Effect of type of cryoprotectant on morphology and developmental competence of in vitro-matured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or vitrification

2008 ◽  
Vol 20 (4) ◽  
pp. 490 ◽  
Author(s):  
S. K. Gautam ◽  
V. Verma ◽  
P. Palta ◽  
M. S. Chauhan ◽  
R. S. Manik
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bubalus Bubalis ◽  
The Other ◽  
Developmental Competence ◽  
Slow Freezing ◽  
Cleavage Rate ◽  
Cryopreservation Method

The present study examined the effects of different cryoprotectants on morphology and developmental competence of in vitro-matured buffalo oocytes after slow freezing or vitrification. After slow freezing in dimethyl sulfoxide (DMSO), ethylene glycol (EG) or 1,2-propanediol (PROH), at 1.0 or 1.5 m each, the proportion of morphologically normal oocytes recovered was significantly higher (P < 0.05) with 1.5 than 1.0 m for all cryoprotectants and was highest (P < 0.05) for 1.5 m DMSO. Following vitrification, the percentage of morphologically normal oocytes recovered was lower (P < 0.01) for 40% EG than for 40% DMSO, 20% EG + 20% DMSO or 20% EG + 20% PROH. The most common damage, irrespective of the cryopreservation method, was loss of cumulus mass. The cleavage rate and the proportion of vitrified–warmed oocytes that developed to morulae/blastocysts were significantly higher (P < 0.01) for 20% EG + 20% DMSO than for the other groups. A higher proportion of oocytes developed to morulae (11.5% v. 4.3%) or blastocysts (5.4% v. 0.6%) after vitrification in 20% EG + 20% DMSO than after slow freezing in 1.5 m DMSO. In conclusion, vitrification was more effective than slow freezing for the cryopreservation of in vitro-matured buffalo oocytes.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
F. Salerno ◽  
M. Rubessa ◽  
B. Gasparrini ◽  
M. Wheeler
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Deuterium Oxide ◽  
Control Group ◽  
Crystal Formation ◽  
Cleavage Rate ◽  
Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

134 EFFECTS OF VITAMIN E AND VITAMIN C ON THE DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION, IN VITRO FERTILIZATION, AND NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
F. Lu ◽  
Z. Zhang ◽  
S. Zhang ◽  
N. Li ◽  
J. Jiang ◽  
...  
Keyword(s):  
Vitamin E ◽  
Nuclear Transfer ◽  
Bubalus Bubalis ◽  
Development Rate ◽  
The Other ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation

The purpose of this study was to explore the effects of vitamin E (VE) and vitamin C (VC) on the in vitro development of embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (NT) in buffalo (Bubalus bubalis). Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22 to 24 h. After maturation, oocytes were separated to 3 groups: one group of oocytes was fertilized in vitro with buffalo sperm; one group of oocytes was parthenogenetically activated by exposing them to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h; the other group of oocytes was enucleated, and fibroblasts in DMEM + 10% FBS for 4 to 5 days were transferred into enucleated oocytes by electronic fusion (100 v mm–1, 15 μs, and 3 pulses). After fusion, the activation of reconstructed embryos was induced by exposure to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h. The embryos of PA, IVF, and NT were respectively cultured in the culture medium (CM) containing different concentrations of VE, VC, or VE + VC for 7 to 9 days to evaluate embryonic development. As a result, when the embryos were cultured in the CM with different concentrations of VE (0, 50, 100, 150, and 200 μM), the blastocyst development rate of the embryos derived from PA, IVF, and NT gradually rose with increasing concentrations of VE and reached the highest amount [PA: 32.9% (81/246); IVF: 21.4% (45/210); and NT: 21.1% (47/223)] in the group containing 150 μM of VE; it was significantly higher than that of other groups (P < 0.05). When the different concentrations of VC (0, 50, 100, 150, and 200 μM) were added to the CM, the blastocyst development rate of the embryos derived from PA, IVF, and NT also enhanced according to the increasing concentration of VC, and more embryos developed to blastocysts in the group containing 150 μM of VC [PA: 31.2% (72/231); IVF: 20.2% (43/213); NT: 19.8% (48/243)] than in the other groups (P < 0.05). Compared with the control group (0 μM), the blastocyst rate of PA and IVF, as well as NT embryos, cultured in the CM with 150 μM VE + 150 μM VC groups was significantly higher (P < 0.05), but there were no significant differences in the percentage of blastocysts among groups of the 150 μM VE, 150 μM VC, and 150 μM VE + 150 μM VC (P > 0.05). These results indicated that adding VE (150 μM), VC (150 μM), or VE (150 μM) + VC (150 μM) in the CM could efficiently enhance the developmental competence of buffalo embryos during in vitro culture. This work was funded by China High Technology Development Program (2007AA100505), Guangxi Science Foundation (0718005-3A), Fok Ying Tung Education Foundation (111034).

60 EFFECT OF CYTOPLASMIC VOLUME ON DEVELOPMENTAL COMPETENCE OF HAND-GUIDED CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
S. K. Panda ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
N. M. Kamble ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cloning Efficiency ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Despite recent successes in the birth of buffalo calves cloned through SCNT or hand-guided cloning (HGC), the cloning efficiency is very low in this species because of lack of information on factors that influence it. The goal of this study was to examine the effects of cytoplasmic volume on the developmental competence of cloned buffalo embryos produced by HGC. In vitro matured oocytes were stripped of their cumulus investment and zona pellucida using hyaluronidase and pronase, respectively. Protrusion cone-guided bisection of zona-free oocytes was performed to remove the nucleus. For reconstructing control HGC embryos, 2 enucleated oocytes (demi-cytoplasts) were fused with a single somatic cell. For reconstruction of embryos with lower or higher cytoplasmic volume, 1 or 3 demi-cytoplasts were fused, respectively, with the donor somatic cell. 2 different cell types, i.e. buffalo fetal fibroblasts (BFF) between passage 10 and 15 and buffalo embryonic stem cell (ESC)-like cells between passage 22 and 25 were used as nuclear donors in 2 different experiments. Data were analysed by 1-way ANOVA after arcsine transformation of percentage values. For BFF, the blastocyst rate for doublet and triplet embryos were significantly higher (P ≤ 0.01) than that for singlet embryos despite the cleavage rate for the 3 groups being similar. For the ESC-like cells, the cleavage and the blastocyst rate were significantly lower (P ≤ 0.01) for the singlet than that for the doublet embryos. The pregnancies were established only in doublet and triplet embryo groups using BFF cells and in the doublet embryo group using ESC-like cells. These results indicate that increasing the cytoplasmic volume could be helpful in improving cloning efficiency in terms of blastocyst production rate in buffaloes. Table 1.Effect of cytoplasmic volume on the developmental competence of cloned buffalo embryos This work was funded by NAIP grant C 2-1-(5)/2007 to SKS.

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 286-294 ◽  
Author(s):  
G. Taru Sharma ◽  
Pawan K. Dubey ◽  
Amar Nath ◽  
G. Saikumar
Keyword(s):  
Growth Factor ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Term Survival ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Epidermal Growth

SummaryThe present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200–250 μm) were isolated and cultured with or without AFs (3–5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus–oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

30 EFFECT OF THE REPEATED USE OF OPEN SYSTEM VITRIFICATION DEVICES ON MII STAGE AND CLEAVAGE RATES OF BOVINE CUMULUS-OOCYTE COMPLEXES

2016 ◽  
Vol 28 (2) ◽  
pp. 145
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Open System ◽  
High Efficiency ◽  
Beef Cows ◽  
Developmental Competence ◽  
Limiting Factor ◽  
Cleavage Rate ◽  
Exact Test ◽  
Repeated Use

Vitrification of mammalian gametes and embryos has become the cryopreservation tool of choice in research and commercial clinical programs because of its high efficiency. Vitrification relies upon high cooling rates. In this regard the use of open system vitrification devices (OSVD) provides the highest cooling-warming rates. A limiting factor of vitrification research in domestic animals is the high cost of OSVD. Reuse of these devices could be a viable alternative for cost reduction in vitrification research projects. The objective of this study was to evaluate the effect of the repeated use of OSVD on the developmental competence of bovine cumulus-oocyte complexes (COC). A 6-treatment factorial arrangement was evaluated, where factor A was number of uses of OSVD (new, one use, and two uses) and factor B was COC type (immature, mature). Cumulus-oocyte complexes were obtained by ovum pickup from crossbred nonlactating beef cows. The vitrification procedure consisted of exposure of COC to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 9 min. Afterward, COC were exposed to 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose and loaded into the tip of the OSVD (Cryolock®) with minimal volume (<1 μL) to be immediately plunged into LN within 60 s. Three to four COC were loaded per OSVD. For warming, the COC were exposed to 0.5-M (37.5°C) and 0.25-M sucrose for 2.5 min each. The base medium for all solutions was D-PBS plus 20% fetal bovine serum. A total of 266 COC were used in the study collected during 6 days (repetitions). Cumulus-oocytes complexes obtained per day were divided for immediate vitrification or for a 22-h maturation period (84.5% MII stage rate) and then vitrified. In each case COC were randomly assigned to treatments (numbers of use of OSVD) before vitrification. From each treatment warmed oocytes were divided for assessment of maturation and cleavage rate evaluation. To assess MII stage rate, COC were exposed to Hoechst 33342 (3.5 μg mL–1) for 15 min at 37.5°C and then observed under an epifluorescence microscope. A standard bovine IVF protocol was used, and cleavage rate was evaluated at 42 h following fertilization. Results of the experiment were tested by chi-square test of independence or Fisher’s exact test when required (P < 0.05). The MII stage rates (mean ± standard error) for immature vitrified COC were 82 ± 2.3, 78.2 ± 5.8, and 75 ± 5.3, and for mature vitrified COC were 69.3 ± 4.3, 88.2 ± 4.1, 96 ± 2.1, for new, one-use, and two-uses OSVD, respectively. The cleavage rates for immature vitrified COC were 31.6 ± 4.8, 38.8 ± 3.4, and 37.7 ± 1.1, and for mature vitrified COC were 31.7 ± 4.8, 34.4 ± 4.7, and 33.3 ± 7.1, for new, one-use, and two-uses OSVD, respectively. No differences in cleavage rate were found when comparing immature vitrified COC with mature vitrified COC (36.1 ± 1.7 v. 33.1 ± 3.2; P > 0.05). Results of the experiment shown that no differences were detected, and similar results in terms of maturation and cleavage rate were obtained when reusing OSVD.

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 151
Author(s):  
H. S. Canesin ◽  
I. Ortiz ◽  
J. G. Brom-de-Luna ◽  
Y. H. Choi ◽  
K. Hinrichs
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Cleavage Rate ◽  
Fetal Bovine ◽  
Vitrification Solution

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&M University.

scholarly journals Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

2011 ◽  
Vol 41 (11) ◽  
pp. 1985-1990 ◽  
Author(s):  
Paula Rodriguez Villamil ◽  
Felipe Ledur Ongaratto ◽  
Daniela Scherer da Silva ◽  
Berenice de Avila Rodrigues ◽  
Jose Luiz Rodrigues
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
The Other ◽  
Developmental Competence ◽  
Time Interval ◽  
Equilibrium Solutions ◽  
Mouse Blastocyst ◽  
In Vitro Development ◽  
Vitro Development

The aim of this study was to determine the effect of dimethylformamide (DF) associated with ethylene glycol (EG) or 1-2 propanediol (PROH) during vitrification, on the in vitro development of mouse blastocysts. Cryoprotectant toxicity was evaluated exposing embryos into three different equilibrium solutions (ES) composed by DF, EG or PROH mixtures (10% v/v of each) in mPBS + 0.5% PVA at different interval times (1, 3 and 10min). In a second experiment, embryos were exposed to the same ES (either 1 or 3min), following for the three respectively vitrification solutions (VS) (20% v/v of each) for 30s. After 72 hours of in vitro culture, embryo hatching and expansion rates were similar for the ES1 and ES2 equilibration solutions during the time interval of 1 or 3min. However embryos exposed for 10 min to the DF equilibration solutions, had lower survival rates than EG-PROH solution (P<0.01). Furthermore, survival rates for embryos exposed to DF-PROH (ES+VS) were lower than embryos exposed to the other solutions (P<0.01). Blastocyst vitrification was performed with the three ES+VS (for 1min and 30s, respectively), using glass micropipettes (GMP). Survival rates were lower for blastocysts vitrified with DF solutions (3%-3/108 and 17.1%-19/111) (P<0.01) than with PROH+EG vitrification solutions (69%-73/105). In conclusion, DF as a cryoprotectant into vitrification solutions have deleterious effects on the in vitro developmental competence of vitrified mouse blastocysts.

Extracts of forage plants affect the developmental competence of ovine oocytes in vitro

2019 ◽  
Vol 59 (10) ◽  
pp. 1814 ◽  
Author(s):  
Anna Aryani Amir ◽  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
Zoey Durmic ◽  
Dominique Blache ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
The Other ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Reproductive Process ◽  
Forage Plants ◽  
Methanolic Extracts ◽  
Blastocyst Rate

Forage plants may contain secondary compounds that disrupt reproduction in ruminants so, as ‘duty of care’, proposed new forage species need to be tested for harmful effects on reproduction before industrial release. We evaluated the effects of Bituminaria bituminosa, Medicago sativa, Chicorium intybus, Trifolium subterraneum, Trifolium pratense, Biserrula pelecinus and Eremophila glabra, on the in vitro developmental competence of ovine oocytes. Crude methanolic extracts of each plant were added to the medium (final concentrations: 0, 50 or 100 μg dry extract per mL) used for in vitro maturation of cumulus-oocyte complexes derived from abattoir-sourced adult ewe ovaries. After in vitro fertilisation, we quantified cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency, and total blastocyst cell number (TCN). Extract from B. pelecinus, at 50 μg/mL concentration, increased cleavage rate at (P &lt; 0.05), and at 100 μg/mL, increased blastocyst rate and efficiency (P &lt; 0.05). The other plant extracts did not affect these measures. TCN was affected by stage of development and treatment, but not by the interaction between stage and treatment. Within treatments, TCN was increased by C. intybus (at both 50 and 100 μg/mL) but decreased by M. sativa (at both 50 and 100 μg/mL; P &lt; 0.05). We conclude that methanolic extracts of forage plants, present during in vitro oocyte maturation, did not disrupt subsequent fertilisation and embryo development until the blastocyst stage. On the contrary, B. pelecinus appears to improve fertilisation and embryo development. Overall, these observations suggest that these plants will not disrupt in vivo oocyte maturation but further testing is still required, especially for the other stages of the reproductive process.

171 PRE-IN VITRO MATURATION OF PORCINE OOCYTES USING PITUITARY ADENYLATE CYCLASE-ACTIVATING PEPTIDE: EFFECTS ON MEIOSIS PROGRESSION AND DEVELOPMENTAL COMPETENCE

2017 ◽  
Vol 29 (1) ◽  
pp. 194
Author(s):  
K.-M. Park ◽  
S. H. Hyun
Keyword(s):  
Adenylate Cyclase ◽  
In Vitro Maturation ◽  
Reduced Glutathione ◽  
The Body ◽  
The Other ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Pituitary Adenylate Cyclase ◽  
Nuclear Stage

Porcine immature oocytes derived from small follicle (SF, ≤3 mm in diameter) are able to resume meiosis, but few oocytes are capable to progress to the metaphase 2 stage. To improve capacitation of oocytes derived from SF and inhibit spontaneous maturation, a pre-in vitro maturation (IVM) system was applied to in vitro culturing of cumulus–oocyte complexes (COC). Pituitary adenylate cyclase-activating peptides (PACAP), increasing cellular cyclic adenosine 3′5′-monophosphate, have pleiotropous actions and multiple functions throughout the body as a neuropeptide. Recently, studies have described the role PACAP play in fertility and reproduction, including follicular development, antiapoptotic effects, and implantation. The purpose of this study is to improve the developmental competence of oocytes derived from SF by exogenous addition of PACAP on pre-IVM. In the conventional IVM group, COC obtained from follicles ≤3 mm in diameter (SF group) and 3 to 6 mm in diameter (medium follicles; MF group) were subjected to IVM for 42 h. In the pre-IVM group, COC obtained from SF were matured with nontreatment [pre-SF(-)PACAP group] and 1μM PACAP [pre-SF(+)PACAP group] for 18 h pre-IVM and were immediately subjected to IVM for 42 h. We examined nuclear stage assessment, intracellular reduced glutathione and reactive oxygen species levels, and embryo developmental competence by parthenogenesis and IVF. After IVM, the result of the nuclear stage assessment of groups showed that the pre-SF(+)PACAP group had the highest metaphase 2 rate in the groups (P < 0.05). Reduced glutathione levels in MF and pre-SF(+)PACAP groups showed significantly higher levels than that of the other groups (P < 0.05). After parthenogenesis, the cleavage rates were significantly (P < 0.05) higher than the others in pre-SF(+)PACAP group. In the IVF experiment, the embryo cleavage rate was significantly higher in that of MF and pre-SF(+)PACAP groups compared with that of SF and pre-SF(-)PACAP groups (P < 0.05). Moreover, no significant differences were found in the cleavage rate between MF and pre-SF(+)PACAP groups. In all groups derived from SF, the pre-SF(+)PACAP group rate of blastocyst formation and total cell number of blastocysts was significantly higher than that of the other groups (P < 0.05). These results indicated that pre-IVM system using PACAP is able to improve meiotic maturation and developmental competence although the oocytes were derived from SF.

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