217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS-OOCYTE COMPLEXES

2016 ◽  
Vol 28 (2) ◽  
pp. 239
Author(s):  
N. G. Canel ◽  
M. Suvá ◽  
R. J. Bevacqua ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
In Vitro Maturation ◽  
Critical Role ◽  
Standard Condition ◽  
Bottom Part ◽  
Low Levels ◽  
Vitro Development ◽  
Sperm Decondensation

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 106 sperm mL–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos. Table 1.Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)

scholarly journals Combinations of Trolox and ascorbic acid have a beneficial effect on in vitro maturation of pig oocytes

2021 ◽  
Author(s):  
Ileana Miclea ◽  
Marius Zahan
Keyword(s):  
Ascorbic Acid ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Membrane Damage ◽  
High Concentration ◽  
First Polar Body ◽  
Morula Stage ◽  
Vitro Development

Abstract: The poor in vitro development of pig oocytes and embryos has been blamed on oxidative stress. We sought to find out if combinations of Trolox (T), a synthetic and cell-permeable derivative of vitamin E, and ascorbic acid (AA) could improve the maturation rates of in vitro cultured pig oocytes. Pig oocytes underwent maturation for 44–45 h in medium M 199 supplemented with 0 μM T + 0 μM AA, 100 μM T + 250 μM AA, 300 μM T + 250 μM AA, 100 μM T + 750 μM AA or 300 μM T + 750 μM AA. These combinations were chosen based on previous research conducted in our laboratory and on the available literature. After maturation, several parameters were assessed: cumulus oophorus expansion, oocyte viability (based on the presence of metabolic activity versus membrane damage), extrusion of the first polar body, mitochondrial membrane potential (MMP), pronucleus formation, and embryo development after fertilization. All antioxidant combinations significantly improved cumulus expansion and formation of the first polar body. The best was 300 μM T + 250 μM AA for the first characteristic and 300 μM T + 750 μM AA for the second. Antioxidant presence in the maturation media increased the percentages of viable oocytes but not significantly. MMP was not significantly modified by the addition of antioxidant combinations. We also found that a low concentration of T (100 µM) mixed with a high concentration of AA (750 µM) in the oocyte maturation media led to significantly higher rates of both female and male pronuclei formation and also enhanced embryo development to the morula stage. Therefore, we recommend this combination to improve the in vitro maturation media of pig oocytes.  

198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Before And After ◽  
Vitro Development ◽  
Bovine Oocytes

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

146 HEPARAN SULFATE IS INVOLVED IN NUCLEAR SPERM DECONDENSATION AFTER FERTILIZATION IN BOVINE

2017 ◽  
Vol 29 (1) ◽  
pp. 181
Author(s):  
N. G. Canel ◽  
M. Romanato ◽  
M. Suva ◽  
L. Calvo ◽  
D. Salamone ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Disulfide Bond ◽  
Intracytoplasmic Sperm Injection ◽  
Critical Role ◽  
Cumulus Cells ◽  
Final Concentration ◽  
Exact Test ◽  
Pronuclear Formation ◽  
Sperm Decondensation

Reduced glutathione (GSH) is an endogenous disulfide bond reducer present in mammalian oocytes. It plays a critical role in sperm decondensation following fertilization, disrupting the protamine bonds that sustain the hypercondensed state of sperm DNA. However, disulfide bond reduction needs to be followed by protamine removal to achieve male pronuclear formation. In humans, heparan sulfate (HS) has been shown to exert this role (Romanato et al. 2008 Hum. Reprod. 23, 1145–1450). Although there are no reports in bovine, we recently demonstrated the presence of HS in cow oocytes by indirect immunofluorescence, using a specific anti-HS monoclonal antibody (Canel et al. 2015, Proc. SSR 48th Annual Meeting). Heparinases are known to cleave HS chains selectively, leading to its depolymerization. In the present work, we analysed the possible role of HS as protamine acceptor after fertilization in cattle. To this aim, we directly injected heparinase into the cytoplasm of IVF presumptive zygotes, and analysed its effect on pronuclei formation. Cumulus-oocyte complexes were collected from slaughtered cow ovaries and matured in vitro under standard conditions (Canel et al. 2012 Cell. Div. 7, 23–33). After 21 h, IVF was performed following Brackett and Oliphant’s protocol (1975 Biol. Reprod. 12, 260–274), using frozen–thawed semen from 1 or 2 bulls at a final concentration of 15 × 106 spermatozoa/mL (5 replicates). After 5 h of incubation, cumulus cells and sperm bound to zona pellucidae were removed from presumptive zygotes. Heparinase III solution (H8891, Sigma, St. Louis, MO, USA) was diluted in 50% (vol/vol) polyvinylpyrrolidone solution in PBS-(polyvinylpyrrolidone) at a final concentration of 50 U mL−1 and ~30 pL was mechanically injected into the cytoplasm of each IVF presumptive zygote (Hep group) using a 9-μm inner diameter injection pipette. A group of zygotes was injected with the same volume of 10% polyvinylpyrrolidone (sham), whereas others were not subjected to injection (control). All zygotes were cultured for 16 h from the beginning of IVF in SOF medium (Holm et al. 1999 Theriogenology 52, 693–700). For pronuclear formation assessment, presumptive zygotes were permeabilized with 0.2% Triton X-100 for 15 min at room temperature, and their DNA content was stained with 5 µg mL−1 propidium iodide and observed under an epifluorescence microscope. Zygotes showing 2 pronuclei (PN) were considered as synchronically fertilized, whereas those showing one PN and one condensed sperm head were considered as asynchronically fertilized. Data were analysed by Fisher’s exact test (P < 0.05). The rate of IVF zygotes showing 2 PN was lower for the Hep group (60.3%, n = 131) than those from sham (94.1%, n = 119) and control groups (98%, n = 101), which did not differ between them (P < 0.05). In conclusion, our results show for the first time that HS is involved in bull chromatin sperm decondensation and allow us to propose HS as a putative protamine acceptor during male pronucleus formation after IVF in cattle. Given the high frequency of sperm decondensation failure observed in bovine after intracytoplasmic sperm injection, this work provides new insights for the development of novel sperm/egg treatments that might improve intracytoplasmic sperm injection outcomes in cattle.

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

scholarly journals Effect of adding growth factors during in vitro maturation on the developmental potentials of ewe oocytes selected by brilliant cresyl blue staining

2021 ◽  
Vol 14 (2) ◽  
pp. 452-456
Author(s):  
Mohamed Fathi ◽  
Amr F. Elkarmoty
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Nuclear Maturation ◽  
Maturation Medium

Aim: Several factors had been concerned with the developmental competence of the sheep oocyte. This study aims to investigate the effect of adding growth factors (insulin-like growth factor 1 [IGF-1] and epidermal growth factor [EGF]) in the maturation medium of ewe oocytes selected based on brilliant cresyl blue (BCB) screening on in vitro maturation (IVM), fertilization, and pre-implantation embryo development. Materials and Methods: Cumulus-oocyte complexes (COCs) were obtained from the ovaries of slaughtered ewes by either aspiration or slicing techniques. COCs were in vitro matured in a medium containing IGF-1 and EGF (control group). For BCB screening, oocytes were stained and divided into BCB+ oocytes that matured in the same maturation conditions without adding growth factors (Group 2) or in the presence of growth factors (Group 3), and BCB– oocytes that matured in medium without growth factors (Group 4) or with growth factors (Group 5). Results: The supplementation of the maturation medium with growth factors during IVM of (BCB+) oocytes resulted in a significant increase in nuclear maturation rate (90.9%), fertilization rate (75.6%), and embryo developmental rates (60.0%, 46.7%, and 33.3% for cleavage, morula, and blastocyst, respectively). Conclusion: Culturing BCB+ oocytes in a maturation medium containing both EGF and IGF-1 showed a significant improvement in nuclear maturation, fertilization, and pre-implantation embryo development in vitro.

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