129 IN VITRO PRODUCTION OF EQUINE EMBRYOS FROM YOUNG AND OLD MARES BY INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 145 ◽  
Author(s):  
C. Herrera ◽  
M. Revora ◽  
L. Vivani ◽  
M. H. Miragaya ◽  
C. Quintans ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Age Groups ◽  
Reproductive Status ◽  
Developmental Competence ◽  
Fisher Exact Test ◽  
In Vitro Production ◽  
The Mean

High merit mares obtain their utmost productive value at the same time their reproductive soundness diminishes. The aim of our study was to compare the developmental competence of equine oocytes from young and old mares after intracytoplasmic sperm injection (ICSI) and in vitro culture. Ovaries from young and old mares were obtained from a pool of slaughterhouse animals that have been previously selected by overall good body condition, reproductive status, and age. Young mares were 3 to 8 years old, and old mares were more than 15 years old. The age of all mares was determined by teeth observation and reproductive status by ultrasonography. Oocytes were obtained from ovaries 1 h postmortem by individual dissection of follicles between 10 and 25 mm and scraping of the follicle wall with a bone curette. Recovered oocytes were matured in vitro for 24–30 h, and all oocytes with an intact cytoplasm and a visible polar body were subject to ICSI and cultured for 7.5 days in SOFm. The maturation rate, cleavage, and embryo development rate and mean number of blastomeres at 7.5 days of culture were compared between oocytes and embryos from young and old mares. Maturation, cleavage, and developmental rates were analyzed by Chi Square and Fisher exact test, whereas the mean number of blastomeres at 7.5 days of culture was compared by one way ANOVA and t-test. A total number of 54 oocytes from young mares and 37 oocytes from old mares were obtained. There were no significant differences between the maturation, cleavage, or embryo development rates between young (79.63, 56.41, and 18.18%) and old (91.89, 63.33, and 15.79%) mares. In addition, the mean number of cells on embryos from each group did not differ significantly (57.75 v. 81; young v. old). Our preliminary results show that similar in vitro rates are achieved when oocytes from young or old mares are matured and fertilized in vitro by ICSI. This does not correlate with the reproductive senescence in old mares and the result obtained with other reproductive techniques. Further studies will determine if pregnancies are equally achieved using in vitro produced embryos from both age groups.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

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 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE

2015 ◽  
Vol 27 (1) ◽  
pp. 95
Author(s):  
M. B. Rodríguez ◽  
A. Gambini ◽  
R. J. Bevacqua ◽  
D. F. Salamone
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Electric Pulse ◽  
Polar Body ◽  
Chemical Activation ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Chi Squared ◽  
Second Polar Body ◽  
Pronuclear Formation

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.

255. In vitro maturation of bovine oocytes in serum-free media

2005 ◽  
Vol 17 (9) ◽  
pp. 102
Author(s):  
S. Zhang ◽  
A. J. French ◽  
R. T. Tecirlioglu
Keyword(s):  
Embryo Development ◽  
Bovine Serum ◽  
Culture Medium ◽  
Polar Body ◽  
In Vitro Production ◽  
Serum Free ◽  
Cell Numbers ◽  
Defined Culture ◽  
Vitro Production

Culture medium supplemented with sera is commonly used for the in vitro production (IVP) of livestock embryos. However, serum induced complications including batch variation, the potential risk of virus and mycoplasma contamination and the implication in the large offspring syndrome in domestic animals impels the development of a serum-free culture system. In this study, we investigated whether replacement of fetal bovine serum (FBS) with bovine serum albumin (BSA) in three maturation media, tissue culture medium-199 (TCM-199), a modified synthetic oviduct fluid (mSOF) routinely used in our laboratories and a commercially available SOF-VC (Vitro Cleave, Cook Australia). Harvested oocytes were matured, parthenogenetically activated and in vitro cultured (Day 7) to measure maturation efficiency, embryo development and quality with the aim of developing a simplified and defined culture medium for the in vitro production of bovine embryos. Abattoir derived cumulus oocyte complexes were matured in TCM-199, mSOF and SOF-VC media supplemented with LH and beta-estradiol in the presence of 15% FBS or 0.08% BSA at 39ºC in 5% CO2 in air. Polar body extrusion was assessed twenty-two hour post maturation and selected MII occytes were activated using calcium ionophore/6-dimethylaminopurine and cultured for seven days in SOF medium supplemented with 0.8% BSA. On day seven, blastocyst development was assessed and randomly selected blastocysts were stained to determine inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN). Supplementation with either BSA or FCS did not significantly affect the maturation efficiency, blastocyst rates or differential cell numbers within each maturation media tested. However, maturation efficiency and blastocyst rates were significantly lower (P < 0.01) when oocytes were matured in either mSOF or SOF-VC regardless of FBS or BSA supplementation. From this study, we conclude that BSA effectively replaces FCS and TCM-199 is superior to SOF (mSOF or SOF-VC) in terms of oocyte maturation regardless of protein source. Once matured SOF and TCM-199 parthenogenetically blastocysts were equivalent in terms of embryo development and quality.

scholarly journals Superovulation following follicular synchronization with GnRH at random stages of the oestrous cycle in heifers: oocyte competence and in vitro embryo production

2010 ◽  
Vol 55 (No. 5) ◽  
pp. 190-194
Author(s):  
H. Kohram ◽  
V. Vahedi ◽  
A. Farahavar
Keyword(s):  
Late Phase ◽  
Oestrous Cycle ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Embryo Production ◽  
Oocyte Competence ◽  
The Mean ◽  
Follicular Puncture ◽  
Vitro Production

The objective of this study was to develop a superovulatory program based on the synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. In this experiment, GnRH was given to 30 heifers in lactation between Days 0 and 7 (n = 13), 8 and 12 (n = 12), 13 and 16 (n = 5) of the oestrous cycle. Twenty-four heifers were used as controls and did not receive any GnRH. All follicles &ge; 6 mm were punctured 4 days after GnRH treatment in treated animals and between Days 8 and 12 of the oestrous cycle in control heifers. Two days after the follicular puncture, all heifers were superstimulated with 160 mg Folltropin-V given twice daily over 2 days. Oocytes were collected 42 h after the last FSH treatment. The oocytes were subjected to IVM/IVF and the developmental competence of embryos was compared. In vitro production of embryos was affected only by the stages of the oestrous cycle when the GnRH treatment was given and not by the GnRH treatment. No difference (P &gt; 0.1) in the mean number of oocytes, cleavage and embryo production was noted between the control animals and the animals treated with GnRH in the late phase of the oestrous cycle. The mean number of blastocysts was higher (P &lt; 0.05) in heifers treated with GnRH in the mid and the late phase of the oestrous cycle than in the early phase. In conclusion, the in vitro production of embryos was compromised in the present study with heifers following the follicular synchronization with GnRH. This procedure is advantageous for the in vitro production of bovine embryos since the spontaneous oestrus is eliminated. However, more investigations are needed to increase the competence of oocytes obtained following this procedure.

285BIRTH OF PIGLETS AFTER NON-SURGICAL TRANSFER OF PORCINE EMBRYOS CULTURED IN PZM-4 WITH ALTERED CONCENTRATIONS OF AMINO ACIDS

2004 ◽  
Vol 16 (2) ◽  
pp. 262
Author(s):  
C. Suzuki ◽  
K. Yoshioka ◽  
S. Iwamura
Keyword(s):  
Amino Acids ◽  
Embryo Development ◽  
Intrauterine Insemination ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Developmental Competence ◽  
Double Dose ◽  
In Vitro Production ◽  
Cell Numbers

We previously developed an in vitro production (IVP) system for porcine embryos and obtained piglets after surgical transfer of blastocysts cultured in Porcine Zygote Medium (PZM)-4. However, the developmental competence of pig IVP embryos to the blastocyst stage is still low and further improvement of IVC medium is needed. In the present study, we evaluated the effects of the addition of glutamine (Gln), hypotaurine (HT), taurine (Tau), BME-essential (EA) and MEM-nonessential (NA) amino acids solutions to PZM-4, and the replacement of polyvinyl alcohol (PVA) with BSA on embryo development to blastocysts. Moreover, the developmental competence of IVP blastocysts after nonsurgical embryo transfer (NS-ET), using a flexible catheter (FC) for deep intrauterine insemination, was investigated. Porcine COC from prepubertal gilts were matured and fertilized in vitro, using frozen-thawed ejaculated boar semen. Presumptive zygotes were cultured in PZM-4, as a basal culture medium, until Day 5 after IVF. Data from six replicates were analyzed by ANOVA. Addition of 0.25 to 4mM Gln to PZM-4 (containing 5mM HT) significantly increased the percentage of embryos that developed to blastocysts (15 to 31%), with addition of 2mM Gln significantly increasing the total cell numbers in blastocysts (43±17 cells) compared with no addition (3% and 20±4 cells, respectively). Addition of 1.25 to 10mM HT to HT-free PZM-4 supplemented with 2mM Gln (named PZM-5) significantly increased the percentage of embryos that developed to blastocysts (22 to 28%) compared with control (no HT;; 4%). In the culture with HT-free PZM-5, addition of 5mM Tau significantly increased blastocyst yield (17%) compared with control (4%). However, Tau addition in the presence of 5mM HT had no effect on development to the blastocyst stage. In combinations of EA and NA added to PZM-5, a single dose of EA significantly increased the percentage of embryos that developed to blastocysts (27%) compared with no dose (19%) or with a double dose of EA (20%), while a double dose of NA significantly increased the total cell numbers in blastocysts (43±16 cells) compared with no NA (37± 6 cells). Replacement of PVA with BSA in PZM-5 had no effect on embryo development to the blastocyst stage. Crossbred sows were used as recipients for NS-ET, and had their estrous cycle synchronized by a described previously method (Yoshioka et al., 2002 Biol. Reprod. 66, 112–119). Five days after hCG injection, a FC was introduced via the cervix into the uterine horn of recipients without sedation. Day-5 blastocysts cultured in PZM-5 were then transferred together with 5ml of TALP-Hepes (45 to 50 blastocysts/recipient). Of 6 recipients, one sow became pregnant and farrowed 7 piglets. Our results indicate that the addition of amino acids to PZM-4 can improve porcine embryo development to the blastocyst stage, and that blastocysts cultured in a chemically defined medium, PZM-5, can develop to full-term following NS-ET.

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

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

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

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