143 EFFECTS OF BOAR SEMINAL PLASMA IN IN VITRO CULTURE OF PORCINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 219
Author(s):  
J. H. Moon ◽  
S. J. Kim ◽  
J. T. Kang ◽  
S. J. Park ◽  
J. Y. Choi ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Seminal Plasma ◽  
Harmful Effect ◽  
Developmental Rate ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Significant Difference ◽  
Protective Medium ◽  
La Jolla

Seminal plasma consisting of carbohydrates, proteins, and lipids not only serves as a nutritive and protective medium for sperm cells but also play a pivotal role in inducing the tolerance to pre-existing immune cells as well as improving the intra-uterine conditions for implantation of fertilized embryos (Guerin et al. 2009 Hum. Reprod. Update 15, 517–535). However, the effects of seminal plasma in in vitro culture of fertilized embryos are unknown. In the present study, the seminal plasma was separated from the second fraction of a normal farm boar (n = 1) by centrifugation and filtered seminal plasma was stored at –30°C until use. In a preliminary experiment, the optimal activity of seminal plasma was evaluated by incubating the embryos for different time intervals. To investigate the developmental rates, electrically (EA) (triplicates, n = 490) or chemically (CA) (quintuplicates, n = 599) activated 2-day-old porcine embryos were incubated for 3 h in PZM-5 medium (Funakoshi Co., Tokyo, Japan, Catalog no. IFP0410P) containing 0% (EA: n = 122 and CA: n = 152), 0.1% (EA: n = 123 and CA: n = 148), 0.5% (EA: n = 122 and CA: n = 150), or 1% (EA: n = 123 and CA: n = 149) seminal plasma. Similarly, the developmental rate of chemically activated 2-day-old somatic cell nuclear transferred porcine embryos (quadruplicates, n = 239) was studied after incubation with 0% (n = 119) or 0.1% (n = 120) seminal plasma for 3 h. A significant difference was noticed only in the rate of blastocyst formation in the chemically activated embryos treated with 0.1% seminal plasma (31.7 v. 24.8% in the 0% group, ANOVA; P < 0.05; Prism5, GraphPad Software Inc., La Jolla, CA, USA). None of the treatments showed a significant effect on the cleavage rate and cell numbers of blastocysts. In conclusion, the seminal plasma did not show any harmful effect on early embryos development. Furthermore, the seminal plasma (0.1%) improved the rate of blastocyst formation among the chemically activated nuclear transferred embryos. The results of this preliminary study suggest that the addition of seminal plasma during embryo transfer could increase the rate of pregnancy in pig. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

2008 ◽  
Vol 20 (1) ◽  
pp. 109 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
X. L. Jin ◽  
Y. Y. Lee ◽  
Y. J. Cho ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cumulus Cells ◽  
Complete Medium ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Frozen Semen ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL–1 hCG, and 10 ng mL–1 epidermal growth factor (EGF) at 38.5�C and 5% CO2 in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 � 106 sperm mL–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean � SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 � 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 � 2.7%, 38/45), and iSCNT (89.3 � 2.7%, 100/110, monkey; 89.3 � 3.3%, 45/49, pig; and 86.0 � 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 � 3.0%, 2/49, and 7.9 � 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 � 2.9%, 38/117, and 26.7 � 2.8%, 12/88, respectively; P < 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 � 2.7%, 78.1 � 2.1%, and 82.3 � 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

2018 ◽  
Vol 30 (1) ◽  
pp. 205 ◽  
Author(s):  
R. Emmerstorfer ◽  
K. Radefeld ◽  
V. Havlicek ◽  
U. Besenfelder ◽  
H. Yu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Significant Difference

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

Latrunculin a Enhances the Parthenogenetic Developmental Potentials of Electrically Activated Porcine Oocytes Matured In Vitro

2011 ◽  
Vol 236-238 ◽  
pp. 2548-2553
Author(s):  
Jian Hui Wang ◽  
Dong Min Liu ◽  
Yong Le Liu
Keyword(s):  
Polar Body ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
Latrunculin A ◽  
The Individual ◽  
20 Nm

The present study was carried out to determine the effect of latrunculin A (Lat-A) and FBS on the developmental potentials of electrically activated porcine oocytes maturedin vitro. Electrically activated MII oocytes were incubated for 2 hr in mPZM-3 supplemented either with 0、20 nM、1 μM Lat-A or 2.2 μg/ml cytochalasin B (CB). They were then removed and placed in mPZM-3 with or without 10% FBS (addition) on Day 4 for further development. The nuclear activation rate was significantly improved when activated oocytes were cultured in mPZM-3 with Lat-A (P<0.05). At higher dosage (1μM) of Lat-A, the extrusion of second polar body was inhibited while the cleavage rate increased significantly (P<0.05). Significant interaction between Lat-A and FBS supplementation on Day 4 was observed duringin vitrodevelopment (P<0.05). The hatched blastocyst rates and cell numbers per blastocyst were significantly enhanced with the supplementation of FBS (P<0.05). The blastocyst formation, hatched blastocyst rates and mean cell numbers per blastocyst of 1 μM Lat-A treatment with FBS supplementation were significantly higher than those of 20 nM Lat-A and CB treatments with FBS supplementation (P<0.05), but the individual effect of FBS supplementation was not observed on blastocyst formation in 20 nM and 1 μM Lat-A treatments. While significant difference was seen in blastocyst formation and cell numbers per blastocyst among all the treatments without FBS supplementation (P<0.05). These results indicate that Lat-A especially at the dosage of 1 μM can significantly improve the developmental potential of electrically activated porcine oocytes, and the FBS supplementation on Day 4 of development have an additional effect on porcine embryo development.

131 EFFECT OF BREED AND FROZEN - THAWED RAM SEMEN ON IN VITRO FERTILIZATION AND OVINE EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 170 ◽  
Author(s):  
K. C. Lehloenya ◽  
N. Mahoete ◽  
J. P. C. Greyling ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Developmental Stages ◽  
Germplasm Conservation ◽  
Egg Yolk ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Significant Difference

Ovine embryonic development was evaluated 8 days following in vitro fertilization, after using fresh or frozen–thawed Merino and indigenous (Pedi and Zulu) sheep semen. Semen used was collected twice weekly over a 3-month period with the aid of an electro-ejaculator. Following collection, semen samples were evaluated and semen with acceptable sperm motility and a percentage live sperm of 60% diluted with an egg yolk-based extender (Egg-Yolk Citrate). Semen samples were cryopreserved in straws with a programmable freezer to –130°C and then plunged into liquid nitrogen (–196°C) until used for IVF. Fresh and frozen–thawed semen was used to fertilize the matured oocytes in vitro. A total of 791 oocytes were fertilized using fresh semen and 802 oocytes fertilized using frozen–thawed semen. No significant differences were recorded between the fresh and frozen–thawed semen regarding the embryonic developmental stages. The performance of fresh and frozen–thawed semen followed the same trend, with the cleavage rate gradually declining with the progression in time and the embryonic developmental stage. The lowest developmental rate recorded was the occurrence of blastocyst formation, ranging between 0.4 ± 0.4% and 2.6 ± 1.0%. Regarding breed, no significant difference was observed from cleavage to the 2- to 4-cell stages. The use of fresh and frozen–thawed Zulu semen resulted in a significantly (P < 0.05) higher percentage of 8-cell development compared with the Pedi semen. However, the 8-cell embryonic stage recorded with the use of the Zulu ram semen (fresh and frozen–thawed), did not differ significantly from that of the Merino breed. No significant difference between the breeds regarding blastocyst formation was recorded. The overall cleavage rate, 2- to 4-cell, and blastocyst embryonic developmental stages following the use of fresh and frozen–thawed semen from the different rams were generally lower than those recorded by other researchers. The low blastocyst rates obtained warrant more research regarding the in vitro embryo production technique in order to improve the ovine blastocyst formation rate. The study was funded by the University of the Free State and conducted at the Germplasm Conservation and Reproduction Biotechnologies ARC.

165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM

2012 ◽  
Vol 24 (1) ◽  
pp. 194
Author(s):  
S. Miyashita ◽  
Y. Inaba ◽  
T. Somfai ◽  
M. Geshi ◽  
T. Nagai ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cleavage Rate

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P < 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.

226 EVALUATION OF IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO MYCOPLASMA BOVIS

2011 ◽  
Vol 23 (1) ◽  
pp. 212 ◽  
Author(s):  
D. Pavao ◽  
M. Alves ◽  
R. Qureiroz ◽  
F. Souza ◽  
M. D. Angelo
Keyword(s):  
Statistical Analysis ◽  
Zona Pellucida ◽  
Control Group ◽  
Mycoplasma Bovis ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Animal Reproduction ◽  
Significant Difference

Reproductive biotechnologies are known to improve the quality and productivity of herds. Research on cattle health and on the conditions of oocytes and embryos produced in vitro and in vivo are carried out worldwide to prevent the transmission of infectious agents, particularly those associated with ovarian tissues, the uterine tubes, and the uterus. Thus, there is concern regarding the possibility of transmitting Mycoplasma bovis through animal reproduction procedures. The aim of this study was to evaluate morphological changes by means of optical microscopy, cleavage rate, and blastocyst formation during the in vitro development of bovine embryos experimentally exposed to M. bovis. Oocytes were aspirated from ovaries of slaughtered cows, and oocytes with an intact zona pellucida were selected and matured. After 20 h, the oocytes were divided in 2 groups: a control group (n = 260), and oocytes exposed to 2.5 × 104 cfu mL–1 of M. bovis (n = 261). The semen was treated by a discontinuous Percoll gradient technique, and the sperm concentration was adjusted to approximately 100 000 sperm for each oocyte. Statistical analysis of the experiments was conducted using Student’s t-test (P < 0.05). Embryos exposed to M. bovis showed a cleavage rate of 21.07% (55/261), whereas the control group showed a cleavage rate of 64.6% (168/260). In relation to blastocyst formation, control embryos had a rate of 39.60% (103/260) v. 12.20% (32/261) for the exposed group. Statistical analysis of the experiments revealed a significant difference in the final results. Regarding morphological aspects, we observed failures in the division and asymmetry of blastomeres, cytoplasmic shrinkage, and degeneration and disruption of the zona pellucida in embryos exposed to the pathogen. These results are important to support the development of parameters for evaluating oocytes and in vitro-produced bovine embryos and to establish more efficient methods to control the spread of disease by animal reproduction biotechnologies. Further studies will be done with oocytes exposed to the pathogen and subsequently evaluated according to the Manual of the International Embryo Transfer Society.

140 THE EFFECTS OF INTERVALS OF MECHANICAL VIBRATION DURING IN VITRO CULTURE ON THE DEVELOPMENT OF BOVINE EMBRYO DERIVED FROM LOW-QUALITY OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 183
Author(s):  
M. Takehisa ◽  
S. Kondo ◽  
K. Imai ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
In Vitro Culture ◽  
Vibration Control ◽  
Mechanical Vibration ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate

Mechanical vibration enhances the cytoplyasmic maturation of in vitro-matured (IVM) pig oocytes (Mizobe et al. 2010 J. Reprod. Dev. 56, 285–290), as well as the development of in vitro-cultured (IVC) bovine embryos (Fujita et al. 2010 Rakuno Gakuen University Graduation thesis,1–36). In this study, the effects of intervals of mechanical vibration during in vitro culture, after IVF, on the development of embryos derived from low-quality oocytes were examined. Cumulus-oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter = 2 to 6 mm) obtained from a local abattoir. In this experiment, only grade 3 oocytes (i.e. those with one layer or partially remaining cumulus cells and normal cytoplasm) were used. Groups of 20 COC were matured in 100-μL droplets of in vitro TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. Matured COC were inseminated with 5 × 106 sperms mL–1 for 18 h. After 18 h of gamete co-culture, the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). Presumptive zygotes were cultured in vitro without mechanical vibration (control; n = 467) and with mechanical vibration for 5 s at 5 min (n = 180), 10 min (n = 180), 15 min (n = 180), and 60 min (n = 200) for 9 days. Embryo development was evaluated for cleavage and blastocyst rates, on Days 3 and 7 to 9 after IVF, respectively. The cleavage and blastocyst formation rates were analysed by the chi-squared test. Vibration at 15-min intervals increased (P < 0.05) cleavage rate compared to 5 min, 60 min, and control (control: 66.2 ± 22.1%; 5 min: 49.4 ± 10.2%; 10 min: 70.0 ± 7.7%; 15 min: 86.2 ± 6.6%; and 60 min: 64.0 ± 8.5%).The highest (P < 0.05) blastocyst rate among the experimental groups was found with 15-min intervals for vibration (control: 21.6 ± 9.2%; 5 min: 15.0 ± 5.3%; 10 min: 22.8 ± 1.8%; 60 min: 21.5 ± 5.0%). These results indicated that the cleavage and blastocyst formation rates of IVM-IVF-IVC bovine embryos derived from low-quality oocytes can be improved by physical stimulus during IVC. In addition, it was shown that 15-min intervals of mechanical vibration elicited the highest benefit for the development of embryos.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

52 INITIATION OF PREGNANCIES IN SOUTH AFRICAN RIVERINE RABBIT (BUNOLAGUS MONTICULARES) BY INTERSPECIES NUCLEAR TRANSFER USING ADIPOSE-DERIVED SOMATIC CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 106
Author(s):  
M. J. Sansinena ◽  
D. Owiny ◽  
R. S. Denniston ◽  
D. Salamone ◽  
D. Barry
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Uv Light ◽  
Uv Exposure ◽  
Domestic Rabbit ◽  
Donor Cells ◽  
Significant Difference ◽  
Preliminary Study ◽  
Interspecies Nuclear Transfer

The riverine rabbit (Bunolagus monticulares), one of South Africa's most threatened mammals, with an estimated population size under 250, was upgraded from endangered to critically endangered in 2002. The low number of riverine rabbits precludes any attempts of nuclear transfer (NT) using intraspecific oocytes; therefore, the overall aim of this study was to assess the ability of the domestic rabbit (Oryctolagus cuniculus) oocyte to reprogram the somatic cell of the endangered riverine rabbit by interspecies NT. A preliminary study evaluated the effect of timing of enucleation after induction of ovulation (h post-hCG). A second study assessed the effects of two activation protocols. In addition, since the unique characteristics of the rabbit zona pellucida affect the speed of micromanipulation, different exposure periods to UV light at enucleation were evaluated. Adult domestic Californian rabbits were treated with eCG for 72 h, and ovulation was induced by hCG administration. Oocytes were collected by retrograde flushing at 12–14 h or 16–18 h post-hCG administration and stripped of cumulus investments with 0.5% hyaluronidase in Ca-Mg-free PBS. Metaphase-II oocytes were selected by visualizing the first polar body. Oocytes were stained with 2 mg mL–1 Hoechst 33342 for 5 min, and metaphase plates were removed with a 25–30 μm (O.D.) borosilicate beveled, spiked pipette after exposure to <5 or 30–40 s of UV light. Adult adipose-derived riverine rabbit fibroblasts grown to confluency in DMEM with 10% FCS were used as donor cells and fused with 2 consecutive DC pulses (3.2 kV cm–1, 45 μs). After reconstruction, couplets were randomly assigned for activation by either a second set of electrical pulses or incubation with ionomycin, followed by 1 h of incubation in 2 mm 6-DMAP. Embryos were co-cultured with a bovine oviductal cell monolayer in DMEM with 10% FCS and assessed for cleavage after 36 h of in vitro culture. There was a significant difference in the number of cleaved embryos from oocytes collected at 12–14 h post-hCG (n = 50) or 16–18 h post-hCG (n = 51) administration (57% v. 0% cleaved; P < 0.05). No significant difference was detected in embryos developing after electrofusion v. ionomycin activation treatments. However, a significantly greater number (P < 0.05) of embryos cleaved from oocytes exposed to <5 s UV than from oocytes exposed to 30–40 s UV (Table 1). A total of 20 embryos (4-cell to 16-cell stages) were surgically transferred to the oviducts of 4 adult New Zealand white synchronized recipients after 48 h of in vitro culture. Two recipients (<5 s UV exposure treatment group) were diagnosed pregnant by abdominal palpation at 15 days post-transfer; pregnancies were subsequently lost by Day 30, with placental tissues recovered. This preliminary study indicates the domestic rabbit oocyte is capable of reprogramming riverine rabbit donor cells. In addition, the time of oocyte collection after ovulation induction and the UV exposure period during enucleation have an effect on the efficiency of interspecies NT and embryo development in this species. Table 1. Effect of UV exposure during enucleation on the in vitro development of interspecies nuclear transfer riverine rabbit embryos

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

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