scholarly journals 257 EFFECTS OF GLUTAMINE AND HYPOTAURINE ON OXIDATIVE STRESS OF PORCINE EMBRYOS CULTURED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 278 ◽  
Author(s):  
C. Suzuki ◽  
K. Yoshioka
Keyword(s):  
Oxidative Stress ◽  
Subsequent Development ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
H 41 ◽  
H2o2 Level ◽  
Vitro Development ◽  
Vitro Production ◽  
Number Of Cells

We previously developed an in vitro production system for porcine embryos and reported that the addition of glutamine and hypotaurine during in vitro culture improved blastocyst yield and the total number of cells in the blastocysts. Glutamine and hypotaurine might reduce oxidative stress, allowing the development of embryos cultured in vitro, because glutamine reportedly protects embryos against oxidative stress by helping to maintain intracellular levels of cysteine, a precursor of glutathione (GSH), and hypotaurine is a potent antioxidant. In the present study we evaluated the effects of the presence of glutamine and hypotaurine from Day 2 (Day 0 = the day of in vitro fertilization) to Day 3 on oxidative stress during in vitro development of porcine embryos. Porcine cumulus-oocytes complexes from prepubertal gilts were matured and fertilized in vitro using frozen-thawed ejaculated boar semen (Yoshioka et al. 2003 Biol. Reprod. 69, 2092–2099). Presumptive zygotes were cultured in porcine zygote medium (PZM)-5 (Suzuki et al., 2002) containing 2 mM of glutamine and 5 mM of hypotaurine as a basal culture medium until Day 2. The cleaved embryos were then transferred into one of four media prepared as follows: (1) containing no glutamine or hypotaurine (G−H−), (2) containing glutamine (G+H−), (3) containing hypotaurine (G−H+), (4) containing glutamine and hypotaurine (G+H+) (= PZM-5), and cultured for 24 h. After culture, the total number of cells, intracellular GSH content, and level of hydrogen peroxide (H2O2), which is a reactive oxygen species, in the cleaved embryos were examined. Some cleaved embryos were cultured in PZM-5 from Day 3 until Day 5 and the percentage of embryos that developed into blastocysts and the total number of cells in the blastocysts were investigated. Intracellular GSH content and H2O2 level on Day 3 were determined by a dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay and dichlorohydrofluorescein diacetate (DCHFDA)-based assay, respectively. Data were statistically analyzed by ANOVA and Fisher's PLSD test. The total number of cells (4.3 to 4.4 cells) and intracellular GSH content (2.3 to 2.9 pmol/embryo) in the cleaved embryos on Day 3 did not differ among treatments. On Day 3, the intracellular H2O2 level of the cleaved embryos cultured in G+H+ decreased by 49% compared with those cultured in G−H− (100%) (P < 0.05). On Day 5, the percentage of embryos that developed into blastocysts in G+H+ (52%, 47/90) and G+H− (41%, 36/88) was significantly higher than in G−H− (11%, 11/90) and G−H+ (21%, 19/89) (P < 0.05). The total number of cells in the Day 5 blastocysts from G+H+ (34.5 cells) was significantly (P < 0.05) greater than in those from G−H− (25.8 cells). These results suggest that the presence of glutamine and hypotaurine in PZM-5 from Day 2 to Day 3 improves the subsequent development of porcine embryos into blastocysts by reducing intracellular H2O2 levels. This work was supported by MAFF, Japan.

scholarly journals Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

2020 ◽  
Vol 18 (2) ◽  
pp. 249-255
Author(s):  
Nguyen Viet Linh ◽  
Nguyen Thi Hiep
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
Vitro Production ◽  
Normal Fertilization

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

scholarly journals In vitro development of sugarcane seedlings using ethephon or gibberellin

2018 ◽  
Vol 8 (2) ◽  
pp. 389-395
Author(s):  
Luciane De Siqueira Mendes ◽  
Marcia Eugenia Amaral Carvalho ◽  
Willian Rodrigues Macedo ◽  
Paulo Roberto de Camargo e Castro
Keyword(s):  
Gibberellic Acid ◽  
Dry Mass ◽  
In Vitro Production ◽  
In Vitro Development ◽  
Reduced Height ◽  
Potential Strategy ◽  
Vitro Development ◽  
Vitro Production ◽  
Different Doses

The use of plant growth regulators is directly related to the success of in vitro propagation, which is an advantageous alternative to obtain seedlings on a commercial scale. This study aimed to evaluate the in vitro development of ‘IAC 95-5000’ sugarcane seedlings after the addition of different doses of ethephon (0, 25, 50, 100 and 200 mg L-1) or gibberellic acid (0, 2.5, 5.0, 7.5 and 10.0 mg L-1) to the culture medium. Ethephon increased the number of tillers (up to 231.70%), reduced height of the main tiller (44.66 to 60.47%), and did not affect the shoot´s fresh and dry mass. On the other hand, gibberellin decreased the number of tillers and negatively changed biomass partitioning. It is concluded that the use of ethephon is a potential strategy to enhance in vitro production of ‘IAC 95-5000’ sugarcane seedlings, since it increased the number of usable shoots in subsequent subcultures, and its effects on height reduction can be reversible. However, the use of the tested doses of gibberellic acid is not recommended, because it impaired seedling development of this sugarcane variety.

scholarly journals Modern achievements of the development of scientific research at obtaining in vitro sheep embryos

2020 ◽  
Vol 22 (93) ◽  
pp. 60-68
Author(s):  
O. M. Sharan ◽  
V. Yu. Stefanyk ◽  
S. G. Shalovylo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Culture ◽  
Biologically Active ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Key Factor ◽  
Vitro Production

New literature data on research aimed at improving the in vitro production of sheep embryos presents in the article. An analysis of the achievements of scientists from different countries to increase the efficiency of the main stages of embryo production in vitro: maturation of oocytes in vitro, their in vitro fertilization and in vitro embryo culture. In the literature experience has shown that the efficiency of oocyte maturation in vitro is significantly influenced by the experience and qualifications of scientists, the age of the egg donor, the improvement of the environment by adding roscovitin to inhibit meiosis, α-linolenic acid, cerium dioxide nanoparticles (CeO2 NPs) and sericin to accelerate nuclear maturation and increase the number of oocytes of the second meiotic metaphase (MII). The main factors influencing the effectiveness of in vitro fertilization have been identified, and the parameters of the limited time of fertilization ability of sperm and the ability of oocytes to fertilize, which is called the “fertile span”, have been determined. The main effective medium that increases the effectiveness of in vitro fertilization – synthetic oviduct fluid (SOF) with the addition of heparin and serum of cattle or sheep. The main parameters of sheep embryo culture in vitro are presented with the definition of the most commonly used media and their influence on embryonic development. Potential ways to improve the production of sheep embryos in vitro with the determination of morphological evaluation of categories of oocytes, methods of synchronization of their maturation in vitro are also highlighted. At the same time, literature data on the synchronization of oocyte-cumulus complexes with the use of a large number of inhibitors of meiotic division are presented, which according to many scientists may be a key factor in improving the efficiency of sheep embryo production in vitro. In addition, the results of studies of many scientists on the expansion of the fertile gap of oocytes of sheep cultured in vitro using certain biologically active substances were analyzed. In conclusion, the prospect of using the technology of in vitro production of sheep embryos in biomedical research is highlighted.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

scholarly journals 281REDUCTION OF POLYSPERMY IN PORCINE IN VITRO FERTILIZATION BY MODIFIED SWIM-UP METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 260
Author(s):  
C.-H. Park ◽  
B.-S. Koo ◽  
J.-I. Yun ◽  
M.-G. Kim ◽  
S.-G. Lee ◽  
...  
Keyword(s):  
Sperm Concentration ◽  
Fertilization In Vitro ◽  
In Vitro Production ◽  
Common Procedure ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
A Cell ◽  
The Difference ◽  
Vitro Production

In vitro production (IVP) of porcine embryos facilitates research related to biotechnology and biomedicine. Even though many attempts have been made to optimize the IVP of porcine embryos, the outcome is still unsatisfactory compared to other species, such as mouse and cattle. The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for fertilization in vitro involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Porcine oocytes were aspirated from ovaries and matured. Sperm pellet was prepared in the tube and mature oocytes were placed on a cell strainer with 70-μm pore size (Falcon 2350) at the top of the tube. After fertilization, the oocytes were fixed and stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While penetration rates were similar in both methods (86.67±2.36% to 83.33±1.36%), there was a significant reduction of polyspermy in the modified swim-up method (17.50±1.60%) compared to the control (44.1±3.70%) (P&lt;0.05). Subsequent culture showed higher rate of blastocyst formation in the modified swim-up method (20.44±0.99%) than in the control (15.73±3.26%) (P&lt;0.05), even though the difference was not significant. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure is required to impliment this method in routine porcine IVF.

scholarly journals EFFECT OF THE USE OF TWO SPERM SELECTION TECHNIQUES FOR IN VITRO PRODUCTION OF ALPACA EMBRYOS

SPERMOVA ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 67-72
Author(s):  
Mijail Contreras Huamani ◽  
◽  
Mary Naveros ◽  
Cesar Olaguivel
Keyword(s):  
In Vitro Fertilization ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Medium ◽  
Blastocyst Rate ◽  
Vitro Production

The objective of this research was to evaluate the effect of the use of two sperm selection techniques for in vitro production of alpaca embryos. The ovaries and testis were collected from the local slaughterhouse and transport to 37 ° C in saline solution (0.9%) supplemented with gentamicin. Quality I, II and II oocytes were incubated in a maturation medium for 32 h at 38.5 ° C and 5% O2 and 5% CO2. For in vitro fertilization, sperm from the epididymis were selected using the Percoll gradient and Swim up technique. 18h after the oocytes were incubated with the sperm, these were denuded from the cumulus cells and cultured in SOFaa culture medium for 7 days. Morula and blastocyst rate and their morphological quality are evaluated at day 7 of culture. From a total of 370 ovaries, 1,137 oocytes were recovered, making an average of 3.6 oocytes / ovary. After the maturation and fertilization process and in vitro culture, the blastocyst rate was 8.43 ± 6.04% and 3.89 ± 1.75%, for oocytes fertilized with sperm selected with Percoll gradient and Swim up, respectively, not finding significant statistical differences (p> 0.05), between the groups. In conclusion, the in vitro fertilization of alpaca oocytes with spermatozoa selected with two selection techniques (percoll and swim up) did not significantly influence the quantity and quality of morulae and blastocysts at day 7 of embryo culture.

​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

2021 ◽  
Author(s):  
H.Z. Guerrero-Gallego ◽  
G. Calderon-Leyva ◽  
O. Angel-Garcia ◽  
J.M. Guillen-Muñoz ◽  
C. Leyva ◽  
...  
Keyword(s):  
Holstein Cows ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Follicular Aspiration ◽  
Four Seasons ◽  
Aspiration Technique ◽  
Vitro Production ◽  
Intense Heat

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

scholarly journals N-(2-mercaptopropionyl)-glycine enhances in vitro pig embryo production and reduces oxidative stress

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
J. M. Cambra ◽  
C. A. Martinez ◽  
H. Rodriguez-Martinez ◽  
E. A. Martinez ◽  
C. Cuello ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Culture Medium ◽  
Control Culture ◽  
Related Gene ◽  
In Vitro Production ◽  
Embryo Culture Medium ◽  
Porcine Embryo ◽  
Vitro Production ◽  
Oxidative Stress Related Gene

Abstract This study evaluated the effects of different concentrations (1, 10, 25, 50, and 100 µM) of the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG), during the culture of in vitro-fertilized porcine oocytes. While the highest concentrations of NMPG (50 and 100 µM) were toxic to the developing embryos during the first two days of culture, 25 µM NMPG achieved cleavage rates that were similar to those achieved by the control but did not sustain blastocyst production by Day 7 of culture. Compared to the control culture medium, the culture medium supplemented with 10 µM NMPG increased (P < 0.05) the rates of blastocyst formation, decreased (P < 0.05) the intracellular levels of reactive oxygen substances, and downregulated (P < 0.05) the expression of the oxidative stress related gene GPX1. In conclusion, these results demonstrated that supplementation of porcine embryo culture medium with 10 µM NMPG can attenuate oxidative stress and increase the yield of in vitro production of blastocysts.

scholarly journals In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

2017 ◽  
Vol 20 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A.E. Abdelkhalek ◽  
Sh.A. Gabr ◽  
W.A. Khalil ◽  
Sh.M. Shamiah ◽  
L. Pan ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Epididymal Spermatozoa ◽  
Vitro Production

Abstract Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

scholarly journals Melatonin, Its Beneficial Effects on Embryogenesis from Mitigating Oxidative Stress to Regulating Gene Expression

2021 ◽  
Vol 22 (11) ◽  
pp. 5885
Author(s):  
Dmitry Ivanov ◽  
Gianluigi Mazzoccoli ◽  
George Anderson ◽  
Natalia Linkova ◽  
Anastasiia Dyatlova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hormonal Regulation ◽  
Embryo Implantation ◽  
Antioxidant Properties ◽  
Subsequent Development ◽  
Vitro Fertilization ◽  
Beneficial Effects ◽  
Multi Stage ◽  
Key Factor

Embryogenesis is a complex multi-stage process regulated by various signaling molecules including pineal and extrapineal melatonin (MT). Extrapineal MT is found in the placenta and ovaries, where it carries out local hormonal regulation. MT is necessary for normal development of oocytes, fertilization and subsequent development of human, animal and avian embryos. This review discusses the role of MT as a regulator of preimplantation development of the embryo and its implantation into endometrial tissue, followed by histo-, morpho- and organogenesis. MT possesses pronounced antioxidant properties and helps to protect the embryo from oxidative stress by regulating the expression of the NFE2L2, SOD1, and GPX1 genes. MT activates the expression of the ErbB1, ErbB4, GJA1, POU5F1, and Nanog genes which are necessary for embryo implantation and blastocyst growth. MT induces the expression of vascular endothelial growth factor (VEGF) and its type 1 receptor (VEGF-R1) in the ovaries, activating angiogenesis. Given the increased difficulties in successful fertilization and embryogenesis with age, it is of note that MT slows down ovarian aging by increasing the transcription of sirtuins. MT administration to patients suffering from infertility demonstrates an increase in the effectiveness of in vitro fertilization. Thus, MT may be viewed as a key factor in embryogenesis regulation, including having utility in the management of infertility.

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