133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 210
Author(s):  
P. Hugon ◽  
J. Lamy ◽  
E. Corbin ◽  
P. Mermillod ◽  
M. Saint-Dizier
Keyword(s):  
Corpus Luteum ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Developmental Stages ◽  
Early Embryo ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos

2019 ◽  
Vol 31 (12) ◽  
pp. 1758 ◽  
Author(s):  
Elaine M. Carnevale ◽  
Elizabeth S. Metcalf
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Inner Cell Mass ◽  
Early Stage ◽  
Cell Mass ◽  
Quality Parameters ◽  
Early Embryo ◽  
Developmental Competence ◽  
Limited Information

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for &gt;15 years.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Alessandra Corallo Nicacio ◽  
Renata Simões ◽  
Fabiola Freitas de Paula-Lopes ◽  
Flavia Regina Oliveira de Barros ◽  
Maria Angelica Peres ◽  
...  
Keyword(s):  
Culture Conditions ◽  
Slow Freezing ◽  
Control Group ◽  
Hatching Rate ◽  
Rapid Freezing ◽  
Bovine Embryos ◽  
Group 10 ◽  
Controlled Freezing ◽  
Hatching Rates

SummaryThe aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

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