260 BREED INFLUENCES OUTCOME OF IN VITRO PRODUCTION OF EMBRYOS IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 287 ◽  
Author(s):  
M. C. Abraham ◽  
A. Ruete ◽  
Y. C. B. Brandt
Keyword(s):  
Body Fat ◽  
Age Distribution ◽  
Time Of Day ◽  
Developmental Competence ◽  
Carcass Weight ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference

Fertility among cattle breeds can vary. The Swedish Red and White dairy breed (SRB) has been systematically bred for good reproductive traits since 1970 and might therefore have retained a better oocyte quality than other dairy breeds. The aim of this study was to determine if the breed of oocyte donor affects the development of embryos using IVM, IVF, and IVC. Oocyte developmental competence in vitro was compared between the SRB (n = 77 animals), the Swedish Holstein breed (SLB, n = 49), and beef breeds (mixed breeds, n = 97). The oocytes (n = 1380, 18 batches) were aspirated from abattoir-derived ovaries from healthy animals with known identity. Statistical analyses were performed using Student’s t-tests and generalized linear mixed models with random effects. The time of collection in relation to slaughter and time of day, as well as aspiration and the following in vitro procedures, were consistent throughout the experiment. The oocytes were matured, fertilized (frozen semen), and cultured according to conventional protocols without serum. Data are presented as mean ± SEM. The SRB and SLB groups were comparable in age [SRB: 66% cows (over 3 years of age), 27% young cows (calved at least once but not over 3 years of age), and 7% heifers; SLB: 63% cows, 20% young cows, and 17% heifers], carcass classification (scale 1-15, where 15 = highest amount of muscle; SRB: 3.8 ± 0.2, SLB 3.5 ± 0.3), body fat (scale 1-15, where 15 =highest amount of fat; SRB: 8.4 ± 0.4, SLB 8.8 ± 0.5) and kilograms of carcass weight (SRB: 297.3±7.4, SLB: 311.6 ± 9.0). The beef group had a significantly higher mean carcass classification (6.2 ± 0.2) and a different age distribution with a higher proportion of heifers (38% cows, 12% young cows, and 50% heifers), but was comparable in body fat content (8.5 ± 0.4) and kilograms of carcass weight (310.9 ± 7.9). Cleavage rate, number of embryos developed beyond the 2-cell stage by 44 h post-fertilization, and the number of blastocysts developed by Days 7 and 8 were noted. All blastocysts were graded and stained with Hoechst 33 342 and the number of nuclei was determined. Cleavage rate was not different among the breeds (SRB: 71.9 ± 0.03%, SLB: 72.5 ± 0.02%, beef: 73.9 ± 0.03%). The percentage of embryos developed beyond 2-cells (from cleaved) did not differ between the beef and SRB (beef: 65.1 ± 6.1%; SRB: 70.4 ± 4.9%) but SLB was significantly greater than than the other breeds (75.4 ± 4.5%). The percentage of blastocysts developed by Day 8 was significantly higher in the beef (21.1 ± 2.7%) and SRB (23.3 ± 3.5%) breeds compared with the SLB (12.5 ± 2.4%). There was no significant difference in blastocyst grades among breeds (scale 1-4, where 1 = highest grade; SRB: 2.4 ± 0.1, SLB: 2.4 ± 0.2, beef: 2.1 ± 0.2), but the number of nuclei in Day 8 blastocysts was significantly lower in the SLB (SRB: 98.9 ± 7.7, SLB: 79.2 ± 8.7, beef: 101.4 ± 6.9). In conclusion, the breed of origin of the oocytes is an important factors affecting the development during in vitro embryo production in cattle. Funded by Formas.

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis

2017 ◽  
Vol 8 (2) ◽  
pp. 61-67
Author(s):  
Harsha K Bhadarka ◽  
Nayana H Patel ◽  
Kruti B Patel ◽  
Nilofar R Sodagar ◽  
Yuvraj D Jadeja ◽  
...  
Keyword(s):  
Time Lapse ◽  
Mean Value ◽  
Primary Objective ◽  
Sperm Cryopreservation ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference ◽  
Cryopreserved Sperm ◽  
Day 3 Embryo

ABSTRACT Aim In recent past, many studies had come up with the combination of time-lapse (TL) imaging of embryo morphokinetics as a noninvasive means for improving embryo selection and in vitro fertilization (IVF) success. The primary objective of the study was to find out if there is significant variation in morphokinetics of embryos with different implantation potential and also to study the effect of sperm freezing on time points of embryogenesis events in embryos with implantation potential. Materials and methods Kinetic data and cycle outcomes were analyzed retrospectively in 142 patients who had undergone IVF/intracytoplasmic sperm injection (ICSI) cycles using semen with normal parameters and embryo transfer (ET) on day 3. For the surety of specificity of morphokinetics, only cases with single ET cycles were included in the study. Timing of specific events, from the point of ICSI, was determined using TL imaging. Kinetic markers like time to syngamy (t-pnf), t2, time to two cells (c), 3c (t3), 4c (t4), 5c (t5), 8c (t8), tMor, CC2, CC3, t5–t2, t5–t4, s1, s2, and s3 were calculated. The cleavage synchronicity from the 2–8 cell stage (CS2–8), from 4 to 8 cell stage (CS4–8), and from 2 to 4 cell stage (CS2–4) were calculated as defined elsewhere. Deoxyribonucleic acid replication time ratio (DR) was also included in the comparison. Analysis of variance test was used for comparison of the mean timing of cell division and cell cycle intervals. Results Morphokinetics t-pnf, t2, t8, CC2, S2, S3, CS2–8, CS4–8, and CS2–4 differed significantly between embryos with and without implantation potential, when embryos were developed using fresh semen, while t3, t4, t5, CC2, S2, t5–t2, CS2–4, and DR differed significantly between the embryos with and without implantation potential when frozen semen was used. No significant difference was found in mean value of any of the above-stated parameters when comparison was done between implanted embryos fertilized by either fresh or cryopreserved sperm. Conclusion Many morphokinetics parameters of embryogene­sis vary significantly between embryos with different ability to implant; therefore, the criteria developed in our IVF lab can be useful for selection of suitable embryo even at day 3 of development with more chances of implantation. Clinical significance Study indicates necessity of development of individualized selection model based on morphokinetics for every IVF lab and also confirms freezing as an important tool for fertility preservation of males as it does not affect events of embryogenesis. How to cite this article Bhadarka HK, Patel NH, Patel KB, Sodagar NR, Jadeja YD, Patel NH, Patel MN, Patel AV, Patel DH, Patel JS. Study of Morphokinetics in Day 3 Embryo with Implantation Potential and Effect of Sperm Cryopreservation on Embryogenesis. Int J Infertil Fetal Med 2017;8(2):61-67.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Hruda Nanda Malik ◽  
Dinesh Kumar Singhal ◽  
Shrabani Saugandhika ◽  
Amit Dubey ◽  
Ayan Mukherjee ◽  
...  
Keyword(s):  
Culture Media ◽  
Combined Treatment ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Quantitative Expression ◽  
Pattern Of Variation ◽  
Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

55 Donor age has the least influence on recovery, quality, and in vitro developmental competence of ovum pickup–based Holstein Friesian oocytes under subtropical conditions

2021 ◽  
Vol 33 (2) ◽  
pp. 134
Author(s):  
M. Yaseen ◽  
M. Saleem ◽  
M. Nawaz ◽  
N. Ahmad ◽  
A. Riaz
Keyword(s):  
Transvaginal Ultrasound ◽  
Developmental Competence ◽  
Donor Age ◽  
Adult Group ◽  
Cleavage Rate ◽  
Holstein Friesian ◽  
Frozen Semen ◽  
Chi Squared ◽  
Blastocyst Rate

The use of oocytes obtained from younger donors for invitro fertilization followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation interval. The present study was designed to evaluate the effect of age of donor on the follicular population, recovery, quality and invitro developmental competence of ovum pickup based Holstein Friesian oocytes under subtropical conditions. A total of eight (n=8) Holstein Friesian (with proper oestrus cyclicity) were selected for the study and divided into 2 groups based on animal age: (1) heifers (n=4), 1.5 to 2 years of age, and (2) adults (n=4), 5 to 6 years of age. The study was conducted near Lahore (31°33′ N, 74°19′ E), Punjab, Pakistan, from November 2019 to February 2020. The animals were wave synchronized using the physiological method of wave synchronization. After 4 days of second dominant follicle puncture, the first ovum pickup was carried out and a total of nine (n=9) OPU sessions were held for each group. The COCs from the follicles were aspirated using a transvaginal ultrasound–guided needle. Following searching and grading, COCs of grade A, B and C were processed for IVM in 100-µL droplets of BO-IVM under mineral oil at 37°C, 5% CO2, and 95% humidity for 24h. The frozen semen of a high-pedigree bull was thawed at 37°C and observed for post-thaw sperm motility. The semen samples of the same bull having motility &gt;50% were processed using the sperm swim-up method throughout the study. The IVF was carried out by placing the COCs and required amount of sperm in 100-µL droplets of BO-IVF at similar conditions for a maximum of 18h. The presumptive zygotes were denuded by gentle pipetting and cultured for a period of 7 days after placing in 100-µL drops of BO-IVC at 37°C, 5% CO2, 5% O2, and maximum humidity. The presumptive zygotes were observed for cleavage rate and blastocyst rate on Days 2 and 7 following COCs-sperm co-incubation. Data on the follicular population, oocytes recovered, and viable oocytes were analysed by the PROC GLIMMIX of SAS (SAS Institute Inc.), and proportional data were analysed by the Chi-squared method using SAS 9.1. COCs of grade AB (35.2 vs. 25.4%) were higher (P&gt;0.05) in the adult group than in the heifer group, respectively. Similarly, COCs with grade CD (57.5 vs. 71.9%) were lower (P&lt;0.05) in the adult group compared with the heifer group, respectively. However, the total follicles (6.55±0.42 vs. 6.39±0.39), number of COCs recovered (3.33±0.32 vs. 3.17±0.41), viable oocytes (3.08±0.29 vs. 3.08±0.39), cleavage rate (60.3 vs. 68.7%), and blastocyst rate (38.7 vs. 48.8%) did not differ (P&gt;0.05) between the groups. To conclude, donor age up to third lactation, under subtropical conditions, does not affect invitro embryo production in Holstein Friesian undergoing repeated OPU.

scholarly journals Pengaruh Penambahan Chorionic Gonadotrophin pada Medium Maturasi terhadap Kemampuan Maturasi, Fertilisasi, dan Perkembangan Embrio secara In Vitro Kambing Peranakan Ettawa (The Effect of Chorionic Gonadotrophin Addition Into Maturation Medium on The Abili

2012 ◽  
Vol 34 (1) ◽  
pp. 8
Author(s):  
Nurvina Septi Adifa ◽  
Pudji Astuti ◽  
Diah Tri Widayati
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Chorionic Gonadotrophin ◽  
Fertilization In Vitro ◽  
Cleavage Rate ◽  
Group I ◽  
Maturation Medium ◽  
Frozen Semen ◽  
Significant Difference

<p>This research was conducted to investigate the effect of chorionic gonadotrophin addition into maturation medium on oocyte maturation, fertilization, and embryo development in vitro of Ettawa crossbred. Oocytes were divided into 3 groups, group I: maturation medium without addition of chorionic gonadotrophin (0), group II: 10 μl/10 ml chorionic gonadotrophin was added into maturation medium (1), group III: 20 μl/10 ml chorionic gonadotrophin was added into maturation medium (2). Oocytes were transferred into 50 μl maturation medium, then covered by mineral oil. Oocyte was incubated at 39oC, 5% CO2, 95% humidity for 24 hours for maturation. Matured oocytes were inseminated with frozen semen–thawed concentration 12.5 x 106/ml. Process of fertilization were carried out on incubator 39oC, 5% CO2, 95% humidity for 5 hours. The fertilized oocytes were transferred into 50 μl drop G–1, then incubated at 39oC, 5% CO2, 95% humidity. Embryo development was monitored every 24 hours. Culture medium was changed every 48 hours. G–2 medium used second day after culture. The variables measured involved oocyte maturation, fertilization, and in vitro cleavage rate. The data were analyzed by chi–square, using SPSS 15.0 program. The result showed no significant difference on the percentage of mature oocytes and fertilization rate were 78.0%, 72.8%, 75.0% and 76.6%, 74.5%, 77.8% respectively. But cleavage rate showed significant difference (P≤0.05) with<br />the values of 40.8%, 11.4%, and 12.2% respectively. Based on the result it could be concluded that chorionic gonadotrophin addition into maturation medium had not increased ettawa crossbred oocytes maturation, fertilization, and in vitro cleavage rate. The best maturation, fertilization, and in vitro cleavage rate were found using maturation medium without any addition of chorionic gonadotrophin.</p><p>(Key words: Does oocyte, Chorionic gonadotrophin, In vitro maturation, In vitro fertilization, In vitro embryo development)<br /><br /></p>

77 EFFECT OF HEAT SHOCK DURING IN VITRO MATURATION ON HETEROCHROMATIN COMPACTION IN BOVINE EMBRYOS AT 4- AND 8-CELL STAGES: PRELIMINARY STUDY

2015 ◽  
Vol 27 (1) ◽  
pp. 132
Author(s):  
L. S. A. Camargo ◽  
T. Aguirre-Lavin ◽  
P. Adenot ◽  
T. D. Araujo ◽  
E. D. Souza ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Gene Activation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Morphological Criteria ◽  
Preliminary Study

High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P > 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P < 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P > 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P < 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P < 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).

88 EFFECTS OF REMOVAL OF CUMULUS CELLS AND pb1 PRESENCE OF IN VITRO-MATURED BUFFALO OOCYTES PRIOR TO IVF ON CLEAVAGE RATE AND SUBSEQUENT EMBRYO DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 158
Author(s):  
J.-H. Shang ◽  
H.-Y. Zheng ◽  
C.-Y. Yang ◽  
F.-X. Huang ◽  
B.-Z. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Natural Science ◽  
Polar Body ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Lower Efficiency ◽  
Significant Difference

The efficiency of oocyte maturation and embryo production in vitro in buffalo is relatively poor when compared with that in cattle. The percentage of oocytes selected by pb1 (the 1st polar body) presence for somatic cell nuclear transfer (SCNT) ranged from 50 to 70% in our laboratory, which meant that 30 to 50% oocytes have been abandoned. The present study was designed to identify the effect of cumulus cells removal and pb1 presence or absence before the IVF of matured buffalo oocytes on cleavage rate and subsequent embryo development and to try to reuse those oocytes without pb1 for embryo in vitro production. In vitro-matured oocytes enclosed with cumulus cells were randomly selected and denuded mechanically, then the denuded oocytes (DO) were divided into 3 groups by non-selection (pb1 ± ), selection of pb1 presence (pb1+) and absence (pb1–). Intact cumulus–oocyte complexes (COC, control) and pb1 ± , pb1+, and pb1– DO (treatments) were inseminated with motile buffalo sperm in Tyrode's medium for 24 h. The presumed zygotes were washed 3 times and transferred into 50-μL droplets of IVC medium (TCM 199 + 10% fetal bovine serum) and co-cultured with buffalo cumulus cells monolayer for more than 10 days to evaluate the developmental ability of embryos. Cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 h and 240 h after fertilization (0 h). The results indicated that CR and BR for COC (61.69 ± 9.22% and 34.07 ± 7.61%) and pb1+ (66.59 ± 15.50% and 35.96 ± 10.87%) were significantly higher (P < 0.01) than those for pb1 ±  (49.11 ± 6.83% and 21.88 ± 8.17%) and pb1– (35.09 ± 9.17% and 13.16 ± 5.38%). In addition, there was a significant difference (P < 0.05) in the CR and BR between pb1 ±  and pb1– but no difference (P > 0.05) between COCs and pb1+ DO. These data show that removal of cumulus cells before IVF significantly reduces the overall developmental competence to cleavage and blastocyst stage and this negative effects mainly caused by the immature oocytes (indicated by the absence of pb1), but there was no effect on mature oocytes (presence of pb1). However, the oocytes without pb1 can still be used for in vitro embryo production even with lower efficiency when compared with intact COC. This research was supported by grants from the National Natural Science Foundation of China (31160456), the Natural Science Foundation of Guangxi, China (2011GXSFB018045, 2013GXNSFAA019075).

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

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