scholarly journals Effect of Bali cattle ovarian status on oocytes nuclear maturation and in vitro fertilization rate

2018 ◽  
Vol 22 (4) ◽  
pp. 173 ◽  
Author(s):  
Herry Sonjaya ◽  
M. Yusuf ◽  
A. Hamdana ◽  
Renny Fatmyah Utamy ◽  
Sri Gustina ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fertilization Rate ◽  
Reproductive Status ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Fetal Bovine ◽  
Bali Cattle ◽  
Pronuclear Formation

<p>The aim of this study was to investigate whether the reproductive status influences the nuclear maturation and fertilization rates of bali cattle oocytes in vitro. Several pairs of ovary were classified into four groups: 1) ovaries with Corpus Luteum (CL) and Dominant Follicle (DF), 2) ovaries without CL and with DF, 3) ovaries with CL and without DF, 4) ovaries without both CL and DF. In the first experiment, oocytes were collected by slicing method in Phosphate Buffer Saline (PBS) medium supplemented with 10% Fetal Bovine Serum (FBS) and 100 IU/ml penicillin streptomycin. Oocytes were matured in tissue culture medium (TCM)-199 supplemented with 10% Fetal Bovine Serum (FBS), 10 IU/ml Follicle Stimulating Hormone (FSH), 10 IU/ml Luteinizing Hormone (LH), and 50 μg/ml gentamycin. Oocytes were matured in 5% CO2 incubator, 38oC for 24 h. In the second experiment, oocytes were matured and then fertilized in vitro to observe pronuclear formation. The first experiment showed that the percentage of oocytes reached methaphase-II (MII) stage on ovaries with CL and without DF (89.47%) were higher (P&lt;0,01) compared to ovaries without both CL and DF (75,47%), ovaries without CL and with DF (74.,41%), or ovaries with CL and DF (65,52%). The result of second experiment showed that the ovarian reproductive status was not significantly different (P&gt;0.05) on fertilization rate.</p>

scholarly journals Fetal bovine serum is associated with polar body degeneration after in vitro maturation of bovine oocytes

2018 ◽  
Vol 68 (3) ◽  
pp. 279
Author(s):  
B. MACÍAS-GARCÍA ◽  
S. MACEDO ◽  
A. ROCHA ◽  
L. GONZÁLEZ-FERNÁNDEZ
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Polar Body ◽  
Chromatin Conformation ◽  
Prolonged Incubation ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
Bovine Oocytes

In vitro fertilization (IVF) in cattle is commonly used worldwide. Although extensive research has been conducted using different additives in the different IVF steps, little is known regarding how protein type may affect bovine oocytes during the fertilization period. In addition, unlike Tissue Culture Medium 199 (TCM), fertilization medium may induce oocytes’ chromatin degeneration during prolonged incubation in the horse (Modified Whitten’s medium). Thus, in the present work TCM-199 supplemented with either 7 mg/ml of Bovine Serum Albumin (TCM+BSA) or 10% Fetal Bovine Serum (v/v; TCM+FBS) was used. Bovine oocytes were matured in vitro and placed in the previously mentioned media for further 18 hours, in the absence of added sperm (sham fertilization) and their chromatin conformation was evaluated. After IVM, 78.9% of the initial oocytes had reached the MII stage. After sham fertilization, 58.6% of the oocytes in TCM+BSA while just 28.3% in TCM+FBS maintained the MII chromatin conformation (p < 0.05). Subsequent experiments run using PB extruded oocytes and incubated in TCM+BSA and TCM+FBS during sham fertilization, demonstrated that FBS was consistently associated with polar body dissolution or degeneration.

177 EFFECT OF DIFFERENT FERTILIZATION MEDIA ON IN VITRO BOVINE EMBRYO DEVELOPMENT USING FLOW-CYTOMETRICALLY-SORTED FEMALE SPERM

2014 ◽  
Vol 26 (1) ◽  
pp. 203
Author(s):  
L. B. Ferré ◽  
Y. S. Bogliotti ◽  
J. L. Chitwood ◽  
P. J. Ross
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Cumulus Cell ◽  
Bovine Embryo ◽  
Vitro Fertilization ◽  
Female Sex ◽  
Fetal Bovine

High demand exists for in vitro-derived bovine embryos fertilized with female sex-sorted sperm by seedstock and commercial cattle producers. The aim of this study was to evaluate different fertilization media on in vitro fertilization performance using female sex-sorted semen. Ovaries were collected from a slaughterhouse and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 cumulus-oocyte complexes in 400 μL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 μg mL–1), epidermal growth factor (50 ng mL–1), oFSH (50 ng mL–1), bLH (3 μg mL–1), cysteamine (0.1 mM), and 10% fetal bovine serum for 22 to 24 h. Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm mL–1. Three different fertilization media, M199 (Gibco 11043–023, Grand Island, NY, USA), SOF (Tervit et al. 1972 J. Reprod. Fertil. 30, 493–497), and TALP (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180), were assayed along with 3 female sex-sorted bulls. All fertilization media were supplemented with fructose (90 μg mL–1), penicillamine (3 μg mL–1), hypotaurine (11 μg mL–1), and heparin (20 μg mL–1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-μL drops of KSOM-BSA for 9 days. On Day 3, 3% fetal bovine serum was added. Low oxygen tension (5% O2) was used for the entire culture period. On Days 7 and 9 blastocysts and hatched embryos, respectively, were morphologically evaluated according to IETS standards and recorded. Results are shown in Table 1. Data was compared by chi-squared analysis. Fertilization media affected cleavage rate and subsequent embryo development, quality, and hatching ability. The SOF and TALP fertilization media produced significantly more and higher quality embryos than M199. Table 1.In vitro fertilization performance after oocyte fertilization using sex-sorted sperm

In vitro maturation and fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of oocyte quality and type of serum

1998 ◽  
Vol 10 (2) ◽  
pp. 173 ◽  
Author(s):  
M. S. Chauhan ◽  
S. K. Singla ◽  
P. Palta ◽  
R. S. Manik ◽  
M. L. Madan
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Grade 3

In Experiment 1, to determine the developmental potential of buffalo oocytes of different qualities, compact cumulus–oocyte complexes (COCs) with an unexpanded cumulus mass, and with homogeneous ooplasm were classified as Grade 1 (with 5 layers of cumulus cells) and Grade 2 less than 4 layers of cumulus cells). Grade-3 oocytes were either without cumulus cells or with expanded cumulus mass, and with irregular ooplasm. The oocytes were matured for 24 h at 38·5°C, 5% CO2 in air in maturation medium (10% fetal bovine serum (FBS) in TCM-199 supplemented with 5 µg mL-1 follicle stimulating hormone-P). The nuclear maturation and cleavage rates, and the proportion of cleaved embryos which developed to morula and blastocyst stage were in the order Grade 1>Grade 2>Grade 3 (P < 0·05). For Experiment 2, the maturation medium consisted of TCM-199 supplemented with one of the following sera at 10% concentration: (1) buffalo oestrus serum (BOS), (2) superovulated buffalo serum (SBS), (3) fetal bovine serum (FBS) and (4) steer serum (SS). After in vitro fertilization (IVF), the oocytes were co-cultured with buffalo oviductal epithelial cells in TCM-199 containing the respective sera at 10% concentration for the subsequent 9 days. The extent of cumulus expansion and nuclear maturation were not different among different groups. The cleavage rates were lower (P < 0·05) with FBS than with BOS, SBS and SS. The proportion of cleaved embryos which developed to blastocyst stage was higher (P < 0·05) with SBS than with BOS, FBS and SS.

117 EFFECT OF STEPWISE DILUTION ON THE VIABILITY OF FROZEN - THAWED BOVINE OOCYTES MATURED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 176 ◽  
Author(s):  
A. Hamawaki ◽  
S. Hamano ◽  
M. Yoshikawa ◽  
K. Matsukawa
Keyword(s):  
Bovine Serum ◽  
Room Temperature ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Fertilization Rate ◽  
Single Step ◽  
Normal Morphology ◽  
Fetal Bovine ◽  
Bovine Oocytes

The purpose of this study was to evaluate the effect of stepwise dilution on the viability of frozen–thawed bovine oocytes matured in vitro. Oocytes matured in vitro were denuded and equilibrated in modified TCM-199 (m199: 11 mmol L-1 HEPES, 9 mmol L-1 Na-HEPES, 5 mmol L-1 NaHCO3, 20% (v/v) calf serum) supplemented with 10% (v/v) glycerol for 15 min at room temperature (RT). Then they were exposed to m199 with 10% glycerol and 0.25 mol L-1 sucrose and loaded into 0.25-mL plastic straws. The straws were sealed and seeded at -6�C, cooled at the rate of 0.33�C min-1 to -25�C, and plunged into LN2. For thawing, the straws were first held in air at RT for 10 s, followed by immersion in 30�C water for 10 s. In the first experiment, frozen-thawed oocytes were subjected to cryoprotectants in 5 different manners of dilution. In the non-step dilution, the oocytes (n = 60) were put into m199 for 5 min. In the single-step dilution, the oocytes (n = 37) were transferred to 0.25 mol L-1 sucrose in m199 for 5 min. In the two-step dilution, the oocytes (n = 56) were transferred to 0.5 and then 0.25 mol L-1 sucrose in m199 for 5 and 5 min, respectively. In the three-step dilution, the oocytes (n = 57) were transferred to 0.75, 0.5, and 0.25 mol L-1 sucrose in m199 for 1, 5, and 5 min, respectively. In the four-step dilution, the oocytes (n = 52) were transferred to 1.0, 0.75, 0.5, and 0.25 mol L-1 sucrose in m199 for 1, 1, 5, and 5 min, respectively. After dilution, all of the oocytes were washed twice in TCM-199 supplemented with 5% fetal bovine serum for 5 min and cultured for 1 h to assess the morphology. The rate of morphological normal oocytes in the four-step dilution (94.2%) was significantly (P &lt; 0.05) higher than that in other groups (non-, single-, two-, and three-step dilution: 61.7%, 73.0%, 78.6%, and 77.2%). In the second experiment, non-frozen (control, n = 170) and frozen–thawed oocytes (n = 145) with four-step dilution were fertilized and cultured in vitro (Kuwayama 1992 J. Reprod. Fert. 96, 187–193). To assess fertilization, some of the oocytes were fixed at 10 h after insemination. Cleavage and blastocyst rates were determined on Day 2 and Day 8 after fertilization (Day 0), respectively. There was no difference (P &gt; 0.05) between control and frozen–thawed oocytes in the fertilization rate (88.0% vs. 93.1%). Some of the frozen–thawed oocytes cleaved and developed to blastocysts (44.0% and 11.2%), although the rates were significantly (P &lt; 0.01) lower than those in control (71.7% and 35.0%). These results indicate that stepwise dilution of frozen–thawed oocytes improves the recovery of oocytes with normal morphology, and that the oocytes maintain the abilities to be fertilized and develop to blastocysts.

219 IN VITRO FERTILIZATION RATE OF MATURED PIG OOCYTES BY FROZEN - THAWED KOLBROEK PIG SPERM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 208
Author(s):  
M. H. Mapeka ◽  
K. C. Lehloenya ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Fertilization ◽  
South African ◽  
Fetal Bovine Serum ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Sperm Cells ◽  
Department Of Agriculture ◽  
Vitro Fertilization ◽  
Fetal Bovine

No studies have investigated the IVF rate of South African indigenous Kolbroek sperm cells following cryopreservation. The objective of this study was to test if frozen–thawed Kolbroek pig sperm cells could penetrate pig oocytes matured in vitro. Pig ovaries were collected from a local abattoir and cumulus–oocytes complexes were obtained by aspiration and were then in vitro matured in TCM-199 supplemented with 10% pig follicular fluid, 10% fetal bovine serum, and 1 μg mL–1 of FSH and LH. Following 44 h of incubation, 200 matured pig oocytes were randomly assigned to 2 treatments with frozen–thawed and fresh (control) Kolbroek pig sperm cells. For IVF, Kolbroek sperm cells were in vitro capacitated using Brackett and Oliphant’s sperm wash medium. Matured pig oocytes and sperm cells were co-incubated for 24 h in Brackett and Oliphant’s IVF medium. Following fertilization, presumptive zygotes were in vitro cultured at 39°C in 5% CO2, 5% O2, and 90% N2. Rate of fertilization was identified by the number of cleaved zygotes. Data were analysed by ANOVA. The total motility of Kolboek pig sperm cells used for IVF was 40% for frozen–thawed sperm cells and 80% for fresh sperm cells. The results showed that Kolbroek pig sperm cells were able to penetrate pig oocytes in vitro. However, no significant (P < 0.05) difference was observed in the percentage of cleavage of pig oocytes fertilized with either frozen–thawed (13.25%) or fresh (13.0%) Kolbroek pig sperm cells. The percentage of embryos that developed to the morulae stage was 2% in frozen–thawed sperm cells and was 0% in fresh Kolbroek sperm cells. Furthermore, oocytes fertilized with Kolboek sperm cells did not develop to the blastocyst stage in either treatment. In conclusion, this study demonstrated that frozen–thawed Kolbroek sperm cells are able to fertilize matured pig oocytes in vitro. This study was funded by the Department of Agriculture Forestry and Fishery, ARC, DST-PDP (RT19000), and National Research Foundation (NRF, Grant No. RT21 and 24000).

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum

2006 ◽  
Vol 65 (2) ◽  
pp. 374-386 ◽  
Author(s):  
Misae Suzuki ◽  
Koji Misumi ◽  
Manabu Ozawa ◽  
Junko Noguchi ◽  
Hiroyuki Kaneko ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fetal Bovine

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 Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media

2018 ◽  
Vol 19 (11) ◽  
pp. 3538 ◽  
Author(s):  
Brandon Lehrich ◽  
Yaxuan Liang ◽  
Pooya Khosravi ◽  
Howard Federoff ◽  
Massimo Fiandaca
Keyword(s):  
Cell Growth ◽  
Extracellular Vesicles ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cellular Growth ◽  
Primary Astrocyte ◽  
Fetal Bovine ◽  
Cell Growth And Viability

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

scholarly journals Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays

2021 ◽  
Author(s):  
Xenia Dolde ◽  
Christiaan Karreman ◽  
Marianne Wiechers ◽  
Stefan Schildknecht ◽  
Marcel Leist
Keyword(s):  
Neural Crest ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Developmental Toxicity ◽  
Video Tracking ◽  
Large Panel ◽  
Human Proteins ◽  
Fetal Bovine ◽  
Targeted Approach

Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chemotactic for NCCs in animal models (e.g. VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined in a broad screening approach, whether human cells would produce any factor able to trigger NCC migration. We found that HepG2 hepatoma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease-sensitivity suggested a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

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