123 CHANGES IN EXPRESSION OF GENES ASSOCIATED WITH GENETIC VARIATION IN PRE-IMPLANTATION DEVELOPMENT OF THE BOVINE EMBRYO

2014 ◽  
Vol 26 (1) ◽  
pp. 175
Author(s):  
M. S. Ortega ◽  
J. B. Cole ◽  
T. S. Sonstegard ◽  
P. J. Hansen
Keyword(s):  
Genetic Variation ◽  
Intracellular Transport ◽  
Blastocyst Stage ◽  
Acid Oxidation ◽  
Lethal Gene ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Glycoprotein Synthesis ◽  
Stage Of Development

The objective was to identify patterns of expression during the pre-implantation period of several genes associated with genetic variation in fertility (CWC15) or development to the blastocyst stage (C1QB, MON1B, PARM1, PCCB, PMM2, TBC1D24, and WBP1). These genes are involved in cellular processes such as mRNA splicing, immune protection, fatty acid oxidation, resistance to apoptosis, glycoprotein synthesis, and intracellular transport. Embryos were produced in vitro from slaughterhouse oocytes and semen using a mix of Bos taurus and Bos indicus cows and bulls. Pools of 40 matured oocytes or embryos at the 2-cell [27–31 h post-insemination (hpi)], 3- to 4-cell (46–52 hpi), 5- to 8-cell (49–59 hpi), 9- to 16-cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The RNA was purified and synthesised into cDNA for real-time qPCR analysis. The YWHAZ, GAPDH, and SDHA were used as steady-state controls of expression. A total of 5 pools were analysed for each of the 6 stages. The C1QB was not detected at any stage; however, transcript amounts for the other genes were affected by stage of development (P < 0.05). The WBP1 remained low from the oocyte to the 5- to 8-cell stage (fold-change relative to matured oocytes: 1.0 ± 0.2 v. 1.4 ± 0.2), increased at the 9- to 16-cell stage (14.8 ± 0.2), and decreased to the blastocyst stage (7.1 ± 0.2). The expression pattern of PARM1 was similar, with greatest expression at the 9- to 16-cell stage. In contrast, expression of PMM2 and TBC1D24 was highest at the 2-cell stage and decreased at the morula and blastocyst stages. Expression of CWC15, MON1B, and PCCB decreased steadily from the oocyte to the blastocyst stage. Given that the major round of embryonic genome activation occurs at the 8- to 16-cell stage, it is possible that PARM1 and WBP1 play important roles around this time. The PMM2 and TBC1D24 may represent genes activated before the 8- to 16-cell stage. The CWC15 has been identified as a lethal gene; results suggest lethality occurs after the blastocyst stage. Further research will clarify the role and importance of these genes in the early development of the bovine embryo. The authors acknowledge support from AFRI Grant No. 2013–68004–20365 from USDA NIFA.

The effect of bovine embryo culture without proteins supplements until day 4 on transcription level of hyaluronan synthases, receptors and mtDNA content

Zygote ◽  
2009 ◽  
Vol 18 (2) ◽  
pp. 121-129 ◽  
Author(s):  
A.T. Palasz ◽  
P. Beltrán Breña ◽  
J. De la Fuente ◽  
A. Gutiérrez-Adán
Keyword(s):  
Embryo Culture ◽  
Receptor Gene ◽  
Blastocyst Stage ◽  
Low Molecular Weight ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Hyaluronan Synthases ◽  
Copy Numbers ◽  
Surface Active

SummaryThe effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration andin vitrodevelopment. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to ≤8-cell stage by day 4 were not affected by lack of BSA in the medium, but increased numbers of blastocysts developed in medium supplemented with BSA from days 1 or 4 with or without HA than in medium that had HA only. Addition of both HA and BSA at day 4 increased mtDNA copy numbers at the blastocyst stage. Data suggest that the addition of BSA and/or HA at 96 hpi increased expression ofRHAMMandHas2genes, but notCD44,Has1orHas3genes. Higher expression levels of Has2 than Has1 and the three isoforms indicate that high- rather than low-molecular-weight HA should be used for preimplantation bovine embryo culture.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems

Reproduction ◽  
2001 ◽  
pp. 601-610 ◽  
Author(s):  
MA Yaseen ◽  
C Wrenzycki ◽  
D Herrmann ◽  
JW Carnwath ◽  
H Niemann
Keyword(s):  
Growth Factor ◽  
Polyvinyl Alcohol ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Culture Systems ◽  
Igf I ◽  
Oviduct Fluid

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

scholarly journals Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

scholarly journals Identification of Beta Human Chorionic Gonadotrophin (bHCG) Expression in Frozen Cleavage Embryo of In Vitro Fertilization (IVF) Patients: An Essential Approach for Better Outcome towards Human Being Inheritance in Line with Maqasid Syari’ah Concept

2018 ◽  
Vol 17 (2) ◽  
Author(s):  
Lokman Md Isa ◽  
Afzan Mat Yusof ◽  
Roszaman Ramli ◽  
Syamsul Ahmad Arifin ◽  
Fatin Emalina ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Implantation ◽  
Predictive Marker ◽  
Blastocyst Stage ◽  
Embryo Viability ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Frozen Embryos

Expression of beta-human chorionic gonadotropin (bHCG) mRNA in early cleavage cell stages is important in predicting embryo viability at blastocyst stage of development. Embryo viability is crucial for in vitro fertilization (IVF) treatment to be successful in terms of increment of implantation and pregnancy rate. In order to establish fertilization outside the human body, optimum condition mimicking the natural body environment like hormones and growth factors as well as impeccable timing must be established. Imam Muslim narrated from Hudhayfa ibn Asad that the Prophet Muhammad (SAW) said: “After the sperm-andovum drop (nut.fa) has been [in the uterus] forty-two days, Allah sends it an angel that gives it form and fashions its hearing, sight, skin, flesh, and skeleton”. Therefore the aim of this study is to find the significant association of bHCG expression with early cell stage cleavages and its relationship as predictive marker for potential embryo implantation. Our study focuses on leftover frozen embryos from eight patients consists of six pregnant patients and two non-pregnant patients. We assessed the human bHCG mRNA expression at different cell cleavage stages in these frozen embryos using reverse transcriptasepolymerase chain reaction (RT-PCR). Our results have shown three out of eight patients with five to ten cells of blastomeres were expressed with bHCG. This study indicated that bHCG was expressed on frozethawed late cleavage stage of embryos in IVF patients.

138 FATTY ACID β-OXIDATION REGULATES BOVINE EMBRYO OXYGEN METABOLISM

2013 ◽  
Vol 25 (1) ◽  
pp. 216
Author(s):  
P. J. McKeegan ◽  
F. G. Guerif ◽  
R. G. Sturmey
Keyword(s):  
Oxygen Consumption ◽  
Fatty Acid ◽  
Atp Synthesis ◽  
Oxygen Metabolism ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Metabolic Energy ◽  
Acid Oxidation ◽  
Bovine Embryo

Mammalian oocytes are rich in endogenous lipid, which provides a potential source of metabolic energy during oocyte maturation and early embryo development after fertilisation. Despite this, studies on early embryo metabolism have focussed on consumption of substrates from the culture medium, with comparatively little consideration for endogenous stores. Additionally, the interaction between fatty acid β-oxidation and oxygen consumption during pre-implantation development has not been investigated. This study has investigated the relationship between embryo fatty acid metabolism and oxygen consumption rate (OCR), a marker of overall oxidative metabolic activity. The amount of oxygen consumption by bovine blastocysts coupled to ATP synthesis was assessed by measuring embryo OCR following acute treatment with oligomycin, which inhibits ATP synthase. Bovine embryos were then produced in vitro and cultured from zygote to blastocyst stage (8 days) in the presence of β-mercaptoacetate (BMA), a competitive inhibitor of fatty acid β-oxidation, or L-carnitine, a cofactor that promotes β-oxidation. OCR of individual embryos was measured using a noninvasive, highly sensitive oxygen probe (Unisense), whereas OCR of embryos in groups was determined using the Oxygen Biosensor (OBS) fluorimetric assay (BD Biosciences, Erembodegem, Belgium). Mean OCR measured by each technique correlated well. Data are presented as means ± SEM. The effect of acute inhibition were compared by a paired t-test, with pre-treated embryos acting as their own control, whereas the effect of L-carnitine and BMA on OCR was compared across treatment groups using ANOVA with Dunn’s test for unequal groups post hoc, using Sigmaplot (Systat Software Inc., San Jose, CA, USA). Treatment with oligomycin caused blastocyst OCR to fall from 23.5 ± 3.3 to 7.9 ± 2.5 pmol/embryo/h (n = 3; P = 0.03). Inhibition of β-oxidation by BMA led to a modest, but consistent increased OCR in cleavage stage embryos: 4-cell: 14.8 ± 7.2 (n = 12) v. 8.9 ± 1.1 pmol/embryo/h (n = 3) control; 8-cell: 15.8 ± 2.2 (n = 6) v. 11.9 ± 2.3 pmol/embryo/h (n = 3) control. At the morula stage, BMA led to a significant rise in OCR (26.8 ± 3.6 (n = 7) v. 7.8 ± 1.7 pmol/embryo/h (n = 3) control; P = 0.003) and at the blastocyst stage, OCR was significantly elevated in the presence of BMA; 41.3 ± 2.6 (n = 19) v. 16.9 ± 1.3 pmol/embryo/h (n = 14) control (P = 0.01). By contrast, when groups of embryos were cultured in the presence of L-carnitine, blastocyst OCR was reduced by 45% from 24.7 ± 5.1 (group control) to 15.9 ± 3.7 pmol/embryo/h (n = 30; P < 0.06). We report that 66.5% of oxygen consumption by bovine blastocysts is oligomycin sensitive, meaning that 33.5% of oxygen consumption is used for processes other than ATP synthesis. Surprisingly, inhibition of fatty acid β-oxidation caused total bovine embryo OCR to rise, although the reasons for this are unclear. It is possible that inhibiting β-oxidation leads to an accumulation of free fatty acids in the mitochondrion, which can disrupt the inner membrane and lead decoupling of ATP synthesis from oxygen consumption. Thus, manipulating fatty acid oxidation in the early embryo dysregulates oxidative metabolism; the implications of this are under further investigation.

156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

2017 ◽  
Vol 29 (1) ◽  
pp. 187
Author(s):  
A. Goldacker ◽  
E. Winn ◽  
J. Z. Current ◽  
B. D. Whitaker
Keyword(s):  
Embryonic Development ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Of Development ◽  
Fluid Supplementation ◽  
Chi Square Analysis

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P < 0.05) incidence of polyspermic penetrations and a significantly higher (P < 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P < 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P < 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

Sign in / Sign up

Export Citation Format

Share Document