scholarly journals 46 VITRIFICATION OF IMMATURE AND MATURE BOVINE OOCYTES

2017 ◽  
Vol 29 (1) ◽  
pp. 130
Author(s):  
P. T. Hardin ◽  
F. A. Diaz ◽  
B. A. Foster ◽  
E. J. Gutierrez ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Maturation Stage ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Exact Test ◽  
Immature Oocytes ◽  
Commercial Source ◽  
Bovine Oocytes

While vitrification has become a valuable system used in oocyte and embryo preservation, there is still much to be learned in optimizing this protocol. Both mature and immature oocytes can be vitrified but each presents challenging aspects. Mature oocytes have microfilaments that are not yet developed in immature oocytes, which are fragile and may be disrupted by ice crystal formation during freezing. Further, currently many different cryoprotectants are used in different concentrations, most being combinations of dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. This study aimed to determine if vitrification solutions composed of ethylene glycol and either dimethyl sulfoxide or glycerol resulted in more-competent post-thaw oocytes, and to determine if maturation stage affected optimal vitrification solution. As validation of the IVF protocol, fresh mature oocytes from a commercial source were fertilized and proportion, with pronuclei formation 48 h post-IVF was recorded. Two experiments evaluated 2 cryoprotectant solutions by analysing post-vitrification and thaw competence of in vitro-fertilized oocytes to form pronuclei. Oocytes in both studies were exposed to 2 sequential vitrification solutions containing 10% DMSO or glycerol, 10% ethylene glycol and 0.5 M sucrose, and then 20% DMSO/glycerol and ethylene glycol and 0.5 M sucrose, before vitrification on cryolocks. In the first study, immature bovine oocytes (n = 200) were vitrified. Following thawing and IVM, they were analysed for pronuclei formation, with 8.49% and 0% fertilization following vitrification in DMSO and glycerol, respectively (P < 0.01). In the second study, mature oocytes were vitrified (n = 200), thawed, and fertilized using the same methods as in study 1. In total, 12.62% and 3.4% of the mature oocytes were successfully fertilized following vitrification in DMSO and glycerol, respectively (P < 0.05). Fisher’s exact test was used for all statistics in both studies. These results suggest that DMSO in combination with ethylene glycol may be superior to glycerol for vitrification of both immature and mature bovine oocytes.

71 VITRIFICATION OF BOVINE OOCYTES IN MEDIA WITH GLYCEROL + ETHYLENE GLYCOL BEFORE AND AFTER IN VITRO MATURATION

2008 ◽  
Vol 20 (1) ◽  
pp. 116
Author(s):  
L. G. Devito ◽  
C. B. Fernandes ◽  
H. N. Ferreira ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Quiescent State ◽  
Developmental Potential ◽  
Immature Oocytes ◽  
Nitrogen Vapor ◽  
Bovine Oocytes

The cryopreservation process aims to keep the cellular metabolism in a quiescent state for an indeterminate length of time. In mammals, oocyte cryopreservation success is important for the establishment of genetic banks. The objective of the present experiment was to evaluate the effect of vitrification on oocyte meiotic ability and the integrity of the metaphase plate in immature and in vitro-matured bovine oocytes. Bovine cumulus–oocytes complexes (COCs) were harvested from slaughterhouse ovaries and randomly divided into 3 groups: (G1) non-vitrified oocytes subjected to in vitro maturation, (G2) immature oocytes vitrified and then subjected to in vitro maturation after warming, and (G3) in vitro-matured oocytes subjected to vitrification. For in vitro maturation, oocytes were incubated for 22 h in 5% CO2 in air in TCM-199 with fetal calf serum, estradiol, LH, FSH, pyruvate, and gentamicin. For vitrification, the oocytes were exposed to the cryoprotectors in three steps: solution 1 containing 1.4 m glycerol in PBS for five min, and then solution 2 containing 1.4 m glycerol and 3.6 m ethylene glycol in PBS for another five min. After exposure to the second solution, the oocytes were transferred to 30-µL drops of solution 3 containing 3.4 m glycerol and 4.6 m ethylene glycol, loaded (5 oocytes per straw) in less than 1 min into 0.25-mL straws between two columns of 0.5 m galactose in PBS separated by two air bubbles, and immediately set in liquid nitrogen vapor. After 1 min of equilibration in liquid nitrogen vapor, the straws were immersed in liquid nitrogen. Warming was performed by holding the straws for 10 s in air, followed by 10 more s in a water bath at 20–22�C. The straws were then shaken 5 to 8 times to mix the bubbles (movement similar to that for a thermometer) and left horizontally for 6 to 8 min at room temperature. The rates of metaphase II and degeneration were analyzed by ANOVA followed by the Student t-test. The oocytes were stained with 100 µg mL–1 Hoechst 33342 and examined in an inverted microscope equipped with fluorescent light (UV filters 535 and 617 mm). Three different routines were realized with a total of 90 oocytes per group. The metaphase II rates in G1 (48/90, 53.3%) and G3 (42/90, 46.6%) were statistically the same (P e 0.05), but were higher (P d 0.05) than in G2 (0/90, 0%). The degeneration rates were: G1 (18/90, 20%), G2 (77/90, 85.6%), and G3 (7/90, 7.8%). The vitrification procedure damaged mainly the immature oocytes, since in the G2 the degeneration rate was higher and the oocytes were not able to resume meiosis. Meanwhile, when oocytes were vitrified after in vitro maturation, the metaphase II rate was similar to the one observed in IVM oocytes not subjected to vitrification. This indicates that the vitrification procedure performed in this experiment did not damage the structure of the metaphase II plate. However, more studies are necessary to predict the developmental potential of these in vitro-matured oocytes.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
F. Salerno ◽  
M. Rubessa ◽  
B. Gasparrini ◽  
M. Wheeler
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Deuterium Oxide ◽  
Control Group ◽  
Crystal Formation ◽  
Cleavage Rate ◽  
Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

96 RECOVERY RATES OF BOVINE IVP EMBRYOS CRYOPRESERVED BY SLOW FREEZING AND VITRIFICATION (OPS AND SSV) METHODS

2010 ◽  
Vol 22 (1) ◽  
pp. 207
Author(s):  
L. P. Landim Junior ◽  
L. T. S. Yamazaki ◽  
O. Watanabe ◽  
E. C. D. Benzi ◽  
D. P. Corneglian ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Slow Freezing ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Gas Atmosphere ◽  
Atmosphere Control ◽  
New Methodologies ◽  
Embryo Stage ◽  
Conventional Systems

In current commercial bovine in vitro embryo production (IVP) systems, the majority of costs are related to acquisition and preparation of recipient cows, which must be proportional to the produced embryos. Nevertheless, most of the time this relation is not obtained, and one option is the cryopreservation of extra embryos. Unfortunately, there is a large difference in the pregnancy rates of fresh and cryopreserved IVP embryos or the rates of recovery of frozen-thawed embryos according to the technique used for cryopreservation. The aim of this work was to compare recovery taxes (re-expansion and eclosion) of bovine IVP embryos produced following conventional systems, without any specific media or supply for cryopreservation, simulating one condition of extra produced embryos. The COC obtained from abattoir ovaries were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. At Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS), and at Day 7, grade I embryos were submitted to 3 different cryopreservation methods: slow freezing (ethylene glycol 1.5 M from 6°C to -35°C, 1°C min-1) and vitrification (DMSO, ethylene glycol, and sucrose) by OPS or SSV After the cryopreservation process, all embryos were specifically thawed according to their method and re-cultured in SOFaa for 48 h when the re-expansion and eclosion taxes were evaluated (number of viable embryos after the cryopreservation process). According to results, the taxes of viable embryos cryopreserved by vitrification methods were better than slow freezing, except expanded blastocysts cryopreserved by slow freezing, disproving literature data that show more ability for ice crystal formation in this embryo stage than others due to liquid storage. When the total of embryos is considered in the different methods (n = 1464), the vitrification method was superior to the others, but when new methodologies could be applied aimed at less lipid storage in structures (oocytes and embryos) by gas atmosphere control or total-defined cultured media, better rates can most likely be obtained with the OPS method as SSV or slow freezing. Table 1.Viable embryo rates after different cryopreservation methods WTA-Watanabe Applied Technology.

scholarly journals 103A NEW PAPER CONTAINER FOR THE VITRIFICATION OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 173 ◽  
Author(s):  
Y.M. Kim ◽  
D.H. Ko ◽  
S.J. Uhm ◽  
K.S. Chung ◽  
H.T. Lee
Keyword(s):  
Survival Rates ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Sucrose Solutions ◽  
Mammalian Embryos ◽  
Bovine Oocytes

Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P&lt;0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos. Table 1 The viability of vitrifield-thawed bovine embryos using various containers

scholarly journals Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 861-874 ◽  
Author(s):  
Korakot Nganvongpanit ◽  
Heike Müller ◽  
Franca Rings ◽  
Michael Hoelker ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Free Water ◽  
Double Stranded Rna ◽  
Immature Oocytes ◽  
Selective Degradation ◽  
First Polar Body ◽  
Bovine Oocytes

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2,in vitroproduced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

1 REPLICATION OF SOMATIC MICRONUCLEI IN BOVINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 112
Author(s):  
N. G. Canel ◽  
R. J. Bevacqua ◽  
M. I. Hiriart ◽  
D. F. Salamone
Keyword(s):  
Sham Group ◽  
Cytochalasin B ◽  
Karyotype Analysis ◽  
Egfp Expression ◽  
Dapi Staining ◽  
Exact Test ◽  
Chromosome Manipulation ◽  
A New Technique ◽  
Bovine Oocytes

Microcell-mediated chromosome transfer was developed to introduce a low number of chromosomes into a host cell (Fournier and Ruddle 1977 PNAS 74, 319–323). As a first approach to individual chromosome manipulation, we designed a new technique that consists of injecting a micronucleus into an enucleated oocyte to replicate a low number of chromosomes. Additionally, we studied the capability of such micronuclei to be marked with a transgene. To this aim, micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg mL–1 of demecolcine for 48 h followed by 2 mg mL–1 of mitomycin for 2 h. Cells were finally treated with 10 μg mL–1 of cytochalasin B for 1 h. The cumulus-oocyte complexes aspirated from slaughtered cow ovaries were in vitro-matured under standard conditions for 21 h. MII oocytes were mechanically enucleated and injected with somatic micronucleus, which were previously exposed (Mi*) or not (Mi) to 50 ng μL–1 of pCX-EGFP in 10% PVP. Sham and parthenogenetic (PA*) controls were injected with 50 ng μL–1 of pCX-EGFP in 10% PVP. A PA control was also included. After 2 h, oocytes and reconstructed embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Embryos were cultured in SOF. Cleavage stage and egfp expression were evaluated at Day 2 and 4 of IVC, respectively. At Day 2, some Mi and PA embryos were fixed and stained with DAPI. Nuclei were visualised under blue light (488 nm). Cleaved embryos with more than one nucleus were considered to have replicated their DNA. At Day 2, Mi and PA embryos were karyotyped. An IVF group was also included (Brackett and Oliphant protocol, 1975). Briefly, cleaved embryos were treated with 1.25 μg mL–1 of colchicine for 6 h. After Carnoy fixation, they were stained with Giemsa to determine the chromosomal complement of each blastomere. Embryos were classified as follows: less than 15 chromosomes, euploid (1n and 2n) and others (4n, mixoploid and aneuploid). Differences among treatments were determined by Fisher's exact test (P ≤ 0.05). The Mi*, PA* and PA groups showed higher cleavage rates than the Mi treatment [93/108 (86.1%), 111/136 (81.6%) and 160/186 (86%), respectively vs 89/131 (67.9%); P ≤ 0.05]. Cleavage rates of the Sham* group [78/105 (74.3%)] did not differ from Mi and PA* treatments (P ≤ 0.05). Interestingly, a low number of Mi* embryos showed egfp expression (2.2%). Expression levels were significantly lower than those of the PA* group (38.7%) and did not differ from the Sham control (0%; P ≤ 0.05). Although rates of Mi embryos with more than 1 nucleus (63.6%, n = 22) were lower than those for the PA group (100%, n = 28), DAPI staining confirmed replication of micronuclei. Karyotype analysis revealed that 100% of Mi evaluated embryos (n = 11) had less than 15 chromosomes per blastomere (varying from 1 to 13), whereas none of IVF and PA controls showed such results (P ≤ 0.05). Rates of euploid embryos were 75 (n = 20) and 45% (n = 20) for IVF and PA groups, respectively. In conclusion, we have developed a new method for somatic micronuclei, which could be a useful tool to transfer a small number of specific chromosomes and to target transgenesis to a reduced area of the genome.

38 Vitrification of prepubertal lamb spermatogonia using a novel vitrification system

2019 ◽  
Vol 31 (1) ◽  
pp. 145 ◽  
Author(s):  
S. Ledda ◽  
S. Pinna ◽  
S. Nieddu ◽  
D. Natan ◽  
A. Arav ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
In Vitro Culture ◽  
Dimethyl Sulfoxide ◽  
Fertility Preservation ◽  
Testicular Tissue ◽  
Gonadal Tissue ◽  
New Device ◽  
Vitrification Solution ◽  
Tissue Cryopreservation

Vitrification is a method extensively used for preserving oocytes and embryos and is also gaining acceptance for preserving gonadal tissue. Cryopreservation of spermatogonial stem cells is an applicable method for young males seeking fertility preservation before starting a treatment or can be a tool for genetic preservation of rare or high-value animals. The aim of this work was to evaluate the cryopreservation of testicular tissue from young lambs by vitrification using a new device named E.Vit (FertileSafe, Ness Ziona, Israel) that permits all cryopreservation procedures to be performed in straw. The new device consists of a 0.3-mL straw (Cryo Bio System, IMV, L’Aigle, France) with a capsule containing 50-µm pores inserted at one end. Testicular tissue extracts were prepared from testes of slaughtered lambs (n=10, 40 days old), opened by sagittal sectioning with a microblade and collecting small pieces of testicular tissue (1mm3) from the middle part of the rete testis. Three pieces of gonadal tissue were inserted into each E.Vit device. Each straw was sequentially loaded vertically in two 1.5-mL microtubes, which contained the following solutions: first, the equilibrating solution (7.5% dimethyl sulfoxide+7.5% ethylene glycol+20% FCS in TCM-199) for 6min, followed by 90min in the vitrification solution (18% dimethyl sulfoxide+18% ethylene glycol+0.5M Trehalose+BSA in TCM-199). After exposure to the equilibrating solution and vitrification solution, the solutions were removed and the straws were directly loaded into LN2. The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, and 25% warming solution (1M sucrose in TCM-199+20% FCS) at 38.6°C for 5min each before arrival into the holding medium. Samples were recovered from the straws incubated at 38.6°C in 5% CO2 in air in TCM 199+5% FCS and evaluated at 0 and 2h post-warming for viability using trypan blue staining. Expression of a panel of specific genes (SOD2, HSP90b, BAX, POUF5/OCT4, TERT, CIRBP, KIF11, AR, FSHR) was analysed by real-time PCR in cryopreserved tissue in vitro cultured for 2h post-warming (2hV), in fresh controls immediately after tissue dissection (0hF), and after 2h of in vitro culture (2hF). The majority of cells survived after vitrification, although viability immediately after warming (0hV: 56%±1.45) or after 2h of in vitro culture (IVC) (2hV: 54±7%) was significantly lower compared with non-cryopreserved fresh controls (0hF: 89%±1.45; ANOVA P&lt;0.05). Expression analysis showed specific patterns for the different genes. Notably, BAX transcript abundance was not affected by vitrification or IVC, indicating an acceptable level of stress for the cells. The genes HSP90b and CIRBP were down-regulated in 2hF but increased in 2hV, as expected. Expression of SOD1 and OCT4 was altered by vitrification but not by IVC. Conversely, expression of TERT, KIF11, and AR was affected by both IVC and cryopreservation (ANOVA P&lt;0.05). This novel protocol for testicular tissue cryopreservation of prepubertal animals may be a promising strategy for fertility preservation and can contribute as a new approach in the development of large-scale biodiversity programs.

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
H. S. Canesin ◽  
J. G. Brom-de-Luna ◽  
Y.-H. Choi ◽  
A. M. Pereira ◽  
G. G. Macedo ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Exposure Time ◽  
In Vitro Maturation ◽  
Initial Exposure ◽  
Exact Test ◽  
Fetal Bovine ◽  
Follicle Aspiration ◽  
Meiotic Competence

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P < 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P > 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P < 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P > 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&M University.

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 120
Author(s):  
E. Girka ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Recovery Period ◽  
Normal Chromosome ◽  
Meiotic Spindle ◽  
Control Group ◽  
Chromosome Arrangement ◽  
Epothilone B ◽  
Significant Difference ◽  
Bovine Oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P&gt;0.05). The 2.0μM taxol treatment improved chromosome configuration (P&lt;0.05) with no effect on microtubule distribution compared with the control group (P&gt;0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P&lt;0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP&lt;0.05;. bP&lt;0.001: Different superscripts within a column indicate a significant difference.

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