Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows


  • Mihai Cenariu University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca
  • Mihai Borzan University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca
  • Sorin Dan University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca
  • Remus Chiorean Stațiunea de Cercetare și Dezvoltare pentru Creșterea Bubalinelor Șercaia
  • Emoke Pall University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca



bovine oocytes, ICSI, embryo, slaughtered cows


Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.


Fishel, S.; Aslam, I.; Lisi, F.; Rinaldi, L.; Timson, J.; Jacobson, M.; Gobetz, L.; Green, S.; Campbell, A.; Lisi, R. Should ICSI be the treatment of choice for all cases of in-vitro conception? Hum. Reprod. 2000, 15, 1278–1283. DOI: 10.1093/humrep/15.6.1278

Goto, K.; Kinoshita, A.; Takuma, Y.; Ogawa, K. Fertilisation of bovine oocytes by the injection of immobilised, killed sper-matozoa. Vet. Rec. 1990, 127, 517–520.

Magata, F.; Tsuchiya, K.; Okubo, H.; Ideta, A. Application of intracytoplasmic sperm injection to the embryo production in aged cows. J. Vet. Med. Sci. 2019, 81(1), 84–90. DOI: 10.1292/jvms.18-0284

Wang, B.; Baldassarre, H.; Pierson, J.; Cote, F.; Rao, K.M.; Karatzas, C.N. The in vitro and in vivo development of goat embryos produced by intracytoplasmic sperm injection using tail-cut spermatozoa. Zygote 2003, 11, 219–227. DOI: 10.1017/s0967199403002260

Stein, P.; Schultz, R.M. ICSI in the mouse. Method Enzymol. 2010, 476, 251–262. DOI: 10.1016/S0076-6879(10)76014-6

Uehara, T.; Yanagimachi, R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol. Reprod. 1976, 15, 467–470.

Lanzendorf, S.E.; Maloney, M.K.; Veeck, L.L.; Slusser, J.; Hodgen, G.D.; Rosenwaks, Z. A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil. Steril. 1988, 49, 835–842. DOI: 10.1016/s0015-0282(16)59893-8

Unnikrishnan, V.; Kastelic, J.; Thundathil, J. Intracytoplasmic Sperm Injection in Cattle. Genes 2021, 12, 198. DOI: 10.3390/ genes12020198

Oikawa, T.; Itahashi, T.; Numabe, T. Improved embryo development in Japanese black cattle by in vitro fertilization using ovum pick-up plus intracytoplasmic sperm injection with dithiothreitol. J. Reprod. Dev. 2016, 62(1), 11-16. DOI: 10.1262/jrd.2015-067

Keskintepe, L.; Pacholczyk, G.; Machnicka, A.; Norris, K.; Curuk, M.A.; Khan, I.; Brackett, B.G. Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol. Reprod. 2002, 67, 409–415. DOI: 10.1095/biolreprod67.2.409

Meo, S.C.; Leal, C.L.V.; Yamazaki, W.; Garcia, J.M. Influence of simple and combined ionomycin, strontium and 6-DMAP treatments on activation and parthenogenetic development. Theriogenology 2002, 57, 706.

Chung, J.T.; Keefer, C.L.; Downey, B.R. Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Theri-ogenology 2000 53, 1273–1284. DOI: 10.1016/S0093-691X(00)00271-5

Horiuchi, T.; Emuta, C.; Yamauchi, Y.; Oikawa, T.; Numabe, T.; Yanagimachi, R. Birth of normal calves after intracytoplas-mic sperm injection of bovine oocytes: a methodological approach. Theriogenology 2002, 57, 1013–1024. DOI: 10.1016/s0093-691x(01)00701-4

Horiuchi, T.; Emuta, C.; Oikawa, T.; Numabe, T. Comparison of bovine embryo yield following intracytoplasmic sperm in-jection and in vitro fertilization in individual bulls. Theriogenology 2000, 53, 393.

Hwang, S.; Lee, E.; Yoon, J.; Yoon, B. K.; Lee, J. H.; Choi, D. Effects of electric stimulation on bovine oocyte activation and em-bryo development in intracytoplasmic sperm injection procedure. J. Assist. Reprod. Genet. 2000, 17(6), 310–314. DOI: 10.1023/a:1009496726343

McNutt, T.L.; Johnson, L.A. Flow cytometric sorting of sperm: influence on fertilization and embryo/fetal development in the rabbit. Mol. Reprod. Dev. 1996, 43, 261–267. DOI: 10.1002/(SICI)1098-2795(199602)43:2<261::AID-MRD16>3.0.CO;2-6

Doyle, S.P.; Seidel, G.E, Jr; Schenk, J.L.; Herickhoff, L.A.; Cran, D.G.; Green, R.D. Artificial insemination of lactating Angus cows with sexed semen. J. Anim. Sci. 1999, 77(suppl.1), 99.

Medvedev, S.; Bossak, N.; Eckert, J.; Lucas-Hahn, A.; Niemann, H.; Johnson, L.A. Intra-cytoplasmic sperm injection (ICSI) with flowcytometrically sorted Y-chromosome bearingbovine sperm. Theriogenology 1997, 47, 270.

Skrzyszowska, M.; Shioya, Y.; Nagai, T.; Geshi, M.; Takenouchi, N. Development of cloned bovine embryos from nuclei of cumulus and muscle cell origin. Theriogenology 2000, 53, 244.




How to Cite

Cenariu, M., Borzan, M., Dan, S., Chiorean, R. and Pall, E. (2021) “Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows”, Cluj Veterinary Journal, 26(1), pp. 7–14. doi: 10.52331/cvj.v26i1.8.