Animal Reproduction (AR)
Animal Reproduction (AR)

In vitro embryo production in buffaloes: from the laboratory to the farm

Diego Fernando Dubeibe Marin, Eduardo Baia de Souza, Vanessa Cunha de Brito, Carlos Vinicius Nascimento, Anelise Sarges Ramos, Sebastião Tavares Rolim Filho, Nathalia Nogueira da Costa, Marcela da Silva Cordeiro, Simone do Socorro Damasceno Santos, Otavio Mitio Ohashi

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Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.


buffalo, breeding programs, in vitro embryo production.


Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. 2009. L-Carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos. Fertil Steril, 91(2):589-596.

Abdoon AS, Gabler C, Holder C, Kandil OM, Einspanier R. 2014. Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes. Theriogenology, 82(8):1055-1067.

Balao da Silva C, Macías-García B, Morillo Rodriguez A, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Peña FJ. 2012. Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96. Anim Reprod Sci, 131(3-4):165-71.

Baruselli PS, Mucciolo RG, Visintin JA, Viana WG, Arruda RP, Madureira EH, Molero-Filho JR. 1997. Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis). Theriogenology, 47(8):1531-1547.

Baruselli PS, Soares JG, Gimenes LU, Monteiro BM, Olazarri MJ, Carvalho NAT. 2013. Control of buffalo follicular dynamics for artificial insemination, superovulation and in vitro embryo production. Buffalo Bulletin, 32:160-176.

Boccia L, Attanasio L, Monaco E, Rosa A, di palo R, Gasparrini B. 2006. Effect of progesterone on capacitation of buffalo (Bubalus bubalis) spermatozoa in vitro . Reprod Domest Anim, 41: 311-311.

Boccia L, Francesco S, Di Neglia G, Blasi M, De Longobardi V, Campanile G, Gasparrini B. 2013. Osteopontin improves sperm capacitation and in vitro fertilization ef fi ciency in buffalo ( Bubalus bubalis ). Theriogenology, 80(3): 212–217.

Boni R, Santiella L, Dale B, Roviello S, Di Palo R, Barbieri V. 1992. Maturazione in vitro di oociti buffalini: indagine ultrastrutturale. Acta Med Vet, 38:153-161.

Boni R, Di Palo R, Barbieri V, Zicarelli L. 1994. Ovum pick-up in deep anestrus buffaloes. In: Proc IV world Buffalo Congress, 1994, Napoli, Italy. pp.480-482.

Cavalieri FLB, Morotti F, Seneda MM, Colombo AHB, Andreazzi MA, Emanuelli IP, Rigolon LP. 2018. Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick-up. Theriogenology, 117:57-60.

Danell B. 1987. Oestrus behaviour, ovarian morphology and cyclical variation in follicular system and endocrine pattern in water buffalo heifers. Uppsalá, Suécia: Swedish University of Agricultural Sciences. Thesis.

Dantas JK. 2002. Desenvolvimento de embriões bubalinos (bubalus bubalis) cultivados in vitro em diferentes meios. [In portuguese]. Belém, Brazil: Universidade Federal do Pará. Thesis.

Di Francesco S, Novoa MVS, Vecchio D, Neglia G, Boccia L, Campanile G, Gasparrini B. 2012. Ovum pick-up and in vitro embryo production (OPU-IVEP) in Mediterranean Italian buffalo performed in different seasons. Theriogenology, 77(1):148-54.

Drost M. 2007. Advanced reproductive technology in

the water buffalo. Theriogenology, 68(3): 450-453.

Ferraz ML, Sá Filho MF, Batista EOS, Watanabe YF, Watanabe MR, Dayan A, Baruselli PS. 2015. Paradoxical effects of bovine somatotropin treatment on the ovarian follicular population and in vitro embryo production of lactating buffalo donors submitted to ovum pick-up. Anim Reprod Sci, 154:1-7.

Galli C, Duchi R, Crotti G, Lazzari G. 1998. Embryo production by ovum pick up in water buffalo. Theriogenology, 49(1):400.

Galli C, Crotti G, Notari C, Turini P, Duchi R, Lazzari G. 2001. Embryo production by ovum pick up from live donors. Theriogenology, 55(6):1341-1357.

Galli C, Duchi R, Crotti G, Turini P, Ponderato N, Colleoni S, Lazzari G. 2003. Bovine embryo technologies. Theriogenology, 59(2):599-616.

Galli C, Duchi R, Lazzari G, Lagutina I, Colleoni S, Turini P, Berdugo J. 2012. Pregnancies and calves after transfer of in vitro -produced river buffalo embryos after cryopreservation. Reprod Fertil Dev, 24(1):190.

Galli C, Duchi R, Colleoni S, Lagutina I, Lazzari G. 2014. Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. Theriogenology, 81(1):138-51.

Gasparrini B. 2002. In vitro embryo production in buffalo species: state of the art. Theriogenology, 57(1):237-256.

Gasparrini B, Boccia L, Marchandise J, Di Palo R, George F, Donnay I, Zicarelli L. 2006. Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology, 65(2):275-87.

Gasparrini B. 2007. In vivo embryo production in buffalo: current situation and future perspectives. Ital J Anim Sci, 6(sup2): 92-101.

Gasparrini B, De Rosa A, Attanasio L, Boccia L, Di Palo R, Campanile G, Zicarelli L. 2008. Influence of the duration of in vitro maturation and gamete co-incubation on the efficiency of in vitro embryo development in Italian Mediterranean buffalo (Bubalus bubalis). Anim Reprod Sci, 105(3-4):354-64.

Gasparrini B. 2013. In vitro embryo production in buffalo: Yesterday, today and tomorrow. Buffalo Bulletin, 32(SPEC. ISSUE 1):188-195.

Gasparrini B, Elkhawagah AR, Longobardi V, Sosa GA, Salzano A, Aboul-roos MEA, Zicarelli L. 2014a. Effect of Methyl-B-Cyclodextrin (MBCD) on In vitro Capacitation of Buffalo Frozen/Thawed Sperm. Journal of Buffalo Science, 3(1):12-17.

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

Gimenes L. 2010. Taxa de recuperação in vivo e competência in vitro de oócitos bubalinos, zebuinos e taurinos, aspirados em diferentes fases da onda de crescimento folicular [In portuguese]. São Paulo, Brasil: Universidade de São Paulo. Thesis.

Held-Hoelker E, Klein SL, Rings F, Salilew-Wondim D, Saeed-Zidane M, Neuhoff, C, Hoelker M. 2017. Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids. Theriogenology, 96:145-152.

Hufana-Duran D, Pedro PB, Venturina HV, Hufana RD, Salazar AL, Duran PG, Cruz LC. 2004. Post-warming hatching and birth of live calves following transfer of in vitro -derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology, 61(7-8):429-1439.

Jagan Mohanarao G, Atreja SK. 2012. Identification of NO induced and capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) spermatozoa. Res Vet Sci, 93(2):618-623.

Konrad J, Clérico G, Garrido MJ, Taminelli G, Yuponi M, Yuponi R, Sansinena M. 2017. Ovum pick-up interval in buffalo (Bubalus bubalis) managed under wetland conditions in Argentina: Effect on follicular population, oocyte recovery, and in vitro embryo development. Anim Reprod Sci, 183:39-45.

Kumar D, Anand T. 2012. In vitro Embryo Production in Buffalo: Basic Concepts. Journal of Buffalo Science, 1: 50-54.

Kumar P, Verma A, Roy B, Rajput S, Ojha S, Anand S, Datta TK. 2012. Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo. Reprod Domestic Anim, 47(2):269-273.

Mahmoud KGM, Scholkamy TH, Darwish SF. 2015. Improvement of vitrification of in vitro produced buffalo embryos with special reference to sex ratio following vitrification. Iran J Vet Res, 16(4):325-330.

Mandawala AA, Harvey SC, Roy TK, Fowler KE. 2016. Cryopreservation of animal oocytes and embryos: Current progress and future prospects. Theriogenology, 86(7):1637-1644.

Neglia G, Gasparrini B, Caracciolo di Brienza V, Di Palo R, Campanile G, Antonio Presicce G, Zicarelli L. 2003. Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration. Theriogenology, 59(5-6):1123-1130.

Neglia G, Gasparrini B, Vecchio D, Boccia L, Varricchio E, Di R, Campanile G. 2011. Long term effect of Ovum Pick-up in buffalo species. Anim Reprod Sci, 123(3-4):180-186.

Oba O, Camargos AS. 2011. Produção in vitro de embriões bubalinos. Rev Bras Reprod Anim, 35(2):80-87.

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

Ohashi OM, Nogueira N, Cordeiro S, Filho TR, Francisco H, Ribeiro L, Guimarães TV. 2017. Produção in vitro de embrião ( PIVE ) na espécie bubalina. Rev Bras Reprod Anim, 14(1):195-200.

Palta P, Chauhan MS. 1998. Laboratory production of buffalo (Bubalus bubalis) embryos. Reprod Fertil Dev, 10(5):379-391.

Parnpai R, Liang Y, Ketudat-Cairns M, Somfai T, Nagai T. 2016. Vitrification of buffalo oocytes and embryos. Theriogenology, 86(1): 214-220.

Parrish JJ. 2014. Bovine in vitro fertilization: In vitro oocyte maturation and sperm capacitation with heparin.

Theriogenology, 81(1):67-73.

Perera BMAO. 2011. Reproductive cycles of buffalo. Anim Reprod Sci, 124(3-4):194-199.

Rath D, Moench-Tegeder G, Taylor U, Johnson LA. 2009. Improved quality of sex-sorted sperm: a prerequisite for wider commercial application. Theriogenology, 71(1):22-29.

Sales JNS, Iguma LT, Batista RITP, Quintão CCR, Gama MAS, Freitas C, Baruselli PS. 2015. Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows. J Dairy Sci, 98(5):3086-3099.

Saliba W, Gimenes L, Drumond R, Bayão H, Alvim M, Baruselli P, Gasparrini B. 2013. Efficiency of OPU-IVEP-ET of Fresh and Vitrified Embryos in Buffaloes. Buffalo Bulletin, 32(2):385-388.

Santos S do SD, Dantas JK, Miranda M dos S, Biondi FC, Ohashi OM. 2002a. Cinética da maturação nuclear in vitro de oócitos bubalinos. Braz J Vet Res Anim Sci, 39(5):266-270.

Santos SSD, Costa SHF, Dantas JK, Ohashi OM. 2002b. Maturação in vitro de oócitos bubalinos. Rev Bras Reprod Anim, 26(1):37-42.

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

Singhal S, Prasad S, Singh B, Prasad JK, Gupta HP. 2009. Effect of including growth factors and antioxidants in maturation medium used for in vitro culture of buffalo oocytes recovered in vivo. Anim Reprod Sci, 113(1-4):44-50.

Suárez Novoa MV, Di Francesco S, Rubessa M, Boccia L, Longobardi V, De Blasi M, Gasparrini B. 2011. Effect of reducing glucose concentration during in vitro embryo culture in buffalo (Bubalus bubalis). Reproduction, Fertility and Development, 23(1):168.

Suresh KP, Nandi S, Mondal S. 2009. Factors affecting laboratory production of buffalo embryos: a meta-analysis. Theriogenology, 72(7):978-985.

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

Verma M, Pandey S, Bhat IA, Mukesh B, Anand J, Chandra V, Sharma GT. 2018. Impact of l-carnitine on lipid content and post thaw survivability of buffalo embryos produced in vitro . Cryobiology, 82:99-105.

Wadhwa N, Kunj N, Tiwari S, Saraiya M, Majumdar SS. 2009. Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning. Cloning and Stem Cells, 11(3):387-395.

Yang CY, Pang CY, Yang BZ, Li RC, Lu YQ, Liang XW. 2012. Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer. Theriogenology, 78(7):1437-1445.

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