Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 57 issue 10 (october 2023) : 1279-1284

The Role of Ionomycin in the Improvement of in vitro Fertilization and Increasing the Rate of Bovine Embryo Development

Ramzi A. Amran1,*, Mohammed S. Aleissa2, Muath G. Al Ghadi1, Aiman A. Ammari1, Nawal M. Al-Malahi1, Ahmad R. Alhimaidi1
1Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
2Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia.
Cite article:- Amran A. Ramzi, Aleissa S. Mohammed, Ghadi Al G. Muath, Ammari A. Aiman, Al-Malahi M. Nawal, Alhimaidi R. Ahmad (2023). The Role of Ionomycin in the Improvement of in vitro Fertilization and Increasing the Rate of Bovine Embryo Development . Indian Journal of Animal Research. 57(10): 1279-1284. doi: 10.18805/IJAR.BF-1664.

Background: The utilization of in vitro fertilization (IVF) and in vitro embryo production (IVEP) has increased significantly as an effective tool of assisted reproductive technology. Sperm capacitation is a process of membrane maturation allowing the acrosome reaction to take place. Ionomycin is a calcium ionophore that induces the acrosome reaction by increasing calcium cycling across phospholipid membranes. The aim of this study was to enhance the cleavage rate and development of bovine embryos. 

Methods: Bovine ovaries were collected from the central slaughter house. Oocytes were obtained from follicles and were let to mature for 24 h. Frozen bull semen thawed in 37°C water and transferred to a discontinuous percoll gradient (Sigma GE17-0891-01). The spermatozoon pellet was separated into four groups after resuspension in bovine in vitro fertilization media (BO-IVF): control without additives; with DMSO and with ionomycin (25 and 50 nM) and kept in an incubator for three hours. The oocytes matured under commonly used media (TCM-199) for 24 h. The mature oocytes were then fertilized for 18-24h. The embryos were cultured in SOF medium until the seventh day to study cleavage rate and blastocyst rate. 

Result: The results indicated that 50 nM ionomycin supplementation considerably reduced cleavage while DMSO and 25 nM ionomycin supplementation had no effect on cleavage percentage. Moreover, when compared to the other groups, ionomycin at 25 nM significantly increased blastocyst development. In conclusion, the highest rate of blastocyst development was observed in the capacitation medium supplemented with 25 nM ionomycin, apparently due to the importance of calcium and metabolism in spermatozoa for fertilization and embryo development.

In vitro embryo production is a crucial technique in cow breeding that has seen a substantial increase in utilization. Commercial companies produce bovine embryos throughout the world (Abd El-Aziz et al., 2016; Stoecklein et al., 2021; Blaschka et al., 2021).
A certain protocol is taken during the in vitro production of embryos to mimic the in vivo conditions in various types of animals, beginning with in vitro maturation of oocytes within 20 to 24 hours following retrieval (Wrenzycki, 2018; Damayanti et al., 2020) and the first polar body extrusion, which is required for fertilization and the beginning of embryonic development (Sirard, 2016; Turhan et al., 2021). Therefore, the oocyte’s maturation is essential for fertilization and preimplantation development (Barakat et al., 2018).
The second stage of in vitro production, known as in vitro fertilization, is characterized by the creation of male and female pronuclei and the extrusion of the second polar body (Parrish, 2014). For the in vitro fertilization process to begin, capacitated acrosome reaction of spermatozoa must be found beside mature oocytes, such sperm has the capacity to break through the zona pellucida of the oocyte and merge with the ooplasm during the fertilization process (Stival et al., 2016).
Capacitation is believed to be primarily brought on by changes in cholesterol concentration, lipid composition, calcium permeability, fluidity and surface characteristics of membranes (Davis, 1981). Most of these alterations are connected to modifications in spermatozoa’s plasma membrane, supporting the theory that capacitation is a process of membrane maturation (Sukardi et al., 2001).
The in vitro capacitation of the spermatozoa is achieved by washing the sperm in a media containing equal levels of electrolytes, metabolic energy sources and compounds that affect the sperm’s vitality and motility as well as the induction and pace of the acrosome reaction process. Some of the substances that contribute to the motility and vitality of sperm include β-amino acids (e.g. hypotaurine and taurine), catecholamines (e.g. adrenaline), as well heparin and pentoxifylline, while caffeine stimulates hyperactivation and the acrosome reaction (Leibfried and Bavister, 1982; Parrish et al., 1988; Taketo et al., 1991; Pereira et al., 2000; Barakat et al., 2015). The calcium ionophore has been the most widely used agent for triggering the acrosome reaction in vitro. Triana et al., (1980) were the first to discover that calcium ionophore A23187 causes changes in bovine sperm depending on the calcium ion.
Ionomycin is a calcium ionophore that increases Ca2+ cycling across phospholipid membranes to induce the acrosome reaction. (Ball et al., 1983). With the help of the calcium ionophore A23187, spermatozoa from a number of species, including horses, can be made to go through the acrosome reaction. (Varner et al., 1987; Zhang et al., 1991; Magistrini and Palmer, 1990; Farlin et al., 1992).
The in vitro culture of bovine embryos, which initiates with zygote culture and typically takes around 7 to 8 days, is the last stage of in vitro production (IVP). The embryo advances through its first cleavage division, the embryonic genome is activated and the morula and blastocyst are formed at this stage (Wrenzycki, 2018; Ramos-Deus et al., 2020; Nogueira et al., 2021). This study aims to improve the cleavage rate and development of bovine embryos in order to increase the efficiency of in vitro fertilization by investigating the effects of applying DMSO and (25, 50 nM) ionomycin to the sperm capacitation medium. 
Chemicals and reagents
All chemicals and media used in this study were from Sigma Corporation (St. Louis, MO, USA) unless otherwise noted.
Experimental design
All of the experiments were conducted in the Zoology Department of the College of Science at King Saud University from January to June. We had four different groups to be examined; each group had seven replicates. All groups had the same final volume and final sperm cell concentration (8×106 sperm per ml) and the only difference between them was the final composition of the capacitation medium. The standard capacitation medium consisted of a Bracket-Oliphant medium (112 mM NaCl, 4 mM KCl, 2.25 mM CaCl2.2H2O, 0.5 mM MgCl2.6H2O, 37 mM NaHCO3 and 0.83 mM NaH2PO4.2H2O, 1.25 mM Sodium Pyruvate and 0.05 mM Phenol Red) supplemented with 10 μg/ml Heparin sodium salt, 3 mg/ml BSA and 0.5 μg/ml gentamicin sulfate. The first group was the control group; the sperm were capacitated using a standard capacitation medium without any additives. The second group was the DMSO group; the sperm were capacitated using a standard capacitation medium supplemented with DMSO solution. The third group was the low-treatment group; the sperm were capacitated using a standard capacitation medium supplemented with ionomycin at 25 nM final concentration. The sperm were capacitated using a standard capacitation medium supplemented with ionomycin at a final concentration of 50 nM in the last group, development assessment was used at the end of seven days of culturing. 
Ionomycin solution
A vial of ionomycin calcium salt (Sigma I0634) containing 1 mg was dissolved using 134 μl DMSO solution to make a 10 mM stock solution and then stored at -20°C until use.
Frozen semen
Frozen semen of Holstein Friesian cattle was obtained from the Center of Asset Conservation and Genetic Improvement in Al-Kharj City, which is affiliated with the Saudi Ministry of Environment, Water and Agriculture.
Oocyte collection and in vitro maturation (IVM)
Ovaries were collected from the central slaughterhouse in Riyadh (Saudi Arabia) and transported to the laboratory in 0.9% sodium chloride (NaCl) within 2 hours. The follicles were separated into three groups according to their diameter: small (S: 3 mm), medium (M: 3-6 mm) and large (L: >6 - <10 mm). Through the use of the aspiration method, the oocytes were extracted from each of the three types of follicles. using a sterile syringe on 0.5 ml of TCM199 handling medium (Hank’s salts) supplemented with 50 μg/ml gentamicin sulfate, 0.5 mM sodium pyruvate and 10% Fetal Bovine Serum (FBS). Oocytes with three or more layers of intact cumulus cells and uniform cytoplasm were collected using a mouth pipette under a stereomicroscope. The cumulus-oocyte complexes were then washed three times in 100 μl of TCM-199 maturation medium (Earle’s salts) (Sigma M4530; Merck KGaA, Darmstadt, Germany) with 0.5 mM Na-Pyruvate, 0.023 IU/ml luteinizing hormone (LH) (Sigma L5269), 0.02 IU/ml follicle-stimulating hormone (FSH) (Sigma F8174), 1 μg/ml Estradiol (E2) (Sigma E2758), 50 μg/ml gentamicin sulfate and 10% FBS. Then, groups of ten cumulus-oocyte complexes were transferred to 60 mm Petri dishes in 50 μl maturation drops which were covered with mineral oil and incubated for 24 hours in a humidified atmosphere of 5% CO2 at 39°C. (Ammari et al., 2022a).
In vitro fertilization (IVF) of oocytes
The sperm capacitation medium used in this trial consisted of a Bracket-Oliphant medium (112 mM sodium chloride, 4 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.5 mM magnesium chloride hexahydrate, 37 mM sodium bicarbonate, 0.83 mM sodium dihydrogen phosphate dehydrate and 0.05 mM phenol red) supplemented with 10 μg/ml heparin sodium salt, 3 mg/ml BSA, 1.25 mM sodium pyruvate and 0.5 μg/ml gentamicin sulfate and was subsequently incubated at least two hours before being used. 
Frozen bull semen thawed in 37°C water and transferred to a discontinuous percoll gradient (Sigma GE17-0891-01) [45 and 90% (vol/vol) Percoll] (Maziero et al., 2020; Nogueira et al., 2021). The semen was placed in a 15 mL tube containing 5 ml of capacitation medium. The pooled semen was washed twice to get rid of ionomycin residues present in the capacitation medium before fertilization with capacitation medium by centrifugation at 1800 rpm for 5 min. The resultant pellet was diluted to give a concentration of 8×106 sperm per ml and then split into four different experimental treatments: The first treatment was the control, the sperm were capacitated using a capacitation medium without any additives; the second was the DMSO group, the sperm were capacitated using a capacitation medium supplemented with DMSO solution; the third was the low-treated group, the sperm were capacitated using capacitation medium supplemented with ionomycin as 25 nM final ionomycin concentration; the last group was the high-treated group, the sperm were capacitated using capacitation medium supplemented with ionomycin as 50 nM final ionomycin concentration. Before transfer to fertilization drops, the oocytes were washed four times in the previous capacitation medium (BO-IVF). Insemination was carried out by adding 8×106 into the fertilization medium that included 10 oocytes per 50 ìl fertilization drop in an atmosphere of 5% CO2 and humidified air at 39°C for 24 h.
In vitro culture (IVC) of embryos
A glass Pasteur pipette (Thermo Fisher, 15202699) was used to repeatedly pipette zygotes in a hyaluronidase enzyme solution at 24 hours after fertilization to completely eliminate cumulus cells. The zygotes were subsequently washed in an in vitro culture medium (IVC-SOF) (Caisson IVL05) enriched with 1 mM L-glutamine, 0.34 mM sodium pyruvate, 1% (v/v) 50X MEM-essential amino acids (Sigma B6766), 1% (v/v) 100X MEM nonessential amino acids (Sigma M7145), 25 μg/ml gentamicin, 3 mg/mL BSA (Sigma A6003), 1 μg/ml EDTA and 1.5 mM glucose. They were then assigned to 50 μl drop of SOF medium (25 embryos/drop) in a 35 mm Petri dish (BD Falcon, 351008) and the drops were covered with mineral oil (Sigma M 5310). Embryos were maintained in incubation for 7-8 days at 39°C in a high-humidity environment with 5% CO2, 5% O2 and 90% N(Ammari et al., 2022b). The cleavage rate and blastocyst rate were calculated using the following equations at the end of the seventh day of embryo culture:

Statistical analysis
The Mine Tab INSTAT program was used to analyze all results while Chi-square test was carried out to analyze the cleavage and blastocyst rates (Ammari et al., 2022b).
Embryo cleavage rate
The impact of incorporating DMSO and ionomycin into the spermatozoa’s in vitro fertilization medium on the embryo cleavage rate following oocyte fertilization was the focus of the current investigation.
The embryo cleavage rate was significantly (P<0.05) higher in control (67.1%), DMSO (66.3%) and ionomycin 25 nM (69.5%) groups compared to ionomycin 50 nM (56.8%), as shown in Table 2.
The average number of embryos during the stage of development
The number of embryos at each cleavage stage was determined at the end of the seventh day of embryo culture. Degenerated oocytes in the 50 nM ionomycin group (17.8%), were significantly higher (P<0.05) than in control (13.3%), DMSO (13.5%) and ionomycin 25 nM (13.6%) groups. Additionally, the experimental 25 nM ionomycin group had a significantly (P<0.05) lower percentage of non-cleavage oocytes (one cell) than the 50 nM ionomycin group (16.9% vs. 25.4%). Furthermore, the percentage of fragmented embryos in both the control group (18.2%) and the DMSO group (18.1%) was significantly higher (P<0.05), than both 25 nM ionomycin (9.8%) and 50 nM ionomycin (11.7%) groups. Most of the embryos in the 50 nM ionomycin group (14.0%) were in the 2-cell stage but the blastocysts were on the same level, which was significantly higher (P<0.05) compared to the control (3.7%), DMSO (4.0%) and 25 nM ionomycin (2.1%) groups. As for the 4-cell stage, it was significantly (P<0.05) lower in the experimental 25 nM ionomycin group (2.1%) compared to the experimental 50 nM ionomycin group (6.0%). In addition, the blastocyst was significantly (P<0.05) higher in the 25 nM ionomycin group (71.5%), compared to the control (50.6%), DMSO (51.4%) and 50 nM ionomycin (36.9%) groups. As demonstrated in Table 1,  there was no statistically significant difference between any of the groups regarding the percentage of embryos at the 8 and 16-cell stages, as well as at the morula stage.

Table 1: Effect of DMSO and ionomycin addition to the IVF medium on embryo development of bovine embryos (seven replicates).

Embryo arrival rate to the blastocyst
The current study examined the impact of the addition of DMSO and ionomycin to IVF medium on the cleavage rate and blastocyst rate on the seventh day from embryo culture.
In this experiment, the blastocyst rate was significantly (P<0.05) higher in the 25 nM ionomycin group (71.5%), compared to the control (50.6%), DMSO (51.4%) and 50 nM ionomycin (36.9%) groups. In addition, the expanded blastocyst rate was significantly (P<0.05) higher in the 25 nM ionomycin group (34.5%), compared to control (22.3%), DMSO (22.7%) and 50 nM ionomycin (14.0%) groups. In addition, the rate of hatching blastocyst was significantly (P<0.05) higher in the 25 nM ionomycin group (20.9%), compared to control (13.3%), DMSO (13.0%) and 50 nM ionomycin (11.2%) groups. In addition, as shown in Table 2 and Fig 1, the control (15.0%), DMSO (15.7%) and 25 nM ionomycin (16.2%) groups all had significantly (P<0.05) greater rates of hatched blastocysts than the 50 nM ionomycin group (11.7%).

Table 2: Impact of DMSO and ionomycin addition to the IVF medium on cleavage rate and blastocyst rate of bovine oocytes (seven replicates).


Fig 1: The different stages of embryo blastocyst development in the experiment. A: Early blastocyst, B: Expanded blastocyst, C: Before hatching blastocyst, D: Hatching blastocyst, E: Hatched blastocyst.

The capacitation process requires the removal of inhibitors from the sperm head, an increase in intracellular pH and a decrease in the cholesterol/phospholipid ratio of the membranes, allowing them to be more permeable to calcium ions (Langlais and Roberts, 1985). Our study objective is to improve the efficiency of in vitro fertilization by increasing the cleavage rate and development of bovine embryos by studying the effect of adding; DMSO, 25 nM and 50 nM ionomycin to the sperm capacitation medium. The results of this study contradict those of Hong et al., (2020), who utilized 5 mM ionomycin to the in vitro maturation medium and goat ova embryo development and discovered no significant differences between the experimental group and the control group. This might be a result of the differing ionomycin concentrations they employed in their investigation or because they added the ionomycin to the maturation medium rather than the capacitation medium. Although it was discovered that ionomycin activated mouse oocyte and embryo development (Heytens et al., 2008). Additionally, according to Navarrete (2017), the calcium and metabolism in sperm are thought to play a key role in fertilization and embryo development. While our result agrees with Slonina et al., (1995) who used the Ca2+ A23187 for stallion sperm capacitation and found that the sperm exposed to A23187 had a significantly higher occurrence of acrosome reactions than sperm capacitated without Ca2+ A23187 (p<0.01).
Increased Ca2+ influx brought on by the acrosome reaction (AR) leads to higher Ca2+ concentrations inside cells. This increase in Ca2+ enables dephosphorylation/activation of p-gelsolin resulting in the dispersion of F-actin, allowing the plasma membrane PM and outer acrosome membrane OAM to come into close proximity and undergo fusion (Breitbart and Shabtay, 2018).
The sperm capacitation process requires protein kinase A activation (PKA), changes in membrane potential and an increase in intracellular calcium (Ca2+). Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. According to Nakagawa et al., (2001) and Lu et al., (2006), the greater concentration (50 nM) of ionomycin may increase the likelihood of danger in sperm in vitro capacitation, IVF and/or embryo development, necessitating further research. Despite the lack of conclusive proof of calcium ionophore toxicity on gametes and embryos, given the widespread application of the treatment in human oocytes, there could still be some concerns. However, according to Spandorfer et al., (2001), the main cause of IVF failure seems to be the sperm’s incapability to penetrate the oocyte membrane, which demands for further research.
Ionomycin, at a dose of 25 nM in capacitation medium, performed better when compared to the other implemented groups, resulting in a faster rate of early embryo cleavage development. Moreover, the medium treated with 25 nM ionomycin showed the highest rate of blastocyst formation. In contrast to DMSO and control, which had no effect on the percentage of cleavage at the end of the experiment, 50 nM.
The authors sincerely acknowledge the Researcher Support Project (RSP-2023/R232) for funding this work at King Saud University, Riyadh, Saudi Arabia. This research was supported by the Deanship of Scientific Research, Imam Mohammad Ibn Saud Islamic University, Saudi Arabia, Grant No. (18-11-12-009).

  1. Abd El-Aziz, A.H., Mahrous, U.E., Kamel, S.Z. and Sabek, A.A. (2016). Factors influencing in vitro production of bovine embryos: A review. Asian J. Anim. Vet. Adv. 11(12): 737- 756. 

  2. Ammari, A.A., ALghadi, M.G., ALhimaidi, A.R. and Amran, R.A. (2022b). The role of passage numbers of donor cells in the development of Arabian oryx-cow interspecific somatic cell nuclear transfer embryos. Open Chem. 20(1): 342-349. 

  3. Ammari, A.A., Amran, R.A., Al Ghadi, M.G. and Alhimaidi, A.R. (2022a). Morphometric assessment of the bovine ovary for in vitro matured oocyte quality to determine developmental competence. Indian J. Anim. Res. 56(5): 557-562. 

  4. Ball, G.D., Leibfried, M.L., Lenz, R.W., Ax, R.L., Bavister, B.D. and First, N.L. (1983). Factors affecting successful in vitro fertilisation of bovine follicular oocytes. Biol Reprod. 28(3): 717-725. 

  5. Barakat, I.A.H., Alajmi, R.A., Zoheir, K.M.A., Salem, L.M. and Al- Hemidiy, A.R. (2018). Gene expression and maturation evaluation of sheep oocytes cultured in medium supplemented with natural antioxidant source. South African J. Anim. Sci. 48(2): 261-270. 

  6. Barakat, I.A.H., Danfour, M.A., Galewan F.A.M. and Dkhil, M.A. (2015). Effect of various concentrations of caffeine, pentoxifylline  and kallikrein on hyperactivation of frozen bovine semen. Biomed Res. Int. 2015: 1-7. 

  7. Blaschka, C., Diers, S., Aravina, M., Geisler, S., Schuler, G. and Tetens, J. (2021). Evaluation of a small volume oil free in vitro production system for bovine embryos. Vet. Med. Sci. 7(3): 868-875. 

  8. Breitbart, H. and Shabtay, O. (2018). CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization. Sperm Acrosome Reaction. 415(1): 64-74. doi: 10.1016/j.ydbio.2016.05.008.

  9. Damayanti, E., Sonjaya, H., Hasbi, H. and Baco, S. (2020). Strategies  to increase growth early embryo stages of bovine in achieving blastocysts in vitro: A review. IOP Conf. Ser. Earth Environ. Sci. 492: 012082. DOI: 10.1088/1755-1315/492/1/012082.

  10. Davis, B.K. (1981). Timing of fertilization in mammals: Sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval. Proc. Natl. Acad. Sci. 78(12): 7560-7564. 

  11. Farlin, M.E., Jasko, D.J., Graham, J.K. and Squires, E.L. (1992). Effects of calcium ionophore A23187 on stallion spermatozoa. Theriogenology. 37(1): 209.

  12. Heytens, E., Soleimani, R., Lierman, S., De Meester, S., Gerris, J., Dhont, M., Van der Elst, J. and De Sutter, P. (2008). Effect of ionomycin on oocyte activation and embryo development in mouse. Reproductive Biomedicine Online. 17(6): 764-771. 

  13. Hong, S., Vettical, B.S. and Wani, N.A. (2020). Development of prepubertal goat oocytes after their in vitro maturation and chemical activation. Zygote. 28(6): 447-452. 

  14. Langlais, J. and Roberts, K.D. (1985). A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res. 12(2): 183-224. 

  15. Leibfried, M.L. and Bavister, B.D. (1982). Effects of epinephrine and hypotaurine on in vitro fertilization in the golden hamster.  Reproduction. 66(1): 87-93. 

  16. Lu, Q., Zhao, Y., Gao, X., Li, Y., Ma, S., Mullen, S., Critser, J.K. and Chen, Z.J. (2006). Combination of calcium ionophore A23187 with puromycin salvages human unfertilized oocytes after ICSI. Eur. J. Obstet. Gynecol. Reprod Biol. 126(1): 72-76. 

  17. Magistrini, M. and Palmer, E. (1990). Motility, triple stain and electron microscopic analysis of spermatozoa treated with ionophore  A23187 for in vitro fertilization. J. Reprod. Fertil. Suppl. 44: 661-663. 

  18. Maziero, R.R.D., Guaitolini, C.R.D.F., Paschoal, D.M., Crespilho, A.M., Monteiro, B.A., Lima, J.S. De, Sestari, D.A.O. and Landim-Alvarenga, F.D.C. (2020). Treatment with roscovitine  and butyrolactone I prior to in vitro maturation alters blastocyst production. Zygote. 28(1): 24-31. 

  19. Nakagawa, K., Yamano, S., Moride, N., Yamashita, M., Yoshizawa, M. and Aono, T. (2001). Effect of activation with Ca ionophore  A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection. Fertil Steril. 76(1): 148-152. 

  20. Nogueira, B.G.R., de Souza, L.F.A., Puelker, R.Z., Giometti, I.C., Firetti, S.M.G., Dias, T.S.D.S.B. and Castilho, C. (2021). Factors affecting the in vitro production of bovine embryos in a commercial program. Res. Soc. Dev. 10(2): e16110212264. DOI: 

  21. Parrish, J.J. (2014). Bovine in vitro fertilization: In vitro oocyte maturation and sperm capacitation with heparin. Theriogenology. 81(1): 67-73. 

  22. Parrish, J.J., Susko-Parrish, J., Winer, M.A. and First, N.L. (1988). Capacitation of bovine sperm by heparin. Biol. Reprod. 38(5): 1171-1180. 

  23. Pereira, R.J.T.A., Tuli, R.K., Wallenhorst, S. and Holtz, W. (2000). The effect of heparin, caffeine and calcium ionophore A 23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa. Theriogenology. 54(2): 185-192. 

  24. Ramos-Deus, P., Santos Nascimento, P., Vieira, J.I.T., Chaves, M.S., Albuquerque, K.A., Ferreira-Silva, J.C., Grázia, J.G.V., Santos Filho, A.S., Batista, A.M., Teixeira, V.W. and Oliveira, M.A.L. (2020). Application of platelet-rich plasma in the in vitro production of bovine embryos. Trop. Anim. Health Prod. 52: 2931-2936. 

  25. Sirard, M.A. (2016). Somatic environment and germinal differentiation  in antral follicle: The effect of FSH withdrawal and basal LH on oocyte competence acquisition in cattle. Theriogenology. 86(1): 54-61. 

  26. Slonina, D., Okólski, A. and Barañska, K. (1995). Effect of capacitation method and calcium lonophore A23187 on stallion spermatozoa acrosome changes. Biol. Reprod. 52(monograph_series1): 719-727. 

  27. Spandorfer, S.D., Navarro, J., Levy, D., Black, A.R., LIU, H.C., Veeck, L., Witkin, S.S. and Rosenwaks, Z. (2001). Autologous endometrial coculture in patients with in vitro fertilization (IVF) failure: Correlations of outcome with leukemia inhibiting factor (LIF) production 1. Am. J. Reprod. Immunol. 46(6): 375-380. 

  28. Stival, C., Molina, L.D.C.P., Paudel, B., Buffone, M.G., Visconti, P.E. and Krapf, D. (2016). Sperm capacitation and acrosome reaction in mammalian sperm. Sperm Acrosome Biogenesis  and Function During Fertilization. 93-106.

  29. Stoecklein, K.S., Ortega, M.S., Spate, L.D., Murphy, C.N. and Prather, R.S. (2021). Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF and IGF1. PLoS One. 16(2): e0243727.

  30. Sukardi, S., Elliott, R.M., Withers, J.O., Fontaine, U., Millar, J.D., Curry, M.R. and Watson, P.F. (2001). Calcium-binding proteins from the outer acrosomal membrane of ram spermatozoa: Potential candidates for involvement in the acrosome reaction. Reproduction. 122(6): 939-946. 

  31. Taketo, M., Schroeder, A.C., Mobraaten, L.E., Gunning, K.B., Hanten, G., Fox, R.R., Roderick, T.H., Stewart, C.L., Lilly, F., Hansen, C.T. and Overbeek, P.A. (1991). FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. 88(6): 2065-2069. 

  32. Triana, L.R., Babcock, D.F., Lorton, S.P., First, N.L. and Lardy, H.A. (1980). Release of acrosomal hyaluronidase follows increased membrane permeability to calcium in the presumptive capacitation sequence for spermatozoa of the bovine and other mammalian species. Biol. Reprod. 23(1): 47-59. 

  33. Turhan, A., Pereira, M.T., Schuler, G., Bleul, U. and Kowalewski, M.P. (2021). Hypoxia-inducible factor (HIF1alpha) inhibition  modulates cumulus cell function and affects bovine oocyte maturation in vitro. Biol. Reprod. 104(2): 479-491. 

  34. Varner, D.D., Ward, C.R., Storey, B.T. and Kenney, R.M. (1987). Induction and characterization of acrosome reaction in equine spermatozoa. Am. J. Vet. Res. 48(9): 1383-1389.  

  35. Wrenzycki, C. (2018). Gene expression analysis and in vitro production  procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects. Reprod. Domest Anim. 53: 14-19. 

  36. Zhang, J.J., Muzs, L.Z. and Boyle, M.S. (1991). Variations in structural  and functional changes of stallion spermatozoa in response to calcium ionophore A23187. J. Reprod. Fertil. Suppl. 44: 199-205.

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