233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

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.

351 EFFECTS OF FETAL CALF SERUM, GROWTH FACTOR, AND HORMONE SUPPLEMENTATION DURING IN VITRO MATURATION ON PARTHENOGENETIC ACTIVATION AND EMBRYO DEVELOPMENT OF FOLLICULAR RABBIT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 290
Author(s):  
Z. Polgar ◽  
T. Somfai ◽  
V. Angeli ◽  
X. H. Tang ◽  
W. Ji ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Complete Medium ◽  
Treatment Groups ◽  
Significant Difference

Improvement of the in vitro maturation (IVM) system for rabbit oocytes could play a role in rabbit biotechnology. Our goal was to improve IVM to have an efficient source of rabbit oocytes for further studies on nuclear transfer. The effects of FCS, growth factors, and hormone supplementation on oocyte maturation, activation, and embryo development rates were evaluated. Rabbit ovaries were transferred from the slaughterhouse to the laboratory in PBS. Oocytes were collected by aspiration and subjected to IVM in 3 types of media based on TCM-199. In one group, growth factors (50 ng mL−1 of insulin-like growth factor-I and 10 ng mL−1 of epidermal growth factor) and hormones (5 IU mL−1 of hCG and 5 IU mL−1 of pregnant mare serum gonadotropin) and BSA were added to the IVM medium (IVM+); in the other groups, IVM medium was supplemented with either 10% (IVM + 10% FCS) or 20% FCS (IVM + 20% FCS). Maturation was assessed by the presence of a polar body after 16 h. Matured oocytes were activated twice by electric stimuli (3 DC pulses, 1.6 kV cm−1, 60 µs) and twice by chemical activation (incubation with 2.5 mM 6-DMAP for 30 min, second time for 2 h) and cultured in vitro in Earle's balanced salt solution complete medium at 38.5°C under 5% CO2 in air. Cleavage rates were recorded 16 h after activation and the blastocyst rates were recorded at Day 5 of in vitro culture. Data were analyzed by ANOVA. Maturation rates did not differ between the treatment groups (Table 1). There was no significant difference in cleavage rates between the IVM+ and the IVM + 10% FCS groups; however, the cleavage rate of the IVM + 20% FCS group was significantly lower compared with the others (Table 1; P &lt; 0.05). Development to the blastocyst did not differ significantly between the treatment groups (Table 1; P &lt; 0.05). The results showed that high (20%) FCS supplementation during IVM had a detrimental effect on oocyte cleavage. Hormonal and growth factor supplementation had no beneficial effects on maturation, activation rates, or blastocyst formation, and in fact were not essential for in vitro embryo production in the rabbit. Table 1.Effect of hormonal and growth factor supplementation on embryo development in rabbit oocytes The project was supported by RABIOTECH OMFB-00330/2004, EU FP6 (MEXT-CT-2003-509582, and 518240), Wellcome Trust (Grant No. 070246), and TET CH-28/04.

scholarly journals 132 BLASTOCYST DEVELOPMENT OF EQUINE OOCYTES WITH LOW MEIOTIC COMPETENCE HELD IN ROSCOVITINE BEFORE IN VITRO MATURATION

2005 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
Y.H. Choi ◽  
L.B. Love ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
Epithelial Cells ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Exact Test ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Chromatin Configuration ◽  
Meiotic Competence

At the time of recovery, immature equine oocytes may be separated into those with either expanded cumuli (Ex) or compact cumuli (Cp). The Cp oocytes originate from viable follicles but are largely juvenile, with low meiotic competence (20 to 30% maturation to MII), and possibly reduced developmental competence. We previously found that in Cp oocytes recovered immediately after slaughter, suppression of meiosis with roscovitine for 24 h before maturation increased embryo development at 4 days after intracytoplasmic sperm injection (ICSI; Franz et al. 2003 Reproduction 125, 693–700). The present study was conducted to evaluate the effect of roscovitine suppression on nuclear maturation and blastocyst formation of Cp oocytes recovered after transport of ovaries from the abattoir (i.e. recovered 5–9 h after slaughter). Compact oocytes recovered from transported ovaries were cultured in M199 with 10% FBS containing 66 μM roscovitine with or without an oil cover. After 16–18 or 24 h, oocytes were fixed to examine the chromatin configuration. Treatment for 16–18 h without oil resulted in the lowest rate of meiotic resumption (0%); thus this treatment was utilized in further studies. Resumption in other treatments ranged from 3 to 6%. Following roscovitine suppression, oocytes were cultured for 30 h in M199 with 10% FBS and 5 μU mL−1 FSH for maturation; control oocytes were cultured for 30 h in the same medium immediately after recovery. Mature oocytes were subjected to ICSI, then cultured in DMEM/F-12 with 10% FBS with or without co-culture with equine oviductal epithelial cells under mineral oil in 5% CO2 in air at 38.2°C, and then evaluated at 7.5 days. Progression to MII (82/376, 22%) after maturation of roscovitine-treated oocytes was similar to that for control oocytes (74/395, 19%). There was no significant difference in cleavage rates after ICSI (72–78%) among treatments. Development to blastocyst was highest in roscovitine-treated oocytes in DMEM/F-12 with co-culture (11/30, 37%); this was significantly higher than that of non-treated oocytes in DMEM/F-12 alone (5/36, 14%), but similar to that of non-treated/DMEM/F-12/co-culture (10/37, 27%) and roscovitine/DMEM/F-12 alone (8/39, 21%). These data indicate that roscovitine induces a fully reversible meiotic suppression in Cp equine oocytes recovered 5–9 h after slaughter, and that this suppression does not harm subsequent developmental competence. This treatment may be used to manipulate the time of onset of maturation of equine oocytes for ease of subsequent procedures. Co-culture with oviductal epithelial cells tended to increase blastocyst rate (P = 0.1, Fisher's exact test) in contrast to our previous findings with embryos from Ex oocytes (Choi et al. 2004 Biol. Reprod. 70, 1231–1238). Further work is needed to determine whether this is related to differences in intrinsic developmental competence between oocyte types. This work was supported by the Link Equine Research Endowment Fund (Texas A&M University).

Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 135-145 ◽  
Author(s):  
Ma. Ninia L. Gomez ◽  
Jung Taek Kang ◽  
Ok Jae Koo ◽  
Su Jin Kim ◽  
Dae Kee Kwon ◽  
...  
Keyword(s):  
Cell Function ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Secreted Factors ◽  
Significant Difference

SummaryThe oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 178
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Nile Red ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Treatment Groups ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Conventional Freezing

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

225 EFFECTS OF PEDIGREE, MEAT QUALITY, AND MEAT QUANTITY OF SLAUGHTERHOUSE DONOR COWS ON OOCYTE RECOVERY, EMBRYO DEVELOPMENT, AND PREGNANCY AFTER EMBRYO TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
Y. S. Park ◽  
H. D. Park
Keyword(s):  
Pregnancy Rate ◽  
Meat Quality ◽  
Embryo Development ◽  
Development Program ◽  
Developmental Rate ◽  
Blastocyst Development ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
Donor Cows

In conventional in vitro embryo production, oocytes are obtained from various ovaries, and donor characteristics, such as pedigree type, meat quality, or meat quantity, are not considered. The aim of this study was to examine the effects of pedigree type and meat quality and quantity of slaughterhouse donor cows on oocyte recovery, embryo development, and pregnancy rates. The ovaries of individual Korean native cows were obtained from a slaughterhouse. The ear tag numbers were obtained 24 h after slaughter, and the donor data were compared according to the pedigree type (no registration to third bloodline registration), meat quantity according to the percentage of carcass weight (more than 69%, 66 to 69%, and below 66%; Grades A, B, and C, respectively), and meat quality according to the marbling grade (marbling grade 8 to 9, 6 to 7, 4 to 5, 2 to 3, and 1; Grades 1++, 1+, 1, 2, and 3, respectively). There were 390 donors in total, and each group had 30 donors. Recipients (Holstein heifers; n = 222) were synchronized with a progesterone device, and a single blastocyst was transferred nonsurgically on Day 7 from the onset of oestrus. Data on embryo development were compared by Duncan’s multiple range tests, and pregnancy rate was analysed by chi-square test. Values of P < 0.05 were considered to indicate a significant difference. No significant difference was detected in the average number of oocytes recovered (17.5 ± 2.9 to 22.9 ± 1.2%) or blastocyst development (16.4 ± 2.6 to 19.5 ± 3.4%) from cows of different pedigrees. A higher number of oocytes were recovered from Grade A (22.9 ± 1.0) than from Grade B (19.8 ± 0.7; P < 0.05) cows. However, the meat quantity grade had no significant effect on blastocyst development (14.3 ± 1.2 to 15.0 ± 0.8%). A greater number of oocytes were recovered from the Grade 1++ (25.0 ± 1.6), 1+ (27.7 ± 2.4), and 2 (23.6 ± 1.5) groups than from the Grade 3 group (19.7 ± 1.0; P < 0.05). The developmental rate to the blastocyst stage was significantly higher in the Grades 2 and 3 groups (22.1 ± 2.2 and 19.4 ± 1.7) than in the Grade 1++, 1+, and 1 groups (9.0 ± 1.2 to 13.8 ± 2.0). Pregnancy rate was not affected by pedigree type, meat quantity, or meat quality (41.7 to 58.6%). We concluded that 1) pedigree type had no effect on oocyte recovery and embryo development; 2) meat quantity affected oocyte recovery but did not affect embryo development; 3) high meat quality (Grades 1++ and 1+) resulted in greater oocyte recovery but lower embryo development; and 4) pregnancy rate remained unaffected by pedigree type, meat quantity, and meat quality. This research was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 184
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Limiting Factor ◽  
Time Interval ◽  
Activation Process ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Chi Square ◽  
Group I

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.

208 CLONING AND CHARACTERIZATION OF BUFFALO INTERFERON-TAU AND EFFICACY OF RECOMBINANT BUFFALO INTERFERON-TAU FOR IN VITRO EMBRYO DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 194
Author(s):  
S. Saugandhika ◽  
H. N. Malik ◽  
S. Saini ◽  
V. Sharma ◽  
S. Bag ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Embryo Development ◽  
Large Scale ◽  
Signal Sequence ◽  
Development Rate ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Significant Difference ◽  
Pregnancy Recognition

Interferon tau (IFN-tau) is known as maternal pregnancy recognition factor in ruminants. IFN-tau not only acts as a signalling molecule of pregnancy recognition but also performs various functions for successful implantation and pregnancy establishment. The aim of the present study was to produce recombinant buffalo interferon-tau (BuIFN-Tau) and observe if it has any effect on in vitro embryo development. The BuIFN-Tau gene was obtained through polymerase chain reaction (PCR) from hatched buffalo blastocysts and was cloned into pJET cloning vector. Screening of the recombinant colonies gave 8 distinct buffalo IFN-tau isoforms, out of which the predominant buffalo IFN-t tau1 isoform (gene bank accession number JX481984), was subcloned into expression vector pET22b without signal sequence. The recombinant plasmid was induced to express the recombinant protein by isopropyl b-D-1-thiogalactopyranoside. Analysis of the products of recombinant BuIFN-tau without signal sequence by SDS–PAGE revealed a new 20-kDa protein coinciding with the molecular weight of IFN-tau as reported earlier in literature. The purified recombinant BuIFN-tau was confirmed by Western blot using anti-HIS antibody and was subjected to three steps of large-scale purification using HIS affinity chromatography, anion exchange chromatography, and gel filtration chromatography. Finally, a relatively pure histidine-tagged recombinant protein, which had a purity of at least 90%, was generated as confirmed through SDS. The concentration of recombinant BuIFN-tau was 1 mg mL–1 by Bradford assay. The purified recombinant BuIFN-tau was used as supplement of the culture medium for IVF early buffalo embryos at the following concentrations: control, 1, 2, and 4 µg mL–1. Sixty oocytes each in 4 groups (with 20 oocytes/drop in three replicates for each group) were used for in vitro maturation. After 24 h, the matured oocytes were incubated with in vitro capacitated sperm cells for 18 h; thereafter, the presumptive zygotes were cultured in IVC medium supplemented with 0, 1, 2, or 4 µg mL–1 of the purified recombinant BuIFN-tau. The experiment was repeated 3 times. The data were analysed using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values. The differences were analysed by one-way ANOVA followed by Fisher's least significant difference test. Out of 3 concentrations of recombinant BuIFN-tau, the 2 µg mL–1 concentration significantly promoted the rate of blastocyst development, 45.55% against 31.1% (control; P < 0.01). Blastocyst development rate for low and high concentrations was 29.97% and 10.18% respectively. It is concluded that the addition of 2 µg mL–1 of recombinant BuIFN-tau enhances the blastocyst development rate in buffalo, and hence there is some evidence that BuIFN-tau has not only a role in maternal recognition of pregnancy but also in embryonic development.

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