scholarly journals In vitro production of zygote from slaughterhouse driven buffalo oocyte

2020 ◽  
pp. 127-132
Author(s):  
GK Deb ◽  
MFH Miraz ◽  
SMJ Hossain ◽  
MF Afroz ◽  
MA Kabir ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Meat Production ◽  
In Vitro Production ◽  
Embryo Production ◽  
Cell Stage

Buffalo is a highly potential animal species in terms of milk and meat production but traditionally they are regarded as poor breeder. In vitro embryos production technology has been introduced in many countries to improve reproductive efficiency of buffalo. Considering the above fact, the present study was undertaken aiming to produce in vitro buffalo embryo in the laboratory. Ovaries of slaughtered buffaloes were collected from abattoir and transported to the laboratory within 4 to 5 hr of slaughter. Cumulusoocyte- complexes (COCs) possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells was selected for in vitro maturation (IVM) for 24 hr (5% CO2 in air at 38.5°C with maximum humidity). After IVM, the presumptive matured COCs were co-cultured with capacitated fresh spermatozoa for 18 hr After IVF, the presumptive zygote were denuded, washed and transferred in to in vitro culture medium (IVC 1) for 3 days. After three days cleavage were recorded and 4 cell embryos were transferred in to in vitro culture media II for next 2 days. The development of embryos was evaluated on day 6. A total of 227 buffalo ovaries were collected from the slaughterhouse and categorized into 2 groups based on presence (n=83) or absence (n=144) of corpus luteum (CL). A total of 1464 follicles were counted on the ovarian surface, 1066 being from CL absent and 398 from CL-containing ovaries. A significantly higher (P<0.01) number of follicles, aspirated follicles, normal COCs and total COCs (7.4 ± 0.21, 5 ± 0.00, 1.98 ± 0.77 and 2.98 ± 0.16 respectively) were observed in CL-absent ovaries than those aspirated from CL-containing ovaries (4.80 ± 0.17, 3.92 ± 0.95, 0.88 ± 0.60 and 1.88 ± 0.16 respectively). Total 358 normal COCs were set for in vitro maturation and underwent for IVF and IVC. Results showed that cleavage rates were 56.42%. Among the cleaved embryos, 137 were at 2-cell stage and 65 were at 4-cell stage. Therefore, development rate to 2 cell and 4-cell stage was 38.27% and 18.15% respectively. No embryo developed beyond 4-cell stage. This result indicates that follicle and oocyte numbers and oocyte quality are associated with CL of ovaries and current culture system support in vitro embryo production upto 4-cell stage. The in vitro culture condition may be improved for increasing efficiency of embryo production. Bangladesh J. of Livestock Res. 21-25: 127-132, 2018

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

scholarly journals EVALUATION OF TWO CULTURE MEDIA IN IN VITRO PRODUCTION OF ALPACA (Vicugna pacos) EMBRYOS

SPERMOVA ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 46-52
Author(s):  
Irving Laines-Arce ◽  
◽  
Mijail Contreras ◽  
Cesar Olaguivel
Keyword(s):  
Saline Solution ◽  
Culture Media ◽  
Physiological Saline ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Fluid Medium ◽  
Vicugna Pacos ◽  
Blastocyst Rate ◽  
Rate Evaluation

The present study aims to evaluate the effect of two culture media on the production of in vitro embryos in alpacas (Vicugna pacos). The ovaries were transported at 10.52° C in 0.9% saline solution supplemented with gentamicin. The ovaries were transported at 10.52° C in 0.9% physiological saline solution supplemented with gentamicin. 492 ovaries were used throughout the experiment. 2142 oocytes of quality I, II and III were recovered. The oocytes were matured in vitro for 32 h and were subsequently fertilized (incubated for 18 h) with sperm obtained from the tail of the epididymis and selected with a 45/90 percoll gradient. Then, the presumed zygotes were denuded from the cumulus cells, to later be cultured in two culture media: synthetic oviductal fluid medium (SOFaa) and simple optimized potassium medium (KSOMaa) and incubated at 38.5 ° C, 5 % CO2, 5%, O2, and 90% relative humidity for 7 days. Morula and blastocyst rate evaluation was performed at the end of embryo culture. The morula rate at 7 days was 41.49 ± 10.52 and 41.51 ± 6.50% for KSOMaa and SOFaa, respectively (P <0.05). The blastocyst rate for the two culture media KSOMaa and SOFaa, was 14.08 ± 5.17 and 11.73 ± 5.69 %, respectively, and there were no statistical differences (P˃0.05). The embryonic quality in KSOMaa and SOFaa media did not show statistical differences. In conclusion, the KSOMaa and SOFaa culture medium can be used in the production of in vitro embryos of alpacas

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS

2009 ◽  
Vol 21 (1) ◽  
pp. 203
Author(s):  
Y. Y. Liang ◽  
D. N. Ye ◽  
C. Laowtammathron ◽  
T. Phermthai ◽  
R. Parnpai
Keyword(s):  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Success Rates ◽  
In Vitro Production ◽  
Cell Stage ◽  
Sham Injection ◽  
Swamp Buffalo ◽  
Second Polar Body

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO2 in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates. This work was supported by the Thailand Research Fund and Suranaree University of Technology.

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