Comparison of Culture Media for In Vitro Maturation of Oocytes of Indigenous Zebu Cows in Bangladesh

Joydev Kumar Singha,Mohammad Musharraf Uddin Bhuiyan,Mohammad Moshiur Rahman,Farida Yeasmin Bari 한국수정란이식학회 2015 한국동물생명공학회지 Vol.30 No.4

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at 39℃ with 5% CO2 in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was 75.5±3.9 and 62.2±20.2% in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher (76.6±13.2%) in FSH supplemented medium than gonadotrphin supplemented counterpart (69.7±10.8%) (P<0.05). The maturation rates of oocytes were 81.7±12.9 and 85.7±12.7% in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

Optimization of in vitro fertilization technique for oocytes of indigenous zebu cows

Mohammad Moshiur Rahman,Md. Masudur Rahman,Nasrin Sultana Juyena,Mohammad Musharraf Uddin Bhuiyan 사단법인 한국동물생명공학회 2020 한국동물생명공학회지 Vol.35 No.2

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode’s albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.

Monitoring the Sonographic Ovarian Dynamics and Pregnancy Rate in Cyclic Murrah Buffalo Cows Synchronized with Prostaglandin F2α

Mohammad Harun-or-Rashid,SK Phulia,Mir Md. Iqbal Hasan,Mohammad Musharraf Uddin Bhuiyan,Nasrin Sultana Juyena,Rakesh Kumar Sharma 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.1

The objective of this research work was to know ovarian dynamics and pregnancy rate of cyclic Murrah buffalo cows with induced estrus by administration of prostaglandin F2α (PGF2α) and timed artificial insemination (TAI) with frozen thawed semen. A total of 31 female buffaloes were selected for the study. The buffalos having matured CL observed by ultrasonography were given one intra muscular injection of cloprostenol 500 μg and TAI was performed using frozen thawed semen of Indian Murrah buffalo bull. Results showed that 90.32% (significantly, at p < 0.05) cows explore the sign of heat after injection of PG and 67.85% (significantly, at p < 0.05) cows were become pregnant out of 28 inseminated (TAI) cows. In the 28 inseminated (TAI) cows, average number of smaller and larger size of follicles were non-significantly (p > 0.05) higher at day 3 post PG injection, but the medium size of follicles was non-significantly (p > 0.05) higher at PG injection. At day 3 post PG injection the diameter of follicles was significantly (p < 0.05) higher, but the diameter of CL was significantly (p < 0.01) lower compared at PG injection. At PG injection the diameter of largest follicle was non-significantly differences (p > 0.05) in between pregnant and non-pregnant cows. But at day 3 post PG injection it was significantly (p < 0.01) higher in pregnant cows compared to non-pregnant cows. The number of small, medium, and large follicles at PG injection and at day 3 post PG injection were non-significantly (p > 0.05) difference in between pregnant and non-pregnant buffalo cows. Finally, it is concluded that the CL was effectively regresses and induced the sign of heat in buffalo cows and after AI the cows were become pregnant with significant rate. The study will help to the veterinarian and researcher to know the efficacy of PG injection and AI for reproductive efficiency in buffalo cows.

Optimization of in vitro fertilization technique for oocytes of indigenous zebu cows

Mohammad Moshiur Rahman,Md. Masudur Rahman,Nasrin Sultana Juyena,Mohammad Musharraf Uddin Bhuiyan 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.2

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode’s albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes

이은송,Mohammad Musharraf Uddin Bhuiyan,Hiroyuki Watanabe,Kohji Matsuoka,Yoshihiro Fujise,Hajime Ishikawa,Yutaka Fukui 대한수의학회 2009 JOURNAL OF VETERINARY SCIENCE Vol.10 No.4

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60∼81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Parthenogenetic Activation of Black Bengal Goat Oocytes

Haque, Aminul,Bhuiyan, Mohammad Musharraf Uddin,Khatun, Momena,Shamsuddin, Mohammed 韓國受精卵移植學會 2011 한국동물생명공학회지 Vol.26 No.2

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at with 5% in humidified air. The matured oocytes (n = 248) were activated with 5 ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at with 5% in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was . The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was . Parthenogenetic activation, evidenced by formation of pronucleus, occurred in of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

Parthenogenetic Activation of Black Bengal Goat Oocytes

Aminul Haque,Mohammad Musharraf Uddin Bhuiyan,Momena Khatun,Mohammed Shamsuddin 사단법인 한국동물생명공학회 2011 한국동물생명공학회지 Vol.26 No.2

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at 39℃ with 5% CO_2 in humidified air. The matured oocytes (n =248) were activated with 5 μM ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at 39℃ with 5% CO_2 in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was 3.5 ± 0.5. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was 42.1 ± 4.7%. Parthenogenetic activation,evidenced by formation of pronucleus, occurred in 37.2 ± 15.8% of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats’ oocytes

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Momena Khatun,Mohammad Musharraf Uddin Bhuiyan,Jalal Uddin Ahmed,Aminul Haque,Mohammed Shamsuddin,Mohammad Bozlur Rahman 대한수의학회 2011 Journal of Veterinary Science Vol.12 No.1

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle’s salt with L-glutamine and sodium bicarbonate) for 27 h at 39oC under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean ± SD) per prepubertal and pubertal goats were 5.2 ± 0.6 and 6.8 ± 0.6, and 77.3 ± 0.1% and 80.5 ± 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 ± 0.8 vs. 2.6 ± 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode’s albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 ± 0.9% vs. 32.7 ± 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 ± 1.7 vs. 32.2 ± 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.

Comparison between Two Cryo-devices for Vitrification of Immature Oocytes of Indigenous Zebu Cows in Bangladesh

Sk Mohiuddin Choudhury,Mohammad Musharraf Uddin Bhuiyan,Mohammad Moshiur Rahman,Md. Masudur Rahman,Md. Newaz Sharif,Jayonta Bhattacharjee,Farida Yeasmin Bari,Nasrin Sultana Juyena 사단법인 한국동물생명공학회 2017 한국동물생명공학회지 Vol.32 No.4

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen (LN2). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in 50 μl droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop (47.1±6.9%) than that of French mini straw (15.9±12.5%). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) (84.5±14.2%) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes

Comparison between Two Cryo-devices for Vitrification of Immature Oocytes of Indigenous Zebu Cows in Bangladesh

Choudhury, Sk Mohiuddin,Bhuiyan, Mohammad Musharraf Uddin,Rahman, Mohammad Moshiur,Rahman, Md. Masudur,Sharif, Md. Newaz,Bhattacharjee, Jayonta,Bari, Farida Yeasmin,Juyena, Nasrin Sultana The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.4

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.