81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 199
Author(s):  
L.-Y. Sung ◽  
C.-H. Chen ◽  
T.-A. Lin ◽  
L.-J. Sung ◽  
H.-Y. Su ◽  
...  
Keyword(s):  
Polar Body ◽  
Fluorescent Microscopy ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Adult Rabbit ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
First Polar Body

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

81 MOUSE CLONING BY USING A LASER-ASSISTED ZONA OPENING AND ELECTRO-FUSION TECHNIQUE

2007 ◽  
Vol 19 (1) ◽  
pp. 158
Author(s):  
R. Rungsiwiwut ◽  
A. Thongphakdee ◽  
P. Numchaisrika ◽  
P. Virutamasen ◽  
M. Techakumphu
Keyword(s):  
Zona Pellucida ◽  
Polar Body ◽  
National Research Council ◽  
Donor Cell ◽  
Developmental Rate ◽  
Fusion Rate ◽  
Cleavage Rate ◽  
Fusion Technique ◽  
First Polar Body ◽  
Chi Square Analysis

Mouse cloning can be performed by a direct microinjection of donor nuclei using a conventional or a piezo-actuated technique (Rybouchkin et al. 2002 Reproduction 124, 197–207; Wakayama et al. 1998 Nature 394, 369–374). However, a high percentage of lysed oocytes was observed during the pipette penetration of the cytoplasmic membrane through the zona pellucida. The aim of this experiment was to investigate the possibility of a combination of a laser-assisted zona opening and electro-fusion for mouse cloning. Mature oocytes were obtained from FSH-superovulated B6D2F1 female mice. Enucleation and transfer of donor cell were performed in HEPES-buffered CZB medium. Spindle-chromosome complexes (SCCs) together with first polar body were removed by blunt-end pipette via a small hole in the zona pellucida which was cut by a laser beam. An adult fibroblast cell was introduced into the perivitelline space and fused to the enucleated oocyte by using a single DC pulse of 1.5 kV cm-1, 20 �s, in a fusion medium (Liu and Aoki 2003 Animal Sci. J. 75, 125–129). The fusion rate was checked 30 min later and only the fused oocytes were subjected to activation by 6 h culture in Ca2+-free CZB medium supplemented with 10 mM Sr2+ and 5 �g mL-1 cytochalasin B. The oocytes which presented the pseudo-pronuclei were considered as the activated oocytes and were cultured in CZB medium at 37�C, 5% CO2 in humidified atmosphere. The developmental rate was observed every 24 h for 4 days. The diploid parthenogenetically activated embryos serving as a control were obtained using the same activation protocol but without SCC removal. The percentages of survival after enucleation and after fusion were recorded. The formation of pseudo-pronuclei and the embryos developing to a particular stage were determined by chi-square analysis. The results show that most of the oocytes survived after enucleation (92.5%, 172/186) and the fusion rate was 71.9% (105/146). The formation of pseudo-pronuclei and the cleavage rate of cloned embryos was lower than in the control (87.6% (92/105) vs. 100% (90/90) and 69.6% (64/92) vs. 92.2% (83/90), respectively). The developmental rate to morula–blastocyst stage of cloned embryos was significantly lower than in the control [1.1% (1/92) vs. 44.4% (40/90); P &lt; 0.05]. These results indicate that using laser-assisted zona opening and electro-fusion technique is practical for mouse cloning and provides an alternative method when injection of donor nuclei into the recipient oocytes using a conventional or a piezo-driven method is technically difficult. This study was supported by grants from The National Research Council of Thailand and The Thailand Research Fund (Loyal Golden Julilee Ph.D. program).

130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 145
Author(s):  
H. J. Kim ◽  
S. R. Cho ◽  
C. Y. Choe ◽  
S. H. Choi ◽  
D. S. Son ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Non Invasive ◽  
First Polar Body ◽  
Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

scholarly journals 66 IN VITRO DEVELOPMENT OF YAK (POEPHAGUS MUTUS) CLONED EMBRYOS BY INTERSPECIES SOMATIC NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 183
Author(s):  
L. Su ◽  
F.L. Du ◽  
L.Y. Sung ◽  
S. Yang ◽  
B.S. Jeong ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Bos Taurus ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Cell Number ◽  
Developmental Potential ◽  
Somatic Nuclear Transfer ◽  
Humidified Air

Interspecies nuclear transfer (NT) is an important tool for preservation of endangered animal species. This study was carried out to clone Yak (Poephagus mutus) embryos by using Yak skin fibroblasts and bovine (Bos taurus) recipient cytoplasts, and to compare the efficiency of YAK interspecies NT (bovine cytoplast-Yak donor cell) and bovine somatic NT (bovine cytoplast-bovine donor cell). Recipient oocytes were extracted from antral follicles of bovine ovaries, and subsequently cultured in maturation medium for 18–20 h in 5% CO2 and 95% humidified air at 39°C. Cumulus cells were removed from the oocytes by vortexing also facilitated further enucleation. Yak skin fibroblast cells were prepared from cultured ear explants of an adult 5-year-old female. Fibroblasts were cultured at passage 6–9 in 10% FBS DMEM at 37°C in 5% CO2 humidified air. The donor cell at a diameter of 19–20 μm was inserted into the perivitelline space of an enucleated oocyte. A bovine female cell line at similar passage number was used for bovine somatic NT as control. Somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV/cm for a duration of 6–10 μs/pulse. Fused embryos were activated in 10 μg/mL cycloheximide and 2.5 μg/mL cytochalasin D in M199 plus 7.5% FBS for 5 h. Reconstructed Yak embryos were cultured in CR1aa plus 6 mg/mL BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2, and 90% N2, and then in 7.5% FBS CR1aa medium for 5 successive days on bovine cumulus monolayers. Expanding and hatching blastocysts on Day 7 were recorded and cryopreserved for further embryo transfer trials. The percentage of cleavage and the development to morulae and blastocysts were statistically analyzed using a General Linear Model (GLM, Univariate, SPSS 9.0, SPSS Inc, Chicago, IL, USA). As indicated in Table 1, the results demonstrated that the efficiencies of fusion rate as well as developmental potential in vitro were significantly higher in the bovine somatic NT group compared to those of the Yak interspecies NT group. However, the morphology and cell number per embryo of interspecies Yak cloned embryos were indistinguishable from those of bovine NT embryos. Our data suggest that bovine oocytes possess the capability of reprogramming/reactivation of the genome from differentiated somatic Yak nuclei. Table 1. Comparison of yak interspecies and bovine somatic nuclear transfer

156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 192
Author(s):  
L. Cai ◽  
E. Kim ◽  
S. U. Hwang ◽  
J. D. Yoon ◽  
Y. Jeon ◽  
...  
Keyword(s):  
Polar Body ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
First Polar Body

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Antioxidant β-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro

2018 ◽  
Vol 30 (9) ◽  
pp. 1204 ◽  
Author(s):  
Yun-Gwi Park ◽  
Seung-Eun Lee ◽  
Yeo-Jin Son ◽  
Sang-Gi Jeong ◽  
Min-Young Shin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Control Group ◽  
Mrna Levels ◽  
Cleavage Rate ◽  
Antioxidant Genes ◽  
Developmental Potential

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant β-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of β-cryptoxanthin (0, 0.1, 1, 10 or 100 μM). Treatment with 1 µM β-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that β-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

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.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

197 INHIBITION OF CYSTEINE PROTEINASES DURING IN VITRO OOCYTE MATURATION IMPROVES DEVELOPMENT OF PORCINE CUMULUS - OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
A. M. Lichtenauer ◽  
L. D. Spate ◽  
R. S. Prather ◽  
J. A. Green
Keyword(s):  
Oocyte Maturation ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Developmental Potential

Biochemical differences exist between oocytes that give rise to viable blastocysts and oocytes that give rise to embryos that are developmentally compromised. For example, specific proteolytic enzymes (e.g. cathepsin B) are transcriptionally abundant in in vitro-matured bovine oocytes from prepubertal heifers that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus–oocyte complexes (COC) that represented both poor- and good-quality oocytes. Those reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development. This project sought to determine if similar results would be obtained in a porcine oocyte/embryo culture system. Inclusion of 10 and 20 μM E-64 in maturation medium was performed. Maturation rates of porcine COC in 20 μM E-64 were elevated compared to those incubated in 10 μM E-64 (74% vs 53%; P < 0.05) or without E-64 (55%; P < 0.05: N = 1750 oocytes tested). Successful maturation to metaphase II was based on the presence of a polar body and a uniform cytoplasm 44 h after follicular aspiration. Based on these preliminary results and the earlier bovine work, it was hypothesized that the E-64 was having little influence on normal oocytes, but was promoting maturation of low-quality oocytes, possibly those that were beginning to degenerate. Consequently, 20 μM of E-64 was added to the maturation media of COC segregated based on morphological characteristics of the oocytes. Good COC had a homogeneous cytoplasm and greater than 3 layers of cumulus cells; the COC were considered poor if they displayed a nonhomogeneous cytoplasm and 1 layer or less of cumulus cells, yet were still considered fertilizable. Without E-64, an increase in maturation was measured when good oocytes were compared to poor oocytes (52% vs 29%; P < 0.05: N = 1600). No significant differences in maturation were observed between good oocytes incubated in the presence or absence of E-64. Likewise, no significant differences were observed between poor oocytes incubated in the presence or absence of E-64. The percentage of maturation of good oocytes cultured in E-64 was significantly higher than that of poor oocytes cultured with E-64 (67% vs 43%; P < 0.05). Maturation with the inhibitor did not significantly affect the subsequent cleavage or blastocyst rates of embryos that arose from these oocyte groups after fertilization. These experiments suggest that inhibition of cysteine proteinases significantly promotes oocyte maturation, as was seen in previous bovine work. Our data did not support the hypothesis that cysteine proteinase inhibition was selectively improving maturation of poor oocytes within the pool. It remains possible that increased maturation in good oocytes is a result of cysteine inhibition on juvenile oocytes that morphologically appeared good and the effect was less on already degenerated oocytes that appeared poor. Differences between treatments were determined by ANOVA with post-test by Tukey's multiple comparison test.

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