128 N-METHYL-D-ASPARTIC ACID CAN BE USED TO PARTIALLY REPLACE BOVINE SERUM ALBUMIN IN CULTURE MEDIUM

2009 ◽  
Vol 21 (1) ◽  
pp. 164
Author(s):  
L. D. Spate ◽  
B. K. Bauer ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Aspartic Acid ◽  
Bovine Serum ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Chemically Defined Media ◽  
Defined Media

One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop chemically defined media based off the embryo’s biological and physiologic makeup. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3-fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mm) would improve development of embryos in an already chemically defined medium. Slaughterhouse derived ovaries were aspirated, cumulus–oocyte complexes were identified and then matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris buffered medium with 0.25 × 106 mL–1 frozen–thawed porcine semen for 5 h. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: (1) PZM3, (2) PZM4, or (3) PZM4 + 0.5 mm NMDA. Embryos were cultured in 5% CO2, 5%O2, 90% N2 until Day 7. For this experiment the number of cleaved embryos cultured in each treatment group were 260 for group 1, 220 for group 2 and 300 for group 3. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure (a,b P < 0.05). The percentage developed to blastocyst was (1) 47.5% a, (2) 29.6% b, and (3) 36.1% a,b, respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was (1) 25.8 a, (2) 19.6 b, and (3) 22.9 a,b, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number; however, with the addition of 0.5 mm NMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media. Funded by a grant from the USDA NRI 2006-35203-17282.

Effects of amino acid supplements and replacement of polyvinyl alcohol with bovine serum albumin in porcine zygote medium

2006 ◽  
Vol 18 (7) ◽  
pp. 789 ◽  
Author(s):  
Chie Suzuki ◽  
Koji Yoshioka
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bovine Serum Albumin ◽  
Polyvinyl Alcohol ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Essential Amino Acids ◽  
Total Cell ◽  
Cell Numbers

The effects of glutamine, hypotaurine, taurine and premixed solutions of essential amino acids (EAA) and non-essential amino acids (NEAA) on in vitro development of porcine zygotes were evaluated. The effects of refreshing the medium and replacing polyvinyl alcohol (PVA) with bovine serum albumin (BSA) on embryonic development were also investigated. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilisation (IVF) were cultured in porcine zygote medium (PZM), as the basal culture medium, for 5 days after IVF. The total number of cells in blastocysts was significantly increased by the addition of 2 mm glutamine to PZM, as was blastocyst yields after supplementation with 0.25 to 4 mm glutamine. Addition of 1.25 to 10 mm hypotaurine to PZM significantly increased blastocyst yields. Addition of 5 mm taurine to PZM significantly increased blastocyst yield, whereas taurine had no effect on blastocyst yield in cultures already containing 5 mm hypotaurine. Adding 1× EAA significantly increased the rate of blastocyst formation compared with no or 2× EAA, whereas 2× NEAA significantly increased the total cell numbers in blastocysts compared with no NEAA. Refreshing the medium at Day 3 had no effect on blastocyst yields, whereas medium change significantly reduced the total cell numbers in blastocysts. Adjusting the amino acid concentrations of a chemically defined medium can improve the developmental competence of porcine embryo.

Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 601 ◽  
Author(s):  
PA Batt ◽  
DK Gardner ◽  
AW Cameron
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Oxygen Concentration ◽  
Bovine Serum ◽  
Calf Serum ◽  
Cell Number ◽  
The Mean ◽  
Number Of Cells

The effect of oxygen concentration and the source of protein in culture medium on the development of 2- to 4-cell goat embryos in vitro was investigated. Embryos were collected from superovulated Angora-Cashmere-cross goats 48 h after ovulation and cultured for 6 days in synthetic oviduct fluid (SOF) medium under one of two oxygen concentrations (20% or 7%) and in the presence of one of five protein sources; Miles bovine serum albumin (Miles BSA), Commonwealth Serum Laboratory bovine serum albumin (CSL BSA), goat serum (GS), fetal calf serum (FCS) and human serum (HS). In the presence of 20% oxygen the percentage of embryos reaching the expanded and/or hatched blastocyst stage in SOF medium containing Miles BSA was 29%, with a mean cell number per embryo of 28.1 +/- 6.0 (+/- s.e.m.). Use of an oxygen concentration of 7% significantly increased the percentage of embryos reaching this stage (80%, P less than 0.01) and the mean number of cells per embryo (65.3 +/- 8.2, P less than 0.01). The mean number of cells of the early-cleavage-stage embryos was significantly lower when the medium contained CSL BSA, GS or FCS (42.7 +/- 5.6, 29.0 +/- 6.1 and 21.3 +/- 3.2, respectively) than with Miles BSA (92.8 +/- 6.4) or HS (104.8 +/- 17.2) (P less than 0.01). Under 7% oxygen and with Miles BSA or HS, embryos were morphologically comparable to those developed in vivo, but the mean cell numbers in vitro were only approximately half those obtained in vivo.

136 N-METHYL-D-ASPARTIC ACID AND HOMOCYSTEINE CAN BE USED TOGETHER AS A REPLACEMENT FOR BOVINE SERUM ALBUMIN IN EARLY PORCINE EMBRYO CULTURE

2011 ◽  
Vol 23 (1) ◽  
pp. 172
Author(s):  
L. D. Spate ◽  
B. K. Bauer ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Dna Methylation ◽  
Bovine Serum Albumin ◽  
Surface Area ◽  
Embryo Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Cell Number ◽  
Global Dna Methylation ◽  
Treatment Groups ◽  
Defined Culture

Most mammalian embryo culture media contain some form of unidentifiable biological contaminant, usually associated with fetal bovine serum (FBS) or bovine serum albumin (BSA). Such factor(s) confound experiments attempting to evaluate culture media composition and decrease the repeatability of experiments when different lots or batches of FBS or BSA are used. The goal of this study was to formulate a completely chemically defined culture media for development of early porcine embryos based on adding ligands for which there is the presence of mRNA for the corresponding receptors in the blastocyst. Cumulus–cell oocyte complexes were matured for 42 h in M199 supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris-buffered media for 4 h. Presumptive zygotes were then placed into porcine zygote media with 0.3% BSA (PZM3) or 0.1% polyvinyl alcohol (PZM4). At 28 h post-fertilization, 2- to 4-cell stage embryos were selected and placed into treatment groups for 5 days. Fifteen embryos were put into 25 μL of media and cultured in 5% CO2, 5% O2, and 90% N at 38.5°C. The treatment groups were as follows: 1. PZM3, 2. PZM4, 3. PZM4 + 0.5 mM N-methyl D-aspartic acid (NMDA), 4. PZM4 + 0.5 mM NMDA + 10 μM homocysteine (HC), and 5. PZM4 + 10 μM HC. There were 120, 135, 120, 135, and 120 embryos for each treatment, respectively. The percentages of embryos that developed to the blastocyst stage were 63.3%a, 29.7%b, 46.1%c, 55.6%ac, and 49.2%ac, respectively [SAS; SAS Institute, Cary, NC, USA) Proc GLM (a,b,cP < 0.05)]. Total cell number was determined using bisbenzimide to stain the nuclei, and the data were analysed by SAS Proc GLM. There was no difference in cell number among treatments with a mean cell number of 31.4. To further investigate the equality of the chemically defined media, the surface area of the blastocysts was measured by using Nis Elements BR3.0 software under 20× magnification. There was less surface area in treatment 5 compared with 4 [296 180ab, 295 114ab, 303 451ab, 271 913b, and 316 773a arbitrary units (a,bP < 0.05), with n = 33, 26, 37, 31, and 32 embryos in each treatment, respectively]. Because HC has been shown to affect global DNA methylation of bovine embryos, we stained our embryos for 5-methylcytidine (Eurogentec anti 5-MECY-0100) and embryos were visualised by UV light with a Texas red filter, and intensity was measured by Nis Elements BR 3.0 software under 20X magnification. The mean intensities were lower for the NMDA treatment [26.7a, 19.4a, 13.6b, 17.0a, and 19.4a arbitrary units (a,bP < 0.05)] compared with the other treatments. When embryos were cultured without BSA development decreases, but adding NMDA and HC returns development to control levels as measured by percentage of blastocysts, surface area, and global DNA methylation. We conclude that PZM4 supplemented with 0.5 mM NMDA and 10 μM HC may replace PZM3 as a chemically defined culture media for early porcine embryos. Embryo transfer experiments will be necessary to confirm that these embryos have equal developmental competence. Funded by the NRI (2006-35203-17282) and Food for the 21st Century.

scholarly journals Sequence of residues 400–403 of bovine serum albumin

1980 ◽  
Vol 191 (3) ◽  
pp. 867-868 ◽  
Author(s):  
R G Reed ◽  
F W Putnam ◽  
T Peters
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Aspartic Acid ◽  
Bovine Serum ◽  
Tryptic Peptide ◽  
Edman Degradation ◽  
Protein Residues

A large tryptic peptide of bovine serum albumin (residues 377–582) was subjected to 32 cycles of Edman degradation to determine the sequence of the last remaining unknown segment of this protein. Residues 400–403 were identified as gly-Phe-Gln-Asn. Amide assignments were also made at positions 388 (glutamine), 389 (asparagine), 391 (aspartic acid) and 392 (glutamine).

Self-assembling of poly(aspartic acid) with bovine serum albumin in aqueous solutions

2017 ◽  
Vol 95 ◽  
pp. 412-420 ◽  
Author(s):  
L.E. Nita ◽  
A.P. Chiriac ◽  
M. Bercea ◽  
M. Asandulesa ◽  
Bernhard A. Wolf
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Aqueous Solutions ◽  
Aspartic Acid ◽  
Bovine Serum ◽  
Self Assembling

scholarly journals Tailorable polyelectrolyte protein complex based on poly(aspartic acid) and bovine serum albumin

2016 ◽  
Vol 19 (7) ◽  
pp. 596-606 ◽  
Author(s):  
Loredana E. Nita ◽  
Aurica P. Chiriac ◽  
Elena Stoleru ◽  
Alina Diaconu ◽  
Nita Tudorachi
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Aspartic Acid ◽  
Protein Complex ◽  
Bovine Serum

scholarly journals 26 ASSESSMENT OF CHROMOSOME ABNORMALITIES IN SHEEP PARTHENOGENETIC AND NUCLEAR TRANSFER EMBRYOS: EFFECT OF 6-DMAP AND CYCLOHEXIMIDE ON PLOIDY

2005 ◽  
Vol 17 (2) ◽  
pp. 163
Author(s):  
B. Alexander ◽  
G. Coppola ◽  
D. Di Berardino ◽  
D.H. Betts ◽  
W.A. King
Keyword(s):  
Nuclear Transfer ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Total Cell Number ◽  
Parthenogenetic Activation ◽  
Treatment Groups ◽  
Reconstructed Embryos ◽  
The Mean ◽  
Painting Probes

In current somatic cell nuclear transfer (NT) protocols, the reconstructed embryos are activated by incorporation of secondary oocyte activation compounds such as 6-DMAP or cycloheximide (CHX). The effects of these compounds on the chromosome complement of sheep NT embryos have not been studied in detail. Therefore, the aim of this study was to assess the chromosome abnormalities using sex chromosome specific probes of Day 6 blastocyst-stage sheep embryos produced from parthenogenetic activation and NT. Following 20–22 h of IVM, the oocytes were activated by electric pulsing followed by 30-min culture in cytochelasin B. They were reactivated using ionomycin (5 min) followed by 2-h culture in 6-DMAP or CHX. In contrast, NT embryos were produced using standard NT procedures using male sheep fetal fibroblasts. Reconstructed embryos were activated using the same methods described earlier. The embryos (compact morulae and blastocysts) were fixed and subjected to FISH analysis using cattle X and Y chromosome painting probes. The data were analyzed using Fisher's exact test. Of the parthenogenetic embryos (6-DMAP, n = 28; CHX, n = 32) analyzed, none of the embryos was totally haploid (X) or totally polyploid. When all of the nuclei per embryo were considered, normal (XX) genotype embryos were 6.2% and 0.0% in CHX and 6-DMAP groups, respectively. The rest of the embryos were abnormal due to mixoploidy (100% vs. 93.8%, P < 0.05) in 6-DMAP and CHX treatment groups, respectively. The abnormal nuclei per embryo ranged from 7.3% to 72.2%. The mean total cell number of parthenogenetic blastocysts was 91.2 ± 4.3 and 81.8 ± 6.2 (mean ± SE) in 6-DMAP and CHX, respectively. Among NT embryos analyzed, (6-DMAP, n = 30; CHX, n = 32) only 40.0% and 43.8% of embryos were completely normal for XY chromosomes in 6-DMAP- and CHX-treated groups, respectively. The rest of the embryos were abnormal due to mixoploidy (60.0% vs. 56.2%, P > 0.05) in 6-DMAP and CHX groups, respectively. Monosomy (XO or OY), trisomy (XXY), and tetrasomy (XXYY) were the common abnormalities detected in mixoploid embryos. The abnormal cells per embryo ranged from 3.8% to 41.8% in both treatment groups. The mean total cell number of NT blastocysts was 71.2 ± 9.8 and 63.8 ± 8.4, in 6-DMAP and CHX treatment groups, respectively. In conclusion, the 6-DMAP-treated embryos derived from parthenogenetic activation had significantly higher chromosomal abnormalities than CHX-treated embryo groups (P < 0.05). In contrast, the NT embryos derived from either 6-DMAP or CHX treatment did not show any significant difference in producing chromosomally abnormal embryos at the blastocyst stage. This study also highlights the feasibility of using bovine chromosome painting probes on ovine embryo spreads. This work was supported by NSERC, OMAFRA, and ICCS.

scholarly journals Penambahan Bovine Serum Albumin pada Beltsville Thawing Solution Dapat Mempertahankan Kualitas Semen Babi yang Disimpan pada 15°C (ADDITION OF BOVINE SERUM ALBUMIN TO BELTSVILLE THAWING SOLUTION COULD MAINTAIN QUALITY OF PIG SEMEN STORED AT 15OC)

2019 ◽  
Vol 20 (3) ◽  
pp. 330
Author(s):  
Wayan Bebas ◽  
Wayan Gorda
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Plasma Membranes ◽  
Treatment Groups ◽  
Progressive Motility ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
Optimal Quality

This study aims to maintain the quality of pig semen for longer during storage at 15oC, in an effort to support artificial insemination programs with the addition of Bovine Serum albumin (BSA) to diluent Beltsville Thawing Solution (BTS). This study uses a completely randomized design with five treatment groups, each To = semen was diluted with BTS without the addition of BSA ; T1 = with the addition of 5 mg BSA/mL diluent; T2 = with the addition of 10 mg BSA/mL diluent; T3 = with the addition of 15 mg BSA/mL diluent; T4 = with the addition of 20 mg BSA/mL diluent. Each treatment was repeated five times so that the number of samples used was twenty-five. The diluted cement is stored at 15oC for 72 hours then observing the quality of cement includes: progressive motility, dead spermatozoa, abnormalities, and intact plasma membranes. The data obtained were analyzed by analysis of variance, if there were differences followed by Duncan’s test. The results showed, addition of BSA concentration of 10 mg/mL and 15 mg/mL of diluent gives the same effect on the quality of cement during storage and significantly better (p <0.05) when compared to the addition of 0 mg/mL, 5 mg/mL and 20 mg/mL diluents. It can be concluded, the addition of BSA 10 mg/mL BTS diluents can maintain the most optimal quality of pig semen against progressive motility, dead spermatozoa, abnormalities and intact plasma membranes.

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 207
Author(s):  
Y. Jeon ◽  
S.-S. Kwak ◽  
S.-A. Jeong ◽  
R. Salehi ◽  
Y. H. Seong ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Vitis Amurensis ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P < 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P < 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, Republic of Korea.

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