Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times (original) (raw)
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Theriogenology, 2000
Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol, FicoIl using open-pulled straws. Oocytes from slaughterhouse ovaries (N=SO/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 set and then to 5 M ethylene glycol in EFS for 25 to 30 set at 37°C. Oocytes were loaded into straws in -2 uL of cryoprotectant and plunged directly into LN,. Warming straws and dilution of cryoprotectant was at 3 7 "C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Nonvitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342.
Effects of vitrification of immature bovine oocytes on in vitro maturation
The aim of this study was to evaluate the effect of ethylene glycol (EG) concentration, equilibration time, and the use of two disaccharides in the vitrification solution used on immature bovine oocytes that were thawed and then matured in vitro. Factorial combinations of the following were tested: three equilibration solutions (ES), containing 3, 20 or 40% EG; three equilibration times, 0.5, 5 or 15 min; and two vitrification solutions (VS), containing 40% EG + 1.0 M trehalose or 40% EG + 1.0 M sucrose. The control treatment had fresh, non-vitrified oocytes maturated in vitro. The total number of immature oocytes distributed across the 19 treatments was 2103. The combination VS with sucrose, an equilibration time of 5 min, and a ES with 20% of EG had the highest MII (metaphase II) rate (44.5%) that was more than twice the rate of the next best combination of vitrification treatments but less (P<0.05) than the non-vitrified control (74.6%). The lowest MII rates in treatments with sucrose were found in combinations of ES with 40% EG and equilibration times of 5 and 15 min (0.0 and 0.9%, respectively). The highest MII rate for treatments with trehalose was 5.3%. High chromatin condensation rates were found in treatments with trehalose. In conclusion, the use of trehalose in the vitrification solution impaired the oocyte maturation. Better results were obtained with sucrose. A high concentration (40%) of EG in addition to a long equilibration time (5 or 15 min) was detrimental to oocyte maturation. Vitrification of immature bovine oocytes using 20% EG in the ES, an equilibration time of 5 min, and a VS containing 40% EG + 1.0 M sucrose, yielded acceptable in vitro maturation rates.
Studies on Survival of Horse Oocytes after Rapid-I Method Vitrification
Journal of Equine Veterinary Science, 2013
Intracytoplasmatic sperm injection of in vitro maturated equine oocytes. Biol. Reprod. 1997;57:1495-1501 4. Li X., Morris L.H.A., Allen W.R. Influence of co-culture during maturation on the developmental potential of equine oocytes fertilised by intracytoplasmic sperm injection (ICSI). Reproduction 2001;121:925-932 5. Squires E.L., Wilson J.M., Kato H. A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro. Theriogenology 1996;45:306 6. Galli C., Lagutina I., Crotti G., Colleoni S., Turini P., Ponderato N., Duchi R., Lazzari G. Pregnancy: a cloned horse born to its dam twin. Nature 2003;424-635 7. Hinrichs K., Choi Y.H., Varner D.D., Hartman D.L. Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/ or ionomycin. Reproduction 2007;134:319-325 8. Chen S.U., Lien Y.R., Cheng Y.Y. Vitrification of mouse oocytes using closed pulled straws ( CPS) achives a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws and grids.
Journal of Reproduction & Infertility, 2012
Background Vitrification has proven to be more effective than slow freezing methods to cryopreserve mammalian oocytes. The objectives of this study were to evaluate the effects of vitrification on immature and in vitro matured, denuded and cumulus compact goat oocytes and their subsequent fertilization. Methods Oocytes were either cryopreserved as immature cumulus compact (IMCC) (n=98 Exp 1; 102 Exp 2) and immature denuded (IMDN) (n=127 Exp 1; 109 Exp 2) or were first matured in vitro for 28 h and then cryopreserved as mature cumulus compact (MCC) (n=109 Exp 1; 89 Exp 2) or mature denuded (MDN) (n=112 Exp 1; 110 Exp 2) oocytes in four groups. The vitrification solution comprised of Dulbecco's phosphate buffered saline supplemented with 0.5% sucrose, 0.4% bovine serum albumin and 8 M propylene glycol. After 7 days of cryopreservation in liquid nitrogen, oocytes in all groups were evaluated for normal morphologic survival and in vitro maturation (Experiment 1) and fertilization in...
An improved vitrification protocol for equine immature oocytes, resulting in a first live foal
Equine Veterinary Journal, 2017
Background: The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objectives: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Study design: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilization and culture. Embryo transfer was performed in a standard manner. Results: Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and non-vitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (p = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (p<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (p<0.001), transfer of 5 embryos resulted in one healthy foal. Main limitations: The relatively low number of equine oocytes and embryo transfer procedures performed. Conclusions: For vitrification of immature equine oocytes, the use of (1) CR oocytes, (2) a high concentration of CPAs and (3) a short exposure time may be key factors for maintaining developmental competence.
Theriogenology, 2005
This study was designed to evaluate the effects of the cryopreservation of oocytes obtained from prepubertal calves or adult cows on chromosome organization, spindle morphology, cytoskeleton structures, and the ability of fertilized oocytes to develop to the blastocyst stage. Once in vitro matured (IVM), the oocytes were divided into three groups according to whether they were: (1) left untreated (control); (2) exposed to cryoprotectant agents (CPAs); or (3) cryopreserved by the openpulled-straw (OPS) vitrification method. After thawing, oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. After vitrification or CPA exposure, significantly higher proportions of oocytes showed changes in spindle morphology compared to the control group. The spindle structure of the adult cow IVM oocytes was significantly more resistant to the OPS vitrification process. Vitrification of oocytes from calves or adult cows led to significantly increased proportions of oocytes showing discontinuous or null actin staining of the cytoskeleton compared to non-treated controls. Oocytes only exposed to the cryoprotectants showed a similar appearance to controls. A normal distribution of actin microfilaments was observed in both calf and adult cow oocytes, irrespective of the treatment. Cleavage and blastocyst rates were significantly lower for vitrified versus non-treated oocytes. Oocytes obtained from adult cows were more sensitive to CPA exposure, while the vitrification procedure seemed to have more detrimental effects on the calf oocytes.
Factors affecting the survivability of bovine oocytes vitrified in droplets
Theriogenology, 2000
Vitification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine oocytes subjected to vitrification according to the minimum drop size approach. In total, 748 bovine, in vitro matured oocytes were vitrified using VS14 vitrification solution, containing 5.5-M ethylene glycol and 1.0-M sucrose after different pre-equilibration and equilibration protocols performed at 35" to 37°C. Experiment 1 showed no significant toxic effect during pre-equihbration treatments of oocytes in 2%, 4% or 6% ethylene glycol solutions, except the lower cleavage rate of oocytes exposed to 6% ethylene glycol(77.2% vs. 93.9% in control, P&.05). In Experiment 2, 12 to 15 &in of pre-equilibration treatments in O%, 1% or 2% ethylene glycol solutions were tested, followed by 30 or 45 set of equilibration in VS14 solution and vitrification in droplets of medium dropped directly into liquid nitrogen. The development rate of vitrified oocytes to the blastocyst stage tended to be higher after 30-set equilibration treatment (9.5%, 13.9% and 13.8% in groups of oocytes pre-equilibrated in O%, 1% or 2% ethylene glycol solutions, respectively). Expenment 3 tested pre-equilibration treatments m 0%, I%, 2%, 3%, 4%, 5% or 6% ethylene glycol solutions, followed by 30-set equilibration and vitrification in droplets. The highest cleavage, blastocyst and hatched blastocyst rates, which were not significantly different from control, were achieved in a group of oocytes pre-equilibrated in 3% ethylene glycol solution (76%, 30% and 15% vs. 89%, 42% and 21% in control, respectively). A healthy calf was born on Feb 22 1999, after transfer of 4 morula/blastocyst stage embryos developed from oocytes vitrified in droplets after pre-equilibration in 3% ethylene glycol solution. We conclude that gentle preequilibration of bovine oocytes in diluted, 3% ethylene glycol solution is an important factor improving the effectiveness of vitrification in droplets of bovine oocytes.
Viability of bovine in vitro matured oocytes following ultra-rapid vitrification
Medical Journal of Cell Biology
The aim of the study was to examine viability of cattle oocytes after cryopreservation. Oocytes after in vitro maturation (IVM) were vitrified in minimum volume on the nickel electron microscopy grids by ultra-rapid cooling technique. After warming and subsequent in vitro fertilization the presumptive zygotes were cultured to reach the stage of the blastocyst (Bl). Several devitrified oocytes were processed for electron microscopy assay. Although, embryo cleavage and Bl percentages in the vitrified group were slightly lower than in the control group (P < 0.05), the Bl total cell number (TCN), apoptosis and dead cell percentages did not differ between both groups. However, significant difference was found between day 7 (D7) and day 8 (D8) Bl in the TCN in control (108.0 vs. 90.5) and vitrified group (103.75 vs 98.14). Electron microscopy of frozen oocytes revealed slight reversible injuries in mitochondria and the smooth endoplasmic reticulum (SER), nevertheless, the development o...
Theriogenology, 2018
Most wild donkey breeds are severely threatened by poaching for meat, habitat loss, and competition with livestock for food resources. Moreover, due to the mechanization in agriculture and in transport, most domestic donkey breeds are at risk of extinction. Considering the importance of biodiversity and preservation of genetic resources, the creation of genetic banks for endangered donkey breeds is urgently needed. Cryopreservation of immature jennies oocytes would be an efficient tool to allow storage of female genetics. The aim of the present study was to establish conditions for immature donkey oocyte vitrification, using equine oocytes as a control. Asine and equine immature cumulus-oocyte complexes were collected by transvaginal ultrasound-guided follicular aspiration and flushed to obtain oocytes surrounded by only corona radiata. Oocytes were vitrified after exposure to increasing concentrations of dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectants in a solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin. Oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation. The recovery rate was 48% for jennies oocytes (4.8 oocyte per female) and 42% for mares oocytes (3.5 oocyte per female). When oocytes were exposed to cryoprotectants in INRA-Freeze™ medium none of the jennies re-warmed oocytes matured, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation. When oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured. In conclusion, we developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. Their competence for fertilization and development has to be ascertain.