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Review (Published online: 18-04-2016)

10. Advances in reproductive biotechnologies - K. K. Choudhary, K. M. Kavya, A. Jerome, and R. K. Sharma

Veterinary World, 9(4): 388-395



   doi: 10.14202/vetworld.2016.388-395



K. K. Choudhary: ICAR-National Dairy Research Institute, Karnal - 132 001, Haryana, India;

K. M. Kavya: ICAR-Indian Veterinary Research Institute, Bareilly - 243 122, Uttar Pradesh, India;

A. Jerome: ICAR-Central Institute for Research on Buffaloes, Hisar - 125 001, Haryana, India;

R. K. Sharma: ICAR-Central Institute for Research on Buffaloes, Hisar - 125 001, Haryana, India;


Received: 14-12-2015, Accepted: 11-03-2016, Published online: 18-04-2016


Corresponding author: A. Jerome, e-mail:

Citation: Choudhary KK, Kavya KM, Jerome A, Sharma RK (2016) Advances in reproductive biotechnologies, Veterinary World, 9(4): 388-395.

In recent times, reproductive biotechnologies have emerged and started to replace the conventional techniques. It is noteworthy that for sustained livestock productivity, it is imperative to start using these techniques for facing the increasing challenges for productivity, reproduction and health with impending environment conditions. These recent biotechniques, both in male and female, have revolutionized and opened avenues for studying and manipulating the reproductive process both in vitro and in vivo in various livestock species for improving tis efficiency. This review attempts to highlight pros and cons, on the recent developments in reproductive biotechnologies, both in male and female in livestock species.

Keywords: assisted reproductive technologies, biotechnology, livestock, reproduction.

1. Johnson, L.A. (2000) Sexing mammalian sperm for production of offspring: The state-of-the-art. Anim. Reprod. Sci., 60-61: 93-107.
2. Seidel, G.E. Jr. (2014) Update on sexed semen technology in cattle. Animal, 8(1): 160-164.
PMid:24680061 PMCid:PMC3951379
3. Garner, D.L. (2006) Flow cytometric sexing of mammalian sperm. Theriogenology, 65: 943-957.
4. Garner, D.L. (2001) Sex-sorting mammalian sperm: Concept to application in animals. J. Androl., 22(4): 519-526.
5. Campanile, G., Gasparrini, B., Vecchio, D., Neglia, G., Senatore, E.M., Bella, A., Presicce, G. and Zicarelli, L. (2011) Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis). Theriogenology, 76(3): 500-506.
6. Campanile, G., Vecchio, D., Neglia, G., Bella, A., Prandi, A., Senatore, E.M., Gasparrini, B. and Presicce, G.A. (2013) Effect of season, late embryonic mortality and progesterone production on pregnancy rates in pluriparous buffaloes (Bubalus bubalis) after artificial insemination with sexed semen. Theriogenology, 79(4): 653-659.
7. Gaviraghi, A., Puglisi, R,, Balduzzi, D., Severgnini, A., Bornaghi, V., Bongioni, G., Frana, A., Gandini, L.M., Lukaj, A., Bonacina, C. and Galli, A. (2013) Minimum number of spermatozoa per dose in Mediterranean Italian buffalo (Bubalus bubalis) using sexed frozen semen and conventional artificial insemination. Theriogenology, 79(8): 1171-1176.
8. Warriach, H.M., McGill, D.M., Bush, R.D., Wynn, P.C. and Choha, K.R. (2015) A review of recent developments in buffalo reproduction - A review. Asian Aust. J. Anim. Sci., 28(3): 451-455.
PMid:25656203 PMCid:PMC4341092
9. Blondin, P., Beaulieu, M., Fournier, V., Morin, N., Crawford, L., Madan, P. and King, W.A. (2009) Analysis of bovine sexed sperm for IVF from sorting to the embryo. Theriogenology, 71: 30-38.
10. Carvalho, J.O., Sartori, R., Machado, G.M., Mourao, G.B. and Dode, M.A. (2010) Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology, 74(9): 1521-1530.
11. Gosálvez, J., López-Fernández, C., Fernández, J.L., Gouraud, A. and Holt, W.V. (2011a) Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol. Reprod. Dev., 78: 951-961.
12. Gosálvez, J., Nú-ez, R., Fernández, J.L., López-Fernández, C. and Caballero, P. (2011b) Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors. Andrologia, 43: 373-377.
13. Faustini, M. (2011) New aspects of boar sperm encapsulation. Reprod. Domest. Anim., 46(2): 52-4.
14. Feugang, J.M., Rodriguez-Osorio, N., Kaya, A., Wang, H., Page, G., Ostermeier, G.C., Topper, E.K. and Memili, E. (2010) Transcriptome analysis of bull spermatozoa: Implications for male fertility. Reprod. Biomed. Online, 21(3): 312-324.
15. Srivastava, J., Premi, S., Kumar, S. and Ali, S. (2008) Organization and differential expression of the GACA/GATA tagged somatic and spermatozoal transcriptomes in buffalo Bubalus bubalis. BMC Genomics, 9: 132.
PMid:18366692 PMCid:PMC2346481
16. Feugang, J.M. (2010) Transcriptome analysis of bull spermatozoa: Implications for male fertility. Reprod. Biomed. Online, 21(3): 312-324.
17. Miller, D. (2014) Sperm RNA as a mediator of genomic plasticity. Adv. Biol., 2014: Article ID: 179701, 13.
18. Bissonnette, N., Lévesque-Sergerie, J.P., Thibault, C. and Boissonneault, G. (2009) Spermatozoal transcriptome profiling for bull sperm motility: A potential tool to evaluate semen quality. Reproduction, 138(1): 65-80.
19. Arangasamy, A., Singh, L.P., Ahmad, N., Ansari, M.R. and Ram, G.C. (2005) Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa. Anim. Reprod. Sci., 90: 243-254.
20. Harshan, H.M., Sankar, S., Singh, L.P., Singh, M.K., Sudharani, S., Ansari, M.R., Singh, S.K., Majumdar, A.C. and Joshi, P. (2009) Identification of PDC-109-like protein(s) in buffalo seminal plasma. Anim. Reprod. Sci., 115(1-4): 306-311.
21. Srivastava, N., Jerome, A., Srivastava, S.K., Ghosh, S.K. and Kumar, A. (2013) Bovine seminal PDC-109 protein: An overview of biochemical and functional properties. Anim. Reprod. Sci., 138(1-2): 1-13.
22. Peddinti, D., Nanduri, B., Kaya, A., Feugang, J.M., Shane, C.B. and Memili, E. (2008) Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Syst. Biol., 2: 19.
PMid:18294385 PMCid:PMC2291030
23. Galli, C., Crotti, G., Notari, C., Turini, P., Duchi, R. and Lazzari, G. (2001) Embryo production by ovum pick up from live donors. Theriogenology, 55(6): 1341-1357.
24. Verma, O.P., Kumar, R., Kumar, A. and Chand, S. (2012) Assisted reproductive techniques in farm animal - From artificial insemination to nanobiotechnology. Vet. World, 5(5): 301-310.
25. Boni, R. (2012) Ovum pick-up in cattle: A 25 year retrospective analysis. Anim. Reprod., 9(3): 362-369.
26. Prasad, S., Singh, B., Singhal, S., Khan, F.A., Prasad, J.K. and Gupta, H.P. (2013) Production of the first viable ovum pick-up and in vitro embryo produced (OPU-IVEP) buffalo calf in India. Asian Pac. J. Reprod., 2(2): 163-165.
27. Purohit, G. N., Duggal, G.P., Dadarwal, D., Kumar, D., Yadav, R.C. and Vyas, S. (2003) reproductive biotechnologies for improvement of buffalo: The current status. Asian Aust. J. Anim. Sci., 16(7): 1071-1086.
28. Machado, S.A., Reichenbach, H.D., Weppert, M., Wolf, E. and Gonçalves, P.B. (2006) The variability of ovum pick-up response and in vitro embryo production from monozygotic twin cows. Theriogenology, 65(3): 573-583.
29. Van Wagtendonk-de Leeuw, A.M. (2006) Ovum pick up and in vitro production in the bovine after use in several generations: A 2005 status. Theriogenology, 65: 914-925.
30. Brackett, B.G., Bousquet, D., Boice, M.L., Donawick, W.J., Evans, J.F. and Dressel, M.A. (1982) Normal development following in vitro fertilization in the cow. Biol. Reprod., 27(1): 147-158.
31. Hasler, J.F. (2003) The current status and future of commercial embryo transfer in cattle. Anim. Reprod. Sci., 79(3-4): 245-264.
32. Betteridge, K.J. (2006) Farm animal embryo technologies: Achievements and perspectives. Theriogenology, 65: 905-913.
33. Madan, M.L., Singla, S.K., Jailkhani, S. and Ambrose, J.D. (1991) In vitro fertilization in buffalo and birth of first ever IVF buffalo calf. Vol. 7. Proceeding of the 3rd Buffalo Congress. Verma, Bulgaria. 15-19 May. p11-17.
34. Galli, C. and Lazzari, G. (2008) The manipulation of gametes and embryos in farm animals. Reprod. Domes. Anim., 43: 1-7.
35. Drost, M. (2007) Advanced reproductive technology in the water buffalo. Theriogenology, 68: 450-453.
36. Lin, T.P. (1966) Microinjection of mouse eggs. Science, 151(3708): 333-337.
37. Liang, Y.Y., Ye, D.N., Laowtammathron, C., Phermthai, T., Nagai, T., Somfai, T. and Parnpai, R. (2011) Effects of chemical activation treatment on development of swamp buffalo (Bubalus bubalis) oocytes matured in vitro and fertilized by intracytoplasmic sperm injection. Reprod. Domest. Anim., 46(1): 67-73.
38. Ohlweiler, L.U., Brum, D.S., Leivas, F.G., Moyses, A.B., Ramos, R.S., Klein, N., Mezzalira, J.C. and Mezzalira, A. (2013) Intracytoplasmic sperm injection improves in vitro embryo production from poor quality bovine oocytes. Theriogenology, 79(5): 778-783.
39. Mapletoft, R.J. (2013) History and perspectives on bovine embryo transfer. Anim. Reprod., 10(3): 168-173.
40. Hasler, J.F. (2014) Forty years of embryo transfer in cattle: A review focusing on the journal. Theriogenology, the growth of the industry in North America, and personal reminisces. Theriogenology, 81: 152-169.
41. Mapletoft, R.J. (1985) Embryo transfer in the cow: General procedures. Rev. Sci. Tech. Off. Int. Epiz., 4(4): 843-858.
42. Hasler, J.F. (1998) The current status of oocyte recovery, in vitro embryo production, and embryo transfer in domestic animals, with an emphasis on the bovine. J. Anim. Sci., 76 Suppl 3: 52-74.
43. Drost, M., Brand, A. and Aarts, M.H. (1976) A device for nonsurgical recovery of bovine embryos. Theriogenology, 6(5): 503-508.
44. Nandi, S., Raghu, H.M., Ravindranatha, B.M. and Chauhan, M.S. (2002) Production of buffalo (Bubalus bubalis) embryos in vitro: Premises and promises. Reprod. Domest. Anim., 37: 65-74.
45. Smith, A.K. (2001) Retrospective study of the effect of superovulation on subsequent fertility in embryo-transfer donors. Vet. Rec., 148(4): 114-116.
46. Leibo, S.P. and Mazur, P. (1978) Methods for the preservation of mammalian embryos by freezing. In: Daniel J.C. Jr., editor. Methods in mammalian reproduction. Academic Press, New York. p179-201.
47. Youngs, C.R. (2011) Cryopreservation of preimplantation embryos of cattle, sheep, and goats. J. Vis. Exp., (54): 2764.
48. Hwang I.S., Hochi, S. (2014) Recent progress in cryopreservation of bovine oocytes, review article. Biomed. Res. Int., 2014: Article ID: 570647, 11.
49. Sirisha, K., Selokar, N.L., Saini, M., Palta, P., Manik, R.S., Chauhan, M.S. and Singla, S.K. (2013) Cryopreservation of zona-free cloned buffalo (Bubalus Bubalis) embryos: Slow freezing vs open-pulled straw vitrification. Reprod. Domest. Anim., 48(4): 538-544.
50. Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at - 196 degrees C by vitrification. Nature, 313(6003): 573-575.
51. Lonergan, P. (2007) State-of-the-art embryo technologies in cattle. Soc. Reprod. Fertil. Suppl., 64: 315-325.
52. Yavin, S., Aroya, A., Roth, Z. and Arav, A. 2009. Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush. Hum. Reprod., 24: 797-804.
53. Thibier, M. and Nibart, M. (1995) The sexing of bovine embryos in the field. Theriogenology, 43(1): 71-80.
54. Rao, K.B., Pawshe, C.H. and Totey, S.M. (1993) Sex determination of in vitro develoeped buffalo (Bubalus bubalis) embryoes by DNA amplification. Mol. Reprod. Dev., 36: 291-296.
55. Zoheir, K.M.A. and Allam, A.A. (2010) A rapid method for sexing the bovine embryo Anim. Reprod. Sci., 119(1-2): 92-96.
56. Sood, S.K., Chauhan, M.S. and Tomer, O.S. (2001) A direct duplex PCR assay for sex determination of Murrah buffalo embryos. Buffalo J., 17: 113-124.
57. Graf, A., Krebs, S., Heininen-Brown, M., Zakhartchenko, V., Blum, H. and Wolf, E. (2014) Genome activation in bovine embryos: Review of the literature and new insights from RNA sequencing experiments. Anim. Reprod. Sci., 149(1-2): 46-58.
58. Graf, A., Krebs, S., Zakhartchenko, V., Schwalb, B., Blum, H. and Wolf, E. (2014) Fine mapping of genome activation in bovine embryos by RNA sequencing. Proc. Natl. Acad. Sci. USA., 111(11): 4139-4144.
PMid:24591639 PMCid:PMC3964062
59. Niemann, H. and Wrenzycki, C. (2000) Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology, 53: 21-34.
60. Latham, K.E. and Schultz, R.M. (2001) Embryonic genome activation. Front. Biosci., 6: D748-D759.
61. Nath, A., Sharma, V., Dubey, P.K., Pratheesh, M.D., Gade, N.E., Saikumar, G. and Sharma, G.T. (2013) Impact of gonadotropin supplementation on the expression of germ cell marker genes (MATER, ZAR1, GDF9, and BMP15) during in vitro maturation of buffalo (Bubalus bubalis) oocyte. In Vitro Cell Dev. Biol. Anim., 49(1): 34-41.
62. Eswari, S., Sai Kumar, G. and Sharma, G.T. (2013) Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: Markers of successful embryo implantation. Zygote, 21(2): 203-213.
63. Sharma, G.T., Nath, A., Prasad, S., Singhal, S., Singh, N., Gade, N.E., Dubey, P.K. and Saikumar, G. (2012) Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos. Reprod. Dom. Anim., 47(6): 975-983.
64. Rajhans, R., Kumar, G.S., Dubey, P.K. and Sharma, G.T. (2010) Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro. Cell Biol. Int., 34(5): 463-468.
65. Gad, A., Hoelker, M., Besenfelder, U., Havlicek, V., Cinar, U., Rings, F., Held, E., Dufort, I., Sirard, M.A., Schellander, K. and Tesfaye, D. (2012) Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions. Biol. Reprod., 87(4): 1-13.
66. Tesfaye, D., Ponsuksili, S., Wimmers, K., Gilles, M. and Schellander, K. (2004) A comparative expression analysis of gene transcripts in post-fertilization developmental stages of bovine embryos produced in vitro or in vivo. Reprod. Domest. Anim., 39: 396-404.
67. Abdoon, A.S., Ghanem, N., Kandil, O.M., Gad, A., Schellander, K. and Tesfaye, D. (2012) cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6): 1240-1251.
68. Singla, S.K., Selokar, N.L., Saini, M., Palta, P., Chauhan, M.S. and Manik, R.S. (2015) Buffalo cloning: What we have achieved so far. Curr. Sci., 109(4): 670-671.
69. Vajta, G., Lewis, I.M., Trounson, A.O., Purup, S., Maddox-Hyttel, P., Schmidt, M., Pedersen, H.G., Greve, T. and Callesen, H. (2003) Handmade somatic cell cloning in cattle: Analysis of factors contributing to high efficiency in vitro. Biol. Reprod., 68(2): 571-578.
70. Vajta, G. and Gjerris, M. (2006) Science and technology of farm animal cloning: State of the art. Anim. Reprod. Sci., 92: 211-230.
71. Campbell, K.H., McWhir, J., Ritchie, W.A. and Wilmut, I. (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature, 380(6569): 64-66.
72. Shah, R.A., George, A., Singh, M.K., Kumar, D., Chauhan, M.S., Manik, R., Palta, P. and Singla, S.K. (2008) Hand-made cloned buffalo (Bubalus bubalis) embryos: Comparison of different media and culture systems. Cloning Stem Cells, 10(4): 435-442.
73. Shah, R.A., George, A., Singh, M.K., Kumar, D., Anand, T., Chauhan, M.S., Manik, R.S., Palta, P. and Singla, S.K. (2009) Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis). Theriogenology, 71(8): 1215-1219.
74. Avasthi, S., Srivastava, R.N., Singh, A. and Srivastava, M. (2008) Stem cell: Past, present and future - A review article. Int. J. Med. Update, 3(1): 22-30.
75. Tecirlioglu, R.T. and Trounson, A.O. (2007) Embryonic stem cells in companion animals (horses, dogs and cats): Present status and future prospects. Reprod. Fertil. Dev., 9: 740-747.
76. Talbot, N.C. and Blomberg, L.A. (2008) The pursuit of ES cell lines of domestic ungulates. Stem Cell Rev., 4: 235-254.
77. Nowak-Imialek, M., Kues, W.A. and Niemann, H, (2011) Pluripotent stem cells and reprogrammed cells in farm animals. Microsc. Microanal., 17: 474-497.
78. Yadav, P.S., Singh, R.K. and Singh, B. (2012) Fetal stem cells in farm animals: Applications in health and production. Agric. Res., 1(1): 67-77.
79. George, A., Sharma, R., Singh, K.P., Panda, S.K., Singla, S.K., Palta, P., Manik, R. and Chauhan, M.S. (2011) Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts. Cell Reprog., 13(3): 263-272.
80. Selokar, N.L., Saini, M., Palta, P., Chauhan, M.S., Manik, R.S. and Singla, S.K. (2014) Hope for restoration of dead valuable bulls through cloning using donor somatic cells isolated from cryopreserved semen. PLoS One, 9(3): e90755.
81. Wall, R.J. (1996) Transgenic livestock: Progress and prospects for the future. Theriogenology, 45(1): 57-68.
82. Niemann, H. and Kues, W.A. (2003) Application of transgenesis in livestock for agriculture and biomedicine. Anim. Reprod. Sci., 79(3-4): 291-317.
83. Robl, J.M., Wang, Z., Kasinthan, P. and Kuroiwa, Y. (2007) Transgenic animal production and animal biotechnology. Theriogeneology, 67: 127-133.
84. Verma, V., Gautam, S.K., Palta, P., Manik, R.S., Singla, S.K. and Chauhan, M.S. (2008) Development of a pronuclear DNA microinjection technique for production of green fluorescent protein-expressing bubaline (Bubalus bubalis) embryos. Theriogenology, 69: 655-665.
85. Wells, D.J. (2010) Genetically modified animals and pharmacological research. Handb. Exp. Pharmacol., 199: 213-216.
86. Ebert, K.M., Selgrath, J.P., DiTullio P., Denmen, J., Smith, T.E., Memon, M.A., Schindler, J.E., Monastersky, G.M., Vitale, J.A. and Gordan, K. (1991) Transgenic production of variant human tissue type palsminogen activator in goat milk. Genneration of transgenic goat and analysis of expression. Biotechnology, 9: 835-838.
87. McNeil, E.S. (2005) Nanotechnology for the biologist. J. Leukoc. Biol., 78: 1-10.
88. Beebe, D., Wheeler, M., Zerigue, H., Walters, E. and Raty, S. (2002) Microfluidic technology for assisted reproduction. Theriogenology, 57: 125-135.
89. Knowlton, M., Sadasivam, M. and Tasoglu, S. (2015) Microfluidics for sperm research. Trends Biotech., 33(4): 221-229.
90. Rishpon, J. and Ivnitski, D. (1997) An amperometric enzyme channeling immunosensor. Biosens. Bioelectron., 12(3): 195-204.