261 EVALUATION OF TWO GAS MIXTURES, ADDITION OF N-ACETYLCYSTEINE, AND LIPOPEROXIDATION LEVELS IN PORCINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 287
Author(s):  
A. L. Alvarez ◽  
Y. C. Ducolomb ◽  
H. Vera ◽  
A. Villa-Godoy ◽  
J. F. De la Torre ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Thiobarbituric Acid ◽  
Gas Mixtures ◽  
Embryo Viability ◽  
Morula Stage ◽  
Antioxidant Agent ◽  
Humidified Air ◽  
532 Nm

In vitro embryo development can be affected by many different factors, such as incubation atmosphere, which can cause oxidative stress. In this study the effect of 2 gas mixtures and the addition of the antioxidant agent N-acetylcysteine (NAC) on porcine embryos produced in vitro and lipoperoxidation (LPO) were evaluated. Ovaries collected from slaughtered gilts were aspirated to obtain oocytes, to be matured and fertilized in vitro. In experiment 1 (E1), putative zygotes were cultured in NCSU-23 medium at 38.5°C in humidified air and randomly allocated to each of the following atmospheres: conventional atmosphere (CA: 5% CO2 in air) and modified atmosphere (MA: 5% CO2, 5% O2, 90% N2). In experiment 2 (E2), zygotes were incubated in CA or MA and cultured in NCSU-23 with 2.5 mM NAC. In experiment 3 (E3), embryos produced in E1 and E2 were used to evaluate LPO with the TBARS method: embryos were sonicated, then 300 μL of distilled water and 680 μL of thiobarbituric acid were added and heated at 95°C for 20 min in a water bath. The resulting reaction was read by spectrophotometry at 532 nm. Final concentration was expressed in micromoles of TBARS/embryo (μMTE). Five days after IVF, embryos were evaluated to determine their development to the morula stage. Data from each experiment were transformed using arcsine square root function before analysis, and means were compared using ANOVA. For E1, 954 oocytes were used, 475 for CA and 479 for MA, in 5 replicates. The percentage of cleaved embryos was significantly higher in MA (300/479, 63%) compared with CA (245/475, 52%) (P < 0.05). The percentage of morulae obtained was higher in MA (127/479, 27%) than in CA (80/475, 17%) (P < 0.05). In E2, 936 oocytes were used, 470 for CA NAC and 466 for MA NAC, in 5 replicates. There were no differences in the percentages of cleaved embryos in MA NAC (250/466, 54%) compared with CA NAC (243/470, 52%), and morulae in MA NAC (86/466, 18%) compared with CA NAC (81/470, 17%) (P > 0.05). In E3, during the first 24 h of culture the embryos in CA NAC had lower LPO concentrations (0.029μMTE) (P < 0.05) than in CA (0.059μMTE), MA (0.057μMTE), and MA NAC (0.054μMTE). In morulae, a higher concentration of LPO was observed in the CA group (0.103μMTE) compared with MA (0.083μMTE), CA NAC (0.085μMTE), and MA NAC (0.081μMTE). A correlation analysis between morulae development and LPO in CA and MA, with or without NAC, did not show a significant relationship (r = 0.20; P > 0.05). It is concluded that MA improves the development of in vitro-derived porcine embryos; that NAC treatment did not cause differences in any stage of embryo development, probably meaning that the embryo has its own defensive mechanisms against LPO, and therefore that NAC is not a good antioxidant for porcine embryos, or that it does not work well at the dosage and/or in the culture medium used; that the embryos exposed to CA and MA showed higher LPO that the groups with NAC, indicating that the addition of NAC tends to decrease LPO; and that LPO could be a good indicator of embryo viability. We thank CONACYT Mexico for the graduate student’s scholarship.

203 EFFECT OF FRUCTOSE ON THE DEVELOPMENT OF BOVINE SEXED EMBRYOS IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 199
Author(s):  
S. A. Chaubal ◽  
J. Xu ◽  
X. Yang ◽  
F. Du
Keyword(s):  
Embryo Transfer ◽  
Culture Medium ◽  
Subsequent Development ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Humidified Air ◽  
Vitro Development

It is important to optimize the condition of culture to promote the development of embryos fertilized by sex-sorted sperm. Fructose is a monosaccharide hexose that can be used as a substrate for glycolysis. Recently, there has been interest in exploring the embryotrophic properties of fructose during bovine embryo culture. The present study was undertaken to ascertain the effect of supplementation of fructose in culture on the development of sexed embryos in vitro. A total of 2310 slaughterhouse dairy oocytes, spread over 3 experimental replicates, were subjected to routine in vitro maturation (IVM) for 22 h. The maturation medium was M-199 with Earl’s salts, L-glutamine, 26.19 mm sodium bicarbonate, and 25 mm HEPES plus 10% fetal bovine serum. Fertilization of IVM oocytes was performed in Brackett and Oliphant’s medium by using frozen and sorted X-sperm from a single bull. Groups of 50 matured oocytes were added to a final volume of 100 μL of fertilization medium, with a final concentration of 0.3 × 106 spermatozoa mL–1. The oocytes were incubated with X-sperm for 6 h in 5% CO2 humidified air at 39°C. Presumptive zygotes were randomly allocated to culture either in a control medium (6 mg mL–1 of BSA CR1aa medium) or with 4.5 mm fructose added to CR1aa culture medium for 7 days in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C. Embryo cleavage, and development to the morulae and blastocyst stages were evaluated on Days 2, 5, and 7 of culture, respectively. Embryos were graded according to International Embryo Transfer Society standards. The total number of blastocysts recorded was the sum of grade C1 and C2 embryos. Data were analyzed by using the General Linear Model (SPSS 11.0, SPSS Inc., Chicago, IL, USA). As shown in Table 1, addition of fructose to the culture medium affected neither the rate of cleavage nor subsequent development to the morula stage or total blastocyst development. However, the proportion of C1-grade embryos of the total blastocysts was significantly (P < 0.05) improved. This study suggests that addition of fructose to the culture medium can promote embryo quality and yields that are suitable for embryo transfer. Table 1.Effect of addition of fructose on in vitro development of bovine sexed embryos

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

1990 ◽  
Vol 68 (11) ◽  
pp. 1457-1460 ◽  
Author(s):  
Subhash C. Juneja ◽  
Melvin G. Dodson
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
In Vitro Development ◽  
Stage Embryo ◽  
Morula Stage ◽  
Vitro Development

17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)estra-4,9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 °C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 μg/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 μg/mL culture medium (p < 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 μg/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 μg/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.Key words: RU486, mouse, preimplantation embryos, embryo culture, postcoital contraceptive.

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium

2014 ◽  
Vol 26 (4) ◽  
pp. 570 ◽  
Author(s):  
Eva Torner ◽  
Eva Bussalleu ◽  
M. Dolors Briz ◽  
Marc Yeste ◽  
Sergi Bonet
Keyword(s):  
Hyaluronic Acid ◽  
Sex Ratio ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Porcine Embryo ◽  
Preferential Loss

In the present study, the effects of replacing glucose with pyruvate–lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0 mg mL–1 HA, and with either 5.55 mM glucose (IVC-Glu) or pyruvate (0.17 mM)–lactate (2.73 mM) from 0 to 48 h post insemination (h.p.i.) and then with glucose from 48 to 168 h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7 ± 1.5%) than those cultured with IVC-Glu (14.27 ± 2.75%). At 1.0 mg mL–1, HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0 mg mL–1 HA and IVC-Glu (4.28 ± 0.28% vs 11.01 ± 1.42% and 10.14 ± 2.77%, respectively) and IVC-PL (14.37 ± 1.35% vs 20.96 ± 2.85% and 22.99 ± 1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0 mg mL–1 HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL.

175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Embryo Development ◽  
Relative Abundance ◽  
Culture Medium ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Glut 1 ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas
Keyword(s):  
Lipid Peroxidation ◽  
Embryo Development ◽  
Antioxidant Properties ◽  
Thiobarbituric Acid ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

Cytokines and embryo/endometrial interactions

1995 ◽  
Vol 4 (2) ◽  
pp. 87-100 ◽  
Author(s):  
Andrew Sharkey
Keyword(s):  
Growth Factors ◽  
Animal Models ◽  
Embryo Culture ◽  
Culture Medium ◽  
Dramatic Reduction ◽  
Rt Pcr ◽  
Embryo Viability ◽  
Experimental Animal Models ◽  
Implantation Rates

Experimental animal models have shown that the in vitro embryo culture involved in many treatments for infertility results in a dramatic reduction in embryo viability. Recent advances in methodology such as RT-PCR for localization and quantitation of cytokines and their receptors, are revealing the role that this group of growth factors plays in the basic physiology of embryo development and the process of implantation itself. These studies offer the likelihood of dramatically improving in vitro embryo culture in humans and other species by supplementation of culture medium with growth factors or antagonists to improve embryo viability and hence implantation rates.

Sign in / Sign up

Export Citation Format

Share Document