Effect of sperm motility enhancers on in vitro fertilization and embryo development of buffalo oocytes

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1 Effect of sperm motility enhancers on in vitro fertilization and embryo development of buffalo oocytes Eman Mahmoud Abu-El Naga 1, Hossam El-Sheikh Ali 2, Magdy Ramdan Badr 3, Ashraf Mohamed El Desouky 2, and Samy Moawad Zaabel 2 * 1 Theriogenology Department, Faculty of Veterinary Medicine, Aswan University, Egypt 2 Theriogenology Department, Faculty of Veterinary Medicine, Mansoura University, Egypt 3 Artificial Insemination and Embryo Transfer Department, Animal Reproduction Research Institute, Al Haram, Giza, Egypt Abstract The present study was carried out to investigate the effect of adding various concentrations of penicillaminen, hypotaurine and epinephrine (PHE) to the in vitro fertilization (IVF) media on the fertilization rate and embryonic development up to the blastocyst stage of buffalo oocytes. In vitro matured oocytes were fertilized in different PHE concentration (10, 40 and 80 µm/ml) in the presence of 5.0 mm caffeine. After IVF, the fertilization rate, the cleavage rate and the development rates up to the blastocyst stage were assessed. The present results revealed that supplementation of the fertilization media with 40 mg/ml PHE to the fertilization media increased significantly (P<0.05) the fertilization, cleavage rates and the development rates up to the morula and blastocyst stage (73.15±2.19, 59.90±6.19, 48.60±24.88 and 33.13±7.04%, respectively) compared to those of the control groups (38.83±4.82, 37.13±2.43, 7.75± ±1.93 and 2.50±1.04%, respectively). In conclusion, the current results demonstrated that addition of 40 µm/ml PHE to fertilization medium had a positive effect on buffalo oocytes, fertilization and supported embryonic development up to the blastocyst stage. Keywords: In vitro fertilization, Oocye, Embryo development, Buffalo, PHE 1. Introduction Buffalo is the main dairy animal in Egypt, as it the major source of the milk and the beef available in the country. Assisted reproductive technologies such as artificial insemination (AI), multiple ovulation and embryo transfer (MOET) and in vitro fertilization (IVF) have been introduced to improve reproductive efficiency in this species (1). However, in vitro embryo production efficiency in buffalo is still very low and variable compared with cattle (2) due to various factors including poor oocyte recovery rate (3), lack of standardization of cultural conditions (4) and low IVF performance of buffalo bull spermatozoa (5,6). In vitro fertilization results are also affected by individual bulls (7), sperm * Corresponding address: Dr. Samy Moawad Zaabel, Theriogenology Department, Faculty of Veterinary Medicine, Mansoura University, Egypt, jesintyj@yahoo.co.in preparation methods (8) and sperm stimulating motility factors (9). Fertilization is a complex process, which results in the union of two gametes, the restoration of the somatic chromosome number and the start of the development of a new individual. Successful buffalo IVF requires appropriate preparation of sperm and oocyte, as well as culture conditions that are favourable to the metabolic activity of the male and female gametes (10). It is clearly important to have highly motile bull sperm available for IVF. This may be achieved by applying various procedures for isolating motile samples. There are also a number of chemical agents which may be employed to stimulate motility and AR of bull sperm and to maintain motility. A combination of penicillamine, hypotaurine, and epinephrine (PHE) can be added to the fertilization medium to improve cattle sperm motility (11). However, little 6

2 information is available on the effect of PHE on the buffalo sperm motility and its in vitro fertilizing potentials. Therefore, the purpose of this study was to investigate the effect of sperm motility enhancers (PHE) on in vitro buffalo oocyte fertilization rate and subsequent in vitro embryonic development. 2. Materials and Methods 2.1. Oocyte collection Ovaries from sexually mature buffaloes were collected within 30 minutes after slaughter from a local abattoir and transported within 2 hours to the laboratory in a vacuum flask containing sterilized phosphate buffered saline (PBS, ph 7.35) supplemented with 100 iu penicillin G and 100 ug/ml of streptomycin at the 30 C (3). Oocytes were aspirated from medium follicles (2-8 mm in diameter) using an 18-gauge needle attached to a 10 ml disposable syringe. Only oocytes having a dense cumulus cell mass and homogeneous, unfragmated cytoplasm and intact zona were selected for further processing (12) In vitro maturation of oocytes The selected oocytes were washed 3 times with Dulbecco s phosphate-buffered saline. Maturation was performed using 10 to 15 COCs in 10 x 35 mm Petri dishes with 100 μl drops of modified Synthetic Oviduct Fluid (SOF) media containing Earle's salts (GibcoTM, Ref , Invitrogen Corporation,USA) supplemented with mg/ml sodium pyruvate, 0.01 IU r-hfsh/ml, 0.05 mg/ml of slh (Lutrophin-V, Bioniche Animal Health, Ontario, Canada) and 10% of fetal calf serum (FCS). The selected oocytes were washed twice in maturation medium and were incubated in 200 μl drops of TCM- 199 covered with sterile mineral oil (Sigma) and placed in a CO2 incubator at 39 C and 5% CO2 in air, and saturated humidity for 24 hours. After 24 hours, oocytes were examined under stereomicroscope and those having well expanded cumulus cells with extrusion of the polar body were considered matured (13) Sperm preparation and in vitro fertilization (IVF) Several 75μl fertilization drops varying according to absence (control) or presence of PHE at different concentrations (10, 40, and 80 μl/ml) are deposited in sterile disposable petri dish. These drops are covered with mineral oil and incubated at 38.5 C under 5% of CO2 in air. Matured COCs were washed three times in sperm-talp medium then added to the fertilization drops, 10 COCs in each drop. Three straws of frozen semen are thawed for 30 seconds in 37C water bath, evacuated in small test tube. The freezing media was washed out two times by centrifugal sedimentation (500 Xg for 5 minutes) and resuspension twice in 1ml sperm-talp medium. Sperm cell concentration is adjusted after checking motility to 10 7 motile sperm. 20 μl of semen is coincubated with each fertilization drop to achieve motile sperm concentration of 2 x 106/ml IVF media. Gametes were co-incubated at 38.5 C under 5% of CO2 and in air for hr. At the end of gamete coincubation, the presumptive zygotes were either fixed and stained or further cultured (3,14) In vitro culture (IVC) Presumptive zygotes were denuded from cumulus cells and the extra spermatozoa by gentle pipetting, and washed three times in modified Synthetic Oviduct Fluid (SOF) media. Immediately, 20 to 25 zygotes were randomly distributed in 100 μl drops of modified Synthetic Oviduct Fluid (SOF) medium according to (15), with 5% FCS, 20 μl/ml of essential amino acids and 10 μl/ml of non-essential amino acids, under mineral oil in 10 x 35 mm Petri dishes for 7- days at 38.5 C in an atmosphere of 5% CO2 in air with maximum humidity. The proportional of cleaved oocytes was recorded 48 hours after fertilization and the proportional of oocytes developed to the morula and blastocyst stages were recorded at 5-7 days post-fertilization according to (3) as shown in figure.1. Figure 1. Assessment of buffalo embryos development. a; two-cell stage, b; four-cell stage, c; Morula stage and d; Blastocyst stage Experimental design The effect of different concentrations of PHE, on in 7

3 vitro fertilization rate and subsequent embryo development of buffalo oocytes were examined. PHE were added to fertilization medium at different concentrations, (10, 40, 80 µm/ml, respectively) and the fertilization medium without PHE supplement served as control Statistical analysis All data were analysed by using Graphpad prism Version 6 and were compared by ANOVA followed by the least significant difference (LSD). 3. Results The effect of replenishing of the fertilization medium with different concentrations of PHE improved the in vitro fertilization rate compared with the control in a dose-dependent trend. Addition of 40 µm/ml PHE to the fertilization medium improved (P<0.05) significantly the in vitro fertilization rate (73.15± 2.19 %) as compared with the control (38.83± 4.82 %; Table.1). Table 1. Effect of different concentrations of PHE on the in vitro fertilization rate of buffalo oocytes. Treatment No. of oocytes fertilization rate Control ±4.82 b PHE 10 µm/ml ±11.78 a PHE 40 µm/ml ±2.19 a PHE 80 µm/ml ±2.83 ab P: penicillamin, H: hypotaurine, E: Epinephrine Results presented in table 2 and 3 revealed that, supplementation of the fertilization medium with different concentrations of PHE improved the cleavage rate, morula and blastocyst stage development compared with the control in a dosedependent trend. Addition of 40 µm/ml PHE to the fertilization medium increased (P<0.05) significantly the in vitro cleavage rate, morula and blastocyst development as compared with the control. Table 2. Effect of different concentrations of PHE on the number of oocytes and cleavage rate of buffalo oocytes. Treatment No. of Cleavage rate oocytes Control ±2.43 b PHE 10 µm/ml ±7.78 ab PHE 40 µm/ml ±6.19 a PHE 80 µm/ml ±8.62 ab P: penicillamine, H: hypotaurine, E: Epinephrine Table 3. Effect of different concentrations of PHE on the in vitro embryo developmental rate of buffalo oocytes. Treatment Morula Blastocyst Control 7.75±1.93 b 2.50±1.04 b PHE 10 µm/ml 28.65±5.25 ab 17.96±9.38 ab PHE 40 µm/ml 48.60±24.88 a 33.13±7.04 a PHE 80 µm/ml 27.05±8.37 ab 23.64±10.88 ab P: penicillamine, H: hypotaurine, E: Epinephrine 4. Discussion The results in the present study, revealed that fertilization media supplemented with (PHE) causes a significant improvement in the in vitro fertilization and embryos development rates. These findings were similar to results obtained with (11, 16) who found that the addition of PHE to the fertilization media increased the proportion of fertilized oocytes and that developed into the morula and blastocyst stage. Fertilization media supplemented with PHE maintained excellent sperm motility (16). PHE may have served as antioxidants in their fertilization culture system, which used a gas atmosphere of 5 % carbon dioxide in air; an adverse effect of atmospheric oxygen levels on early embryonic development has been attributed to that formation of oxygen free radicals, which accelerate the processes of lipid peroxidation and cells- damaging enzyme activation (11). It is known that two of PHE components, hypotaurine and adrenaline, are able to limit superoxide formation, which may inhibit lipid peroxidation within sperm cell. hypotaurine is a precursors of taurine (17). Taurine (2-aminoethane sulfonic acid) is the main end product of cysteine metabolism (18, 19). It is worth noting that, hypotaurine is an antioxidant in human and bull semen (20). It is believed that taurine in the mammalian oviducts is necessary for sperm capacitation and fertilization and important for protection of spermatozoa against ROS (21, 21). The addition of taurine to the IVF medium increased frozen-thawed hamster sperm motility and penetration to oocytes in vitro (23). Taurine increase the catalase activity, an antioxidant enzyme, of frozen-thawed bovine spermatozoa when added to the medium (22). Moreover, taurine, hypotaurine, epinephrine increased the forward motility and 8

4 inhibited the rate of lipid peroxidation in the spermatozoa, as measured by rate of formation of malondialdehyde (17). Hypotaurine and epinephrine reduces the amount of superoxide production. Since superoxide seems to be the major inducer of lipid peroxidation in spermatozoa, the protective effect of hypotaurine, may be ascribed to scavenging of intracellular superoxide (17). Furthermore, penicillamine can increase the percentage of sperm that undergo the acrosome reaction (AR) when used in presence of adrenaline (16). Penicillamine counteract the action of cytotoxic aldehydes, suppress the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement as a means of ensuring the long-term survival of spermatozoa in vitro (24). The author further indicated that, equine spermatozoa could be preserved for 8 days at ambient temperature when the culture medium was supplemented with penicillamine. 5. Conclusion In conclusion, the current results inferred that PHE has a positive action on buffalo oocytes fertilization and subsequent embryo development in a dose dependent trend. The addition of 40 µm/ml PHE to the fertilization medium containing may stimulate buffalo embryonic development up to the blastocyst stage. Aknowledgment The authors would like to thank all members in Theriogenology Department, Faculty of Veterinary Medicine, Mansoura University. References: 1. S.Nandi, H.M. Raghu,B.M. Ravindranatha and M.S. Chauhan, Production of buffalo (Bubalus bubalis) embryos in vitro: Premises and promises. Reprod. Dom. Anim, 37(2002) K.H.Lu, I. Gordon, M. Gallagher and H.McGovern, Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet. Rec., 121(1987) S.M.Totey,G. Singh,M. Taneja,C.H.Pawshe and G.P.Talwar, In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis)(1992). 4. M.L.Madan,S.K.Das and P. Palta, Application of reproductive technology to(1996). 5. Z.Barandi, I.Solti, S. Cseh, Z.S. Varga, Z. Machaty and G.Vajta, Comparison of in vitro fertilizing ability of sperm from endangered Hungarian Grey bulls. Anim. Reprod. Sci., 31(1993) M.S.Chauhan,S.K. Singla, P.Palta, R.S. Manik and M.L.Madan, In vitro maturation and fertilization and subsequent development of buffalo (Bubalus bubalis) embryos: Effects of oocytes quality and type of serum. Reprod. Fertil. Develop., 10 (1998) A.K.Misra,M.M. Rao, R.Kasiraj,N.S.R. Reddy and H.C.Pant, Bull specific effect on fertilization rate and viable embryo recovery in the superovulated buffalo (Bubalus bubalis). Theriogenology, 52(1999) J.J.Parrish,A. Krogenaes and J.L.Susko-Parrish, Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology, 44(1995) Y.Tsuzuki,H. Toyama1, H. Nabenishi1, T. Morita and K.Ashizawa1, The Effect of Various Concentrations of Taurine during In vitro Fertilization on the Development of Bovine Embryos Fertilized with Spermatozoa from Three Different Bulls. Asian-Aust. J. Anim. Sci.Vol. 23(2010) I.Gordon, Laboratory Production of Cattle Embryos. Cambridge, UK: Cambridge University Press (1993)600 pp. 11. G.F.Miller,D.W. Gliedt,J.M. Rakes and R.W. Rorie, 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,41 (3)(1994) J.F.Hasler, W.B. Henderson, P.J. Hurtgen, Z.Q. Jin, A.D. McCauley, S.A. Mowes, B. Neely,L.S. Shuey, J.E. Stokes and S.A.Trinmer, Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology, 43(1995) M.R.Jainudeen,Y. Takahashi, M. Nihayah and H.Kanagawa, In vitro maturation and fertilization of swamp buffalo (Bubalus bubalis) oocytes. Anim. Reprod. Sci., 31(1993) J.J.Parrish,J.L. Susko-Parrish, M.L.Leibfried- Rutledge, E.S. Critser, W.H. Eyestone and N.L. 9

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