scholarly journals 83 CRYOPRESERVATION OF IN VITRO PORCINE OOCYTES BY SOLID SURFACE VITRIFICATION (SSV)

2005 ◽  
Vol 17 (2) ◽  
pp. 191 ◽  
Author(s):  
A. Dinnyes ◽  
T. Somfai ◽  
D. Sage ◽  
M. Marosan ◽  
J. Carnwath ◽  
...  
Keyword(s):  
Solid Surface ◽  
Electric Pulse ◽  
High Concentration ◽  
Parthenogenetic Activation ◽  
Surface Warming ◽  
National Office ◽  
Significant Difference ◽  
Pre Treatment ◽  
And Control

The effects of the cytosceletal inhibitor cytochalasin in solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) were investigated on in vitro matured (IVM) porcine oocytes. Cumulus-free IVM oocytes were subjected to one of the following: SSV (equilibration in 4% ethylene glycol (EG) for 10 min followed by vitrification in 35% EG, 5% polyvinyl pirrolidone, and 0.3 M trehalose on a cold (about −150°C) surface; warming in 0.4 M trehalose at 37°C), SSV pre-treatment with 5 μg/mL cytochalasin B (SSV + CB), or the steps of SSV without cooling, i.e. toxicity control (TC). Non-lysed oocytes together with the non-treated controls were subjected to parthenogenetic activation and then in vitro cultured (IVC) for six days. The proportion of non-lysed oocytes was higher when pre-treatment with CB was performed compared to SSV. However, both results were significantly lower than that of the TC. After parthenogenetic activation via a combination of a direct current electric pulse (1 kV/cm for 45 μs) followed by 3 h treatment with 2 mM 6-dimethylaminopurine, the proportion of cleaved embryos was higher in the SSV + CB than in the SSV. Nevertheless, significantly more oocytes cleaved in the TC and control groups. On Day 6 no blastocyst, were determined in the SSV group, and one blastocyst was obtained in the SSV + CB, while significantly more blastocysts developed in both the TC and the control (Table 1). There was no significant difference in blastocyst rates and in the number of blastomere nuclei/embryo between the TC and the control. These results indicate that the high concentration of cryoprotectants per se applied in SSV were not detrimental for in vitro development and that CB pre-treatment increased survival and further development of SSV vitrified pig oocytes resulting in one parthenogenetic blastocyst from vitrified pig oocytes. Table 1. Parthenogenetic development of vitrified IVM porcine oocytes This research was supported by a Bilateral Scientific and Technological Collaboration (TET, No. D6/01) grant between Germany and Hungary and by the Hungarian National Office of Research and Technology (OM-KMUFA; BIO-00086/2002).

In vitro biomechanical comparison of load to failure testing of a canine unconstrained medial compartment elbow arthroplasty system and normal canine thoracic limbs

2013 ◽  
Vol 26 (05) ◽  
pp. 356-365 ◽  
Author(s):  
K. L. Wendelburg ◽  
S. Tepic ◽  
S. M. Stover ◽  
T. Garcia-Nolen ◽  
P. B. Stearns ◽  
...  
Keyword(s):  
Axial Load ◽  
Failure Modes ◽  
Ex Vivo ◽  
Medial Compartment ◽  
Elbow Arthroplasty ◽  
Total Arthroplasty ◽  
Significant Difference ◽  
Load To Failure ◽  
And Control

SummaryElbow dysplasia, primarily affecting the medial compartment, is the most common cause of lameness in the thoracic limb. Elbow arthroplasty is an option for end stage or severely affected patients. The purpose of this study was to compare ex vivo axial load to failure of an implanted novel elbow arthroplasty system to control limbs. The partial arthroplasty is a medial compartmental, unconstrained system, intended to allow conversion to total arthroplasty. We hypothesized that there would not be any significant difference between implanted and controlled limbs when loaded to failure. Six pairs of medium mixed breed canine cadaveric thoracic limbs were prepared for comparison of failure loading of control and implanted limbs. Axial compression was performed using a mechanical testing system. Failure loads were normalized to bodyweight. The mean normalized failure load (N/kg) for the implanted limbs and control limbs were 2.47 (range: 1.62-3.38) and 2.68 (range: 2.25-3.25), respectively. An implanted to control ratio of 0.93 ± 0.19 was calculated. The difference between paired control and implanted limbs in normalized failure loading was not significant (p = 0.38). There were not any differences noted in the yield load (p = 0.30), stiffness (p = 0.62), or energy (0.58). Failure modes were recorded. We concluded that the differences between implanted and control limbs in supra-physiologic axial load to failure were not significant.

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<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<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<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

scholarly journals The effectiveness of collaborative infertility counseling (CIC) on pregnancy outcome in infertile women undergoing in vitro fertilization: A randomized controlled trial

2019 ◽  
Author(s):  
Mahboobeh Rasoulzadeh Bidgoli ◽  
robab latifnejad roudsari ◽  
ali montazeri
Keyword(s):  
In Vitro Fertilization ◽  
Pregnancy Rate ◽  
Treatment Success ◽  
Intervention Group ◽  
Control Group ◽  
Vitro Fertilization ◽  
Infertile Women ◽  
Significant Difference ◽  
And Control

Abstract Background: Infertility is an emotional tension which influences the whole aspects of relationships in infertile couples. A main objective of infertility treatments is elevation of pregnancy rate. The present study aimed to examine the effect of collaborative counseling on pregnancy rate in infertile women, undergoing in vitro fertilization in Mashhad, Iran. Methods: In this clinical trial, 60 women with primary infertility were selected from an infertility research center and were randomly allocated into intervention (n=29) and control (n=31) groups. The intervention group received individual counseling, based on the collaborative reproductive healthcare model with collaboration of a midwife, a gynecologist and a clinical psychologist in five sessions during a two-month period. The control group received routine care. Positive pregnancy test was considered as a criterion of treatment success at the end of the study. Data were analyzed using statistical tests including independent samples t-test. Results: There was no significant difference in pregnancy rate between intervention and control groups (P = 0.298). Also, there were no significant differences in follicle and embryo numbers between two groups. However, a significant difference was observed between two groups in terms of oocyte numbers where the intervention group had more oocyte (P = 0.014). Conclusion: Overall the findings indicated that the collaborative infertility counseling did not improve treatment success in infertile women undergoing in vitro fertilization

Correction formula for carbamylated haemoglobin in diabetic uraemic patients

2001 ◽  
Vol 38 (2) ◽  
pp. 115-119 ◽  
Author(s):  
A M A Hammouda ◽  
G E Mady
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Free Amino ◽  
Urea Concentration ◽  
Diabetic Patients ◽  
Amino Groups ◽  
Correction Formula ◽  
Significant Difference ◽  
And Control

The measurement of carbamylated haemoglobin is a useful indicator of uraemic state during the preceding few weeks in patients with renal failure. In diabetic uraemic patients with hyperglycaemia, glycation of haemoglobin may interfere with its carbamylation, as both reactions involve the free amino groups of the protein. The aim of this study was to investigate the carbamylation of haemoglobin in the presence of hyperglycaemia. The study included 29 patients with chronic renal failure on regular haemodialysis, 14 diabetic and 15 non-diabetic patients, and 10 healthy controls. We found a significant correlation between the degree of haemoglobin carbamylation and mean blood urea concentration in both uraemic and control subjects. Carbamylation of haemoglobin was higher in both diabetic and non-diabetic chronic renal failure patients, but there were no significant differences between the groups regarding mean blood urea concentration or level of haemoglobin carbamylation. Carbamylated haemoglobin per unit of blood urea concentration was lower in the diabetic patients. Using a correction formula to account for the degree of haemoglobin glycation, there was no longer a significant difference in carbamylation per unit of blood urea concentration. In vitro incubation of red blood cells from six healthy and six diabetic non-uraemic patients in 70mmol/L urea showed a significantly lower carbamylation in the diabetic patients, but there was no significant difference when using corrected carbamylated haemoglobin values. We conclude that glycation of haemoglobin affects its carbamylation and that monitoring of uraemia in a diabetic patient necessitates the use of carbamylated haemoglobin value corrected for the degree of glycation.

Effect of starvation on gonadotrophin secretion and on in vitro release of LRH from the isolated median eminence of the male rat

1983 ◽  
Vol 103 (3) ◽  
pp. 293-301 ◽  
Author(s):  
Michael Warnhoff ◽  
Gunter Dorsch ◽  
Karl M. Pirke
Keyword(s):  
In Vitro Release ◽  
Basic Mechanism ◽  
Male Rat ◽  
Gonadotrophin Secretion ◽  
Significant Difference ◽  
Lh Release ◽  
And Control ◽  
Vitro Release

Abstract. A perfusion system was developed in which isolated median eminences (ME) were stimulated in vitro by depolarizing agents such as potassium and veratridine. Potassium concentrations between 30 and 80 mm released increasing amounts of luteinizing hormone-releasing hormone (LRH) from the MEs of starved and control rats. Veratridine at a concentration of 50 μm caused a more prolonged LRH release in both starved and control animals. LRH secretion in vitro was slightly, though not under all conditions, significantly greater in rats starved for 5 days. The testosterone (T)-LH feedback was studied by castrating the animals and substituting various doses of T through implantation of T-releasing capsules of different sizes. The concentration in plasma, which can prevent the castration-induced much smaller in starved than in control rats. The in vitro release of LRH evoked by 80 mm potassium was not different for starved and fed rats under various feedback conditions. Both groups revealed decreased in vitro release of LRH when castrated animals were not substituted with T. The effect of castration was studied from 1 to 28 days. The plasma LH values rapidly increased in starved and control animals, indicating that the hypothalamic responsestration is not delayed by starvation. The release in vitro of LRH decreased from the first to the fifth day and remained constant thereafter. No significant difference between starved and fed rats was observed. The experiments indicate that the 'releasable pool' of LRH in vitro is greater under conditions of reduced LH release in vivo. The basic mechanism of depolarization-induced exocytosis of LRH from the ME is intact in starved animals.

Accumulation of calcium by normal and dystrophin-deficient mouse muscle during contractile activity in vitro

1992 ◽  
Vol 82 (4) ◽  
pp. 455-459 ◽  
Author(s):  
A. McArdle ◽  
R. H. T. Edwards ◽  
M. J. Jackson
Keyword(s):  
Creatine Kinase ◽  
Contractile Activity ◽  
Calcium Flux ◽  
Mouse Muscle ◽  
Control Muscle ◽  
Significant Difference ◽  
And Control ◽  
Muscle Calcium ◽  
Total Muscle

1. Accumulation of calcium by extensor digitorum longus muscles from dystrophin-deficient mdx and control C57BL/10 mice has been studied in vitro by measurements of total muscle calcium and by following the retention of 45Ca resulting from the incubation of muscles with the isotope for up to 2 h. 2. The rate of influx of calcium, calculated from the retention of 45Ca, was linear over 2 h in muscles at rest with no significant difference between mdx and control muscles. 3. Repetitive tetanic stimuli caused a substantial increase in 45Ca flux into both mdx and control muscles. This elevated rate of influx was maintained by control muscle, but not by mdx muscle after stimulation resulting in a significantly smaller total calcium flux into mdx muscle compared with control muscle by 1 h after stimulation. Similar changes were also seen in the total muscle calcium content of mdx and control muscles. Comparison of these results with those for loss of cytosolic creatine kinase previously reported (McArdle, A., Edwards, R.H.T. & Jackson, M.J. Clin. Sci. 1991; 80, 367-71) [1] indicate that control and dystrophin-deficient muscles release equivalent amounts of intracellular creatine kinase in response to the same accumulation of intracellular calcium. 4. These results therefore do not support the hypotheses that dystrophin deficiency in muscle leads to increased calcium influx during contractile activity, or that dystrophin-deficient muscle shows any inherent increased permeability to cytosolic proteins.

scholarly journals In Vitro Screening Ketahanan Galur Padi (Oryza Sativa) B7 Hasil Rakitan Politeknik Negeri Lampung Terhadap Keracunan Besi (Fe)

2019 ◽  
Vol 19 (3) ◽  
pp. 241
Author(s):  
Onny Chrisna Pandu Pradana ◽  
Siti Novridha Andini
Keyword(s):  
Tissue Culture ◽  
Oryza Sativa ◽  
Iron Toxicity ◽  
In Vitro Screening ◽  
Culture Bottle ◽  
Randomized Design ◽  
Significant Difference ◽  
Completely Randomized Design ◽  
And Control

This research aimed to invstigate the response of paddy culture (B7 strain) assembled by Lampung State Polytechnic to the iron toxicity tolerance. The research was done at Plant Tissue Culture Laboratory, Lampung State Polytechnic, from July to September 2019. Treatments were single arranged in a completely randomized design with three replications. The treatment tried was five levels of Fe concentrations (5,6 ppm 28 ppm, 56 ppm, 84 ppm, 112 ppm, and control). Each replication consisted of three culture bottle containing one explant. The homogenity of data was tested using Barlett test. If the assumption were fulfilled then analysis of variance is executed using STATISTIX 10, and then followed by the Honest Significant Difference (HSD) test in 5% alpha for mean separation and RPA analysis. The result of this research showed that the B7 strain has tolerance to iron toxicity until 56 ppm of Fe concentration, it can be concluded from the PAR value of its strain (>0,50). Meanwhile in  84 and 112 ppm of Fe concentration, the RPA value of B7 starin (<0,50), and it is indicate that its strain is sensitive. 

167 EXPRESSION OF INTERFERON τ IN BOVINE EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 200
Author(s):  
A. Minamihashi ◽  
S. Moriyasu ◽  
H. Takahashi ◽  
H. Hirayama ◽  
M. Geshi ◽  
...  
Keyword(s):  
Electric Pulse ◽  
Embryonic Stem ◽  
Transcript Level ◽  
Reproductive Technologies ◽  
Parthenogenetic Activation ◽  
Similar Expression Pattern ◽  
Development And Differentiation ◽  
Uterine Flushing

Parthenogenetic activation (PA) is a useful technique for reproductive technologies such as somatic cell nuclear transfer. Furthermore, there is a possibility of embryonic stem cell establishment deriving from PA embryos (Cibelli et al. 2002 Science 295, 819). Understanding of the ability of development and differentiation in PA embryos is important for the application. This study was designed to assess the gene expression of octamer-binding transcription factor (OCT-4) and interferon τ (IFNτ) in bovine PA embryos at the blastocyst (BC) and elongated (EL) stages, and the protein secretion of IFNτ at the EL stage. PA embryos were produced from oocytes matured in vitro (24 h), activated with Ca-ionophore (5 min) and electric pulse, and then treated with cytochalasin B and cycloheximide (5 h). In vivo-produced (Vivo) embryos were obtained non-surgically at Day 8 (Day 0 = estrus) from superovulated donor cows. PA or Vivo embryos were transferred to recipient cows (PA: 10 embryos/cow; Vivo: 1 embryo/cow) at Day 8, and then recovered non-surgically with uterine flushings at Day 16. Total RNA in single BC and EL embryos were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. IFNτ secretion of uterine flushings was estimated by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). The cleavage and blastocyst developmental rates of PA oocytes were 65.8 and 29.7%, respectively. Most embryos had positive signals of OCT-4 and IFNτ regardless of the origin and stage of embryos. The relative abundance (mean � SEM) of OCT-4 expression in PA and Vivo embryos dropped to its lowest level (1.78 � 0.32 and 1.14 � 0.45, respectively) at the EL stage, and it was significantly lower than that at the BC stage (1018.87 � 148.69 and 696.29 � 151.80, respectively; P &lt; 0.05). The transcript level of OCT-4 was not significantly different between PA and Vivo embryos at both stages. Although the transcript level of IFNτ in PA and Vivo embryos increased significantly at the EL stage (0.36 � 0.06 and 7.68 � 2.01, respectively) from the BC stage (0.03 � 0.01 and 0.01 � 0.004, respectively; P &lt; 0.05), that in PA embryos was significantly lower than that in Vivo embryos at the EL stage (P &lt; 0.01). The total amount (mean � SEM) of IFNτ in uterine flushings from cows with transferred PA embryos was 3.38 � 0.35 �g (the number of embryos in each uterine flushing was unknown), and it was low compared with that from cows with Vivo embryos (13.40 � 3.03 �g). Our results indicate that bovine PA embryos have the ability to secrete IFNτ in the uteri of recipient cows at the EL stage, and there is a similar expression pattern of OCT-4 for Vivo embryos.

62 COMPARISON OF SUGARS, COMBINATIONS OF PERMEABLE CRYOPROTECTANTS, AND EQUILIBRATION REGIMENS FOR THE SOLID SURFACE VITRIFICATION OF IMMATURE PORCINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 124 ◽  
Author(s):  
T. Somfai ◽  
N. T. Men ◽  
H. Kaneko ◽  
J. Noguchi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Solid Surface ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Morphological Evaluation ◽  
Development Rates ◽  
Significant Difference

Cryotop and solid surface vitrification are frequently used methods for the cryopreservation of porcine oocytes. These methods differ not only in the vitrification carrier but also in the cryoprotectant (CPA) treatment including the type of sugar, permeable CPA (pCPA) combinations, and the equilibration regimen. This study compared the distinct points of CPA treatment of these 2 methods to determine the optimum CPA treatment for the solid surface vitrification of immature porcine oocytes. We vitrified and warmed follicular cumulus-oocyte complexes by our method (Somfai et al. 2014 PLoS One 9, e97731). In each experiment, the vitrification solution consisted of 50 mg mL–1 polyvinyl pyrrolidone, 0.3 M of the actual sugar, and 35% [v/v] in total of the actual pCPA combination (depending on the experiment). After warming, the cumulus-oocyte complexes were subjected to in vitro maturation, IVF, and embryo culture (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Oocyte survival was assessed after IVF by morphological evaluation, and live oocytes were subjected to in vitro embryo culture. Cleavage and blastocyst rates were calculated from cultured oocytes on Day 2 (Day 0 = IVF) and Day 6, respectively. Each experiment was replicated at least 3 times. Results were analysed by ANOVA. In Experiment 1, we compared trehalose (n = 416) and sucrose (n = 440) as supplementations during vitrification and warming (0.3 M and 0.4 M of each, respectively). There was no significant difference between oocytes vitrified with trehalose or sucrose in terms of survival, cleavage, and blastocyst development (83.2% v. 80.3%, 39.7% v. 42.4%, and 3.6% v. 5.9%, respectively). Thus, vitrification and warming media were supplemented with sucrose thereafter. In Experiment 2, we compared 1 : 1 combinations of ethylene glycol with propylene glycol (EG+PG group, n = 452) and ethylene glycol with dimethyl sulfoxide (EG+DMSO group, n = 465) used as pCPA for equilibration (4% [v/v] pCPA in total for 15 min) and vitrification (35% [v/v] pCPA in total for 30 s). Oocyte survival rate was higher (P < 0.05) in the EG+PG group compared with the EG+DMSO group (73.8% v. 51.1%, respectively); however, cleavage and blastocyst development rates of surviving oocytes were not significantly different between the 2 groups (30.5% v. 44.5% and 4.1% v. 6.3%, respectively). In Experiment 3, we compared an equilibration treatment in 4% [v/v] of EG+PG for 13 to 15 min (regimen A, n = 368) with an equilibration in 15% [v/v] of EG+PG for 5 to 7 min (regimen B, n = 363) for oocyte vitrification. Survival, cleavage, and blastocyst development rates were higher (P < 0.01) for oocytes vitrified using regimen A compared with those vitrified using regimen B (82.5% v. 22.7%, 24.0% v. 7.7%, and 3.2% v. 0%, respectively). In conclusion, trehalose and sucrose are equally effective during vitrification and warming, the combination of EG+PG as pCPA is superior to EG+DMSO, and equilibration in 4% pCPA for 13 to 15 min is superior to that in 15% pCPA for 5 to 7 min for the vitrification of immature porcine oocytes.This work was partly supported by JSPS KAKENHI Grant Number 26870839.

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

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