scholarly journals 162INSULIN IMPROVES FREQUENCY OF CLEAVAGE AND IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN DEFINED MEDIUM

2004 ◽  
Vol 16 (2) ◽  
pp. 203
Author(s):  
G. Wirtu ◽  
C.E. Pope ◽  
P. Damiani ◽  
B.L. Dresser ◽  
R.A. Godke ◽  
...  
Keyword(s):  
Amino Acids ◽  
Egf Receptor ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Cell Number ◽  
Bovine Embryos ◽  
Beneficial Effects ◽  
Morula Stage ◽  
Hoechst Staining

Pre-elongation stage bovine embryos contain insulin receptor, ligands and receptors of IGF-I and IGF-II, and possibly EGF ligand. Cumulus cells and blastocysts also express EGF receptor; however, insulin is not produced (Watson AJ et al. 1992 Mol. Reprod. Dev. 31, 87–95; Tetens F. et al. 2000 Anat. Embryol. 201, 349–55; Yaseen MA et al. 2001 Reproduction 122, 601-10). Reported effects of external growth factors during bovine IVC are conflicting. The present study evaluated effects of EGF and insulin on the development of in vitro-produced embryos in a chemically defined IVC medium. IVM medium was TCM199 plus fetal bovine serum, LH and estradiol. IVF was done in Tyrodes solution containing BSA, lactate, pyruvate, heparin, penicillamine-hypotaurine-epinephrine and nonessential amino acids. At 18h post-insemination, ova were vortexed, washed and placed in IVC treatments. Modified KSOM (Yang BK et al. 1995 J. Reprod. Dev. 41, 213–18) with 1X MEM nonessential and 1X BME amino acids was the base IVC medium. Incubations were done at 39°C in a humidified atmosphere of 5% CO2 in air (IVM, IVF) or 5% CO2, 10% O2 and 85% N2 (IVC). Effects of three EGF (0, 50, 100ngmL−1; Experiment 1) or five insulin (0, 5, 10, 15, 20μgmL−1; Experiment 2) doses were tested, in five replicates, by supplementation during the entire IVC period (up to Day 9; Day 0=day of insemination). Embryos were placed in fresh medium on Day 4 post-insemination. Data were analyzed using one-way ANOVA and Tukey’s test. In Experiment 1 (n=646 oocytes), EGF treatment did not affect percentages of Day 2 cleavage (83±3, 82±3, and 81±2%, respectively; mean±SEM.) and Day 9 blastocysts (34±4, 33±4 and 33±5%, respectively) or cell number per blastocyst after Hoechst staining (95±5, 88±4 and 89±5, respectively). In Experiment 2 (n=687 oocytes), insulin improved the frequency of cleavage and percentage of expanding, hatching and total blastocysts. It also increased blastocyst cell number (see Table 1). Lack of effect of EGF on bovine IVC is in contrast to its widely reported beneficial effects in mice. In many bovine IVC studies, the beneficial effects of external insulin were seen at or after the morula stage. The present study demonstrates that insulin can influence embryonic development as early as the initial cleavage stage and before embryonic genome activation. Reseach was funded in part by J. Bennett Johnston Science Foundation. Table 1 Effects of insulin (μgmL−1) on development to >2-cell (Day 2), expanded, hatched and total blastocysts (Day 9: %±SEM) and on blastocyst cell number (Day 9: mean±SEM)

Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure

2003 ◽  
Vol 15 (8) ◽  
pp. 439 ◽  
Author(s):  
G. Wirtu ◽  
C. E. Pope ◽  
P. Damiani ◽  
F. Miller ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Amino Acids ◽  
Osmotic Pressure ◽  
Cell Number ◽  
Basic Medium ◽  
Blastocyst Development ◽  
Beneficial Effects ◽  
Pressure Medium ◽  
Morula Stage ◽  
Mean Differences

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mm) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

scholarly journals 325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 282
Author(s):  
L.J. Royo ◽  
A. Rodriguez ◽  
A. Gutierrez-Adan ◽  
C. Diez ◽  
E. Moran ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Defined Medium ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Atp Production ◽  
Beneficial Effects ◽  
Oocyte Developmental Competence ◽  
Grant Support ◽  
Bovine Oocytes

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP&lt;0.01; a,bP&lt;0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

Effects of supplemental progesterone on the development, metabolism and blastocyst cell number of bovine embryos produced in vitro

2011 ◽  
Vol 23 (2) ◽  
pp. 311 ◽  
Author(s):  
Jamie E. Larson ◽  
Rebecca L. Krisher ◽  
G. Cliff Lamb
Keyword(s):  
Present Experiment ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryos ◽  
Morula Stage ◽  
Metabolic Data ◽  
Embryo Metabolism ◽  
Number Of Cells

The objectives of the present experiment were to determine whether supplementation with progesterone (LO, 1 ng mL–1 or HI, 100 ng mL–1) during either the first (Culture-1, Day 1 to 3) or second (Culture-2, Day 4 to 7) phase of culture of in vitro-produced embryos alters embryo development, embryo metabolism or blastocyst cell number. The percentage of oocytes that cleaved, the percentage of cleaved embryos that developed to the morula stage or greater, the blastocyst stage or greater or the hatched blastocyst stage were similar among treatments. Quantities of glucose metabolised per blastocyst per hour were similar, but when metabolic data was normalised for numbers of cells in each blastocyst, the LO treatment during Culture-2 metabolised more glucose (P = 0.03) compared with all other treatments. Embryos receiving LO progesterone tended to have greater (P = 0.085) metabolism of glucose compared with embryos receiving HI progesterone. Quantities of pyruvate oxidised per blastocyst per hour, and per cell, were similar among treatments. The number of cells per blastocyst in the control group was increased (P = 0.039) compared with cells in progesterone-treated groups. In conclusion, supplementation with progesterone during the culture of in vitro-produced embryos does not appear to improve embryo characteristics.

121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
L. S. Amorim ◽  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Culture Media ◽  
Cumulus Cells ◽  
Similar Efficacy ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Developmental Rates ◽  
Effects Of Media

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium

1996 ◽  
Vol 8 (8) ◽  
pp. 1199 ◽  
Author(s):  
JM Lim ◽  
W Hansel
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Defined Medium ◽  
Basic Medium ◽  
Bovine Embryos ◽  
Tgf Beta ◽  
Cell Stage ◽  
Morula Stage ◽  
Beta 2

Bovine embryos at the 8- or 16-cell stage were cultured singly, or in groups (10-12 embryos), in the presence or absence of bovine oviduct epithelial cells (BOEC) in a defined medium which was used as a basic culture medium. A higher (P < 0.05) proportion of 8-cell embryos (48.3-50.8%) cultured singly developed beyond the 8-cell stage after the addition of platelet-derived growth factor (PDGF)-AB (1 ng mL-1) only, or with PDGF-AB + basic fibroblast growth factor (bFGF; 1 ng ml-1) + transforming growth factor (TGF)-beta 1 beta 2 (1 ng mL-1) than in basic medium alone (30.3%). In contrast, a significantly (P < 0.02) higher percentage (62.6-65.8%) of 16-cell embryos developed to the morula stage after the addition of TGF-beta 1 beta 2 only, or the addition of TGF-beta 1 beta 2 + bFGF + PDGF-AB than in basic medium alone (30.2%). These proportions were not significantly (P > 0.05) different from the proportions obtained when embryos were cultured in groups, but were significantly (P < 0.005) lower than the proportions obtained when embryos were cultured in groups on BOEC monolayers. Arachidonic acid (50 ng mL-1), beta-mercaptoethanol (10 microM) and glutathione (10-1000 microM) stimulated the development of 8-cell embryos in the presence of PDGF and TGF-beta 1 beta 2; blastocyst formation was observed for the first time in 8-cell embryos cultured singly in the presence of these embryotrophic substances (2.2-6.2%).

Metabolic regulation of in-vitro-produced bovine embryos. I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls

2006 ◽  
Vol 18 (5) ◽  
pp. 585 ◽  
Author(s):  
Jose Fernando De La Torre-Sanchez ◽  
Kimberly Preis ◽  
George E. Seidel
Keyword(s):  
Embryo Development ◽  
Sodium Azide ◽  
Metabolic Regulation ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Beneficial Effects ◽  
Response Information ◽  
Phenazine Ethosulfate ◽  
Metabolic Regulators

The toxic and/or beneficial effects of four metabolic regulators on embryo development were evaluated. In-vitro-produced compact morulae were cultured for 3 days in a chemically defined medium + bovine serum albumin (BSA; CDM-2) plus regulators (4991 total embryos). Phenazine ethosulfate (PES), phloretin (PL), pyrroline-5-carboxylate (P5C), and sodium azide (NaN3) were evaluated at four doses each in factorial combinations with four concentrations of glucose: 0, 0.5, 2, and 8 mm. Phenazine ethosulfate at 0.9 μm resulted in poorer development than lower or no PES. Phloretin was, in general, detrimental for embryo development, but most markedly at the highest dose (270 µm). Pyrroline-5-carboxylate had little effect on post-compaction embryos at the doses studied, 9 to 81 μm. Sodium azide at the concentrations used (3, 9, and 27 μm) had little effect on embryo development compared with controls. Concentrations of glucose had little effect on development of embryos. A fifth metabolic regulator, 2,4-dinitrophenol (DNP), was studied at various doses at pre-morula or morula-blastocyst stages cultured in 2 mm glucose. Embryos (2189 total) cultured in 90 µm DNP developed more slowly and were darker than embryos cultured at lower doses. Embryos cultured in 30 µm DNP had a higher blastocyst rate (48.3%) than controls (34.9%). In the last experiment using G1.2/G2.2 media, DNP (30 μm) resulted in a marked decrease in embryonic development when embryos were exposed at the zygote to 8- to 16-cell stages but had little effect when morulae were exposed for 2 days. The dose–response information for these metabolic regulators is crucial for designing future experiments.

90 SUCCESSFUL VITRIFICATION OF PARTHENOGENETIC PORCINE BLASTOCYSTS PRODUCED FROM DELIPATED IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 153 ◽  
Author(s):  
Y. Du ◽  
P. M. Kragh ◽  
X. Zhang ◽  
H. Yang ◽  
G. Vajta ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Survival Rates ◽  
Cumulus Cells ◽  
Cell Number ◽  
Significant Loss ◽  
Developmental Competence ◽  
Average Cell ◽  
Analogue System ◽  
Hoechst Staining

Cryopreservation of cloned porcine embryos may improve the output of somatic cell cloning considerably by alleviating logistic problems. However, the high lipid content of porcine oocytes and embryos compromises their cryotolerance. Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol. Reprod. 71, 432-436). This method was applied to our present work with few modifications to compare the cryosurvival of porcine blastocysts produced from delipated vs. intact oocytes with parthenogenetic activation. In four replicates, a total of 192 oocytes were used for the experiments. After in vitro maturation for 44 h, cumulus cells were removed and oocytes were randomly distributed into two groups. For delipation, oocytes were digested with 1 mg/mL pronase in the presence of 50% cattle serum (CS) for 3 min, and washed in HEPES-buffered TCM-199 medium supplemented with 20% CS. Subsequently, 40-50 oocytes were centrifuged (12 000g, 20 min) in HEPES-buffered TCM-199 medium supplemented with 2% CS, 3 mg/mL polyvinyl alcohol and 7.5 �g/mL cytochalasin B (CB). Zonae pellucidae of both centrifuged and intact oocytes were removed completely by further digestion in 2 mg/mL pronase solution. For activation, a single direct current of 85 kV/cm for 80 �s was applied to both groups, followed by 4-h treatment with 5 �g/mL CB and 10 �g/mL cycloheximide. All embryos were then cultured in modified NCSU37 medium. Day 7 blastocysts were vitrified and warmed by using the Cryotop technique (Kuwayama et al. 2005 RBM Online 11, 300-308) at 38.5�C. Survival of vitrified blastocysts was determined according to re-expansion rates after 24 h recovery in culture medium supplemented with 10% CS. Cell numbers of reexpanded blastocysts from both groups were determined after Hoechst staining. Results were compared by ANOVA. Partial zona digestion and centrifugation resulted in successful delipation in 173/192 (90%) of oocytes. The development to blastocysts was not different between delipated and intact oocytes (28 � 7% vs. 28 � 5%, respectively; P > 0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85 � 6% vs. 32 � 7%, respectively; P < 0.01). There was no difference in average cell number of re-expanded blastocysts derived from either delipated or intact oocytes (36 � 7 vs. 38 � 9, respectively; P > 0.05). Our results prove that the simple delipation technique does not hamper the in vitro developmental competence of activated porcine oocytes, and improves the cryosurvival of the derived blastocysts without significant loss in cell number. Future investigations are required to prove the value of the method in an analogue system with blastocysts produced by somatic cell nuclear transfer.

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