53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during invitro maturation of oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 133
Author(s):  
A. Zegarra ◽  
J. Rivas ◽  
A. Gallegos ◽  
E. Mellisho
Keyword(s):  
Embryonic Development ◽  
Decisive Role ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Advanced Stages ◽  
Commercial Medium ◽  
Antioxidant Effects

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P<0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P>0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.

182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 199
Author(s):  
A. W. Harl ◽  
E. L. Larimore ◽  
A. Al Naib ◽  
L. K. Wooldridge ◽  
A. D. Ealy ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cumulus Expansion ◽  
Oocyte Competence ◽  
Maturation Medium

The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10 mm) and small follicles (≤3 mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n = 250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21 h of maturation). Other COC (n = 770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P < 0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P < 0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P < 0.05) and also less likely to develop to the blastocyst stage (P < 0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42 h after prostaglandin F2α injection. Samples from individual cows were categorized as having high oestradiol (>800,000 pg mL−1; H) or low oestradiol concentrations (<800,000 pg mL−1; L). The FF was retained for use in in vitro experiments, where it was added to maturation media (20% vol/vol). Cumulus-oocyte complexes (n = 1,775) were randomly distributed into treatments across 12 in vitro maturation/fertilization replicates (H and L, balanced within replicate; 4 replicates/cow). Each replicate included the following 3 control groups: maturation medium containing BSA without FF, maturation medium without BSA with abattoir-derived FF, and maturation medium without BSA and without FF. The COC were matured in their assigned medium for 21 h, and then all COC were subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 7 and 8 post-fertilization. Oestradiol content of the FF (H v. L) did not affect oocyte cleavage nor blastocyst rates on Day 7 or 8. The results of these studies indicate that although FF improves cumulus cell expansion during maturation in vitro, it does not result in higher rates of cleavage or blastocyst development regardless of oestradiol content.

scholarly journals Supplementation of 9-cis retinoic acid in the in vitro maturation medium increase blastocyst development rate and quality

2018 ◽  
Vol 3 (4) ◽  
pp. 516-520 ◽  
Author(s):  
Rabiul Islam ◽  
Gautam Kumar Deb ◽  
Md Ahsanul Kabir ◽  
Md Faizul Hossain Miraz ◽  
Talukder Nurun Nahar ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Media Control ◽  
Maturation Medium ◽  
Medium Increase

The 9-cis retinoic acid (9-cisRA) enhances early embryonic development in both in vitro and in vivo conditions. This experiment was conducted to evaluate the effect of supplementation of 9-cisRA in the in vitro maturation (IVM) medium on embryo development efficiency and embryo quality. For this purpose, immature cumulus oocyte complexes (COC) collected from slaughterhouse derived bovine ovaries were matured in three different IVM media (control group, DMSO group and DMSO+RA group). In the control group, base IVM medium were used without supplementation of 9-cisRA and DMSO. In the DMSO group, base IVM medium was supplemented with 0.5 μl DMSO per ml IVM medium without 9-cisRA. In DMSO+RA group, base medium was supplemented with 5 nm 9-cisRA dissolved in 0.5 μl DMSO. Data were analyzed using one way ANOVA method and means were compared using Duncan’s multiple range test. Results showed that, supplementation of 9-cisRA in the maturation medium has no effect on embryonic development uptocleavage stage. However, blastocyst development rates (P>0.01), total blastomere number (P> 0.01), number of apoptotic blastomere per blastocyst (P>0.05) and percent of apoptotic blastomere per blastocyst (P>0.05) were significantly influences by 9-cisRA. In conclusion, 9-cisRA may be supplemented into the maturation medium for increasing bovinein vitro blastocyst development efficiency and blastocyst quality.Asian J. Med. Biol. Res. December 2017, 3(4): 516-520

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

Erratum to: 264 EXOGENOUS LINOLENIC ACID IN OOCYTE MATURATION MEDIA PROMOTES NUCLEAR MATURATION AND PARTHENOGENETIC PREIMPLANTATION EMBRYONIC DEVELOPMENT IN THE GOAT

2013 ◽  
Vol 25 (1) ◽  
pp. 280
Author(s):  
A. Veshkini ◽  
A.-A. Khadem ◽  
M. Soleimani ◽  
R. Jahanbin ◽  
M. Salehi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Linolenic Acid ◽  
Oocyte Maturation ◽  
Mass Spectrometry Analysis ◽  
Control Group ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Wide Range ◽  
And Control

Dietary intakes of polyunsaturated fatty acids are thought to mediate a wide range of actions in reproductive tissues. This includes the effects on ovarian follicle and corpus luteum functions via improved energy efficiency as well as providing precursors for the synthesis of signalling molecules such as steroids and prostaglandins. An appropriate level of α-linolenic acid (ALA) in the oocyte maturation medium has been shown to induce molecular changes associated with oocyte maturation and embryo developmental competence. In that light, we hypothesised that supplementation of exogenous ALA to maturation media could enhance nuclear maturation and embryonic development in the goat. A preliminary experiment was executed to measure the level of ALA in antral follicles by gas chromatography/mass spectrometry analysis. Our results revealed that the concentration of ALA in follicular fluids ranged from 0.006 to 0.02 mg mL–1 (21.5 to 71.8 µM, with a mean of ~50 µM). To test the effect of ALA on the competence of goat oocytes to complete meiotic maturation to metaphase II and sustain embryonic development, ovaries were obtained from a local abattoir. Cumulus–oocyte complexes were recovered by the slicing method followed by selection of oocytes with a homogenous cytoplasm and at least three layers of compact cumulus cells. The cumulus–oocyte complexes were placed in maturation media supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the treatment (n = 170) and control (n = 166) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. Other groups of oocytes from both the treatment (n = 70) and control (n = 61) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After activation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated above. Four replications were performed. Differences in developmental rates were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P < 0.05 to be significant. As a result, supplementation of the maturation media with ALA did not appear to affect cumulus expansion. In contrast, IVM of goat oocytes in the presence of ALA resulted in a significantly higher maturation rate compared with maturation without ALA supplementation (66.4% v. 57.9%). Likewise, addition of ALA to the IVM medium significantly increased the rate of cleavage (60.1% v. 52.4%) and blastocyst formation (22.6% v. 14.9%), calculated from the activated oocytes. Collectively, the results of our study show that supplementation of IVM media with 50 µM ALA promotes nuclear maturation, increases cleavage rate, and results in higher blastocyst rate in goat oocytes after parthenogenetic activation. Thus, providing appropriate levels of ALA in maturation media could have beneficial effects on embryo development and reproductive efficiency in the goat.

291 NOVEL OOCYTE SHIPPING AND MATURATION MEDIUM WITHOUT CO2 GAS PHASE IMPROVES IN VITRO MATURATION AND PREGNANCY OUTCOME IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 234
Author(s):  
M. Barcelo-Fimbres ◽  
L. F. Campos-Chillon ◽  
N. R. Mtango ◽  
L. Bonilla ◽  
J. Verstegen
Keyword(s):  
Embryonic Development ◽  
Pregnancy Rates ◽  
Control Group ◽  
System Control ◽  
Co2 Gas ◽  
Oocyte Competence ◽  
N2 Atmosphere ◽  
Maturation Medium ◽  
Medium System

The aim of the present work was to evaluate embryonic development after shipping and maturation of bovine cumulus oocyte complexes (COC) collected by ovum pick up (OPU) in medium (SMM) that does not require CO2 gas for transport and maturation. Two experiments were conducted, experiment 1 stimulated nonlactating Holstein (n = 4), Jersey (n = 2), Angus (n = 4), and Wagyu (n = 2) donors with 6 pFSH injections (Pluset, MOFA Global LLC, Verona, WI, USA) were used. From each donor, some OPU sessions were delivered the same day (~3 h after collection) for IVM in conventional gas bicarbonate-equilibrated medium system (control), while COC from the other sessions were placed in a portable incubator at 38.5°C, delivered the next day allowing 24 h of maturation in SMM (BoviPro, Mofa Global, WI, USA). The COC were fertilized using commercial semen for each breed, and embryos were cultured in BBH7 medium (BoviPro, Mofa Global, WI, USA) at 38.5°C in 5% O2, 5% CO2, and 90% N2 atmosphere. Embryonic development was evaluated in this experiment. For experiment 2, Day 7 fresh Holstein and Jersey embryos (n = 610) from SMM (n = 550) and controls (n = 60) were transferred in synchronized virgin heifers and pregnancies were diagnosed by ultrasonography at d 35. Data were analysed by ANOVA using GLM, percentages were transformed using arcsin square root, and pregnancy rates were analysed by GenMod using SAS statistical software (Cary, NC, USA). Similar COC numbers were recovered for maturation treatments (P > 0.1; Table 1). The COC matured in SMM had higher cleavage and blastocyst rates than the control group (P < 0.01; Table 1), and this resulted in more transferable embryos per OPU session (P < 0.05; Table 1). We did not find breeds effects or interactions for any variable (P > 0.1; Table 1). After ET, SMM had similar pregnancy rates than control (53.8 v. 58.3%; P > 0.1); however, as more blastocysts were produced per OPU session in the SMM condition, more pregnancies were obtained per session (4.3 v. 2.1; P < 0.01). We conclude that COC matured in SMM had greater oocyte competence than control in commercial settings. The SMM resulted in greater embryonic development, similar pregnancy rates, but more transferable embryos and pregnancies per OPU session than the conventional maturation system. Table 1.Least squares means (± SE) of embryonic development of COC matured in SMM or control

142 EFFECT OF HIGH AND LOW ANTRAL FOLLICLE COUNT IN PUBERTAL BEEF HEIFERS ON IVF

2014 ◽  
Vol 26 (1) ◽  
pp. 184
Author(s):  
C. C. Chase ◽  
E. C. Wright ◽  
A. K. McNeel ◽  
R. A. Cushman ◽  
G. A. Perry ◽  
...  
Keyword(s):  
Prostaglandin F2α ◽  
Antral Follicle ◽  
Antral Follicle Count ◽  
Blastocyst Stage ◽  
Oestrous Cycle ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Frozen Semen ◽  
Oocyte Collection ◽  
Collection Medium

Pubertal heifers can be classified between those with high (n = 25) or low (n = 15) antral follicle counts (AFC). The objective of this study was to determine oocyte development and maturation (e.g. fertility) in an IVF system for high- and low-AFC heifers. From a pool of 120 heifers, 10 high- and 10 low-AFC heifers were determined by transrectal ultrasonography; all heifers with evidence of oestrous cyclicity (i.e. pubertal) were synchronized with two 5-mL injections of prostaglandin F2α 11 days apart. Heifers were euthanized over 4 days on Days 15 to 16 of the synchronized oestrous cycle. A total of 15 heifers (n = 7 high and n = 8 low AFC) were at the appropriate stage of the oestrous cycle. Ovaries were collected and transported to the laboratory. Follicles less than 8 mm in diameter were aspirated. The IVF procedures and media were as previously described (Miles et al. 2004. Biol. Reprod. 71, 1919–1926). Cumulus-oocyte complexes (COC) were identified and washed in oocyte collection medium and then in maturation medium and were cultured (5% CO2; 38.5°C) for 24 h. Following maturation, COC were transferred and washed in fertilization medium. Thawed frozen semen from a crossbred bull was subjected to the swim-up procedure. Motile spermatozoa were collected and added to COC to yield a final concentration of spermatozoa per milliliter of fertilization medium. About 24 h later, presumptive zygotes were washed in development medium, placed in microdrops of development medium, and cultured for 8 days. On Days 3 and 8 after fertilization, cleavage and blastocyst development, respectively, were assessed. Data were analysed using the Proc Mixed procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of day of collection (n = 4), group (n = 7 high- or n = 8 low-AFC heifers), and the interaction. The interaction did not differ (P = 0.10). Day of collection influenced (P < 0.05) the number of COC and the number of oocytes cleaved. High- compared to low-AFC heifers had the greater (P < 0.05) numbers of COC (42.7 ± 4.66 v. 22.1 ± 4.59), oocytes that cleaved (28.1 ± 3.60 v. 15.9 ± 3.55), and developed to blastocysts (13.2 ± 1.71 v. 6.2 ± 1.69). However, there was no difference (P > 0.10) in the percentage of COC that cleaved (65.3 ± 5.58 v. 66.2 ± 5.50%, high v. low, respectively) or that developed to blastocysts (46.7 ± 6.75 v. 42.2 ± 6.65%). In conclusion, AFC did not appear to affect oocyte maturation and development through the blastocyst stage.

scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

scholarly journals Antioxidant Effects of Ocimum Gratissimum Leaf Essential Oils as a Supplement to Extender on Chilled Canine Sperm Quality

2020 ◽  
Author(s):  
Vui Van Nguyen ◽  
Samorn Ponchunchoovong ◽  
Sajeera Kupittayanant ◽  
Pakanit Kupittayanant
Keyword(s):  
Essential Oils ◽  
Sperm Quality ◽  
Antioxidant Properties ◽  
Quality Parameters ◽  
Control Group ◽  
Dependent Manner ◽  
Ocimum Gratissimum ◽  
Significant Difference ◽  
Antioxidant Effects

Abstract Background:Oxidative stress during chilled storage is a major problem withcanine sperm. To improve the quality of chilled canine sperm during storage, many synthetic antioxidants have been examined, but different outcomes were investigated depending on antioxidant properties. The bioactive compounds of essential oils fromOcimum gratissimumleaves are known as a natural antioxidant source. This study aimed to evaluate the antioxidant effects of essential oils from Ocimum gratissimumleavesas a supplement in extender on chilled canine sperm during 12 days of storage. Results:The results showed thatlow concentrations of Ocimum gratissimum essential oils (25, 50, and 100µg/mL) have beneficial effectson sperm quality, whereasOcimum gratissimumessential oils athigh levels (above 200µg/mL) have harmful effects. Specifically, the addition of 100µg/mL ofOcimum gratissimum essential oilsto the extender had the greatestbeneficial effect in improving the quality of chilled canine sperm, and had a significant difference in all sperm quality parameters except motility when compared to the control group (p<0.05). Conclusions:Ocimum gratissimum essential oilshave an impact on chilled canine sperm quality in a dose-dependent manner, and the best results areachieved with a maximum dose ofOcimum gratissimum essential oils of 100µg/mL.

309 ADDITION OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN MATURATION MEDIUM IMPROVED IN VITRO MEIOTIC COMPETENCE OF OVINE OOCYTES AND SUBSEQUENT PARTHENOGENETIC DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 243
Author(s):  
A. H. Abazari-kia ◽  
A. Mohammadi-Sangcheshmeh ◽  
M. Salehi ◽  
M. Zhandi
Keyword(s):  
Embryonic Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Culture Conditions ◽  
Control Group ◽  
P Value ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
And Control

Overall efficiency of in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition, and supplementation. Brain-derived neurotrophic factor (BDNF), which is a physiologically important neurotrophin, has been used to enhance oocyte maturation in some previous studies (Lee et al. 2007; Zhang et al. 2010). However, the efficacy of BDNF to improve oocyte competence has not been fully established especially in ovine. Therefore, the present study aimed to evaluate the effect of BDNF during in vitro maturation (IVM) on maturation rate, intracellular glutathione (GSH) content, and embryonic development in sheep oocytes. Cumulus-oocyte complexes (COC) were obtained from ovaries of ewes. The COC were placed in maturation medium supplemented with either 10 (IVM-B10) or 100 (IVM-B100) ng mL–1 of BDNF (PeproTech, London, UK). Oocytes in control group were incubated in the same maturation medium without BDNF. The IVM was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the IVM-B10 (n = 110), IVM-B100 (n = 124), and control (n = 110) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. To measure GSH content, several oocytes from the IVM-B10 (n = 28), IVM-B100 (n = 33), and control (n = 37) groups were incubated in tyrodes medium containing 10 µM Cell Tracker blue for 30 min and transferred under fluorescence microscope, with digital images analysed by image J software. To evaluate the embryonic development, several oocytes from IVM-B10 (n = 145), IVM-B100 (n = 137), and control (n = 143) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After stimulation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated previously. Four replications were performed. The metaphase-II rate, cleavage, and blastocyst rates were compared by x2 analysis. The GSH content was analysed by one-way ANOVA. A P-value of less than 0.05 was considered significant. The results showed that metaphase-II rate was higher in the IVM-B100 group (88.7%), as compared with the control group (77.3%), but not significant as compared with that in the IVM-B10 group (84.5%). No difference was also found between the IVM-B10 group and control group in terms of the metaphase-II rate. Oocytes in the IVM-B10 group revealed a higher (96.8%) GSH content than both of the IVM-B100 (86.9%) and control (86.3%) groups. There was, however, no difference in the GSH content between the IVM-B100 group and control group. The proportion of cleaved embryos was not different between the groups; however, the blastocyst rate was higher in both the IVM-B10 (37.9%) and IVM-B100 (39.3%) groups compared with the control group (22.4%). Collectively, the results of this study showed that supplementation of IVM media with BDNF promoted nuclear maturation, increased GSH content, and stimulated in vitro embryonic development in ovine.

286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
B. Agung ◽  
T. Otoi ◽  
D. Fuchimoto ◽  
S. Senbon ◽  
A. Onishi ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Control Group ◽  
Rotating System ◽  
Vitro Fertilization ◽  
Maturation Medium

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P &lt; 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P &lt; 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

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