Effects of Roscovitine on In Vitro Development of Porcine Oocyte Using Brilliant Cresyl Blue

Pantu Kumar Roy,Xun Fang,Bahia MS Hassan,Sang Tae Shin,Jong Ki Cho 한국수정란이식학회 2017 한국동물생명공학회지 Vol.32 No.3

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 75 μM of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

Cobalamin Supplementation during In Vitro Maturation Improves Developmental Competence of Porcine Oocytes

Pantu Kumar Roy,Xun Fang,Bahia MS Hassan,Sang Tae Shin,Jong Ki Cho 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

The objective of this experiment was to explore the effects of cobalamin prior to in vitro maturation (IVM) of immature pig oocyte. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 200 pM of cobalamin for 22 h, and afterward cultured for 22 h without exposure of cobalamin. The COCs that were matured in the same IVM for 44 h without cobalamin used as control group. Diameter of cobalamin treated oocytes were significantly greater than control group. GSH was higher in cobalamin group than control oocytes group but reduction of ROS in cobalamin than control group of oocytes. In PA, exposure with cobalamin has been significantly greater than control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly greater in cobalamin group than control group. SCNT embryos treated with cobalamin 22 h in IVM showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with control group. Our results demonstrate that cobalamin treatment prior to IVM improves the development competence of porcine oocyte.

Effects of different post-activation conditions on the developmental competence of in vitro matured porcine oocytes

Pantu Kumar Roy,Xun Fang,Bahia MS Hassan,Jong Ki Cho 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

The objective of this study was to establish the effect of post-activation treatment with cytoskeletal regulators of CB, CB+CHX, CB+DC, CB+6’DMAP on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT embryos were produced by using in vitro matured pig oocytes and treated for 4 h after electric activation with cytochalasins B (7.5 μg/ml), CB+cycloheximide (10 μg/ml), CB+demecolcine (0.4 μg/ml), and CB+2mM 6-Dimethylaminopurine. Post-activation treatment of PA oocytes with CB, CB+CHX, CB+DC and CB+6’DMAP no significant differences were found in embryo cleavage (83.2~91.5%), mean cell number of blastocysts (40.6~ 42.3% cells/blastocyst) but significantly (P<0.05) differences blastocyst formation (28.6~36.4%). When PA oocytes were treated with CB, CB+CHX, CB+DC, CB+6’DMAP, blastocyst formation was significantly (P<0.05) improved by CB (36.6%) compared to CB+CHX (30.9%), CB+DC (28.6%) and CB+6’DMAP (35.2%). In SCNT, was not significantly (P<0.05) increased by post-activation treatment with CB+CHX (81.3%), CB+DC (83.9%) and CB+6’DMAP (90.0%) compared to CB (84.5%) on embryo cleavage, blastocyst formation (19.1%~23.6%) and blastocyst cell number (39.6~41.4% cells/blastocyst) also were not influenced. But increased tendency in CB+6’DMAP. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-4 cell stage SCNT embryos were significantly higher 6’DMAP treated group than other treatment groups of SCNT embryos. These observations suggested that 2-4 cell cleaving embryos at have high developmental competence, and which may be influenced by survivin expression in porcine SCNT embryos. Our results demonstrate that post-activation treatment with CB, CB+CHX, CB+DC, CB+6’DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

Oestrus Synchronization with Ovuprost® and Prostenol® in the Indigenous Ewes of Bangladesh

Pantu Kumar Roy,Begum Fatema Zohara,Ashit Kumar Paul,MMU Bhuiyan,Farida Yeasmin Bari 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.2

The present study was designed to observe the oestrus responses in the indigenous ewe induced by cloprostenolsodium manufactured by two different companies (Ovuprost®, BOMAC, Newzealandand Prostenol®, Techno, Bangladesh). Twelve local ewes were divided into 3 groups (n= 8). The ewes in Group I and II were induced by intramuscular injectionof 100 μg (0.4 ml) of cloprostenol sodium (Ovuprost® and Prostenol®), respectively. The 2nd injection in each groupwas given at 9 days apart. The ewes in Group III were kept as control for observing natural oestrus characteristics andcomparing the responses with induced oestrus. Hundred and 75% ewes showed oestrus following 2nd injection ofOvuprost® and Prostenol®, respectively. The average time of onset of oestrus following 1st and 2nd injection of Ovuprost®and Prostenol® were 50.5 ± 3.5 vs 48.0 ± 0.0 h and 49.9 ± 1.9 vs 49.5 ± 1.7 h, respectively. There was no significantdifference between the two types of cloprostenol sodium group on the onset of oestrus. The average duration of oestruswas 27.5 ± 0.7 vs 27.5 ± 0.0 h and 25.9 ± 3.3 vs 24.2 ± 0.3 h in Ovuprost® and Prostenol® treated ewes, respectively. For natural oestrus, the duration of oestrus was 25.2 ± 3.3 h. There was no significant difference among the cloprostenolsodium produced by two different companies and natural oestrous ewes on the duration of oestrus. The higher percentagesof cornified cells were present in induced oestrus (90 and 85%) compared with natural oestrus (80%), although therewas no significant difference among them. The pregnancy rates were 75, 66.7 and 100% in Ovuprost®, Prostenol® andnatural oestrous ewes, respectively. The above results indicate the suitability of using cloprostenol sodium for synchronizationof oestrus in indigenous ewes in Bangladesh.

Selection of developmentally competence porcine oocytes using the brilliant cresyl blue

Pantu Kumar Roy,Xun Fang,Bahia MS Hassan,Jong Ki Cho 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

Introduction The objective of this study was to investigate the effects of staining of porcine cumulus-oocytes complexes (COCs) by brilliant cresyl blue (BCB) test prior to in vitro maturation may be used to select developmentally competent oocytes. Furthermore, milrinone can be used to promote developmental competence of porcine embryos produced during parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). Materials and Methods Slaughterhouse-derived porcine cumulus-oocyte-complexes (COCs) were exposed to BCB and treated oocytes divided into BCB+ (colored cytoplasm), BCB- (colorless cytoplasm) groups. After division into 2 groups, intracellular glutathione (GSH) and reactive oxygen species (ROS) of matured oocytes were compared. And, preimplantation development of PA and SCNT embryos were also compared between 2 groups. BCB- oocytes were exposed to milrinone with different concentrations (0, 50, 75, and 100μM) for 6 h prior to IVM for further development of embryos. Results and Discussion GSH was higher in BCB+ group than BCB- group whereas ROS was lower in BCB+ than BCBgroup. In parthenogenetic embryos, BCB+ oocytes group was significantly higher on maturation (87.5 vs 80.6, 71.3%), cleavage (88.6 vs 82.9, 76.3%), and blastocyst formation rates (34.3 vs 27.8, 25.3%) than control and BCB- oocytes groups, respectively. Moreover, ratio of ICM:TE cells were higher in BCB+ oocytes group (30.3% vs. 28.6, 26.4%, respectively) than other groups. In cloned embryos, the significant higher blastocyst formation rates were shown BCB+ groups (30.6% vs. 26.0, 20.1%) than BCB- groups. To improve the cytoplasmic maturation in BCB- oocytes, 4 different concentrations of milrinone (0, 50, 75, and 100μM) were supplemented in the IVM media for 6 h. BCB- oocytes supplemented with 75μM milrinone showed the significantly higher rates of blastocyst formation than other groups. Our results demonstrate that staining of porcine oocytes with BCB before IVM may be used for selection of good quality oocytes and milrinone supplementation can be used to improve embryo developmental competence of porcine embryos.

Optimization of Post-Activation Systems to Improve the Embryonic Development in Porcine Parthenogenesis and Somatic Cell Nuclear Transfer

Pantu Kumar Roy,Ghangyong Kim,Xun Fang,Bahia MS Hassan,Mahanama De Soysa,Sang Tae Shin,Jong Ki Cho 한국수정란이식학회 2017 한국동물생명공학회지 Vol.32 No.3

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB (7.5 μg/mL), CB (7.5 μg/mL) + CHX (10 μg/mL), CB (7.5 μg/mL) +DC (0.4 μg/mL), and CB (7.5 μg/mL) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

Oestrus Synchronization with $Ovuprost^{(R)}$ and $Prostenol^{(R)}$ in the Indigenous Ewes of Bangladesh

Roy, Pantu Kumar,Zohara, Begum Fatema,Azizunnesa, Azizunnesa,Paul, Ashit Kumar,Bhuiyan, M.M.U.,Bari, Farida Yeasmin The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.2

The present study was designed to observe the oestrus responses in the indigenous ewe induced by cloprostenol sodium manufactured by two different companies ($Ovuprost^{(R)}$, BOMAC, Newzealandand $Prostenol^{(R)}$, Techno, Bangladesh). Twelve local ewes were divided into 3 groups (n= 8). The ewes in Group I and II were induced by intramuscular injection of $100{\mu}g$ (0.4 ml) of cloprostenol sodium ($Ovuprost^{(R)}$ and $Prostenol^{(R)}$), respectively. The $2^{nd}$ injection in each group was given at 9 days apart. The ewes in Group III were kept as control for observing natural oestrus characteristics and comparing the responses with induced oestrus. Hundred and 75% ewes showed oestrus following $2^{nd}$ injection of $Ovuprost^{(R)}$ and $Prostenol^{(R)}$, respectively. The average time of onset of oestrus following $1^{st}$ and $2^{nd}$ injection of $Ovuprost^{(R)}$ and $Prostenol^{(R)}$ were $50.5{\pm}3.5$ vs $48.0{\pm}0.0h$ and $49.9{\pm}1.9$ vs $49.5{\pm}1.7h$, respectively. There was no significant difference between the two types of cloprostenol sodium group on the onset of oestrus. The average duration of oestrus was $27.5{\pm}0.7$ vs $27.5{\pm}0.0h$ and $25.9{\pm}3.3$ vs $24.2{\pm}0.3h$ in $Ovuprost^{(R)}$ and $Prostenol^{(R)}$ treated ewes, respectively. For natural oestrus, the duration of oestrus was $25.2{\pm}3.3h$. There was no significant difference among the cloprostenol sodium produced by two different companies and natural oestrous ewes on the duration of oestrus. The higher percentages of cornified cells were present in induced oestrus (90 and 85%) compared with natural oestrus (80%), although there was no significant difference among them. The pregnancy rates were 75, 66.7 and 100% in $Ovuprost^{(R)}$, $Prostenol^{(R)}$ and natural oestrous ewes, respectively. The above results indicate the suitability of using cloprostenol sodium for synchronization of oestrus in indigenous ewes in Bangladesh.

Effects of Roscovitine on In Vitro Development of Porcine Oocyte Using Brilliant Cresyl Blue

Roy, Pantu Kumar,Fang, Xun,Hassan, Bahia MS,Shin, Sang Tae,Cho, Jong Ki The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.3

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

Optimization of Post-Activation Systems to Improve the Embryonic Development in Porcine Parthenogenesis and Somatic Cell Nuclear Transfer

Roy, Pantu Kumar,Kim, Ghangyong,Fang, Xun,Hassan, Bahia MS,Soysa, Mahanama De,Shin, Sang Tae,Cho, Jong Ki The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.3

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

Comparison of Reproductive Parameters of Sow at the Rural Areas of Rangamati District of Bangladesh

Chakma, Sumen,Roy, Pantu Kumar,Paul, Ashit Kumar,Alam, M. Golam Shahi,Juyena, Nasrin Sultana 한국수정란이식학회 2014 한국동물생명공학회지 Vol.29 No.3

It is imperative to know the reproductive parameters of the existing swine breed for profitable farming in Bangladesh. This study was designed to determine the reproductive parameters of sows at hilly areas. A total of 245 pigs with 47 breedable sows were included. The data on age at puberty, oestrous cycle length, oestrus duration, gestation length, interval between furrowing and onset of oestrus, first service pregnancy rate, service per pregnancy and number of piglets born per sow in local and cross bred sows were determined. The present study revealed that age at puberty of local and cross breed sows was 232.5±8.4, 221.3±6.9 days, respectively. Oestrous duration was 41.1±3.1 hours. The interval between furrowing and onset of oestrus was shorter in cross bred sows. There was no variation in values of oestrus cycle length and gestation length. The first service pregnancy rate was higher in both the local and cross bred sow. Number of piglets per sow per farrowing was 6.1±2.2 and 60.9% local sows gave birth of 3 to 5 piglets per farrowing whereas 75.0% cross bred sows gave birth of 6 to 9 piglets. These results suggest that reproductive parameters of local sow need to be improved for better production, and cross bred sows should be reared for obtaining expected productivity.