99 Increased expression of microRNAs in sperm of Nelore Bulls with high invitro fertility

2021 ◽  
Vol 33 (2) ◽  
pp. 157
Author(s):  
T. R. S. Hamilton ◽  
C. M. Mendes ◽  
M. D. Goissis ◽  
J. C. Silveira ◽  
M. E. O. A. Assumpção
Keyword(s):  
Embryo Development ◽  
Test Procedure ◽  
Rna Extraction ◽  
Genetic Material ◽  
Mrna Translation ◽  
Experimental Unit ◽  
Bovine Embryo ◽  
Percoll Gradient ◽  
Cleavage Rate ◽  
Small Noncoding Rnas

The spermatozoon is no longer known only as a cell that delivers the male genetic material to the oocyte, because it also provides molecules such as microRNAs (miRNAs) that play a significant role in fertilization and embryonic development. MiRNAs are small noncoding RNAs capable of modulating mRNA translation, thus affecting important biological processes. Sperm miRNAs may influence embryo development and therefore, might be related to invitro production of embryos (IVP), considering that individual bulls have different fertility rates when used for IVF. The aim of this work was to identify miRNAs expressed in semen of bulls with high and low IVP rates. The composition of groups was based on a retrospective database from a reproductive biotechnology company between the years of 2016 and 2018, generating around 7000 IVP manipulations of 430 Nelore bulls. We only considered IVP manipulations that used a minimum of 30 oocytes and conventional semen selection by Percoll gradient. A total of 87 Nelore bulls fit these criteria. We then ranked bulls based on cleavage rate (number of cleaved structures/number of oocytes), blastocyst rate (number of blastocysts/number of oocytes) and embryo development rate (number of blastocysts/number of cleaved structures). Considering these three rates, we allocated bulls to two groups. The top eight were considered to have high IVP fertility (HF) and the bottom eight were grouped together as low IVP fertility (LF). We performed the T TEST procedure (SAS 9.3 software; SAS Institute Inc.) to compare the groups for cleavage (P<0.0001), blastocyst (P=0.0006), and embryo development (P=0.0001) rates. For miRNA analysis, sperm were separated using Percoll gradient and subjected to RNA extraction and cDNA synthesis. First, quantitative real-time PCR was used to evaluate the abundance of 380 bovine-specific miRNAs in a pool of samples for each group, using QuantStudio 6Flex. Then, 48 miRNAs presenting at least a 3-fold change of normalized cycle threshold values between groups were selected: 23 highly detected in HF, 1 highly detected in LF, 18 exclusively detected in HF, and 2 exclusively detected in LF. Last, we evaluated the abundance of these selected miRNAs in each experimental unit. We identified four miRNAs highly abundant in sperm from HF bulls, bta-miR-10a, bta-miR-383, bta-miR-93, and bta-miR-449b. Our results suggest that these miRNAs could play important roles in bovine embryo development. This work was supported by FAPESP (grant# 2018/03871-6).

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

21 EFFECT OF SEVERAL PARAMETERS ON PARTHENOGENETIC BOVINE EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
S. Arat ◽  
H. Bagis ◽  
A. Tas ◽  
T. Akkoc
Keyword(s):  
Embryo Development ◽  
Development Rate ◽  
Electrical Pulse ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
The Third ◽  
Parthenogenetic Embryo ◽  
Development In Vitro ◽  
Parthenogenetic Embryos

The activation of oocytes is one of the most important steps for a successful cloning and has great importance on embryo development in vitro. The objective of this study was to examine the different parameters affecting parthenogenetic embryo development in vitro. In the first experiment, two activation protocols were compared to examine the effect of electrical pulse on activation. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rat insulin-like growth factor (rIGF-1), bovine follicle-stimulating hormone (bFSH), and bovine luteinizing hormone (bLH). A group of oocytes was exposed to a DC pulse of 133 V/500 �m for 25 �s, and then activated by calcium ionophore (5 �M) for 10 min, cytochalasin D (CD) (2.5 �g/mL) + cycloheximide (CHX, 10 �g/mL) for 1 h, and CHX alone for 5 h (Group 1). Another group of oocytes was activated only by chemicals without electrical pulse. Activated oocytes were cultured for 72 h in G1-3 and then 4-6 days in G2-3 medium. In the second experiment, oocytes activated by electrical pulse and chemicals were cultured in Barc medium for 7-9 days or 72 h in G1-3 and then 4-6 days in G2-3 medium. In the third experiment, oocytes activated by electrical pulse and chemicals were cultured for 48 h or 72 h in G1-3 and then 5-7 days or 4-6 days in G2-3 medium. The differences among groups were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, cleavage rate (75.6%), development rate (37.3%), and blastocyst cell number (78.4 � 3.2) of oocytes activated by electrical pulse was higher than for the group without electrical pulse (28.7%, 8.0%, 59.5 � 4.3, respectively; P < 0.05). This result showed that activation was started more effectively by electrical pulse than by chemicals. In the second experiment, there was no significant difference on cleavage rate between the two groups (66.6%, 65.0%, respectively), and the blastocyst development rate of parthenogenetic embryos cultured in G1-3/G2-3 (36.6%) was higher than in the Barc medium group (16.6%; P < 0.05). This result showed that G1-3/G2-3 medium was more effective for parthenogenetic embryo development than Barc medium. In the third experiment, although significant differences could not be found between the two groups in the development rate of parthenogenetic embryos cultured for a total of 7-9 days (30.8%, 39.2%, respectively), the development rate of embryos cultured for 72 h in G1-3 was higher (26.4%) than for the 48-h group (15%; P < 0.05) on Day 7. This result showed that embryos developed more slowly when cultured for a shorter time in G1-3 medium before transfer to G2-3 medium. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 183 ◽  
Author(s):  
T. A. Patrocínio ◽  
C. A. C. Fernandes ◽  
L. S. Amorim ◽  
J. R. Ribeiro ◽  
G. C. Macedo ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Culture Medium ◽  
Synthetic Analogue ◽  
Bovine Embryo ◽  
High Humidity ◽  
Cleavage Rate ◽  
Antioxidant Agents ◽  
Blastocyst Rate

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175–189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24 h and oocytes were in vitro fertilized for 20 h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0 µM (control, n = 347), 0.1 µM (n = 337), 0.5 µM (n = 277), and 2.5 µM (n = 343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P < 0.05. Values are shown as mean ± SEM. Cleavage rate was higher (P < 0.05) for 2.5 µM (69.0 ± 4.4%) than for 0, 0.1, and 0.5 µM AR33 (62.1 ± 2.0%, 60.7 ± 5.9%, and 56.7 ± 5.8%, respectively). At Day 7, the blastocyst rate was similar (P > 0.05) among 0.1, 0.5, and 2.5 µM (18.1 ± 5.4%, 17.5 ± 2.9%, and 19.4 ± 3.3%, respectively) and all of them were higher (P < 0.05) than 0 µM AR33 (12.4 ± 2.5%). At Day 8, there was again no difference (P > 0.05) among 0.1, 0.5, and 2.5 µM AR33 (21.0 ± 5.0%, 18.4 ± 2.1%, and 24.6 ± 3.3%, respectively) but only 0.1 and 2.5 µM showed higher (P < 0.05) blastocyst rate than 0 µM AR33 (15.2 ± 2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5 µM AR33 can be a choice for further studies. This study was supported by Fapemig, CAPES, and CNPq.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

58 STAGE–SPECIFIC EFFECTS OF THE PROGESTERONE RECEPTOR ANTAGONIST, RU486, ON EARLY BOVINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 141
Author(s):  
C. M. O'Meara ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Progesterone Receptor ◽  
Receptor Antagonist ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Significant Decline ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Antagonist Ru486

Progesterone plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating progesterone in the immediate post-conception period are associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Progesterone-induced changes in the uterine environment are thought to be responsible for the reported advancement in conceptus elongation; however, the function of the progesterone receptor in embryos is not known. Therefore, the aim of the current study was to examine the effect of adding a progesterone receptor antagonist (mifepristone, RU486) at various stages of early embryonic development and at varying concentrations to examine the effects on subsequent embryo development in vitro. Bovine zygotes (n = 2902), 2-cell (n = 1991) and 8-cell (n = 1244) embryos, derived by in vitro maturation and fertilization, were cultured in synthetic oviduct fluid medium in the absence or presence of RU486 at concentrations ranging from 0.0004 to 20 μg mL–1. Cleavage rate (of zygotes), 8-cell development rate (of 2-cell embryos) and development to the blastocyst stage (for all cell stages) were recorded at Day 2, 3 and 8 post-insemination (day of IVF = Day 0), respectively. Cultures of zygotes in the presence of RU486 at concentrations of 0.004 and 0.04 μg mL–1 resulted in a decline in cleavage rate (62.5 ± 2.55% and 48.8 ± 5.07% for respective treatments vs controls without RU486 81.9 ± 5.97%; P ≤ 0.05). These same concentrations resulted in a significant decline in blastocyst development on Day 8 (18.8 ± 1.82% and 17.4 ± 4.85% for respective treatments compared to controls 35.1 ± 4.89%; P ≤ 0.05). Cultures at concentrations of 0.4 μg mL–1 resulted in a 10-fold decrease in blastocyst development (3.3 ± 1.3%; P ≤ 0.05) and concentrations in excess of 10 μg mL–1 completely ablated blastocyst development (P ≤ 0.05). Cultures of 2-cell embryos with RU486 at concentrations below 8 μg mL–1 had no effect on 8-cell rate or blastocyst development. However, cultures with RU486 at 10 μg mL–1 resulted in a significant decline in the proportion reaching the 8-cell stage (59.1 ± 4.59% vs 38.1 ± 2.13% for control and treated, respectively) and developing to the blastocyst stage (32.8 ± 4.68% vs 17.8 ± 3.77% for control and treated, respectively; P ≤ 0.05). Cultures with RU486 at a concentration of 20 μg mL–1 resulted in a dramatic effect in 8-cell rate (16.3 ± 2.55%; P ≤ 0.05) and prevented blastocyst development. Similarly, cultures of 8-cell embryos with RU486 at concentrations at or below 10 μg mL–1 had no effect on blastocyst development. However, cultures at concentrations of 15 or 20 μg mL–1 resulted in no blastocyst development. In conclusion, addition of the progesterone and glucocorticoid receptor antagonist RU486 to culture media has a clear stage-specific and concentration-dependent effect on bovine embryo development, which is more pronounced at earlier developmental stages. Supported by Science Foundation Ireland (07/SRC/B1156).

scholarly journals Effect of arachidonic acid supplementation and cyclooxygenase/lipoxygenase inhibition on the development of early bovine embryos

2006 ◽  
Vol 35 (2) ◽  
pp. 422-427 ◽  
Author(s):  
Rosa Maria Pereira ◽  
Carla Cruz Marques ◽  
Maria da Conceição Baptista ◽  
Maria Irene Vasques ◽  
António Eduardo Horta
Keyword(s):  
Arachidonic Acid ◽  
Embryo Development ◽  
Nordihydroguaiaretic Acid ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cell Block ◽  
Bovine Embryos ◽  
Negative Effects ◽  
Cleavage Rate ◽  
Two Phase

The effect of arachidonic acid (AA) cascade on bovine embryo development in a granulosa cell co-culture system was studied. Arachidonic acid (100 µM) was supplemented from 1-cell to 8-16 cell block stage (first three days of co-culture) and from 1-cell to hatching. Specific cyclooxygenase (indomethacin, 28 µM) and lipoxygenase (nordihydroguaiaretic acid - NDGA, 28 µM) inhibitors were used from 1-cell to 8-16 cell block stage with AA. Embryo development was assessed by cleavage, day 7-day 8 and hatched embryo rates and by measuring growth rates through development stages found in days 7-10 of culture (day 0 = insemination day). Embryo quality was scored at day 8. A 6.5-10.4% increase on cleavage rate after AA supplementation was found. This AA supplementation from 1-cell to hatching delayed embryo growth rate beyond day 7 and a reduction on hatching rate was detected. When AA supplementation was restricted to the first three days of co-culture those negative effects were overcome. Also, indomethacin and NDGA prevented the positive effect of AA and induced a significant reduction on cleavage, respectively. NDGA further decreased day 7 embryo rate and quality. Results suggest that AA has a two-phase action on bovine embryos, promoting early development and impairing embryo growth from day 7 onwards and hatching rates. Both cyclooxygenase and lipoxygenase were found to be important pathways to promote cleavage.

scholarly journals 323OPTIMIZATION OF SERUM-FREE IVP PROTOCOL: EFFECT OF SERUM-FREE IVM ON BOVINE EMBRYO DEVELOPMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 281
Author(s):  
M.E. Räty ◽  
E.H. Ketrja ◽  
K.M. Kananen-Anttila ◽  
J.M.H. Peippo
Keyword(s):  
Embryo Development ◽  
Complete Medium ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Serum Free ◽  
Logistic Regression Models ◽  
Explanatory Variables ◽  
Igf I ◽  
Embryo Cleavage ◽  
Bovine Oocytes

The use of serum in IVP medium may cause abnormalities, e.g. reduced post-thaw survival, in bovine IVP embryos (Abe H et al., 1999 Mol. Reprod. Dev. 53, 325–335). Moreover, serum may be a source of contamination and its composition is highly variable. Several reports have shown that serum-free IVF–IVC does not compromise embryo development (see, e.g. Keskinteipe L et al., 1996 Biol. Reprod. 55, 333–339). Here we studied the effect of serum-free IVM on bovine IVP embryo development, aiming for production of good quality Day 7 embryos for freezing. In total, 11734 abattoir-derived bovine oocytes in 13 batches were washed with emCare Complete Medium with BSA (1mgL−1) and matured for 24h in TCM-199 with glutamax-I (Gibco, Paisley, UK), 0.25mM Na-pyruvate, 100IUmL−1 penicillin, 100μgmL−1 streptomycin, 10μgmL−1 LH, 2μgmL−1 FSH and 1μg mL−1 β-estradiol supplemented either with 1) 10% FBS (Gibco, New Zealand), 2) 4mgmL−1 fatty acid-free albumin (FAF-BSA), 3) 4mgmL−1 FAF-BSA+growth factors (GF;; 100ngmL−1 IGF-I+100ngmL−1 EGF), 4) 4mgmL−1 polyvinylpyrrolidone (PVP), 5) 4mgmL−1 PVP+GF and 6) PVP+amino acids (10μLmL−1 MEM+20μLmL−1 BME). After 20h fertilization in FERT-TALP+2mgmL−1 BSA with semen of pre-tested IVF-bull, the oocytes were denuded and cultured in modified SOFaaci+6mgmL−1 FAF-BSA in 5% O2 (Holm P et al., 1999 Theriogenology 52, 683–700). The statistical analyses are based on logistic regression models with IVP batch and treatment as explanatory variables. The estimated probabilities (P) are shown in Table 1. The upper and lower values of 95% confidence intervals varied within P±0.05, P±0.03, and P±0.02 for cleavage, Day 7 embryo development and development of good quality Day 7 embryos, respectively. PVP+GF-IVM supported cleavage equally well as FBS-IVM (P=0.49), whereas the remaining serum-free IVM treatments had lower embryo cleavage rate than FBS-IVM (P&lt;0.01). On Day 7 none of the serum-free IVM treatments supported embryo development and development of good quality embryos as well as FBS-IVM (P&lt;0.01). Addition of GF in FAF-BSA-IVM reduced embryo cleavage, Day7 embryo development and development of good quality embryos compared to that of FAF-BSA alone (P&lt;0.03). PVP-IVM resulted in lower embryo cleavage rate than PVP+GF (P&lt;0.005), whereas according to the two other criteria there were no differences between the treatments (P&gt;0.13). In conclusion, these preliminary results indicate that replacing the FBS as a protein source in IVM needs more optimization. Table 1 Estimated probability (proportion) for embryo cleavage at 34–38 hpi (Y1), Day 7 embryo development (Y2) and development of good quality Day 7 embryos (Y3) after serum-containing and serum-free IVM

scholarly journals Lipidome signatures in early bovine embryo development

2016 ◽  
Vol 86 (2) ◽  
pp. 472-484.e1 ◽  
Author(s):  
Mateus J. Sudano ◽  
Tatiana D.S. Rascado ◽  
Alessandra Tata ◽  
Katia R.A. Belaz ◽  
Vanessa G. Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bovine Embryo
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