scholarly journals Superovulation following follicular synchronization with GnRH at random stages of the oestrous cycle in heifers: oocyte competence and in vitro embryo production

2010 ◽  
Vol 55 (No. 5) ◽  
pp. 190-194
Author(s):  
H. Kohram ◽  
V. Vahedi ◽  
A. Farahavar
Keyword(s):  
Late Phase ◽  
Oestrous Cycle ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Embryo Production ◽  
Oocyte Competence ◽  
The Mean ◽  
Follicular Puncture ◽  
Vitro Production

The objective of this study was to develop a superovulatory program based on the synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. In this experiment, GnRH was given to 30 heifers in lactation between Days 0 and 7 (n = 13), 8 and 12 (n = 12), 13 and 16 (n = 5) of the oestrous cycle. Twenty-four heifers were used as controls and did not receive any GnRH. All follicles ≥ 6 mm were punctured 4 days after GnRH treatment in treated animals and between Days 8 and 12 of the oestrous cycle in control heifers. Two days after the follicular puncture, all heifers were superstimulated with 160 mg Folltropin-V given twice daily over 2 days. Oocytes were collected 42 h after the last FSH treatment. The oocytes were subjected to IVM/IVF and the developmental competence of embryos was compared. In vitro production of embryos was affected only by the stages of the oestrous cycle when the GnRH treatment was given and not by the GnRH treatment. No difference (P > 0.1) in the mean number of oocytes, cleavage and embryo production was noted between the control animals and the animals treated with GnRH in the late phase of the oestrous cycle. The mean number of blastocysts was higher (P < 0.05) in heifers treated with GnRH in the mid and the late phase of the oestrous cycle than in the early phase. In conclusion, the in vitro production of embryos was compromised in the present study with heifers following the follicular synchronization with GnRH. This procedure is advantageous for the in vitro production of bovine embryos since the spontaneous oestrus is eliminated. However, more investigations are needed to increase the competence of oocytes obtained following this procedure.

scholarly journals Modern achievements of the development of scientific research at obtaining in vitro sheep embryos

2020 ◽  
Vol 22 (93) ◽  
pp. 60-68
Author(s):  
O. M. Sharan ◽  
V. Yu. Stefanyk ◽  
S. G. Shalovylo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Culture ◽  
Biologically Active ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Key Factor ◽  
Vitro Production

New literature data on research aimed at improving the in vitro production of sheep embryos presents in the article. An analysis of the achievements of scientists from different countries to increase the efficiency of the main stages of embryo production in vitro: maturation of oocytes in vitro, their in vitro fertilization and in vitro embryo culture. In the literature experience has shown that the efficiency of oocyte maturation in vitro is significantly influenced by the experience and qualifications of scientists, the age of the egg donor, the improvement of the environment by adding roscovitin to inhibit meiosis, α-linolenic acid, cerium dioxide nanoparticles (CeO2 NPs) and sericin to accelerate nuclear maturation and increase the number of oocytes of the second meiotic metaphase (MII). The main factors influencing the effectiveness of in vitro fertilization have been identified, and the parameters of the limited time of fertilization ability of sperm and the ability of oocytes to fertilize, which is called the “fertile span”, have been determined. The main effective medium that increases the effectiveness of in vitro fertilization – synthetic oviduct fluid (SOF) with the addition of heparin and serum of cattle or sheep. The main parameters of sheep embryo culture in vitro are presented with the definition of the most commonly used media and their influence on embryonic development. Potential ways to improve the production of sheep embryos in vitro with the determination of morphological evaluation of categories of oocytes, methods of synchronization of their maturation in vitro are also highlighted. At the same time, literature data on the synchronization of oocyte-cumulus complexes with the use of a large number of inhibitors of meiotic division are presented, which according to many scientists may be a key factor in improving the efficiency of sheep embryo production in vitro. In addition, the results of studies of many scientists on the expansion of the fertile gap of oocytes of sheep cultured in vitro using certain biologically active substances were analyzed. In conclusion, the prospect of using the technology of in vitro production of sheep embryos in biomedical research is highlighted.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 209
Author(s):  
S. Miyashita ◽  
K. Miyata ◽  
C. Tachibana ◽  
Y. Inaba ◽  
H. Koyama ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Large Mass ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Stage Of Development ◽  
The Mean ◽  
Vitro Production

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2013 ◽  
Vol 25 (1) ◽  
pp. 250
Author(s):  
N. Schlüter ◽  
A. Hanstedt ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Relative Abundance ◽  
Clinical Signs ◽  
Follicular Phase ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Vitro Production

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

scholarly journals Synchronization with estradiol benzoate in the presence of the corpus luteum in Wagyu cows increases the number of medium follicles but does not interfere with in vitro production of embryos

2021 ◽  
Vol 42 (3) ◽  
pp. 1147-1158
Author(s):  
Maria Fernanda Zamai ◽  
◽  
Fábio Luiz Bim Cavalieri ◽  
Marcia Aparecida Andreazzi ◽  
Fabio Morotti ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Estradiol Benzoate ◽  
Breeding Systems ◽  
In Vitro Production ◽  
Embryo Production ◽  
Follicular Wave ◽  
In Vitro Embryo Production ◽  
Oocyte Donors ◽  
Vitro Production

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P < 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P > 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.

scholarly journals Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

2020 ◽  
Vol 18 (2) ◽  
pp. 249-255
Author(s):  
Nguyen Viet Linh ◽  
Nguyen Thi Hiep
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
Vitro Production ◽  
Normal Fertilization

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

scholarly journals Kinetic analysis of in vitro production of wild-type Spodoptera frugiperda nucleopolyhedrovirus

2010 ◽  
Vol 53 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Andréa Farias de Almeida ◽  
Gorete Ribeiro de Macedo ◽  
Leslie Chee Loong Chan ◽  
Márcia Regina da Silva Pedrini
Keyword(s):  
Cell Line ◽  
Spodoptera Frugiperda ◽  
Wild Type ◽  
In Vitro Production ◽  
Extracellular Virus ◽  
Cell Productivity ◽  
The Mean ◽  
Body Production ◽  
Vitro Production

In this study, the kinetic behavior of Sf9 and Sf21 cells used in the production of a baculovirus biopesticide to control the pest of corn Spodoptera frugiperda was analyzed. Kinetic variables such as maximum specific growth rate, cell productivity, mean rate of infection, as well as the mean rate of occlusion body production were determined during the infection of these cell-lines with the extracellular virus of the S. frugiperda nucleopolyhedrovirus (SfMNPV). The Sf9 cell-line resulted in better viral production results (5.0 x 10(8) OB/mL) than the Sf21 cell-line (2.5 x 10(8) OB/mL).

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