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.

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 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.

Polyspermic penetration in porcine IVM - IVF systems

2003 ◽  
Vol 15 (3) ◽  
pp. 167 ◽  
Author(s):  
Hiroaki Funahashi
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Final Maturation ◽  
Morphological Differences ◽  
Vitro Production ◽  
Boar Spermatozoa

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

166 MATURATION OF BOVINE OOCYTES IN POLY(DIMETHYLSILOXANE) MICROWELLS AND THEIR SUBSEQUENT DEVELOPMENT FOLLOWING IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 197
Author(s):  
K. Saeki ◽  
D. Iwamoto ◽  
S. Taniguchi ◽  
M. Kishi ◽  
N. Kato
Keyword(s):  
Oocyte Maturation ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
High Humidity ◽  
Bovine Oocyte ◽  
Vitro Fertilization ◽  
Maturation Medium ◽  
Normal Fertilization

During bovine oocyte maturation, a lower density of cumulus cells surrounding oocytes reduces the developmental competence of the oocytes after IVF. Adding more cumulus cells (Hashimoto et al. 1998) rescues the developmental competence of the corona-enclosed oocytes. In this study, we examined the effects of poly(dimethylsiloxane) (PDMS) microwells (MW) for bovine oocyte maturation on the developmental competence of the oocytes following IVF. In experiment 1, MW were produced by making holes on 0.5-mm-thick PDMS plates using a 0.5-mm-diameter biopsy punch. The punched plates were placed on the bottoms of culture dishes. Bovine cumulus oocytes complexes (COC) were collected from slaughterhouse ovaries. Cumulus layers were removed from COC to prepare corona-enclosed oocytes (CEO) and denuded oocytes (DO). Then, COC, CEO, or DO were individually matured in single MW for 24 h at 39°C under 5% CO2 in air with high humidity. Ten oocytes of each group were matured in 50-μL droplets of maturation medium (group culture, GC) as controls. Maturation medium was TCM-199 supplemented with 10% FCS, 0.02 AU mL–1 FSH, and 1 μg mL–1 E2. The matured oocytes were fertilized with frozen–thawed spermatozoa. The embryos were cultured in CR1aa medium for 168 h under 5% CO2, 5% O2 and 90% N2 with high humidity. In experiment 2, effects of depth of MW for maturation on subsequent development following IVF were examined. Microwells were produced by making 0.5-mm-diameter holes on 0.5- or 1.5-mm-thick PDMS plates. Then, COC or CEO were individually matured in the MW for 24 h. Matured oocytes were fertilized in vitro and cultured for 168 h. Oocytes that were matured by GC were used as controls. In experiment 1(N = 4), rates of maturation (76–100%, n = 26 to 38), normal fertilization (53–70%, n = 44 to 49), and cleavage (61–77%, n = 114 to 117) were not different among all groups (P > 0.05; Fisher's PLSD test following ANOVA). Blastocyst rates were the same (P > 0.05) for COC matured in MW (50%) and by GC (43%). The rate for CEO that matured in MW (46%) tended to be higher (P = 0.061) than the rate for CEO that matured by GC (31%), and was comparable to the rate for COC matured by GC (43%). The blastocyst rates for DO that matured in MW and by GC were low (6%). In experiment 2 (N = 3), rates of maturation (86–100%, n = 13 to 28), normal fertilization (60–78%, n = 22 to 40), and cleavage (67–73%, n = 85 to 90) were not different among all groups (P > 0.05). However, the blastocyst rate for COC that matured in 1.5-mm-deep MW (53%) was significantly higher than the rates for COC that matured in 0.5-mm-deep MW (38%) and by GC (31%; P < 0.05). The results indicate that the developmental competence of oocytes that matured individually in PDMS MW was greater than that of oocytes that matured by GC. The deeper (1.5 mm) MW were found to be more effective for oocyte maturation than shallow (0.5 mm) MW and GC. The MW might increase density of cumulus cells surrounding oocytes, and the high cell-density enhanced the developmental competence of the oocytes.

232 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 264
Author(s):  
R. R. D. Maziero ◽  
M. D. Guastali ◽  
M. J. Sudano ◽  
D. M. Paschoal ◽  
P. N. Guasti ◽  
...  
Keyword(s):  
Linear Models ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Negative Effect ◽  
Vitro Production ◽  
Co2 Atmosphere

Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle’s salts + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in the presence of 25 µM BL-I + 6.25 µM ROS. The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO2 atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3 ± 3.74%; BL-I + ROS, 38.0 ± 4.5%. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.

scholarly journals In Vitro Production Of Goat Embryos In Bangladesh

1970 ◽  
Vol 37 (1) ◽  
pp. 1-9
Author(s):  
A Mondal ◽  
MAMY Khandoker ◽  
MA Mondal ◽  
AHMS Rahman ◽  
AS Apu ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Culture Condition ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Type I ◽  
Type Ii ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Vitro Production

The present study was undertaken to collect the quality cumulus-oocyte-complexes (COCs) from ovaries of goat from slaughterhouse by aspiration to establish the suitable culture condition for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Follicular COCs were collected from follicles of 2-6 mm diameter, categorized by microscopic observation and cultured for 22 h in TCM-199 medium supplemented with 5% fetal calf serum (FCS) to determine the success rate of in vitro maturation in a condition of 5% CO2 in air at 38.5°C. The collected ovaries were classified as type-I (corpus luteum absent) and type-II (corpus luteum present). The average numbers of follicles aspirated per ovary were 3.15 and 2.57 in type-I and type-II, respectively. The collected COCs were classified into normal COCs (grade A and B) and abnormal COCs (grade C and D). The number of normal and abnormal COCs collected from two type of ovaries were significantly (P<0.01) differed. Average number of normal COCs per ovary obtained from type-I (1.30) was significantly (P<0.01) higher than that of type-II (0.68). Within the normal COCs significantly (P<0.01) higher maturation was obtained in grade A COCs (71.70%) than that of grade B (51.52%). The matured COCs were cultured for 5 h with fresh buck semen in Brackett and Oliphant (BO) medium and assumed that the COCs were fertilized successfully. In progress, IVC was practiced in TCM-199 supplemented with FCS and bovine serum albumen (BSA) at 38.5°C with 5% CO2 for 6-7 days. The rate of development to compact morula was found significantly (P<0.01) higher in grade A (25.64%) compared to grade B COCs (6.89%) and similar trend of blastocyst was found in grade A COCs (12.82%) than that of grade of B (3.45%). The results suggested that culture condition for IVM, IVF and IVC was found optimum and grade A COCs might be suitable for in vitro production (IVP) of goat embryos.DOI: http://dx.doi.org/10.3329/bjas.v37i1.9859 BJAS 2008; 37(1): 1-9

scholarly journals Stimulatory Role of Glycated Fetal Bovine Serum Along with Iron on In Vitro Production of Insulin by Differentiated Mouse Embryonic Stem Cells

2011 ◽  
Vol 57 (4) ◽  
pp. 356-361
Author(s):  
Ikuo Nishigaki ◽  
Gowri Rangasamy Gunassekaran ◽  
Panjan Nagappan Venkatesan ◽  
Mandupal Chaco Sabu ◽  
Sabu Priya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Embryonic Stem ◽  
In Vitro Production ◽  
Fetal Bovine ◽  
Vitro Production

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.

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