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.

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.

scholarly journals 141 BENEFICIAL EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID COMBINED WITH HEMOGLOBIN ON PRE-IMPLANTATION DEVELOPMENT OF PORCINE IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 221
Author(s):  
J.H. Kim ◽  
G.S. Lee ◽  
H.S. Kim ◽  
S.H. Lee ◽  
D.H. Nam ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Embryo Development ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Porcine Embryo

Developing a porcine embryo culture system is important for increasing the rates of implantation and pregnancy of somatic cell nuclear transfer (SCNT) embryos. Ethylenediaminetetraacetic acid (EDTA) was shown to inhibit glycolytic activity of cleavage stage embryos, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for later-stage embryos as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development. On the other hand, addition of a nitric oxide (NO) scavenger, hemoglobin (Hb), to the culture medium is known to promote embryo development to the blastocyst stage. This study was conducted to evaluate the beneficial effect of EDTA combined with Hb on pre-implantation development of porcine embryos in vitro. Porcine embryos produced by in vitro maturation and fertilization were cultured for 6 days in North Carolina State University (NCSU)-23 medium supplemented with EDTA or/and Hb. All data were subjected to one-way ANOVA and protected least significant difference (LSD) test using the general linear models (GLM) procedure of the statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Statistical significance was determined when the P value was less than 0.05. In Exp. 1, culturing porcine zygotes with 100 mM EDTA (n = 537) significantly increased cleavage rates (85.3%) at 48 h post-insemination compared to supplementing with 0, 1, or 10 mM EDTA (78.9, 79.7, or 78.2%, respectively). However, EDTA at these concentrations did not promote blastocyst formation compared to the control. In addition, no difference was observed in total cell numbers in blastocysts among the experimental groups (41.8, 42.6, 45.8, 44.5, respectively). In Exp. 2, in vitro-fertilized oocytes were cultured with 0, 1, or 10 mg/mL Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the total cell number of blastocysts obtained from 1 mg/mL Hb supplementation (n = 566) compared to that of the control (56.8 vs. 41.6). In Exp. 3, culturing embryos (n = 548) with 100 mM EDTA + 1 mg/mL Hb significantly improved rates of cleavage (84.0% vs. 75.2%) and blastocyst formation (19.2% vs. 12.7%), and the total number of cells in blastocysts compared to those of the control (58.4 vs. 42.3). In conclusion, our results demonstrated that EDTA or Hb have different roles in supporting in vitro pre-implantation development of porcine embryos; EDTA mainly stimulated early cleavage up to the 2- to 4-cell stage, and Hb promoted the total cell number of blastocysts. However, combined supplementation with these two chemicals improved cleavage, blastocyst formation, and total cell number in blastocysts. This study was supported by a grant from Korea Ministry of Science and Technology (Biodiscovery).

97 SUPPLEMENTATION WITH FOLATE IN VITRO INCREASES TROPHECTODERM AND TOTAL CELL NUMBER IN IN VITRO-DERIVED PORCINE BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 161 ◽  
Author(s):  
B. K. Redel ◽  
L. D. Spate ◽  
A. N. Brown ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Transcriptional Profiling ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Total Cell Number ◽  
Culture Systems

It is vital that improvements are made to current culture environments because in vitro culture systems are suboptimal compared with in vivo. A previous transcriptional profiling endeavour conducted by Bauer et al. (2010 Biol. Reprod. 83, 791–798) identified hundreds of mRNA transcripts that were mis-expressed in porcine embryos fertilized in vivo and then cultured in vitro to Day 6 compared with in vivo Day-6 embryos. Enriched in the downregulated transcripts were 4 genes involved with the one carbon pool by folate KEGG pathway. This downregulation of genes involved with folate metabolism may illustrate an impaired folate homeostasis in embryos cultured in the current culture environment. The objective of this study was to determine the effects folate had on embryo development of in vitro fertilized embryos. Porcine cumulus–oocyte complexes were matured for 44 h in M199 supplemented with epidermal growth factor (EGF), FSH and LH. Oocytes with a visible polar body were selected and fertilized in modified tris buffered medium for 5 h and then placed into porcine zygote medium 3 with 0 mM, 0.2 mM, 0.4 mM and 0.8 mM folate to find the optimal concentration of folate. Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25-μL drops of respective culture medium and cultured to Day 6 in a water-saturated atmosphere of 5% CO2, 5% O2, 90% N2, at 38.5°C. To determine the effect folate had on development, the blastocyst rate for each treatment group was measured. Results were log-transformed and analysed by using PROC GLM in SAS (SAS Institute Inc., Cary, NC). A least-significant difference post-test comparison was completed to determine if significant differences existed between treatment groups. The percentage of cleaved embryos on Day 6 that developed to blastocyst was 56.2%, 55.9%, 66.9% and 61.8% (n = 133, 149, 135 and 135) in 0 mM, 0.2 mM folate, 0.4 mM folate and 0.8 mM, respectively. The 0.4 mM folate group tended (P = 0.07) to have a higher number of cleaved embryos that developed to the blastocyst stage. Consequently, this concentration was used for all further embryo culture experiments. Differential staining was completed to compare the number of trophectoderm and inner cell mass nuclei for embryos cultured in 0 mM or 0.4 mM folate concentrations. Staining revealed that embryos cultured with folate had an increase in number of trophectoderm (29.7 ± 1.5 vs 24.4 ± 1.4 cells; P = 0.0058) and total cell (36.9 ± 1.0 vs 31.7 ± 1.0; P = 0.0007) numbers compared with embryos cultured without folate. These results illustrate that the addition of folate to current culture medium doesn't hinder development to blastocyst and by increasing trophectoderm and total cell number may give rise to better-quality in vitro-derived embryos. It is evident that using transcriptional profiling can be a great method of identifying ways to improve embryo culture systems and, in this case, supplementing with folate. Funded by Food for the 21st Century.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

188 THE IMPACT OF OIL SOURCE AND TYPE OF GLUTAMINE ON MEDIA EQUILIBRATION TIMES AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. W. Linck ◽  
D. K. Gardner
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Oil Source ◽  
The Impact

Recently, there has been much debate involving the time necessary to effectively equilibrate an embryo culture system, i.e. dishes, media, and oil. Glutamine present in the culture medium spontaneously deaminates at 37�C to release embryo-toxic ammonium. Additionally, the source of the oil overlay can impact the culture environment. A sub-optimal oil overlay, combined with free glutamine (Gln), could effectively become embryo-toxic over a short time period (&lt;48 h). Therefore, the aim of this study was to determine how times of media equilibration, using various combinations of oil source and type of Gln, affect embryo development. Zygotes were collected from 4-week-old CF1 outbred female mice following superovulation and mating. Embryos were cultured in groups of 10 in 20-�L drops of medium G1.2. Initially, embryos were cultured in one of 4 treatment media. In this 2 � 2 factorial design, the culture medium was pre-equilibrated 18 h prior to embryo retrieval and contained free Gln or the heat-stable dipeptide alanyl-glutamine (AlaGln), combined with an oil source of either Sigma mineral oil (Sigma-Aldrich Corp., St Louis, MO, USA) or Ovoil paraffin oil (Vitrolife, Inc., Englewood, CO, USA). The initial study was then repeated using only the best and worst case groups to determine the effect of incubation time as a variable (either 2 h or 18 h). Blastocyst development and total cell numbers were analyzed after 72 h of culture, and differences between treatments were assessed using Fisher&apos;s exact test and Student&apos;s t-test. After 18 h of pre-equilibration (n e 300 embryos/treatment), blastocyst development in Ovoil + AlaGln (38.6%) was significantly greater when compared to: Ovoil + Gln: 25.5% (P &lt; 0.01), Sigma + AlaGln: 12.8% (P &lt; 0.01), and Sigma + Gln: 11.9% (P &lt; 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 � 10) were significantly decreased: 35.5 � 7 (P &lt; 0.001), 34.9 � 9 (P &lt; 0.001), and 29.9 � 9 (P &lt; 0.001), respectively. In the second experiment, blastocyst development and total cell number between Ovoil + AlaGln (n = 224) and Sigma + Gln (n = 264) after 18 h of pre-equilibration were: 40.4% vs. 9.9% (P &lt; 0.01) and 46.6 � 9 vs. 29.4 � 9 P &lt; 0.001), respectively. However, after 2 h of pre-equilibration, the results between Ovoil + AlaGln (n = 260) and Sigma + Gln (n = 284) were: 42.3% vs. 18.3% (P &lt; 0.01) and 46.9 � 10 vs. 33.6 � 6 (P &lt; 0.001), respectively. Therefore, when comparing blastocyst development and total cell number between pre-equilibration times (2 h vs. 18 h), the Ovoil + AlaGln group, 42.3% vs. 40.4% and 46.9 � 10 vs. 46.6 � 9, showed no significant differences, respectively. In contrast, the Sigma + Gln group produced significant differences for both blastocyst development, 18.3% vs. 9.9% (P &lt; 0.01), and total cell number, 33.6 � 6 vs. 29.4 � 9 (P &lt; 0.05), between pre-equilibration times (2 h vs. 18 h), respectively. Data presented confirm the need for an alternative source of glutamine in embryo culture media. The data also indicate that the source of oil has a profound effect on the experimental outcome. Using the appropriate oil and form of Gln means that media can be safely equilibrated for 18 h.

213 MATURATION OF OOCYTES WITH FOLLICULAR FLUID FROM GILTS CONSUMING HIGH FAT AND FRUCTOSE AFFECTS SUBSEQUENT EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
R. Poole ◽  
V. McCracken ◽  
M. Rhoads ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Follicular Fluid ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
High Fat ◽  
Total Cell Number ◽  
Treatment Groups ◽  
High Fructose

Infertility among women has become a growing issue in the world requiring a significant number to seek treatment by means of assisted reproductive technologies. One suggested reason for the fertility issue, which is known to specifically affect oocyte quality, is the modern diet. Previously, we have demonstrated that feeding a high-fructose diet to gilts led to poor reproductive tract characteristics and infertility. In this study, pre-pubescent gilts were fed either a high-fructose; high-fat diet (HFHF), with 15% beef tallow and 35% fructose; or an industry control diet (IND). Porcine follicular fluid (pFF) collected from these gilts was introduced into in vitro maturation systems to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. Follicles from ovaries, collected at a local abattoir, were aspirated by an 18 G needle attached to a 10-mL sterile syringe. Then selected cumulus‐oocyte complexes were maturated in vitro in a TCM-199 maturation media with cysteine, glucose, sodium pyruvate, epidermal growth factor (EGF), FSH, LH, and 20% pFF from treatment groups. Additionally, another group of oocytes, labelled follicle fluid free (FFF), were maturated in TCM-199 media without pFF. Three replicate experiments were conducted using a total of 365 oocytes, 124 FFF, 121 IND, and 120 HFHF. Oocytes were denuded by exposure to 0.1% hyaluronidase and oocytes that reached metaphase II (MII) were selected for in vitro fertilisation. After 5 h of co-incubation in modified Tween medium B with milk powder (mTBM)-based IVF media, presumable zygotes were transferred to porcine zygote medium-3 (PZM-3). Blastocyst frequency was recorded on Days 5 and 6. Day 6 blastocysts were stained with Hoechst for total cell number evaluation. The frequencies of blastocyst formation among the treatment groups were compared by a chi-squared test, and total cell numbers were compared by Student's t-test. Statistical significance was defined by P < 0.05. The frequency of oocytes reaching metaphase II (MII) were observed as 77.4% FFF, 72.7% IND, and 71.7% HFHF (P > 0.05), indicating the supplementation of pFF did not affect maturation. Day 5 blastocysts were observed at frequencies of 8.3% FFF, 6.8% IND, and 4.7% HFHF and did not differ. However, frequency of Day 6 blastocysts from HFHF group was tended to be lower compared with that of other groups; 12.5% FFF, 11.4% IND, and 4.7% HFHF (P = 0.06 and P = 0.1). Average total cell number of Day 6 blastocysts observed were 41.0 ± 9.1 FFF, 36.0 ± 8.9 IND, and 48.3 ± 10.6 HFHF. The total cell number from HFHF group tended to be higher than only that of IND group (P = 0.07). Based on these results, we concluded that the follicular fluid of females consuming HFHF diets did not have impact on nuclear maturation of oocytes but might affect oocyte competency, thus resulting in detrimental effects on subsequent development of embryos, especially blastocyst formation. Further studies will help us identify more specific effects of nutrition on oogenesis and subsequent embryo development.

88 IS STEREOMICROSCOPY AN EFFICIENT METHOD FOR EVALUATING THE VITRIFIED PORCINE EMBRYO QUALITY?

2006 ◽  
Vol 18 (2) ◽  
pp. 152
Author(s):  
C. Cuello ◽  
F. Berthelot ◽  
B. Delaleu ◽  
C. Almiñana ◽  
J. M. Vázquez ◽  
...  
Keyword(s):  
Cell Death ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Large White ◽  
Porcine Embryo

The development of the open pulled straw vitrification has provided excellent results of in vitro porcine embryo development. Embryo quality evaluation after vitrification has been traditionally focused on morphological assessment performed by stereomicroscopy. The objective of this experiment was to evaluate the efficiency of the stereomicroscopic evaluation of vitrified-warmed (V) porcine blastocysts. Unhatched blastocysts were obtained after slaughter from Large-White gilts (n = 9). Blastocysts (n = 75) were vitrified and warmed using the protocol described by Cuello et al. (2004 Theriogenology 61, 353-361). After warming, vitrified blastocysts were cultured for 24 h. Then blastocysts were morphologically assessed for their progression and morphology by stereomicroscopy. Blastocysts that reformed their blastocoelic cavities showing an excellent appearance were considered viable. Some of the viable blastocysts kept their zonae pellucidae (V viable expanded blastocysts) and others hatched during the in vitro culture (V viable hatched blastocysts). The remaining blastocysts were classified as degenerated embryos. A group of fresh blastocysts was not vitrified and cultured in vitro for 24 h (control group). All of the control blastocysts were considered viable by stereomicroscopy. Some fresh, V viable expanded, V viable hatched, and V degenerated blastocysts (n = 13, n = 19, n = 9, and n = 9, respectively) were processed for ultrastructural study by light and transmission electron microscopy or stained with Hoechst-33342 and TUNEL for cell death evaluation (n = 16, n = 21, n = 11, and n = 6, respectively). All V hatched blastocysts showed ultrastructure similar to that of control hatched blastocysts. However, 26.3% of the V viable expanded blastocysts revealed important ultrastructural alterations in comparison with control expanded blastocysts. These observations suggest that stereomicroscopic evaluation was not efficient enough for V expanded blastocysts. As expected, degenerated blastocysts showed ultrastructural disintegration and disorganization. Hatched V blastocysts did not differ (P < 0.05) from control hatched blastocysts with regard to the total cell number and ratio of death cells (173 � 4.8 vs. 202.1 � 10.9 and 2.8 � 0.5% vs. 1.9 � 0.3%, respectively). However, V expanded blastocysts a had higher (P < 0.01) cell death level (4.3 � 3.4%) than that observed in the control expanded blastocysts (1.1 � 0.3%). Degenerated embryos showed the lowest (P < 0.01) total cell number (45.7 � 4.0). The 66.7% of the degenerated blastocysts exhibited wide TUNEL-labeled areas, and the remaining 33.3% showed TUNEL label over 19.4 � 6.3% of the cells. In conclusion, the hatching rate assessed by stereomicroscopy is a more efficient parameter than assessing the in vitro viability (ratio of blastocysts that reformed their blastocoelic cavities after warming) for estimating the quality of V blastocysts. This work was supported by CICYT (AGL2004-07546) and S�neca (01287/PD/04).

Comparative effects of adding β-mercaptoethanol or L-ascorbic acid to culture or vitrification–warming media on IVF porcine embryos

2014 ◽  
Vol 26 (6) ◽  
pp. 875 ◽  
Author(s):  
Miriam Castillo-Martín ◽  
Sergi Bonet ◽  
Roser Morató ◽  
Marc Yeste
Keyword(s):  
Ascorbic Acid ◽  
Embryo Development ◽  
Survival Rates ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Embryo Survival ◽  
Apoptosis Index ◽  
Yield Quality

The aims of the present study were to; (1) determine the effects of supplementation with two antioxidants during in vitro culture (IVC) on embryo development and quality; and (2) test the effects of adding the antioxidants to vitrification–warming media on the cryotolerance of in vitro-produced (IVP) porcine blastocysts. In Experiment 1, presumptive zygotes were cultured without antioxidants, with 50 µM β-mercaptoethanol (β-ME) or with 100 µM l-ascorbic acid (AC). After culture, blastocyst yield, quality and cryotolerance were evaluated in each treatment group. In Experiment 2, survival rates (3 and 24 h), total cell number, apoptosis index and the formation of reactive oxygen species (ROS) in blastocysts vitrified–warmed with 100 µM AC or 50 µM β-ME or without antioxidants added to the vitrification medium were compared. Antioxidant addition during IVC had no effect on embryo development, total cell number or the apoptosis index, and culturing embryos in the presence of β-ME had no effects on cryotolerance. In contrast, ROS levels and survival rates after vitrification–warming were significantly improved in embryos cultured with AC. Furthermore, addition of AC into vitrification–warming media enhanced embryo survival and embryo quality after warming. In conclusion, our results suggest that supplementing culture or vitrification media with 100 µM AC improves the quality and cryosurvival of IVP porcine blastocysts.

31 INTRAFOLLICULAR TRANSFER OF FRESH AND VITRIFIED IMMATURE BOVINE OOCYTES: AN OPTION FOR EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 145
Author(s):  
J. F. W. Spricigo ◽  
S. B. S. Netto ◽  
C. V. Muterlle ◽  
S. A. D. Rodrigues ◽  
L. O. Leme ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Total Cell ◽  
Cell Counting ◽  
Dominant Follicle ◽  
Total Cell Number ◽  
Embryo Production ◽  
Immature Oocytes ◽  
Oestradiol Benzoate

The association of a technique that guarantees the embryo quality of in vivo blastocyst and otherwise allows the increment of embryo production, such as the in vitro model, would result in a healthier and cheaper embryo. Immature oocytes intrafollicular transfer (IOIFT) is a technique in which immature oocytes obtained by ovum pickup are injected into a dominant follicle of a synchronized recipient. We hypothesised that IOIFT could support embryo development even after oocyte vitrification. We aimed to compare IOIFT or traditional in vitro embryo system using fresh and vitrified immature oocytes. Cumulus-oocyte complexes (COC) were obtained from slaughterhouse ovaries; after selection, half of COC were vitrified by cryotop method. Vitrified and fresh oocytes were either cultured in vitro or transferred to a follicle on the recipient ovary. Four groups were used: (1) fresh immature oocytes (VitroF); (2) vitrified/warmed immature oocyte (VitroV), both (1) and (2) were in vitro matured, fertilized, and cultured; (3) fresh immature oocytes submitted to IOIFT (VivoF), and (4) vitrified/warmed immature oocytes submitted to IOIFT (VivoV). Recipients heifers (n = 12) were synchronized with the following protocol: on Day –10 a progesterone device (P4, Primer) was inserted together with the administration of 2 mL of oestradiol benzoate (RIC-BE); at Day –8 the devices were removed simultaneously to the administration of 2 mL of prostaglandin (Veteglan); Day –1, 1 mL of oestradiol benzoate was administered. The COC from VivoF or VivoV groups were injected into dominant follicle (>10 mm), 58 h after P4 removal. The intrafollicular injections were guided by a 7.5-MHz ultrasound vaginal probe (Aloka) using a modified aspiration system. For the injection, 25 COC were placed into a needle (27 G), with 80 μL of follicular fluid. An insulin syringe served to perform the injections. A single dose of semen was used for AI, soon after IOIFT, and embryos were recovered by uterine flushing 7 days later. The results of embryo development and total cell number and apoptotic cells (TUNEL) are present in Table 1. The results obtained for fresh oocytes suggest that IFIOT technique may be an option for bovine embryo production. Despite, it does not improve embryo development or increase total cell number when vitrified and warmed immature oocytes are used. Table 1.Cleaved and blastocyst rates, total cell number, and apoptotic cell counting of expanded blastocyst of fresh (F) and vitrified (V) cumulus-oocyte complexes that were in vitro (Vitro) or immature oocytes intrafollicular transfer (Vivo) produced

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Sign in / Sign up

Export Citation Format

Share Document