahmoud KGM, Scholkamy TH, Darwish SF. Improvement of vitrification of in vitro produced buffalo embryos with special reference to sex ratio following vitrification. Iran J Vet Res. 2015b;16(4):325-30. PMid:27175197.

Marin DFD, Souza EB, Brito VC, Nascimento CV, Ramos AS, Rolim ST Fo, Costa NN, Cordeiro MS, Santos SSD, Otavio Mitio Ohashi OM. In vitro embryo production in buffaloes: from the laboratory to the farm. Anim. Reprod. 2019;16(2):260-66.

Matos DL, Araújo AA, Roberto IG, Toniolli R. Análise computarizada de espermatozoides: revisão de literatura. Rev Bras Reprod Anim. 2008;32(4):225-32.

Mortimer ST. Casa-practical aspects. J Androl. 2000;21(4):515-24. PMid:10901437.

Moussa M, Marinet V, Trimeche A, Tainturier D, Anton M. Low density lipoproteins: extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology. 2002;57(6):1695-706. PMid:12035979.

Neves MM. Extração das lipoproteínas de baixa densidade da gema do ovo de Gallus domesticus e sua aplicação na criopreservação do sêmen canino [tese]. Belo Horizonte: Universidade Federal de Minas Gerais; 2008.

Ocampo MB, De Assis AT, Ocampo LC, Kanagawa H. Histological observation of follicular atresia in Swamp buffalo. Buffalo Bull. 1994;13(3)

Ohashi OM, Cordeiro MS, Miranda MS. Biotecnologia da reprodução aplicada a bubalinos. Rev. Cienc. Agrar. 2006;45:58-63.

Ohashi OM, Costa Almeida NN, Cordeiro MS, Rolim ST Fo, Ribeiro HFL, Santos AX, Ayala HMD, Brito VC, Ramos AS, Silva TVG, Santos SSD, Miranda MS. Produção in vitro de embrião (PIVE) na espécie bubalina. Rev Bras Reprod Anim. 2017;41(1):195-200.

Ohashi OM, Miranda MS, Souza J, Cordeiro MS, Biondi FC. Produção in vitro de embrião bubalino. Rev Bras Reprod Anim. 2003;27:103-9.

Pereira ECM. Produção de oócitos e embriões bubalinos: efeitos da época do ano e da adição de óleo essencial de Lippia origanoides na maturação in vitro [tese]. Botucatu: Faculdade de Medicina Veterinária e Zootecnia, Campus Botucatu, Universidade Estadual Paulista; 2015.

Perera BMAO. Reproductive cycles of buffalo. Anim Reprod Sci. 2011;124(3-4):194-9. PMid:20869822.

Sá MF Fo, Carvalho NAT, Gimenes LU, Torres-Júnior JR, Nasser LFT, Tonhati H, Garcia JM, Gasparrini B, Zicarelli L, Baruselli PS. Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production. Anim Reprod Sci. 2009;113(1-4):51-9.

Saacke RG, DeJarnette JM, Bame JH, Karabinus DS, Whitman S. Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super and single-ovulating cattle? Theriogenology. 1998;50(1):117-28. PMid:10734480.

Salzano A, De Canditiis C, Della Ragione F, Prandi A, Zullo G, Neglia G, Campanile G, Gasparrini B. Evaluation of factors involved in the failure of ovum capture in superovulated buffaloes. Theriogenology. 2018;122:102-8. PMid:30245332.

Santos SSD, Ferreira MAP, Sampaio RV, Costa NN, Santos DCN, Santana PPB, Sá ALA, Silva BB, Cordeiro MS, Silva TVG, Miranda MS, Ohashi OM. Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (Bos indicus) and buffalo (Bubalus bubalis) fetuses. Anim Reprod. 2013;10(1):55-61.

Severo NC. Influência da qualidade do sêmen bovino congelado sobre a fertilidade. A Hora Veterinária [serial on the Internet]. 2009 [cited 2020 Jan 28];28(167):36-9. Available from:

Singh AK, Singh VK, Narwade BM, Mohanty TK, Atreja SK. Comparative quality assessment of buffalo (Bubalus bubalis) semen chilled (5oC) in egg yolk- and soya milk-based extenders. Reprod Domest Anim. 2012;47(4):596-600. PMid:22017209.

Soliman WTM, Mahmoud KGhM, El-Khawagah ARM, Kandiel MMM, Abouel-Roos MEA, Abdel-Ghaffar AE, El Azab AEI. Impact of in vitro fertilization by fresh and frozen semen on developmental competence and cryotolerance of buffalo embryos. 2018;19(3):178-81. PMid: 30349563.

Statacorp. Stata: Release 12. Statistical Software. College Station, TX: Statacorp LP; 2012.

Surresh KP, Nandi S, Mondal S. Factors affecting laboratory production of buffalo embryos: a meta-analysis. Theriogenology. 2009;72(7):978-85. PMid:19695691.

Totey SM, Singh G, Taneja M, Pawshe CH, Talwar GP. In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus Bubalis). J Reprod Fertil. 1992;95(2):597-607. PMid:1518014.

Verstegen J, Iguer-Ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary pratice. Theriogenology. 2002;57(1):149-79. PMid:11775967.

Vitoria A. Anatomy of female genital tract in the buffalo. In: Course on biotecnology of reproduction in buffaloes. Vol. 4. Caserta, Italy: Journal of Buffalo Science; 1997; Suppl 1, p. 15-20.

Watson PF. Recent development and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 1995;7(4):871-91. PMid: 8711221.

Zicarelli L, Mariotti E, Di Francesco S, Velotto S, Rubessa M, Neglia G. Effect of bull on in vitro sperm capacitation induced by different agents in buffalo species (Bubalus bubalis). Ital J Anim Sci. 2009;8(suppl 2):177-9.

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