92 Using physical parameters of bovine zygotes to predict invitro development success

2020 ◽  
Vol 32 (2) ◽  
pp. 172
Author(s):  
C. L. Timlin ◽  
A. Lynn ◽  
L. K. Wooldridge ◽  
K. Uh ◽  
A. D. Ealy ◽  
...  
Keyword(s):  
Digital Camera ◽  
Blastocyst Stage ◽  
Physical Parameters ◽  
Outer Diameter ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Artificial Activation ◽  
Oviductal Fluid ◽  
Blastomere Number

The objective of this study was to determine if physical parameters of bovine zygotes are correlated with invitro development success. We examined the relationship between a zygote's outer diameter (OD), cell area, and zona pellucida (ZP) thickness on blastocyst development and blastomere number. Bovine cumulus-oocyte complexes, from abattoir-derived ovaries, were matured in tissue culture medium 199-based maturation medium for 21-23h. Invitro fertilisation was performed with frozen thawed semen containing a pool of 4 Holstein bulls. After incubation with sperm for 16-18h, presumptive zygotes (n=875) were denuded and placed in 5µL droplets of synthetic oviductal fluid-bovine embryo 1 medium (SOF-BE1) under oil to be individually photographed using an inverted scope with a digital camera. Zygotes were then group-cultured in 50µL of synthetic oviductal fluid-BE1 droplets in a polyester micromesh to identify individuals. Development to the blastocyst stage was recorded on Day 7 and Day 8 of culture, and Day 8 embryos were fixed and stained with Hoechst 33342 (n=87). ImageJ was used to measure the OD, area, and ZP from images. Data were analysed with a logistic regression using the lme4 package in R with Day 7 or Day 8 blastocyst development as the response; OD, area, and ZP as predictors; and study replicate (n=11) and culture droplet as random effects. Residual estimates of area and ZP calculated from OD were used to account for collinearity. Average OD was 151.2µm, ranging 130.4-171.6µm. Average ZP was 11.8µm, ranging 7.2-17.5µm. Area averaged 9856.7µm2, ranging from 6421.9 to 13 814.8 µm2. There was a tendency for an effect of OD on probability of development on Day 8 (P=0.08) but not Day 7. There was an effect of ZP on the probability of development on Day 8 (P=0.04) but not Day 7. Blastocyst rates on Day 8 for zygotes with ZP <11.8µm were 24.3%, whereas zygotes with ZP >11.8µm were 21.7%. There was an interaction (P<0.05) between OD and ZP thickness on Day 7 and Day 8. Estimated probability of development of zygotes with ZP <10.7µm and OD <146.9µm averaged 28.5%, whereas those with ZP >12.7µm and OD >155.8µm averaged 43.3%. The observed blastocyst rates for these two groups were 26.2% and 25%, respectively. Zygotes with OD between 146.9-155.8µm and ZP between 10.7-12.7µm had an estimated development probability of 22.2%. The obtained Day 8 blastocyst rate for this group was 22.5%. Area did not affect probability of development, but was positively correlated with total blastomere number at Day 8 (P=0.01; marginal R2=0.09). The observed interaction between ZP thickness and OD may be indicative of an optimum ooplasm area before maturation or fertilisation that may enhance development. Area assessment after fertilisation is not correlated with development success. In addition, these data in comparison with our previous data generated in parthenogenetic embryos indicate that artificial activation of oocytes is not an ideal model in place of IVF for studying development. We continue to demonstrate that physical parameters of zygotes may have potential as a noninvasive, objective selection tool of embryos.

158 Predicting embryo development success with physical parameters of oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 204
Author(s):  
C. L. Timlin ◽  
A. Lynn ◽  
R. R. White ◽  
K. Lee ◽  
V. R. G. Mercadante
Keyword(s):  
Polar Body ◽  
Digital Camera ◽  
Blastocyst Stage ◽  
Physical Parameters ◽  
Oocyte Diameter ◽  
Blastocyst Development ◽  
Noninvasive Methods ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Blastocyst Rate

There is a continual search for reliable, noninvasive methods of selecting viable oocytes and embryos. Previous studies have indicated that the physical size of oocytes may reflect their developmental potential. The objective of this study was to observe the correlation between an oocyte’s diameter [including zona pellucida (ZP), cell area, and ZP thickness] and its ability to develop. Bovine cumulus-oocyte complexes were collected from abattoir-derived ovaries and incubated for 24h in TCM-199-based maturation medium. The cumulus-oocyte complexes were denuded by vortexing with hyaluronidase, and mature oocytes were selected based on presence of a visible polar body. Selected oocytes were artificially activated by incubating in 5 μM ionomycin for 5min followed by incubation in 2mM 6-DMAP for 3h (n=723). After activation, oocytes were placed in individual 5-μL culture droplets under oil and photographed using an inverted scope with digital camera. Oocytes were then group cultured in 50-μL droplets in a polyester micromesh for identification of individual oocytes. Development to the blastocyst stage was noted on Days 7 and 8 of culture. ImageJ was used to measure diameter, area, and ZP thickness from images. A logistic regression using the lme4 package in R was run with Day 7 or 8 blastocyst development as the response; diameter, area, and ZP thickness as predictors; and replicate and culture droplet as random effects. The residual estimates of area and ZP thickness from diameter were used to account for correlation between predictors. Only significant interactions were kept in the model. The ZP thickness ranged from 6.1 to 17.7μm with a mean of 12μm. There was a significant correlation between diameter and ZP thickness (P<0.01, R2=0.12) with a correlation coefficient of 0.35. Oocyte diameter had a significant effect on subsequent blastocyst development on Day 7 (P<0.01) and Day 8 (P<0.01), with larger oocytes more likely to develop on both days. Oocytes were also grouped into quantiles by diameter. Larger groups were more likely to develop to the blastocyst stage on Day 7 (P<0.001) and Day 8 (P<0.001). Blastocyst rate on Day 8 for oocytes with diameters <149.5μm was 24.2%, whereas blastocyst rate on Day 8 for those with diameters=159μm was 41.2%. In addition, ZP thickness also had an effect: oocytes with thinner ZP were more likely to develop to the blastocyst stage on Day 7 (P<0.001) and Day 8 (P<0.001). Blastocyst rate for oocytes with a ZP thickness <11μm was 37%, whereas the rate for those with a ZP thickness of 12.9μm was 27.6%. Area did not have an effect on blastocyst formation on Day 7 or 8 (P=0.21). Here we have demonstrated that the oocyte diameter, including the ZP and ZP thickness, affects its probability of development. Larger oocytes and those with thinner ZP are more likely to develop to the blastocyst stage. Differences in the size of the perivitelline space could explain why diameter had a significant effect on development and area did not. Further studies will focus on determining the relationship between these physical parameters of oocytes and embryo quality.

Effect of α-tocopherol on in vitro culture of bovine embryos

2007 ◽  
Vol 87 (4) ◽  
pp. 539-542 ◽  
Author(s):  
A. Marques ◽  
G. Antunes ◽  
P. Santos ◽  
A. Chaveiro ◽  
F. Moreira da Silva
Keyword(s):  
Culture Media ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Media Control ◽  
Oviductal Fluid ◽  
Bovine Oocytes ◽  
Key Words Antioxidant

Bovine oocytes were matured and fertilised under 5% CO2. Presumptive zygotes were co-cultured in synthetic oviductal fluid droplets, supplemented with either 0 (control), 25, 50, 100, or 200 µM of α-tocopherol. Blastocyst development rates were significantly influenced (P < 0.05) by the level of antioxidant in the culture media. Control showed a blastocyst yield of 18.46, 21.11, 27.92, and 31.66%, respectively, at α25; α50 and α100. Blastocyst yield for α200 was severely decreased, to 8.01%. An increase in overall IVF results was also observed as the concentration of α-tocopherol increased (control, 14.21%; α25, 14.35%; α50, 19.52%; α100, 21.11%), decreasing to 5.75% for the concentration of α200. The present study clearly demonstrates that α-tocopherol at a concentration of 100 µM significantly improves the proportion of oocytes that develop to the blastocyst stage. Key words: Antioxidant, α-tocopherol, reactive oxygen species, bovine, embryo

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

142 EFFECT OF HIGH AND LOW ANTRAL FOLLICLE COUNT IN PUBERTAL BEEF HEIFERS ON IVF

2014 ◽  
Vol 26 (1) ◽  
pp. 184
Author(s):  
C. C. Chase ◽  
E. C. Wright ◽  
A. K. McNeel ◽  
R. A. Cushman ◽  
G. A. Perry ◽  
...  
Keyword(s):  
Prostaglandin F2α ◽  
Antral Follicle ◽  
Antral Follicle Count ◽  
Blastocyst Stage ◽  
Oestrous Cycle ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Frozen Semen ◽  
Oocyte Collection ◽  
Collection Medium

Pubertal heifers can be classified between those with high (n = 25) or low (n = 15) antral follicle counts (AFC). The objective of this study was to determine oocyte development and maturation (e.g. fertility) in an IVF system for high- and low-AFC heifers. From a pool of 120 heifers, 10 high- and 10 low-AFC heifers were determined by transrectal ultrasonography; all heifers with evidence of oestrous cyclicity (i.e. pubertal) were synchronized with two 5-mL injections of prostaglandin F2α 11 days apart. Heifers were euthanized over 4 days on Days 15 to 16 of the synchronized oestrous cycle. A total of 15 heifers (n = 7 high and n = 8 low AFC) were at the appropriate stage of the oestrous cycle. Ovaries were collected and transported to the laboratory. Follicles less than 8 mm in diameter were aspirated. The IVF procedures and media were as previously described (Miles et al. 2004. Biol. Reprod. 71, 1919–1926). Cumulus-oocyte complexes (COC) were identified and washed in oocyte collection medium and then in maturation medium and were cultured (5% CO2; 38.5°C) for 24 h. Following maturation, COC were transferred and washed in fertilization medium. Thawed frozen semen from a crossbred bull was subjected to the swim-up procedure. Motile spermatozoa were collected and added to COC to yield a final concentration of spermatozoa per milliliter of fertilization medium. About 24 h later, presumptive zygotes were washed in development medium, placed in microdrops of development medium, and cultured for 8 days. On Days 3 and 8 after fertilization, cleavage and blastocyst development, respectively, were assessed. Data were analysed using the Proc Mixed procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of day of collection (n = 4), group (n = 7 high- or n = 8 low-AFC heifers), and the interaction. The interaction did not differ (P = 0.10). Day of collection influenced (P < 0.05) the number of COC and the number of oocytes cleaved. High- compared to low-AFC heifers had the greater (P < 0.05) numbers of COC (42.7 ± 4.66 v. 22.1 ± 4.59), oocytes that cleaved (28.1 ± 3.60 v. 15.9 ± 3.55), and developed to blastocysts (13.2 ± 1.71 v. 6.2 ± 1.69). However, there was no difference (P > 0.10) in the percentage of COC that cleaved (65.3 ± 5.58 v. 66.2 ± 5.50%, high v. low, respectively) or that developed to blastocysts (46.7 ± 6.75 v. 42.2 ± 6.65%). In conclusion, AFC did not appear to affect oocyte maturation and development through the blastocyst stage.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during invitro maturation of oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 133
Author(s):  
A. Zegarra ◽  
J. Rivas ◽  
A. Gallegos ◽  
E. Mellisho
Keyword(s):  
Embryonic Development ◽  
Decisive Role ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Maturation Medium ◽  
Advanced Stages ◽  
Commercial Medium ◽  
Antioxidant Effects

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P&lt;0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P&gt;0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.

182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 199
Author(s):  
A. W. Harl ◽  
E. L. Larimore ◽  
A. Al Naib ◽  
L. K. Wooldridge ◽  
A. D. Ealy ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cumulus Expansion ◽  
Oocyte Competence ◽  
Maturation Medium

The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10 mm) and small follicles (≤3 mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n = 250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21 h of maturation). Other COC (n = 770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P < 0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P < 0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P < 0.05) and also less likely to develop to the blastocyst stage (P < 0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42 h after prostaglandin F2α injection. Samples from individual cows were categorized as having high oestradiol (>800,000 pg mL−1; H) or low oestradiol concentrations (<800,000 pg mL−1; L). The FF was retained for use in in vitro experiments, where it was added to maturation media (20% vol/vol). Cumulus-oocyte complexes (n = 1,775) were randomly distributed into treatments across 12 in vitro maturation/fertilization replicates (H and L, balanced within replicate; 4 replicates/cow). Each replicate included the following 3 control groups: maturation medium containing BSA without FF, maturation medium without BSA with abattoir-derived FF, and maturation medium without BSA and without FF. The COC were matured in their assigned medium for 21 h, and then all COC were subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 7 and 8 post-fertilization. Oestradiol content of the FF (H v. L) did not affect oocyte cleavage nor blastocyst rates on Day 7 or 8. The results of these studies indicate that although FF improves cumulus cell expansion during maturation in vitro, it does not result in higher rates of cleavage or blastocyst development regardless of oestradiol content.

121 DEVELOPMENTAL CHANGES IN THERMOPROTECTIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR-1 ON THE PREIMPLANTATION BOVINE EMBRYO

2011 ◽  
Vol 23 (1) ◽  
pp. 165
Author(s):  
A. Q. S. Bonilla ◽  
L. J. Oliveira ◽  
M. Ozawa ◽  
E. M. Newsom ◽  
M. C. Lucy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Heat Shock ◽  
Differentially Expressed Genes ◽  
Blastocyst Stage ◽  
Differentially Expressed ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Developmentally Regulated ◽  
Cell Embryo

Insulin-like growth factor-1 (IGF1) is an important endocrine signal for regulation of early embryonic development. It increases the proportion of preimplantation embryos becoming blastocysts, alters blastocyst gene expression, improves resistance of embryos to various stresses and can enhance survival of embryos after transfer to recipients. The present study had 2 objectives. The first was to determine whether the thermoprotective actions of IGF1 on the preimplantation bovine embryo were developmentally regulated, with the 2-cell embryo being refractory to IGF1. The second was to determine the molecular basis for the improved competence of embryos treated with IGF1 to establish pregnancy after transfer to heat-stressed recipients. Heat shock at 41°C decreased (P < 0.005) the percentage of 2-cell embryos becoming a blastocyst at day 8 (39.5 v. 21% for 38.5 and 41°C, respectively), and treatment of embryos with 100 ng mL–1 IGF1 did not provide thermoprotection to 2-cell embryos heat shocked at 41°C (21 v. 21% for control and IGF1-treated embryos, respectively). Heat shock at 41°C had no effect on blastocyst development of day 5 embryos. However, exposure to 42°C reduced (P < 0.001) blastocyst development of day 5 embryos (87 v. 47.6% for 38.5 and 42°C, respectively). Furthermore, treatment of embryos with 100 ng mL–1 IGF1 reduced (P = 0.05) the effect of heat shock at 42°C on day 5 embryos (48 v. 66% control and IGF1-treated embryos, respectively). Failure of IGF1 to alter 2-cell embryo survival after heat shock was not associated with reduced expression of genes involved in IGF1 signaling (IGF1R, RAF1, PI3K, and MAPK), as shown by quantitative real-time RT-PCR assay, or in amounts of immunoreactive IGF1R protein. Treatment with IGF1 had little effect on the transcriptome at the blastocyst stage, with a total of 102 differentially expressed genes identified. Among the differentially expressed genes were several involved in apoptosis, protection against free radicals, and development. Changes in gene expression are consistent with IGF1 acting to induce an anti-apoptotic state and inhibit neurulation. In conclusion, thermoprotective actions of IGF1 are developmentally regulated. Failure of IGF1 to protect the 2-cell embryo from heat shock could reflect the fact that these embryos are maximally sensitive to damage caused by heat shock or reflect the quiescence of the embryonic genome at this early stage in development. Changes in gene expression at the blastocyst stage induced by IGF1 could contribute to the increased survival of IGF1-treated embryos when transferred during periods of heat stress. Support: USDA NRI 2007-35203-18070 and 2009-65203-05732.

58 STAGE–SPECIFIC EFFECTS OF THE PROGESTERONE RECEPTOR ANTAGONIST, RU486, ON EARLY BOVINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 141
Author(s):  
C. M. O'Meara ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Progesterone Receptor ◽  
Receptor Antagonist ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Significant Decline ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Antagonist Ru486

Progesterone plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating progesterone in the immediate post-conception period are associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Progesterone-induced changes in the uterine environment are thought to be responsible for the reported advancement in conceptus elongation; however, the function of the progesterone receptor in embryos is not known. Therefore, the aim of the current study was to examine the effect of adding a progesterone receptor antagonist (mifepristone, RU486) at various stages of early embryonic development and at varying concentrations to examine the effects on subsequent embryo development in vitro. Bovine zygotes (n = 2902), 2-cell (n = 1991) and 8-cell (n = 1244) embryos, derived by in vitro maturation and fertilization, were cultured in synthetic oviduct fluid medium in the absence or presence of RU486 at concentrations ranging from 0.0004 to 20 μg mL–1. Cleavage rate (of zygotes), 8-cell development rate (of 2-cell embryos) and development to the blastocyst stage (for all cell stages) were recorded at Day 2, 3 and 8 post-insemination (day of IVF = Day 0), respectively. Cultures of zygotes in the presence of RU486 at concentrations of 0.004 and 0.04 μg mL–1 resulted in a decline in cleavage rate (62.5 ± 2.55% and 48.8 ± 5.07% for respective treatments vs controls without RU486 81.9 ± 5.97%; P ≤ 0.05). These same concentrations resulted in a significant decline in blastocyst development on Day 8 (18.8 ± 1.82% and 17.4 ± 4.85% for respective treatments compared to controls 35.1 ± 4.89%; P ≤ 0.05). Cultures at concentrations of 0.4 μg mL–1 resulted in a 10-fold decrease in blastocyst development (3.3 ± 1.3%; P ≤ 0.05) and concentrations in excess of 10 μg mL–1 completely ablated blastocyst development (P ≤ 0.05). Cultures of 2-cell embryos with RU486 at concentrations below 8 μg mL–1 had no effect on 8-cell rate or blastocyst development. However, cultures with RU486 at 10 μg mL–1 resulted in a significant decline in the proportion reaching the 8-cell stage (59.1 ± 4.59% vs 38.1 ± 2.13% for control and treated, respectively) and developing to the blastocyst stage (32.8 ± 4.68% vs 17.8 ± 3.77% for control and treated, respectively; P ≤ 0.05). Cultures with RU486 at a concentration of 20 μg mL–1 resulted in a dramatic effect in 8-cell rate (16.3 ± 2.55%; P ≤ 0.05) and prevented blastocyst development. Similarly, cultures of 8-cell embryos with RU486 at concentrations at or below 10 μg mL–1 had no effect on blastocyst development. However, cultures at concentrations of 15 or 20 μg mL–1 resulted in no blastocyst development. In conclusion, addition of the progesterone and glucocorticoid receptor antagonist RU486 to culture media has a clear stage-specific and concentration-dependent effect on bovine embryo development, which is more pronounced at earlier developmental stages. Supported by Science Foundation Ireland (07/SRC/B1156).

scholarly journals Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse

Zygote ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 612-630 ◽  
Author(s):  
Cécile Douet ◽  
Olivia Parodi ◽  
Nicola Antonio Martino ◽  
Giovanni Michele Lacalandra ◽  
Michele Nicassio ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Culture Conditions ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Development Rates ◽  
Maturation Medium ◽  
In Vitro Embryo Production ◽  
Oviductal Fluid

SummaryMost wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.

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