scholarly journals Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 931-946 ◽  
Author(s):  
Salilew-Wondim Dessie ◽  
Franca Rings ◽  
Michael Hölker ◽  
Markus Gilles ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Polar Body ◽  
Mrna Level ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Non Invasive ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Selection Of ◽  
Metaphase Ii Oocytes

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB+) and without (PB−) first polar body and zygotes were subjected to DEP at 4 MHz and 450 μm electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB+and PB−oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P< 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.

Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 900-908
Author(s):  
Yukou Sugano ◽  
Manami Yazawa ◽  
Sachio Takino ◽  
Sueo Niimura ◽  
Hideaki Yamashiro
Keyword(s):  
Chorionic Gonadotropin ◽  
Polar Body ◽  
Fertilization Success ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Rate Of Development ◽  
First Polar Body ◽  
Over Time ◽  
Metaphase Ii Oocytes

SummaryThe objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Polar Body ◽  
Petri Dish ◽  
Developmental Competence ◽  
Basic Medium ◽  
High Survival ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

scholarly journals Oocyte Selection for in Vitro Embryo Production in Livestock Species: Non-Invasive Approaches for the New Challenges of Oocyte Competence

2020 ◽  
Author(s):  
Luis Aguila ◽  
Favian Treulen ◽  
Jacinthe Therrien ◽  
Ricardo Felmer ◽  
Martha Valdivia ◽  
...  
Keyword(s):  
Polar Body ◽  
Developmental Competence ◽  
Oocyte Diameter ◽  
Oocyte Competence ◽  
Non Invasive ◽  
Livestock Species ◽  
Large Oocyte ◽  
First Polar Body ◽  
Major Interest

The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30 to 40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of non-invasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different non-invasive markers associated with oocyte quality in mammalian species, with an emphasis on the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (&amp;gt;120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated to better competence. In addition, the combination of oocyte and zygote selection by BCB test and spindle imaging have the potential to further optimize the identification of oocytes with better developmental competence for in vitro-derived technologies in livestock species.

359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES

2006 ◽  
Vol 18 (2) ◽  
pp. 286 ◽  
Author(s):  
T. Suh ◽  
S. Purcell ◽  
G. Seidel Jr
Keyword(s):  
Growth Factor ◽  
Polar Body ◽  
Follicular Development ◽  
Transitional Period ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
First Polar Body

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI. Table 1. Main effect means of responses after ICSI

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.

102 EFFECT OF DEMECOLCINE PRE-TREATMENT ON VIABILITY, TIMING OF FIRST POLAR BODY EXTRUSION, SPINDLE CONFIGURATION, AND SUBSEQUENT DEVELOPMENT OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 210 ◽  
Author(s):  
A. R. Moawad ◽  
J. Zhu ◽  
I. Choi ◽  
K. H. S. Campbell
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
Polar Body Extrusion ◽  
And Control

Oocyte cryopreservation is a potentially valuable way of preserving female germ cells. However, to date the reported developmental competence of cryopreserved oocytes is low. The objectives of this study were to investigate the effects of demecolcine pretreatment on viability, timing of the first polar body extrusion (PBI), spindle, chromatin organization, and in vitro embryo development of ovine vitrified germinal vesicle (GV) oocytes after in vitro fertilization (IVF) and parthenogenetic activation. Cumulus-oocyte complexes (COC) aspirated from ovine ovaries collected at slaughter were selected and randomly divided into 3 groups: (1) untreated (in vitro matured, IVM) as a control, (2) vitrified (Moawad AR et al. 2009 Reprod. Fertil. Dev. 21, 135 abst), and (3) deme + vitrified (oocytes were incubated with 0.1 μg mL-1 demecolcine for 20 min before vitrification). After vitrification COC were thawed and matured in vitro for 24 h. Following IVM, oocytes from 3 groups were subsequently subjected to (1) immunostaining, (2) IVF, or (3) activation. Presumptive zygotes were cultured in vitro in SOF media for 7 days. Data were analyzed using chisquare and t-test. No significant differences (P > 0.05) were observed in survival rates between deme + vitrified (90.8%, 324/357) and vitrified (87.2%, 211/242). However, the numbers of oocytes with PBI in two vitrified groups at 18 h (20.4 and 8.5 v. 47.1%) and 24 h post IVM (51 and 43.2 v. 88.5%) were significantly lower (P < 0.01) than those in the control. Percentage of normal spindle and chromatin configuration in the two vitrified groups also significantly decreased (P < 0.05) compared with those in the control (42.5 and 41.8 v. 76.5%), whereas missing spindle in the 2 vitrified groups significantly increased (P < 0.001) compared with the controls (47.5 and 32.7 v. 3.9%). Following IVF (pi), cleavage rates at 24.48 hpi and morula development (5 days pi) were significantly lower (P < 0.001) in deme + vitrified (6.1, 43.1, and 28.5%) and vitrified groups (3.3, 30.1, and 22.9%) than control (50.4, 82.4, and 46.4%). Blastocyst development in deme + vitrified (9.8%) and control (33.6%) was significantly higher (P < 0.01) than in vitrified group (1.3%). Hatched blastocysts were observed only in deme + vitrified and control groups (4.9 v. 12.8%). In addition, post activation (pa) cleavage rates in deme + vitrified (10.3 v. 40.7%) and control (52.5 v. 76.7%) at 24 and 48 hpa were significantly higher (P < 0.05) than those in the vitrified group. Blastocyst development in deme + vitrified (4.8%) was higher than that in the vitrified group (1.8%), but not significant (P > 0.05); however, these values were still significantly lower (P < 0.001) than those in the control (24.2%). No significant differences were observed in total cell numbers per blastocyst between all the groups. Taken together, these results suggest that pretreatment of oocytes with demecolcine before vitrification could improve the developmental competence of ovine vitrified-thawed GV-stage oocytes. A. R. Moawad was supported by the Egyptian government.

scholarly journals Oocyte Selection for in vitro embryo Production in Bovine Species: Non-Invasive Approaches for the New Challenges of Oocyte Competence

2020 ◽  
Author(s):  
Luis Aguila ◽  
Favian Treulen ◽  
Jacinthe Therrien ◽  
Ricardo Felmer ◽  
Martha Valdivia ◽  
...  
Keyword(s):  
Polar Body ◽  
Developmental Competence ◽  
Oocyte Diameter ◽  
Oocyte Competence ◽  
Non Invasive ◽  
Livestock Species ◽  
Large Oocyte ◽  
First Polar Body ◽  
Major Interest

The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30 to 40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of non-invasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different non-invasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (&gt;120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated to better competence. In addition, the combination of oocyte and zygote selection by BCB test and spindle imaging have the potential to further optimize the identification of oocytes with better developmental competence for in vitro-derived technologies in livestock species.

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

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.

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