153 HEPARIN-BINDING OVIDUCTAL SECRETORY PROTEINS AS A MEDIUM SUPPLEMENT FOR IN VITRO EMBRYO PRODUCTION IN CATTLE

2012 ◽  
Vol 24 (1) ◽  
pp. 188
Author(s):  
A. K. Sharma ◽  
A. K. Mohanty ◽  
S. K. Das
Keyword(s):  
Embryo Development ◽  
Phosphate Buffer ◽  
Secretory Proteins ◽  
Heparin Binding ◽  
Blastocyst Formation ◽  
Sds Page ◽  
National Dairy Research Institute ◽  
Maximum Humidity ◽  
Dairy Research

The purpose of this study was to determine the effect of heparin-binding oviducal secretory proteins as a media supplement on IVF and embryo development in cattle. The native proteins were isolated from oviducts collected from a slaughterhouse by repeated freeze-thawing and precipitated by ammonium sulfate (60%) followed by dialysis overnight in 10 mM phosphate buffer solution containing 1 mM PMSF at pH 7.0 at 4°C. The dialyzed product was then passed through a high-performance liquid chromatography system with a HiTrap™ heparin prepacked column (GE Healthcare, Piscataway, NJ) equilibrated with 10 mM phosphate buffer containing 1 mM EDTA. The collected protein fractions [heparin-binding protein (HBP), heparin-unbinding protein (HUBP)] and total protein (TP) were run on SDS-PAGE. The SDS-PAGE profile of the eluted HBP fraction contained 5 major protein bands visible between approximately 66 to 97 kDa, the profile of the HUBP fraction contained 2 bands nearer to approximately 66 kDa and in TP, all 7 bands were visible between approximately 60 to 95 kDa. All 3 fractions (i.e. HBP, HUBP and TP) were used in 3 different concentrations (0, 1, 10 and 30 μg mL–1) for in vitro maturation, sperm preparation, IVF and in vitro culture of cleaved embryos. Cumulus–oocyte complexes were collected from slaughterhouse ovaries, washed 4 to 5 times and cultured in maturation media for 24 h in a 5% CO2 incubator at 38.5°C with maximum humidity. In vitro-matured oocytes were co-incubated with capacitated sperm in Fert-BO media at 38.5°C in 5% CO2 in air with maximum humidity. After 18 h, oocytes were washed thoroughly and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. In the HBP group, overall cleavage rates (%) were 63 ± 3.0, 53 ± 2.6, 65 ± 4.7 and 43 ± 0.6 and blastocyst formation (%) was 17 ± 1.6, 23 ± 2.5, 25 ± 2.2 and 24 ± 1.7 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. However, in the HUBP group, the overall cleavage rates (%) were 66 ± 4.3, 56 ± 2.0, 60 ± 2.7 and 32 ± 0.8 and blastocyst formation (%) was 18 ± 1.1, 25 ± 1.8, 24 ± 2.2 and 14 ± 0.8 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. In the TP group, cleavage rates (%) were 67 ± 4.9, 60 ± 4.1, 73 ± 3.2 and 41 ± 1.6 and blastocyst formation (%) was 19 ± 1.1, 18 ± 1.9, 27 ± 2.2 and 20 ± 1.6 in 0, 1, 10 and 30 μg mL–1 concentrations, respectively. These results indicate that 10 μg mL–1 of the total oviductal secretory protein fraction used as a media supplement significantly increased (P < 0.01) the cleavage rate and blastocyst formation as compared with the HBP and HUBP fractions. The authors sincerely acknowledge the director and joint director (R), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, India, for providing the facilities to carry out the work.

103 OVIDUCTAL SECRETORY PROTEINS AS MEDIA SUPPLEMENT FOR IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2012 ◽  
Vol 24 (1) ◽  
pp. 164
Author(s):  
S. K. Das ◽  
A. K. Sharma ◽  
V. Bhatia ◽  
A. K. Mohanty
Keyword(s):  
Embryo Development ◽  
Sodium Dodecyl ◽  
Secretory Proteins ◽  
Cleavage Rate ◽  
Exchange Column ◽  
Diethylaminoethyl Cellulose ◽  
National Dairy Research Institute ◽  
Dairy Research ◽  
Research Institute

The objective of this study was to determine the effect of oviducal secretory proteins (cOSP) as a media supplement on in vitro embryo development in cattle. Oviducal secretory proteins were collected from slaughterhouse oviducts by repeated freeze–thaw process and purified by ammonium sulfate precipitation (30%, 40%, 50% and 60%) followed by dialysis in 50 mM tris-HCl (pH 7.0) buffer. Dialyzed products were further purified by SP Sephadex cation exchange column and diethylaminoethyl cellulose (DEAE) anion exchange column, extensively washed and eluted by 50 mM tris-HCl (pH 7.0) containing 1.5 M NaCl. Both bound and unbound proteins were collected separately, dialyzed in phosphate buffer saline and quantified. Presence of protein was confirmed by running sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and OSP bands between ∼66 kD to ∼97 kD were found. The pooled purified cOSP were used as a media supplement in 3 different concentrations (0, 10, 50 and 100 μg mL–1) for in vitro production (n = 3) of cattle embryos. Cumulus–oocyte complexes (n = 370) were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO2 at 38.5°C with maximum humidity. In vitro-matured oocytes were fertilized with in vitro capacitated sperm in Fert-BO media at 38.5°C in 5% CO2. After 16 to 18 h, oocytes were washed and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. A total of 68.42%, 69.31%, 61.82% and 41.67% cleavage rate and 15.38%, 21.31%, 14.70% and 15.0% blastocyst rate was observed at concentrations of 0, 10, 50 and 100 μg mL–1, respectively. These results indicate that addition of cOSP at 10 μg mL–1 increased blastocyst formation significantly (P < 0.05) compared with 0, 50 and 100 μg mL–1 and increased cleavage rate significantly (P < 0.05) compared with 50 and 100 μg mL–1. The authors acknowledge sincere thanks to the Director, Joint Director (Research), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, for providing the necessary facilities to carry out the work.

22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
H. Bagis ◽  
S. Arat ◽  
H. Odaman ◽  
A. Tas
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Group 4 ◽  
Mouse Embryo Development ◽  
O2 Concentration ◽  
Group 3 ◽  
Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

112 EFFECTS OF GUAIAZULENE ON IN VITRO CULTURE OF BOVINE ZYGOTES, AND ON mRNA TRANSCRIPTS RELATED TO EMBRYO QUALITY

2009 ◽  
Vol 21 (1) ◽  
pp. 156
Author(s):  
E. Dovolou ◽  
M. Clemente ◽  
G. S. Amiridis ◽  
I. Messinis ◽  
A. Kalitsaris ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Early Embryo Development ◽  
Mrna Transcripts ◽  
Oviductal Fluid ◽  
Maximum Humidity

We have previously shown that follicular and oviductal fluid provide greater total protection against lipid peroxidation than the respective media used for the in vitro embryo production. Reactive oxygen species (ROS) production has been implicated as a major cause for the reduced in vitro bovine embryo production; it is believed that they participate in meiotic arrest of oocytes, embryonic block and cell death. The aim of this study was to determine whether guaiazulene (G), an exogenous antioxidant, added in the post fertilization culture medium would affect the early embryo development and the quality of the produced blastocysts in terms of mRNA expression of several important genes. In a previous study we had shown that media modified with 0.01 mm of G provided the same antioxidant protection as the respective in vivo environments (i.e. the follicular and the oviductal fluid). Bovine cumulus–oocyte complexes (COC) were aspirated from ovaries derived from slaughtered cows and matured in groups of 50 in 500 μL in TCM199 with 10% fetal calf serum and 10 ng mL–1 Epidermal Growth factor at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Twenty-four hours later matured oocytes were inseminated with frozen/thawed bull semen and co-incubated in the same conditions as maturation. Presumptive zygotes were divided into 4 groups and cultured in groups of 25 in 25 μL of SOF with 5% FCS (Control–, n = 355), supplemented with 0.01 mm of G (n = 344) or 0.1 mm of G (n = 345) or 0.05% DMSO – the G diluent–(Control+, n = 347) at 39°C in an atmosphere of 5% CO2, 5% O2 and maximum humidity. Blastocyst yield was recorded on Days 6, 7, 8 and 9; Day 7 blastocysts from each group were snap frozen and stored at –80°C for mRNA extraction. Quantification of transcripts for aldose reductase mRNA (AKRIBI), prostaglandin G/H synthase-2 (PGHS-2, COX-2), glyceraldehyde 3-phosphate dehydrogenase (GADPH), facilitated glucose/fructose transporter, member 5 (GLUT-5) genes related to metabolism, glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD) antioxidant enzymes and placenta-specific 8 (PLAC8) related to implantation was carried out by real-time quantitative RT-PCR. Data for embryo development and on transcript abundance were analyzed by chi square and ANOVA, respectively. Cleavage rate tended to be higher in 0.01 mm group than in Control– (77.87% v. 71.41%, P = 0.07). Barring that, no other differences were detected in cleavage rate (Control+: 71.32%; 0.1 mm: 72.75%) or in the overall blastocyst yield on Day 9 (Control–: 25.50%; Control+: 26.71%; 0.1 mm: 25.75%; 0.01 mm: 29.58%). The relative abundance of genes studied varied among groups, but these differences were not significant. We infer that under the current culture conditions, G as an antioxidant has no serious direct effect on early embryo development or on embryo quality at least on the mRNA transcripts studied. Further studies using the same antioxidant in different atmospheric conditions are planed. ED and GSA were sponsored by COST (FAO702) and OECD fellowships, respectively.

scholarly journals 311EFFECT OF OOCYTE MATURATION MEDIA ON THE SPEED OF MEIOTIC PROGRESSION AND BLASTOCYST DEVELOPMENT OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 275
Author(s):  
D. Fischer ◽  
J. Bordignon ◽  
C. Robert ◽  
D. Betts
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Immunofluorescence Microscopy ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Meiotic Progression

Environment is crucial for in vitro development of gametes and embryos. The recent progression of culture media towards defined conditions brought to surface the impact of different medium supplements on oocyte and embryo development. In this work we evaluate the effect of various oocyte culture media on bovine oocyte maturation and subsequent embryo development. Bovine cumulus-oocyte complexes were recovered from slaughterhouse ovaries and matured in vitro in either TCM-199 (Gibco) or SOF (Synthetic Oviduct Fluid) media supplemented with BSA (fatty acid-free) or serum (fetal bovine serum). Oocytes from each treatment group were denuded and fixed at 18, 20, 22, 24, 26 and 28h post-maturation (p.m.). Oocyte meiotic progression was monitored in each of the groups (n=28–40 oocytes/group) by immunofluorescence microscopy of chromatin. Oocytes matured in SOF showed a slower rate of meiotic progression when compared to the other groups, with the highest percentage of oocytes reaching the MII stage by 28h p.m. (60.71% SOF-BSA, 71.43% SOF-Serum). The fastest developmental rate was observed in oocytes matured in TCM-serum (77.15% at 24h p.m.) followed by oocytes matured in TCM-BSA (74.29% at 26h p.m.). In order to evaluate the effect of nuclear maturation on chromosome segregation, chromosomal organization of MII oocytes was evaluated by immunofluorescence microscopy within each media group (n=26–31 oocytes/group) at 18, 22 and 26h p.m.. No chromosomal abnormalities were found at 18h p.m.. Both media supplemented with BSA induced lower frequencies of chromosomal abnormalities (0 to 3.23%) and (3.57 to 7.69%) for SOF and TCM, respectively, when compared to their serum-supplemented counterparts (7.14 to 11.54%) and (10 to 10.71%) for SOF and TCM, respectively at 22 and 26h p.m.. Remarkably, the maturation medium and its supplements influenced the speed of blastocyst development. For this experiment, oocytes were matured in TCM-BSA, TCM-Serum, SOF-BSA or SOF-serum, fertilized in vitro in a TALP-base media supplemented with BSA and cultured in SOF-BSA. Blastocyst development was assessed at 7, 8 and 9 days of culture. Cleavage rates were similar between the groups (84–90%), whereas development rates to blastocyst stage varied among treatment groups. Maturation in SOF-BSA induced a delay in blastocyst formation that reached its highest percentage only on day 9 of culture (30.8%); moreover, blastocyst development was carried over until Day 12. When oocytes were matured in the presence of serum, the number of blastocysts did not increase after Day 8 of culture (26.6%, TCM-serum). These results provide evidence of a severe impact of oocyte culture media on the nuclear maturation of oocytes and their subsequent embryonic development after IVF. Moreover, the difference in the rate of oocyte maturation and blastocyst formation emphasizes the necessity for reviewing and adapting current protocols to new systems such as SOF-BSA. [Research funded by NSERC and OMAF of Canada.]

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Byung Chul Jee ◽  
Jun Woo Jo ◽  
Jung Ryeol Lee ◽  
Chang Suk Suh ◽  
Seok Hyun Kim ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Embryo Development ◽  
Trichostatin A ◽  
Formation Rate ◽  
Control Group ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Extended Culture ◽  
Mouse Oocytes

SummaryWe performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.

scholarly journals 278EFFECT OF GLYCOSAMINOGLYCAN SUPPLEMENT ON PORCINE PREIMPLANTATION EMBRYO DEVELOPMENT AND EXPRESSION OF RECEPTORS FOR GLYCOSAMINOGLYCANS IN PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 259
Author(s):  
H.S. Kim ◽  
G.S. Lee ◽  
S.H. Hyun ◽  
D.H. Nam ◽  
Y.W. Jeong ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Control Culture ◽  
Receptor Expression ◽  
Developmental Competence ◽  
Differential Staining ◽  
Heparin Binding ◽  
Blastocyst Formation ◽  
Heparin Group ◽  
Preimplantation Embryo Development

The present study investigated the effect of glycosaminoglycan (GAG) supplement on developmental competence of porcine in vitro fertilized (IVF) embryos and GAG receptor expression in the porcine embryos. In vitro-matured oocytes were inseminated with frozen-thawed boar semen and cultured in North Carolina State University (NCSU)-23 medium in the absence or presence of various GAGs (hayaluronic acid, heparin or both). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. Differential staining was performed in blastocysts at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). Inseminated oocytes were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 0.5 or 1.0mgmL−1) of hyaluronic acid (in Experiment 1) or heparin (in Experiment 2). Supplementing NCSU-23 with 0.5mgmL−1 hyaluronic acid significantly increased (P&lt;0.05) the total number of cells (55.9) and the number of trophectoderm (TE) cells (41.7) compared with the other culture groups (44.7, 45.0 and 31.3, 31.8, respectively). The rate of blastocyst formation was significantly increased (P&lt;0.05) in the 1.0mgmL−1 heparin-supplemented group (21.8%) compared with that in the control culture group (16.4%). In experiment 3, inseminated oocytes were cultured in NCSU-23 supplemented with 0, 0.1mgmL−1 heparin, 0.5mgmL−1 hyaluronic acid, or 0.1mgmL−1 heparin+0.5mgmL−1 hyaluronic acid. The rate of blastocyst formation was significantly increased (P&lt;0.05) in the 1.0mgmL−1 heparin group (21.5%) and the 1.0mgmL−1 heparin+0.5mgmL−1 hyaluronic acid group (22.8%) compared with that of control culture group (16.6%). In experiment 4, total RNA was prepared from oocytes, 2-, 4-, and 8-cell stages, morulae, and blastocysts and reverse-transcribed using the First-strand cDNA Synthesis kit (Amersham Biosciences, Piscataway, NJ, USA). The cDNA was subjected to polymerase chain reaction with primers specific for hyaluronic acid receptor (CD44) and heparin binding protein (HIP). The expression of CD44 gene was detected in oocytes, and in 2- and 4-cell stages;; HIP gene was detected in all preimplanatation-stage embryos. In conclusion, the present study demonstrated the embryotrophic role of GAG and expression of GAGs receptors in porcine IVF embryos. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).

Male-dependent variability of fertilization and embryo development in two bovine in vitro fertilization systems and the effects of casein phosphopeptides (CPPs)

1997 ◽  
Vol 9 (4) ◽  
pp. 465 ◽  
Author(s):  
U. Kreysing ◽  
T. Nagai ◽  
H. Niemann
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Individual Variability ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Casein Phosphopeptides ◽  
Fertilization System

This study investigated the effects of semen from five different bulls and two different ejaculates of the same bull on penetration, cleavage, blastocyst formation, and cell allocation in bovine blastocysts produced in vitro. Casein phosphopeptides (CPPs) were tested for their ability to enhance fertilization and minimize variability among bulls and ejaculates. In Experiment 1, the BO-fertilization system was employed. Penetration and polyspermy both displayed great variation among bulls and between ejaculates, whereas no significant differences were observed in cleavage and blastocyst-formation rates. Similar variability was observed in penetration, polyspermy, cleavage, blastocyst-formation rates and cell allocation and distribution when the two fertilization systems, TALP and BO, were compared in Experiment 2. The BO-system supported penetration and polyspermy better (P < 0·05) than the TALP-system, whereas the TALP-system was superior (P < 0·05) in supporting cleavage and blastocyst formation. Significant interactions existed between bulls and the fertilization system employed. It is concluded that the success of in vitrofertilization is markedly dependent on individual bulls as well as on ejaculates from the same bull. CPPs are able to enhance penetration and embryo development in certain bulls or ejaculates and thus contribute to reducing the degree of individual variability, but they do not generally improve the success of bovine embryo production in vitro.

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

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