Maha EL Gebaly, Hany Abdalla, Hussein Amer, Abu Bakr Hazza


The present study was designed to investigate the influence of cumulus removal before the in vitro fertilization step and the impact of co-incubation time, sire, and additives to the fertilization medium on the efficiency of in vitro fertilization of buffalo oocytes. In vitro matured oocytes were fertilized either as cumulus-oocyte complex (COCs) or after removal of cumulus cells (denuded). Cumulus-enclosed or denuded oocytes were co-incubated with sperm cells for 6, 12 or 18 h (experiment 1), fertilized with sperm cells from one of three sires (experiment 2) or fertilized in medium supplemented with 20µg∕ml heparin, 5 mM theophylline  or a mixture 20µM penicillamine,  10µM hypotaurine and 1 µM epinephrine  (PHE) (experiment 3). In all experiments, a group of oocytes was fixed and stained to evaluate the fertilization pattern (penetration, normal and abnormal fertilization rates) and the rest were cultured up to 8 days to assess the developmental competence (cleavage and blastocyst yield). In all experiments, removal of cumulus cells before fertilization step significantly retarded the fertilization pattern and the developmental competence. Various co-incubation times did not significantly influence the fertilization pattern or the developmental competence of denuded or COCs. However, 6 h tended to decrease the abnormal fertilization (15.74±1.70 vs 28.46±6.06, P=0.069) and to improve the blastocyst/oocyte (11.70±3.41 vs 5.53±1.75) and the blastocyst/cleavage (27.14±6.19 vs 11.98±3.81, P=0.082) when compared with 18 h. Sperm cells from the three sires resulted in similar fertilization pattern and developmental competence in COCs and denuded oocytes. In COCs and denuded oocytes, PHE tended to improve the blastocyst/oocyte (7.44±2.58 vs 14.67±4.29) and blastocyst/cleavage (18.43±6.08 vs 33.13±9.27) in comparison to heparin (P˃0.05). Thus, none of the investigated factors could counteract the adverse effect of cumulus removal otherwise, the addition of PHE showed promising results but it need further investigations.

Key words: cumulus; co-incubation time; additives; fertilization pattern; developmental competence

Full Text:



(1) Gasparrini B, Tyagi S. In vivo embryo production in buffalo: current situation and future perspectives. Ital J Anim Sci 2007; 6 (Suupl 2): 74–91.

(2) Gasparrini B. In vitro embryo production in buffalo species: state of the art. Theriogenology 2002; 57 (1): 237–56.

(3) Suresh KP, Nandi S, Mondal S. Factors affecting laboratory production of buffalo embryos. Theriogenology 2009; 72 (7): 978–85.

(4) Nandi S, Raghu HM, Ravindranatha BM, Chauhan MS. Production of buffalo (Bubalus bubalis) embryos in vitro: premises and promises. Reprod Domest Anim 2002; 37 (2): 65–74.

(5) Gasparrini B, Rosa A, Attanasio L, Boccia L, Palo R, Campanile G, Zicarelli, L. 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 2008; 105 (3-4): 354–64.

(6) Totey SM, Pawshe CH, Singh GH. Effect of bull and heparin and sperm concentration on in vitro fertilization of buffalo (Bubalusbubalis) oocyte matured in vitro. Theriogeonology 1993; 39 (4): 887-98.

(7) Nandi S, Chauhan MS, Palta P. Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalusbubalis) oocytes matured and fertilized in vitro. Theriogenology 1998; 50 (8): 1251–62.

(8) Ravindranatha BM, Nandi S, Raghu HM, Reddy SM. In vitro maturation and fertilization of buffalo oocytes: Effects of storage of ovaries, IVM temperature, storage of processed sperm and fertilization media. Reprod Domest Anim 2003; 38 (1): 21–6.

(9) Hammam AM, Whisnant CS, Elias A, Zaabel SM, Hegab O, Abu-El Naga EM. Effect of media, sera and hormones on in vitro maturation and fertilization of water buffalos (Bubalus bubalis). J Anim Vet Adv 2010; 9 (1): 27–31

(10) Jamil H, Samad HA, Qureshi ZI, Rehman N, Lodphi, LA. Effect of bull and sperm preparation method on in vitro preparation of buffalo oocyte. Pak Vet J 2007; 27 (1): 29–34.

(11) Husna AU, Azam A, Qadeer S, Awan MA, Nasreen S, Shahzad Q, Fouladi-Nashta A, Khalid M, Akhter S. Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes. Reprod Domest Anim 2018; 53 (2): 377–84.

(12) Boccia L, Francesco S, Neglia G, De Blasi M, Longobardi V, Campanile G, Gasparrini B. Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis). Theriogenology 2013; 80 (3): 212–17.

(13) Kang SS, Koyama K, Huang W, Yang Y, Yanagawa Y, Takahashi Y, Nagano M. Addition of D-penicillamine, hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro. J Reprod Dev 2015; 61 (2): 99–105.

(14) Ward F, Rizos D, Boland MP, Lonergan P. Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress. Theriogenology 2003; 59 (7): 1575–84.

(15) Sattar A, Rubessa M, Di Francesco S, Longobardi V, Di Palo R, Zicarelli L, Campanile G, Gasparrini B. The influence of gamete co-incubation length on the in vitro fertility and sex ratio of bovine bulls with different penetration speed. Reprod Domest Anim 2011; 46 (6): 1090–97.

(16) Saeki K, Nagao Y, Hoshi M, Nagai M. Effects of heparin, sperm concentration and bull variation on in vitro fertilization of bovine oocytes in a protein-free medium. Theriogenology 1995; 43 (4): 751–59.

(17) Nedambale TL, Dua F, Xub J, Chaubalb SA, Dinnyesd A, Groenf W, Faberf D. Prolonging bovine sperm-oocyte incubation in modified medium 199 improves embryo development rate and the viability of vitrified blastocysts. Theriogenology 2006; 66 (8): 1951–60.

(18) Ferré LB, Bogliotti Y, Chitwood JL, Fresno C, Ortega HH, Kjelland ME, Ross OJ. Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa. Reprod Fertil Dev 2016; 29 (4): 805–14

(19) Ward F, Enright B, Rizos D, Boland M, Lonergan P. Optimization of in vitro bovine embryo production: effect of duration of maturation, length of gamete co-incubation, sperm concentration and sire. Theriogenology 2002; 57 (8): 2105–17.

(20) Coy P, Ca´novas S, Monde´jar I, Saavedra MD, Romar R, Grullo´n L, Mata´s C, Avile´s M. Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy. PNAS 2008; 105: 15809–14.

(21) Fatehi AN, Roelen BAJ, Colenbrander B, Schoevers EJ, Gadella BM, Bevers MM, Hurk R. Presence of cumulus cells during invitro fertilization protects the bovine oocyte against oxi-dative stress and improves first cleavage but does not affect further development. Zygote 2005; 13 (2): 177–85.

(22) Fatehi AN, Zeinstra EC, Bevers MM. Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate. Theriogenology 2002; 57 (4): 1347–1355.

(23) Zhang L, Jiang S, Yang PJ, Yang X, Godke RA. Cumulus cell function during bovine oocyte maturation, fertilization, and embryo development in vitro. Mol Reprod Dev 1995; 40 (3): 338–44.

(24) Hawk HW, Nel ND, Waterman RA, Wall RJ. Investigation of means to improve rates of fertilization in vitro matured/in vitro fertilized bovine oocytes. Theriogenology 1992; 38 (6): 989–98.

(25) Ortiz-Escribano N, Smits K, Piepers S, Van den Abbeel E, Woelders H, Van Soom A. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development. Theriogenology 2016; 86 (2): 635–41.

(26) Cox JF, Zabal JH, Santa Maria A. Effect of the cumulus on in vitro fertilization of bovine matured oocyte. Theriogenology 1993; 40 (6): 1259–67.

(27) Attanasio L, De Rosa A, De Blasi M, Neglia G, Zicarelli L, Campanile G, Gasparrini B. The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification. Theriogenology 2010; 74 (8): 1504–08.

(28) Chian RC, Okudo k, Niwa k. Influence of cumulus on in vitro fertilization of bovine oocytes derived from in vitro maturation. Anim Reprod Sci 1995; 38 (1-2): 37–48.

(29) Brackett BG, Oliphant G. Capacitation of Rabbit Spermatozoa in vitro. Biol Reprod 1975; 12 (2): 260-74.

(30) Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972; 30 (3): 493–7.

(31) Takahashi Y, First NL. In vitro fertilization of bovine oocytes in the presence of theophylline. Anim Reprod Sci 1993;34 (1): 1–18.

(32) Miller GF, Gliedt DW, Rakes JM, Rorie RW. Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate. Theriogenology 1994; 41 (3): 689–96.

(33) Ball GD, Leibfried ML, Lenz RW, AX RL, Bavister BD, First NL. Factors affecting successful in vitro fertilization of bovine follicular oocyte. Biol Reprod 1983; 28 (3): 717–25.

(34) Van Soom A, Tanghe S, De Pauw I, Maes D, Kruif A. Funcion of the cumulus oophorus before and during mammalian fertilization. Reprod Domest Anim 2002; 37 (3): 144–51.

(35) Kochhar HS, Kochhar KP, Basrur PK, King WA. Influence of the duration of gamete interaction on cleavage, growth rate and sex distribution of in vitro produced bovine embryos. Anim Reprod Sci 2003; 77 (1-2): 33–49.

(36) Rosenkranz CH, Holzmann A. The effect of sperm preparation on the timing of penetration in bovine in vitro fertilization. Anim Reprod Sci 1997; 46 (1-2): 47–53.

(37) Enkhmaa D, Kasai T, Hoshi K. Long-time exposure of mouse embryos to the sperm produces high levels of reactive oxygen species in culture medium and relates to poor embryo development. Reprod Domest anim 2009;44 (4): 634–37.

(38) Gianaroli L, Fiorentino A, Cristina Magli M, Ferraretti AP, Montanaro N. Prolonged sperm oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum Reprod 1996; 11 (11): 2507–11.

(39) Al Naib A, Hanrahan JP, Lonergan P, Fair S. In vitro assessment of sperm from bulls of high and low field fertility. Theriogenology 2011; 76 (1): 161–7.

(40) Selvaraju S, Ravindra JP, Ghosh J, Gupta PSP, Suresh KP. Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Anim Reprod Sci 2008;106 (3-4): 311–21.

(41) Kroetsch TG. Sire and insemination dose does affect in vitro fertilization of bovine oocytes. Theriogenology 1992; 37: 240

(42) Boatman DE, Bavister D, Cruze E. Addition of hypotaurine can reactivate immotile golden hamster spermatozoa. J Androl 1990; 11 (1): 66–72.

(43) Shaeib F, Khan SN, Ali I, Thakur M, Saed MG, Dai J, Awonuga AO, Banerjee J, Abu-Soud HM. The defensive role of cumulus cells against reactive oxygen species insult in metaphase II mouse oocytes. Reprod Sci 2016; 23 (4): 498–507.



  • There are currently no refbacks.