149 EFFECTS OF FSH ADDED IN DEFINED MEDIUM MATURATION ON THE PRE-IMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax, Bcl-2, AND CONNEXIN 43 GENES IN BOVINE IVP BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
L. V. M. Gulart ◽  
L. Gabriel ◽  
L. P. Salles ◽  
G. R. Gamas ◽  
D. K. Souza ◽  
...  
Keyword(s):  
Internal Control ◽  
Connexin 43 ◽  
Calf Serum ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Blastocyst Rate

FSH at low concentrations affect embryo production. In vitro culture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivo has led to the development of several systems of oocyte in vitro maturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1 and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n = 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1 BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1 of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries.

scholarly journals 265EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO

2004 ◽  
Vol 16 (2) ◽  
pp. 253
Author(s):  
D.S. Arathy ◽  
S. Ashis ◽  
G.T. Sharma ◽  
A.C. Majumdar ◽  
M.S. Chauhan
Keyword(s):  
Specific Gravity ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Cell Stage

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta & Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5μgmL−1 FSH in 5% CO2 incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF)+0.8 %BSA (control), 2) mSOF+0.8 % BSA+gelatin (1mgmL−1) 3) mSOF+0.8% BSA+1mgmL−1 gelatin+10ngmL−1 epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658±0.009 to 1.0331±0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P<0.05) in mSOF+0.8% BSA+1mgmL−1 gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF+0.8% BSA+10ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

88 EXPRESSION OF ENZYMES RELATED TO HOMOCYSTEINE METABOLISM AND HOMOCYSTEINE SENSITIVITY IN BOVINE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 145
Author(s):  
S. Ikeda ◽  
M. Sugimoto ◽  
S. Kume
Keyword(s):  
Internal Control ◽  
Methylenetetrahydrofolate Reductase ◽  
Cumulus Cells ◽  
Competitive Inhibitor ◽  
Preimplantation Embryos ◽  
Cleavage Rate ◽  
Development Data ◽  
Blastocyst Rate ◽  
Oviduct Fluid

Homocysteine is a nonessential amino acid produced through methionine metabolism. Elevation of homocysteine levels (hyperhomocysteinemia) increases intracellular S-adenosylhomocysteine (SAH). S-adenosylhomocysteine binds to methyltransferases (MT) with greater affinity than does S-adenosylmethionine, which is the universal methyl donor used by various cellular MT, including DNA and histone MT. Thus, SAH acts as a potent competitive inhibitor of methylation reactions. Therefore, disorder of homocysteine metabolism may affect cellular homeostasis in part through the process involving methylation reactions (Williams KT and Schalinske KL 2007 J. Nutr. 137, 311–314). Despite its predicted importance, the involvement of homocysteine metabolism in pre-implantation embryonic development remains unaddressed. In the present study, the expression of enzymes related to homocysteine metabolism in bovine pre-implantation embryos and the effects of homocysteine on the post-fertilization development of these embryos in vitro were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% v/v FCS and 0.2 IU mL–1 follicle-stimulating hormone. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from one bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF up to Day 8 (IVF = Day 0). All cultures were performed at 38.5°C under 5% CO2, 5% O2, and 90% N2. Total RNA was extracted from individual blastocysts on Day 7 to 8 and reverse transcribed to cDNA using oligo(dT) primer. Transcripts for methionine adenosyltransferase 2A (MAT2A), MAT2B, adenosylhomocysteinase, methionine synthase, betaine-homocysteine MT, serine hydroxymethyltransferase 1, and 5,10-methylenetetrahydrofolate reductase were examined by qualitative PCR using bovine-specific primers for each gene and the cDNA as templates. β-Actin transcripts were used as an internal control. Moreover, presumptive zygotes after IVF were cultured in mSOF supplemented with 0 (control), 10, and 100 μm homocysteine, and development to cleavage stage and blastocyst was assessed on Day 3 and Day 7 and 8, respectively. The cultures were replicated 4 times using 561 embryos. The development data were statistically analyzed by using the general linear model. Transcripts for all genes examined were detected. Homocysteine added to the culture medium of bovine IVF embryos did not affect the cleavage rate (86.8, 83.3, and 84.3% for control, 10 μm, and 100 μm, respectively); however, blastocyst rate significantly decreased (P = 0.02) on Day 7 (12.8, 9.3, and 7.5%, respectively). The blastocyst rate on Day 8 showed no difference (P = 0.33) among the groups (24.4, 20.4, and 20.1%, respectively). These results indicate that a system for homocysteine metabolism is present in bovine pre-implantation embryos and that high homocysteine levels affect the developmental kinetics of these embryos. Supported by KAKENHI.

PSX-A-23 Late-Breaking: Supplementation of Nerve Growth Factor (β-NGF) in the maturation medium and efficiency of in vitro embryo production in cattle

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 365-365
Author(s):  
Lucas Gonçalves ◽  
Muller C Martins ◽  
Natalia Arle ◽  
Rafaela T Torres ◽  
Luisa Migilo ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Oocyte Maturation ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Nerve Growth ◽  
Maturation Medium ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Abstract The aim of this study was to evaluate the supplementation of Nerve Growth Factor (β-NGF) in the maturation medium in in vitro embryo production routines. Antral follicles were aspirated from ovaries of cows obtained from slaughterhouses and then oocytes were selected for quality (grades I and II) for in vitro maturation and subjected to 4 successive in vitro embryo production routines (IVEP). Supplementation of 100 ng of β-NGF was performed in the oocyte maturation medium 22 hours before in vitro fertilization. 48 hours after fertilization of the oocytes, an analysis was made of their cleavage rate by counting blastomeres with the aid of a stereoscopic microscope (cleavage rate = number of embryos / number of initial oocytes). Seven days after fertilization, the blastocyst rate was determined through the relation to the number of oocytes that started cleavage and reached this stage of development (blastocyst rate = number of blastocyst / number of oocytes that started cleavage). To verify the existence of a difference between the supplemented and the non-supplemented groups, the paired T test was applied, using the Excel / Action software (Microsoft). In vitro embryo production routines supplemented with β-NGF in the maturation medium had, on average, a higher cleavage rate (P = 0.0072) and a higher blastocyst rate (P = 0.0033) compared to non-supplemented routines with β-NGF. In this study was demonstrated that Nerve Growth Factor supplementation in the maturation medium improves the efficiency of in vitro embryo production in cattle, and this protein has a probable action in the oocyte maturation process.

scholarly journals Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production.

1981 ◽  
Vol 90 (2) ◽  
pp. 332-338 ◽  
Author(s):  
R P Mecham ◽  
G Lange ◽  
J Madaras ◽  
B Starcher
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Elastic Fiber ◽  
Calf Serum ◽  
Phenotypic Expression ◽  
Fetal Tissue ◽  
Culture Conditions ◽  
Population Doubling

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.

scholarly journals Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats

2016 ◽  
Vol 19 (10) ◽  
pp. 1091-1095
Author(s):  
Camila Louise Ackermann ◽  
Eduardo Trevisol ◽  
Leticia Ferrari Crocomo ◽  
Tatiana da Silva Rascado ◽  
Rodrigo Volpato ◽  
...  
Keyword(s):  
Treated Group ◽  
Control Group ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher’s exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P&lt;0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

177 PERFORMANCE OF Gyr PREPUBERTAL HEIFERS IN IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 219
Author(s):  
P. M. S. Rosa ◽  
A. J. R. Camargo ◽  
R. V. Serapião ◽  
L. S. A. Camargo ◽  
C. S. Oliveira
Keyword(s):  
Cleavage Rate ◽  
Embryo Production ◽  
Exact Test ◽  
Follicular Wave ◽  
Lactating Cows ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate ◽  
Oocyte Donors ◽  
La Jolla

Bovine in vitro embryo production is highly relevant for dairy systems in Brazil, and Gyr dams are commonly used as oocyte donors. The aim of this study was to evaluate the use of prepubertal Gyr heifers as oocyte donors, an alternative to anticipate reproduction of those animals. For that, 11 Gyr [4 prepubertal (PP) donors and 7 adult cows © donors] were used in ovum pickup (OPU) sessions. The PP cows presented an average of 282.5 kg and 26.75 months, and had never displayed oestrous. Non-lactating cows presenting an average of 492 kg and 136 months were selected for C. Five replicates were performed, totaling 27 OPU sessions (C-17, PP-10) and 2–3 sessions per animal. Follicular wave was synchronised by aspiration of follicles larger than 8 mm 96 h before OPU. Cumulus-oocyte complexes (COC) were classified accordingly to their quality in viable (G1, G2, and G3) or non-viable (G4). Viable oocytes were matured and fertilized, and the presumptive zygotes were cultured in SOF medium at 38.5°C and 5% CO2 in air. Cleavage rate was assessed 48 to 72 h post-insemination (hpi) and blastocyst rate at 168 hpi. Mean number of structures was analysed by t-test, and percentage of viable, G1, G2, G3, G4, cleavage, and blastocyst rates were compared among groups by Fisher’s exact test (GraphPadInstat, La Jolla, CA, USA; P = 0.05). Results are followed by standard error values. All procedures were approved by a local ethics committee. We found that despite higher (P < 0.05) numbers for both viable oocytes (PP: 15 ± 2.6; C: 6.11 ± 0.76) and total oocytes (PP: 23.70 ± 2.83; C: 8.82 ± 1.19) in the PP group, the rate of viable oocytes was similar (P > 0.05) among PP and C groups (PP: 61.5 ± 6.51%, C: 66.79 ± 3.79%). Mean numbers of G1, G2, G3, and G4 oocytes were higher (P < 0.05) in the PP group (G1 = 7.1 ± 1.18; G2 = 4.9 ± 1.74; G3 = 3.9 ± 1.09; G4 = 7.8 ± 1.38) than in the C group (G1 = 2.70 ± 0.740; G2 = 2.47 ± 0.44; G3 = 1.11 ± 0.31; G4 = 2.52 ± 0.39). However, the proportion was similar (P > 0.05) among PP and C groups (PP: G1 = 29.5 ± 4.21%; G2 = 19.5 ± 2.85%; G3 = 15.9 ± 13.5%; G4 = 35.1 ± 6.33%; and C: G1 = 27.24 ± 4.44%; G2 = 29.60 ± 5.08%; G3 = 12.34 ± 3.01%, G4 = 30.79 ± 4.93%). Cleavage rate (PP: 91.3 ± 17.94%; C: 74.09 ± 4.65%), mean blastocyst number per OPU session (PP: 3.3 ± 1.29; C: 1.76 ± 0.28), and blastocyst rate (PP: 19.74 ± 7.40%; C: 27.03% ± 4.07%) were similar (P > 0.05) among groups. We conclude that prepubertal heifers presented increased numbers of viable oocytes per OPU session, but blastocyst yield was similar to adult cows. This data suggests that prepubertal Gyr heifers can be used as oocyte donors. Support from FAPERJ and Embrapa is acknowledged.

scholarly journals BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle

2019 ◽  
Vol 97 (6) ◽  
pp. 2555-2568 ◽  
Author(s):  
Alan D Ealy ◽  
Lydia K Wooldridge ◽  
Sarah R McCoski
Keyword(s):  
Production Systems ◽  
Culture Media ◽  
Methylation Status ◽  
Culture Conditions ◽  
Retention Rates ◽  
Binucleate Cell ◽  
Low Oxygen ◽  
Embryo Production

Abstract In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

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