28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

Download Full-text

70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab70 ◽

2007 ◽

Vol 19 (1) ◽

pp. 152

Author(s):

K. Naruse ◽

Y. M. Shin ◽

Y. S. Quan ◽

C. S. Park ◽

D. I. Jin

Keyword(s):

Cell Number ◽

Fibroblast Cells ◽

Fetal Fibroblast ◽

In Vitro Development ◽

Reconstructed Embryos ◽

Streptolysin O ◽

Donor Cells ◽

Developmental Rates ◽

Vitro Development

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P < 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P < 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos. Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos

Download Full-text

139 TRICHOSTATIN A IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY A NEW ACTIVATION METHOD

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab139 ◽

2011 ◽

Vol 23 (1) ◽

pp. 173

Author(s):

L. C. Sui ◽

W. Wang ◽

Y. S. Li ◽

Y. L. Zhang ◽

S. F. Ji ◽

...

Keyword(s):

Nuclear Transfer ◽

Trichostatin A ◽

Formation Rate ◽

Activation Method ◽

Electrical Activation ◽

Cleavage Rate ◽

In Vitro Development ◽

Vitro Development ◽

Equal Contribution

Recently, it has been reported that a new activation method, brief exposure to cycloheximide before electrical activation, could increase development rates and reduce cell death. In our study, we allocated reconstructed SCNT embryos into 3 groups: electrical activation followed by exposure to cycloheximide (10 μg mL–1) for 4 h (ELE+CHX); exposure to cycloheximide (10 μg mL–1) for 10 min followed by electrical activation (CHX+ELE); and electrical pulse treatment alone (ELE). We found the CHX+ELE (10 min) group had a similar blastocyst formation rate and total blastocyst number with the ELE+CHX (4 h) group, and both groups could increase in vitro development compared with the ELE group. Trichostatin A (TSA), an inhibitor of histone deacetylase, has been reported to potentially enhance cloning efficiency. We examine the effect of TSA on nuclear transfer embryos produced by the CHX+ELE activation method. The reconstructed embryos were treated with 50 nM TSA for 0 and 36 h. We found that 50 nM TSA for 36 h after activation had an increased blastocyst rate compared with the control (15.35 v. 8.84%; P < 0.05), but there was no difference in cleavage rate or in total blastocyst numbers. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a new activation method. S.L.C., W.W. equal contribution; Corresponding author ZH. X.R., ZH. Y.H.; Supported by NSFC (30700574), 863 (2008AA101003).

Download Full-text

160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab160 ◽

2004 ◽

Vol 16 (2) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

W.F. Swanson ◽

A.L. Manharth ◽

J.B. Bond ◽

H.L. Bateman ◽

R.L. Krisher ◽

...

Keyword(s):

Culture Media ◽

Ionic Composition ◽

Cell Number ◽

Blastocyst Stage ◽

Domestic Cat ◽

Embryo Viability ◽

Blastocyst Development ◽

In Vitro Development ◽

Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

Download Full-text

In Vitro Development of Resistance to Telithromycin (HMR 3647), Four Macrolides, Clindamycin, and Pristinamycin inStreptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.2.414-417.2000 ◽

2000 ◽

Vol 44 (2) ◽

pp. 414-417 ◽

Cited By ~ 70

Author(s):

Todd A. Davies ◽

Bonifacio E. Dewasse ◽

Michael R. Jacobs ◽

Peter C. Appelbaum

Keyword(s):

The Other ◽

In Vitro Development ◽

Development Of Resistance ◽

Subinhibitory Concentrations ◽

Erythromycin A ◽

Vitro Development

ABSTRACT The ability of 50 sequential subcultures in subinhibitory concentrations of telithromycin (HMR 3647), azithromycin, clarithromycin, erythromycin A, roxithromycin, clindamycin, and pristinamycin to select for resistance was studied in five macrolide-susceptible and six macrolide-resistant pneumococci containing mefE or ermB. Telithromycin selected for resistance less often than the other drugs.

Download Full-text

Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

Journal of Reproduction and Development ◽

10.1262/jrd.20047 ◽

2009 ◽

Vol 55 (3) ◽

pp. 236-239 ◽

Cited By ~ 5

Author(s):

Kenji NARUSE ◽

Yan-Shi QUAN ◽

Baek-Chul KIM ◽

Su-Min CHOI ◽

Chang-Sik PARK ◽

...

Keyword(s):

Cell Fusion ◽

Nuclear Transfer ◽

In Vitro Development ◽

Streptolysin O ◽

Fetal Fibroblasts ◽

Vitro Development

Download Full-text

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab159 ◽

2006 ◽

Vol 18 (2) ◽

pp. 187

Author(s):

J. De la Fuente ◽

A. Gutiérrez-Adán ◽

P. Beltrán Breña ◽

S. S. Pérez-Garnelo ◽

A. T. Palasz

Keyword(s):

Embryo Development ◽

Cell Number ◽

Apoptotic Cells ◽

Bovine Embryos ◽

Chi Square ◽

Oh Groups ◽

In Vitro Development ◽

Chi Square Analysis ◽

Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

Download Full-text

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab50 ◽

2008 ◽

Vol 20 (1) ◽

pp. 105

Author(s):

E. S. Ribeiro ◽

R. P. C. Gerger ◽

L. U. Ohlweiler ◽

I. Ortigari Jr ◽

F. Forell ◽

...

Keyword(s):

Somatic Cell ◽

Chemical Activation ◽

Primary Cultures ◽

Cell Number ◽

Total Cell ◽

Bovine Embryos ◽

In Vitro Development ◽

Vitro Development ◽

Handmade Cloning

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

Download Full-text

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab53 ◽

2013 ◽

Vol 25 (1) ◽

pp. 174

Author(s):

R. Olivera ◽

C. Alvarez ◽

I. Stumpo ◽

G. Vichera

Keyword(s):

Embryo Development ◽

Polar Body ◽

Nuclear Reprogramming ◽

Chi Square ◽

In Vitro Development ◽

First Polar Body ◽

Group 3 ◽

Group 2 ◽

Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

Download Full-text

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab74 ◽

2011 ◽

Vol 23 (1) ◽

pp. 142

Author(s):

J. Galiguis ◽

M. C. Gómez ◽

C. E. Pope ◽

B. L. Dresser ◽

S. P. Leibo

Keyword(s):

Lipid Content ◽

High Speed ◽

Domestic Cat ◽

Blastocyst Development ◽

Chi Square ◽

In Vitro Development ◽

Intracellular Lipids ◽

Fetal Fibroblasts ◽

Vitro Development

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P < 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P < 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P < 0.05), 38 (P < 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P < 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

Download Full-text