158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS - OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
S.-H. Choi ◽  
S.-R. Cho ◽  
M.-H. Han ◽  
H.-J. Kim ◽  
C.-Y. Choe ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Basic Fuchsin ◽  
In Vitro Production ◽  
Control Groups ◽  
Significant Difference ◽  
Native Cattle ◽  
Vitro Development ◽  
Vitro Production

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 �g/mL apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 �g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 �g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 �g/mL and 10 �g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 �g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

180 DEVELOPMENTAL CAPACITY OF SINGLE CULTIVATED EMBRYOS IN SMALL AND BIG DROPLETS

2007 ◽  
Vol 19 (1) ◽  
pp. 206
Author(s):  
I. G. F. Goovaerts ◽  
J. B. P. De Clercq ◽  
M. Nichi ◽  
P. E. J. Bols
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Significant Difference ◽  
Group Treatments ◽  
Developmental Capacity ◽  
Droplet Sizes ◽  
The Individual ◽  
Vitro Production

An in vitro production system where a single oocyte can be followed from the ovary to the blastocyst stage would be a useful tool for studies concerning developmental competence or follicular environment. Unfortunately, until now, only low blastocyst rates could be obtained after single embryo production, and there is still discussion about the ideal droplet size. The objective of the present experiment was to compare the developmental competence of single cultivated zygotes in 20- and 500-µL droplets. Cumulus–oocyte complexes were obtained from slaughterhouse ovaries and were matured and fertilized in groups of 100 for 22 h; the presumptive zygotes were divided into 4 groups. In treatment 1, 25 zygotes were transferred into 50 µL of SOF medium supplemented with 5% serum under oil, whereas in treatment 2, 25 zygotes were transferred into 500 µL of medium. Zygotes were cultivated separately in treatments 3 and 4: in treatment 3 in 20 µL of medium under oil and in treatment 4 in 500 µL of medium. Cleavage rates and division stages were assessed after 3 days of cultivation (5% CO2, 5% O2, 90% N2); blastocyst rates were determined after 7 days. Statistical analysis was performed by logistic regression using SAS (PROC LOGISTIC). There was no difference in cleavage rates between the 2 group treatments or between the 2 single treatments. Also, the division stages were not different between the 2 single treatments (16-cell: 2.0 vs. 1.3%; 8-cell: 25.8 vs. 31.6%; 4-cell: 41.2 vs. 38.0%; and 2-cell: 31.0 vs. 29.1% for the 20 µL and the 500 µL droplet sizes, respectively). Group cultivation after 7 days in 50 µL was significantly better than in 500 µL; however, both treatments resulted in significantly higher blastocyst rates compared with the individual cultures in 20 or 500 µL, between which no significant difference could be found. Noteworthy, only 4-cell and 8-cell stages on Day 3 resulted in blastocysts on Day 7 of cultivation. In conclusion, these results indicate that cultivation in groups gives higher blastocyst rates, although the same embryo density is used as in individual cultivation (1 embryo 20 µL in treatments 2 and 3). Moreover, no significant difference could be found between single cultivation in small and big droplets. This is confirmed by the cleavage stages on Day 3, which indicate no difference in timing of cleaving between small and big droplets; time of cleaving is indicative of further developmental capacity. Table 1.Cleavage and blastocyst rates after single and group cultivation

Laboratory production of buffalo (Bubalus bubalis) embryos

1998 ◽  
Vol 10 (5) ◽  
pp. 379 ◽  
Author(s):  
P. Palta ◽  
M. S. Chauhan
Keyword(s):  
In Vitro Maturation ◽  
Large Scale ◽  
Follicle Stimulating Hormone ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Fertilization Rates ◽  
Vitro Production

There is an increasing interest in large-scale in vitro production (IVP) of buffalo embryos through in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster multiplication of superior germplasm. The recovery of total and usable quality oocytes from slaughterhouse ovaries is low in this species. The nuclear maturation rates of buffalo oocytes matured in the presence of follicular fluid or serum and hormones like luteinizing hormone, follicle-stimulating hormone and oestradiol vary from 70 to 80% and are comparable to those reported for cattle oocytes. However, with fertilization rates of 40–55%, and the yield of blastocysts at around 10–15%, the efficiency of IVP is much lower than that in cattle. The in vitro sperm preparation procedures and the systems employed for performing IVF and culture of zygotes up to blastocyst stage are suboptimal and need substantial improvements. The quality and viability of blastocysts produced need to be checked by cell count, and after transfer to synchronized recipients, for development of quality control standards.

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.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system

2003 ◽  
Vol 15 (4) ◽  
pp. 249 ◽  
Author(s):  
J. R. Herrick ◽  
M. L. Conover-Sparman ◽  
R. L. Krisher
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
High Incidence ◽  
Porcine Embryo ◽  
Medium System ◽  
Epidermal Growth ◽  
Vitro Production ◽  
Single Medium

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL−1 hyaluronan, 0.6 mM cysteine, 10 ng mL−1 epidermal growth factor (EGF), 0.1 U mL−1 porcine LH and FSH, and 1 × Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 × 105 sperm mL−1) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mm bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

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 In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

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.

163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

2004 ◽  
Vol 16 (2) ◽  
pp. 204 ◽  
Author(s):  
J. Ye ◽  
K.H.S. Campbell ◽  
M.R. Luck
Keyword(s):  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Pre Treatment ◽  
Vitro Production

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P&lt;0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P&lt;0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)

Sign in / Sign up

Export Citation Format

Share Document