372 LASER-ASSISTED LENTIVIRAL TRANSGENESIS IN BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 293
Author(s):  
S. Ewerling ◽  
A. Hofmann ◽  
R. Klose ◽  
K. Rink ◽  
G. Brem ◽  
...  
Keyword(s):  
Incubation Time ◽  
Cell Mass ◽  
Pulse Length ◽  
Egfp Expression ◽  
Virus Concentration ◽  
Virus Suspension ◽  
Cleavage Rate ◽  
Lentiviral Transgenesis ◽  
Maximum Humidity

Lentiviral vectors have been shown to be a powerful tool to create transgenic livestock (Hofmann et al. 2003 EMBO Rep. 4, 1054-1060; Hofmann et al. 2004 Biol. Reprod. 71, 405-409). Due to their inability to pass the zona pellucida (ZP) by themselves, viral particles were delivered by subzonal microinjection of oocytes or zygotes. Here we show that an artificial opening of the ZP, generated by an infrared 1.48 �m microsurgical diode laser (OCTAX Laser Shot", kindly provided by MTG, Altdorf, Germany) and subsequent culture of oocytes in virus suspension, was sufficient to allow virus integration and expression of eGFP. The ZP of denuded in vitro-matured bovine oocytes was microdrilled with 2 to 4 laser shots of 3.0-ms pulse length each to create an opening of at least 40 �m. Oocytes were transferred to microdroplets (20 �L of Fert Talp) containing the virus suspension (lentiviral vector containing the eGFP reporter gene under the control of the human phosphoglycerate-kinase 1 promoter). After incubation for 2 or 4 h (5% CO2, maximum humidity, 39�C), oocytes were washed thoroughly in fresh droplets of medium to remove excessive virus. Sperm was added (0.5 � 106/mL) for 8 h. In vitro culture of embryos (5% CO2, 5% O2, maximum humidity, 39�C) was continued in culture medium (SOF) under oil for up to 8 days. Cleavage rate and number of blastocysts were determined on Days 2 and 8, respectively. Expression of eGFP was analyzed on Day 8 by fluorescence microscopy using an eGFP-specific filter with UV light source under an inverted microscope. In total, 267 oocytes were microdrilled. The range of optimal virus concentration was analyzed by titration. Seven different concentrations in a range of 1.25 � 106 to 5 � 107 IU/mL were used at an incubation time of 4 h. In one experiment using a high virus concentration, the incubation time was reduced to 2 h. Control groups were treated equally except for addition of virus. All experiments resulted in blastocysts showing eGFP expression in both the trophoblast and the inner cell mass. In four experiments we used aliquots of the same virus preparation (1.25 � 107 IU/mL) for infection. The cleavage rate ranged from 50 to 70%, and blastocyst rate from 15 to 35%. In three experiments showing good to average embryo development, 25 to 83% of the resulting blastocysts expressed eGFP, indicating efficient lentiviral transduction of oocytes following laser-assisted zona microdrilling. Studies directly comparing zona microdrilling and subzonal injection with regard to the efficacy of lentiviral transgenesis and the incidence of polyspermy are underway.

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.

353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM

2006 ◽  
Vol 18 (2) ◽  
pp. 283 ◽  
Author(s):  
M. Zhang ◽  
X. W. Liang ◽  
Y. Q. Lu ◽  
K. H. Lu
Keyword(s):  
Mineral Oil ◽  
Motile Sperm ◽  
Percoll Gradient ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Blastocyst Rate ◽  
Maximum Humidity ◽  
Cattle And Sheep

Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos. Table 1. Cleavage and blastocyst rates with different sperm types This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.

In vitro production of cattle-water buffalo (Bos taurus - Bubalus bubalis) hybrid embryos

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 155-162 ◽  
Author(s):  
H.P.S. Kochhar ◽  
K.B.C. Appa Rao ◽  
A.M. Luciano ◽  
S.M. Totey ◽  
F. Gandolfi ◽  
...  
Keyword(s):  
Water Buffalo ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Developmental Potential

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

128 MULTIPLICATION OF 8-CELL EMBRYOS BY AGGREGATION OF A SINGLE ENHANCED GREEN FLUORESCENT PROTEIN-LABELED BLASTOMERE WITH PUTATIVE TETRAPLOID EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 168
Author(s):  
M. I. Hiriart ◽  
R. J. Bevacqua ◽  
R. Fernandez-Martin ◽  
D. F. Salamone
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Egfp Expression ◽  
Simple Method ◽  
Transgenic Embryos ◽  
Green Fluorescent

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.

70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?

2019 ◽  
Vol 31 (1) ◽  
pp. 160
Author(s):  
J. A. Sánchez Viafara ◽  
G. Lopez de Vasconcelos ◽  
R. Maculan ◽  
N. Gomes Alves ◽  
J. Camisão de Souza
Keyword(s):  
Docosahexaenoic Acid ◽  
Cell Mass ◽  
Control Group ◽  
Hatching Rate ◽  
Link Function ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Internal Cell ◽  
Fresh Embryos

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1µM DHA (Sigma, St. Louis, MO, USA; n=437), and without the agonist (control group; n=450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72h. A sample of re-cultivated embryos at 12h of DHA (n=5), and without the agonist (control group; n=6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P&gt;0.05) and the production of blastocysts was lower in the DHA group (P&lt;0.05). The number of cells per embryo was higher (P&lt;0.05) by the addition of 1μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72h after devitrification in the group treated with 1μM DHA were not different (P&gt;0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P&lt;0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study. Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.

scholarly journals Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 345-355 ◽  
Author(s):  
F Moulavi ◽  
S M Hosseini ◽  
M Hajian ◽  
M Forouzanfar ◽  
P Abedi ◽  
...  
Keyword(s):  
Cell Fate ◽  
Embryo Development ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Nuclear Remodeling

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep.In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances ofHSP90AA1(HSP90),NPM2andATPasegenes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents ofPOU5F1(OCT4) with the ZI-NT and ZF-NT methods and ofPAPOLA(PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

84 PAIRS OF BLASTOMERES FROM BOVINE DAY 5 MORULAE ARE ABLE TO CONTRIBUTE TO INNER CELL MASS AND TROPHECTODERM IN CHIMERIC EMBRYOS GENERATED BY AGGREGATION WITH TWO DAY 4 MORULAE

2015 ◽  
Vol 27 (1) ◽  
pp. 135
Author(s):  
K. Simmet ◽  
M. Reichenbach ◽  
S. Jung ◽  
R. Fries ◽  
T. Grupp ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Standard Procedure ◽  
Cell Mass ◽  
Breeding Value ◽  
Egfp Expression ◽  
Promising Alternative ◽  
Donor Embryo

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

57 DEVELOPMENTAL CHARACTERISTICS OF LATER-STAGE PORCINE EMBRYOS PRODUCED IN VIVO OR IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 158 ◽  
Author(s):  
H. Callesen ◽  
P. Holm
Keyword(s):  
In Vitro Culture ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Viable Cell ◽  
Outer Diameter ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage

Kinetics and morphological characteristics during pre-implantation embryo development are well established in most mammals. In porcine, such studies are few because of limited use of in vitro culture, but this has changed in recent years due to increasing use of in vitro production and cloning. Therefore, additional characterisation of especially later stage porcine embryos is needed. Here we studied in vitro development of porcine in vivo- and in vitro-derived zygotes collected from weaned, inseminated sows (slaughtered 2 days after first insemination; in vivo group, n = 112) or produced from immature oocytes from sow ovaries (matured and fertilized: Theriogenology 63, 2040–2052; in vitro group, n = 210). Both types were cultured for 7 days in vitro (Theriogenology 63, 2040–2052) in a time-lapse system (Theriogenology 50, 1285–1299) with images recorded every 0.5 h. Individual embryos were followed and characterised for stage and quality from first cleavage. The following was recorded: (i) zygote: inner (i.e. ooplasma) and outer diameter [i.e. including zona pellucida (ZP)], ZP thickness; (ii) compact morula: areas of compacted inner cells and of cellular debris; (iii) blastocyst: partial or total collapse of blastocoelic cavity and diameter at maximal expansion immediately before hatching. At the 1-cell stage, no difference was found between in vivo v. in vitro zygotes in ZP diameter (approximately 150 µm) and thickness (approximately 15.6 µm). In both groups, cleavage rate was around 65%, but more in vivo (85%) than in vitro (28%) zygotes developed beyond the morula stage. Embryos of both types that did not develop to this stage (n = 212) blocked predominantly at the 1st (50%) or 2nd (21%) cell cycle. Cell cycles were generally shorter in in vivo v. in vitro zygotes from compact morula until the hatched blastocyst stage (mean 128 v. 139 h from the 2-cell stage for in vivo v. in vitro; P < 0.05). Compacted in vivo morulae were 25% larger than in vitro, and the debris area was more than twice as large in in vitro. Hatching occurred after approximately two collapses in both in vivo and in vitro but at a larger ZP diameter in vitro. See Table 1 for further details. This study illustrates differences and similarities between morphology and developmental kinetics of in vivo- and in vitro-derived porcine zygotes, but also how various morphological characteristics indicate some of the possible causes of the reduced developmental ability of in vitro embryos. The in vitro period seems to result in more stressful conditions for the embryos, both during early development, but also during the later stages leading to the hatching process. Thus, further optimization of in vitro culture conditions is still needed in porcine. Table 1.Compact morula (CM) viable cell mass and debris, hatched blastocyst (HB) diameter, and early-hatched blastocyst (EB-XB) and expanded-hatched blastocyst (XB-HB) collapses for in vivo- and in vitro-derived zygotes

95 Successful Ovum Pick-Up-In Vitro Embryo Production in Lidia Cattle in France

2018 ◽  
Vol 30 (1) ◽  
pp. 187
Author(s):  
G. G. Lazo ◽  
S. Lacaze ◽  
D. Di Scala
Keyword(s):  
Transvaginal Ultrasound ◽  
Genetic Material ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
Vitro Production ◽  
Maximum Humidity

Lidia cattle are a breed of Bos taurus that has been selected specially to produce bulls with the temperament and aggressiveness necessary to face a bullfighter in a ring. The genetic wealth of this fighting breed is divided into small lineages, traditionally called encastes, which has resulted in the risk of a loss of genetic variability (Ministerio de Medio Ambiente y Medio Rural y Marino, 2011; http://www.toroslidia.com/wp-content/uploads/2012/01/Programa-de-mejora-de-la-Raza-Bovina-de-Lidia.pdf). The technique to produce embryos in vitro may be a useful tool in the conservation of genetic material from this breed in a selection program. The aims of the study were to demonstrate the effectiveness of in vitro production of Lidia cattle embryos, and to evaluate variation in embryo production among males of the breed. Lidia cows, 7 to 13 years of age (n = 12), were used in an ovum pick-up (OPU)-in vitro production (IVP) program in the south of France. Ovarian superstimulation was induced with decreasing doses of pFSH (Stimufol; Reprobiol, Liège, Belgium) twice daily over 3 days (total dose: 350 µg). Transvaginal ultrasound-guided collection of cumulus–oocyte complexes (COC) was done 12 to 24 h after the last FSH injection. The COC were evaluated immediately after OPU and placed into 2.0-mL tubes (Corning Inc., Corning, NY, USA) containing 500 µL of maturation medium. A gas mix (5% CO2 in air) was injected into each tube and the tube was sealed tightly and placed in a portable incubator (Minitub, Tiefenbach, Germany) at 38.0°C for 12 h. On arrival in the Auriva IVP laboratory, tubes were opened and placed into an incubator with 5% CO2 at 38.5°C at maximum humidity to complete a 24-h maturation period. Semen was collected by electro-ejaculation previously from 5 different Lidia bulls (A, B, C, D, and E) and had been frozen by the same technique. The COC were fertilized with the frozen–thawed semen in TALP medium. Presumed zygotes were cultured in SOF medium (Minitub) to Day 7 (Day 0 = fertilization day) at 38.5°C in a 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. A total of 19 OPU/IVP sessions were performed, 5 cows were collected once, and 7 cows collected twice, and 143 COC were processed for in vitro embryo production. Blastocyst and expanded blastocyst numbers were recorded on Day 7. Oocyte recovery and embryo production by bull were analysed by ANOVA and blastocyst yield by Chi-square. The number (mean ± SEM) of oocytes allocated to each bull per IVP session was (P > 0.05): bull A (4.5 ± 1.9), bull B (5.8 ± 2.1), bull C (9.3 ± 2.5), bull D (6.5 ± 2.1), and bull E (7.0 ± 4.4). The cleavage rate differed among bulls (P < 0.05): bull A (4%), B (80%), C (89%), D (81%), and E (76%). The number (mean ± SEM) of blastocysts was lowest (P < 0.05) for bull A and highest (P < 0.05) for bull C (0, 3.7 ± 1.8, 7.0 ± 1.0, 4.3 ± 1.3, 4.7 ± 2.3 for bulls A to E, respectively). The blastocyst development rate (number of blastocysts/number of oocytes entering the IVF process) was also different among bulls (0, 63, 75, 65, and 67%, respectively; P < 0.05). Although there was a male effect on blastocyst production, our data demonstrate that successful in vitro embryo production in Lidia cattle is possible and suggests that this tool would be useful in a genetic program for the multiplication and the conservation of this breed.

Comparison of different fertilisation media for an in vitro maturation–fertilisation–culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production

2016 ◽  
Vol 28 (11) ◽  
pp. 1695
Author(s):  
Luis B. Ferré ◽  
Yanina Bogliotti ◽  
James L. Chitwood ◽  
Cristóbal Fresno ◽  
Hugo H. Ortega ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Female Sex ◽  
Grade 3 ◽  
Oviductal Fluid ◽  
Hatching Rates

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode’s albumin–lactate–pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus–oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

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