249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 241 ◽  
Author(s):  
G. Wirtu ◽  
C. E. Pope ◽  
M. C. Gomez ◽  
A. Cole ◽  
D. L. Paccamonti ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cell ◽  
Calcium Ionophore ◽  
Male Gamete ◽  
Assisted Reproductive Technique ◽  
First Polar Body

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes. Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments

218 AN EVALUATION OF IN VITRO MATURATION OOCYTE ACTIVATION METHODS FOR IMPROVING OVINE INTRACYTOPLASMIC SPERM INJECTION EFFICIENCY

2016 ◽  
Vol 28 (2) ◽  
pp. 240
Author(s):  
J. E. Hernández ◽  
Y. Ducolomb ◽  
S. Romo ◽  
R. Fierro ◽  
M. E. Kjelland ◽  
...  
Keyword(s):  
Polyvinyl Alcohol ◽  
Follicular Fluid ◽  
Polar Body ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Oocyte Activation ◽  
Sperm Suspension ◽  
Maturation Medium ◽  
Frozen Semen ◽  
First Polar Body

Given previous low sperm decondensation rates and poor oocyte activation in sheep ICSI (10–20%), we evaluated activation techniques for IVM/ICSI. Incubations were performed in a 5% CO2 cell incubator at 38.5°C and saturated humidity. Sheep ovaries were collected at an abattoir and transported <3 h to the laboratory. Follicular fluid was aspirated from 2–8 mm follicles using an 18-gauge needle and syringe with 1 mL of modified Tyrode’s medium supplemented with 10 mM sodium lactate, 10 mM HEPES, and 0.1% polyvinyl alcohol (TL-HEPES-PVA, 7.3–7.4 pH), with 200 IU mL–1 heparin. Cumulus-oocyte complexes (COC) with compact cumulus mass and uniform cytoplasm were selected from the follicular fluid and washed 3× in 500-µL drops of maturation medium (TCM 199) with Earle’s salts and 26.2 mM sodium bicarbonate and l-glutamine with 0.1% polyvinyl alcohol, 0.91 mM sodium pyruvate, 3.05 mM d-glucose, 0.57 mM cysteine, and 10 ng mL–1 epidermal growth factor. Next, 500 µL of maturation medium with 0.5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 10% (vol/vol) of FCS was placed in sterile 4-well plates with 20 to 30 COC/well and with mineral oil for 24 h incubation. The COC were placed in a 500-µL drop of TCM 199-HEPES (TCM 199-H) with 300 IU of hyaluronidase for 3 min and washed (3×) in TCM 199-H. Next, 20 to 30 oocytes were placed in 250-µL droplets of TCM 199-H under a microscope to identify the first polar body (PB). Oocytes with PB were placed in 100-µL droplets of modified Tris-buffered medium (mTBM) for 1 to 4 h of incubation. The groups formed were (1) control: oocytes manipulated as in ICSI but no injection, (2) false injection: oocyte pierced but no sperm insertion, (3) chemical activation (c-a): 7% ethanol (7%Et) × 5 min, (4) c-a: 50 µM calcium ionophore (CaI) × 10 min, (5) c-a: 5 µM ionomicine (Io) × 5 min, (6) ICSI, and (7) 7%Et × 5 min + ICSI. For ICSI, 2 straws of frozen semen from a proven ram were thawed and diluted 1 : 10 with TCM 199-H and 3 mg mL–1 BSA, and then centrifuged 3 min at 200 × g. The sperm pellet was diluted with 100 µL of TCM 199-H, and 2 mL of TCM 199-H was added to a 45° bent tube for a 1-h swim-up. Next, 500 µL of supernatant was diluted to 1 × 106 sperm mL–1 and 10 µL added to 10 µL of 10% polyvinylpyrrolidone (PVP). Five oocytes at a time were placed in a Petri dish with a 10-µL drop of TCM-199-H, with 1% gentamycin, 2% serum, and one 2-µL drop of sperm suspension-PVP. Groups of 10 to 20 oocytes were activated in 100-mL drops of respective chemical in TCM 199-H at 20 to 22°C. Oocytes were washed (3×) in mTBM and set in 200 mL of mTBM for 18 to 20 h of incubation. Oocytes were stained with 10 μg mL–1 Hoechst 33258 for 15 min to assess pronucleus formation. Pearson χ2 tests showed statistical differences (α = 0.05) among the groups (χ2 = 123.165, P < 0.001); for example, groups 1 and 7 (χ2 = 68.179, P < 0.001) and 6 and 7 (χ2 = 42.842, P < 0.001). Results (oocytes, percentage activated) for each group were (1) n = 151, 13.2%, (2) n = 78, 32%, (3) n = 393, 53.6%, (4) n = 350, 46.8%, (5) n = 78, 42.3%, (6) n = 200, 24.5%, and (7) n = 123, 60.9%. The highest percentage of oocyte activation was achieved using 7%Et × 5 min + ICSI.

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

scholarly journals Bushen Tiaojing (II and III) Decoctions Activate MAPK14 and MAPK3/1 to Promote the Expression of Cumulus Expansion-Related Factors in Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Xiao Liang ◽  
Xue Tong ◽  
Hui-lan Du ◽  
Ming He ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Polar Body ◽  
Cumulus Cell ◽  
Serum Levels ◽  
Control Group ◽  
Distilled Water ◽  
Related Factors ◽  
Cumulus Expansion ◽  
Sequential Administration ◽  
First Polar Body

Background. Bushen Tiaojing Decoctions (BSTJ-II-D and BSTJ-III-D) are used to assist pregnancy in clinical practice. In this study, we explored the ability of sequential administration of BSTJ-II-D and BSTJ-III-D to promote cumulus cell (CC) expansion and its underlying mechanisms in controlled ovarian hyperstimulation (COH) mice. Methods. Kunming mice were randomly divided into three groups. The normal group was injected intraperitoneally with saline, and distilled water was administered orally by gavage. As the COH model, mice were injected with GnRHa, eCG, and hCG. Subsequently, the BSTJD group received BSTJ-II-D and BSTJ-III-D orally by gavage, while the control group received distilled water. We evaluated CC expansion and oocyte first polar body (PB1) extrusion under a stereomicroscope. Serum levels of follicle-stimulating hormone (FSH) were detected by radioimmunoassay. The expression of the CC expansion-related factors PTX3 and PTGS2 was detected by immunofluorescence, western blot, and quantitative real-time-polymerase chain reaction analyses (qRT-PCR). Expression of p-MAPK14, p-MAPK3/1, MAPK14, and MAPK3/1 was detected by western blot analysis. Results. Sequential administration of BSTJ-II-D and BSTJ-III-D promoted cumulus expansion and oocyte PB1 extrusion and upregulated PTX3 and PTGS2 expression at the mRNA and protein levels. Furthermore, the levels of p-MAPK14/MAPK14, p-MAPK3/1/MAPK3/1 proteins, and serum FSH in the BSTJD group were higher than those in the normal and control groups. Conclusions. Sequential administration of BSTJ-II-D and BSTJ-III-D promotes cumulus expansion and oocyte maturation in COH mice by increasing FSH expression and activating the MAPK14 and MAPK3/1 signalling pathways, thereby increasing expression of PTX3 and PTGS2.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 265
Author(s):  
M. P. Milazzotto ◽  
W. B. Feitosa ◽  
R. Simões ◽  
C. M. Mendes ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryo Quality ◽  
Calcium Influx ◽  
Polar Body ◽  
Chemical Activation ◽  
Electrical Activation ◽  
Activation Process ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
First Polar Body

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5�C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 �s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5�C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation. This work was supported by FAPESP 03/00156-9.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION

2015 ◽  
Vol 27 (1) ◽  
pp. 249
Author(s):  
M. E. Arias ◽  
R. Sanchez ◽  
R. Felmer
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Standard Procedure ◽  
Chemical Activation ◽  
Developmental Rate ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Assisted Reproductive Technique ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P < 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.

Intracytoplasmic sperm injection in dysmorphic human oocytes

Zygote ◽  
1995 ◽  
Vol 3 (4) ◽  
pp. 283-288 ◽  
Author(s):  
Mina Alikani ◽  
Gianpiero Palermo ◽  
Alexis Adler ◽  
Massimo Bertoil ◽  
Marlena Blake ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Smooth Endoplasmic Reticulum ◽  
Polar Body ◽  
Light Microscopic Level ◽  
Human Oocytes ◽  
Fertilisation Rate ◽  
Polar Bodies ◽  
First Polar Body ◽  
Multiple Abnormalities ◽  
Further Development

SummaryFertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes – most probably a product of controlled ovarian stimulation – are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.

Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 261-265 ◽  
Author(s):  
L. Keskintepe ◽  
P.C. Morton ◽  
S.E. Smith ◽  
M.J. Tucker ◽  
A.A. Simplicio ◽  
...  
Keyword(s):  
Breeding Season ◽  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Egg Yolk ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
First Polar Body ◽  
Defined Culture ◽  
Oviduct Fluid

SummaryExperiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2–6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM 199 supplemented with 10 μg each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5°C) for 4.5 h during transportation. Then, oocytes were transferred into 75 μl of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5°C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37°C water bath for 15s. Motile fractions were selected by swim-up, then incubated for 90 mm in TALP with 10 μg heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 μl) were added to 10 μl medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM 199 + 20% FBS at 37°C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 μl of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

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