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.

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.

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).

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.

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.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

scholarly journals Effect of follicular fluid supplementation during in vitro maturation on total cell number in bovine blastocysts produced in vitro

2014 ◽  
Vol 43 (3) ◽  
pp. 120-126 ◽  
Author(s):  
Maria Helena Coelho Cruz ◽  
Naiara Zoccal Saraiva ◽  
Jurandir Ferreira da Cruz ◽  
Clara Slade Oliveira ◽  
Maite Del Collado ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Fluid Supplementation

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

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