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<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<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.

scholarly journals Effects of different cryoprotectants on the cryopreservation of cattle testicular tissue

2015 ◽  
Vol 58 (2) ◽  
pp. 433-439 ◽  
Author(s):  
S. Hu ◽  
Q.-C. Zhu ◽  
C. Han ◽  
X.-G. Zhang ◽  
B. Y. Song ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Cell Viability ◽  
Dimethyl Sulfoxide ◽  
Fertility Preservation ◽  
Testicular Tissue ◽  
Optimal Concentration ◽  
Effective Protection ◽  
Testosterone Production ◽  
Significant Difference ◽  
Tissue Cryopreservation

Abstract. Cryopreservation of testicular tissue is a new option in fertility preservation for prepubertal male animals. The purpose of this study was to explore the effects of different cryoprotectant agents (CPAs) at various concentrations on testes after the cryopreservation of calf testicular tissue. These experiments selected dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PrOH), and sucrose as CPAs in varying doses (2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 %; v/v) in 8-month-old calf testicular tissue that was frozen and preserved. Then, cell viability, testosterone production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) level were detected and analyzed following cryopreservation. The results showed that the optimal concentrations of DMSO, PrOH, glycerol, and sucrose were 10, 10, 7.5, and 10 %, respectively. Compared to the optimal concentrations of CPAs, cell viability and testosterone production decreased significantly at a lower and higher CPA concentration (P < 0.05). At the optimal concentrations of CPAs, the DMSO group showed higher cell viability and testosterone production than other CPA groups (P < 0.05). Compared to the optimal concentration of CPAs, the MDA level increased and the SOD level decreased at a lower or higher concentration of CPAs, but there was no significant difference (P > 0.05). Cell viability was significantly positively correlated with testosterone production (P < 0.05). In conclusion, DMSO provided the most effective protection for calf testicular tissue cryopreservation and the optimal concentration was 10 %.

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

132 VITRIFICATION SYSTEM (OPEN AND CLOSED) IN NEW INCUBATOR WITH REDUCED OXYGEN

2015 ◽  
Vol 27 (1) ◽  
pp. 158
Author(s):  
O. Watanabe ◽  
E. Schmitt ◽  
F. Meirelles ◽  
A. Oliveira ◽  
A. Bos-Mikich ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
In Vitro Culture ◽  
Dimethyl Sulfoxide ◽  
Oxygen Tension ◽  
Female Reproductive Tract ◽  
High Oxygen ◽  
Hatching Rate ◽  
Scientific Model ◽  
Oxygen Environment

Most IVF laboratories uses high oxygen tension (20%) during embryonic culture. However, it is known that under physiological conditions, oxygen tension in the female reproductive tract ranges between 2 and 8%. Therefore, the aim of this study was to evaluate survival and hatching rate after in vitro culture of vitrified/thawed bovine in vitro-produced blastocysts cultured under different oxygen concentrations. The experiment consisted of comparing 2 culture systems using different concentrations of oxygen: conventional incubator (20% O2, Thermo Scientific, model 3130) and a new incubator (5% O2, WTA Watanabe Tecnologia Aplicada, model Eve). Only Day 7 expanded blastocysts grade 1 were used. Embryos were produced according to conventional IVF protocols. Briefly, cumulus-oocyte complexes were aspirated from postmortem ovarian follicles, matured in TCM199 + 10% FCS + 0.5 mg FSH mL–1 + 50 mg LH mL–1 + 1 mg oestradiol mL–1, for 24 h at 38.5°C, 5% CO2 in air. Live spermatozoa from Nellore bull were obtained by centrifugation in Percoll gradients (45 and 90%) and cultured with cumulus-oocyte complexes at 1 million of sperm mL–1 in TALP medium + 10 mg of heparin mL–1. After 20 h incubation, zygotes were transferred to CR2 + 2.5% FCS + 4 mg of BSA mL–1 and granulosa monolayer for 7 days. Expanded blastocysts were randomly allocated to 2 treatments for vitrification (open system – cryotop and closed system – HSV Kit, IMV-Technologies) using the same vitrification media and protocol (VS1: 10% ethylene glycol + 10% dimethyl sulfoxide and VS2: 20% ethylene glycol + 20% dimethyl sulfoxide for 8 min and 50 s, respectively). After exposure to the vitrification solutions, 2 embryos were loaded/straw, and the straws were plunged into LN. The warming procedure consisted of, immediately after removal from LN2, transferring the embryos in 2 successive warming solutions with decreased concentrations of sucrose (1 M and 0.50 M for 5 min each). The vitrified/rewarmed embryos were transferred to in vitro culture. There were no differences in survival rates (P < 0.05) between the open and closed vitrification system for blastocysts produced in reduced oxygen in the Eve incubator – 5% O2 (96% – 109/114 and 98% – 158/161, respectively) compared with embryos produced in the high oxygen environment in the Thermo incubator – 20% O2 (93% – 214/230 and 92% – 94/102, respectively). Hatching rates were increased for blastocysts cultured in the lower oxygen environment (EVE treatment: 95 and 98%, respectively, for open and closed vitrification protocols) when compared with the high oxygen environment (Thermo treatment: 86 and 87%, respectively, for open and closed systems); P < 0.05. In vitro culture in a reduced-oxygen environment improves blastocysts competence after vitrification. Financial support was received from CNPq-RHAE.

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 151
Author(s):  
H. S. Canesin ◽  
I. Ortiz ◽  
J. G. Brom-de-Luna ◽  
Y. H. Choi ◽  
K. Hinrichs
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Cleavage Rate ◽  
Fetal Bovine ◽  
Vitrification Solution

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&M University.

Fertility Preservation

2018 ◽  
Author(s):  
Chantae S Sullivan-Pyke ◽  
Clarisa Gracia
Keyword(s):  
Fertility Preservation ◽  
Ovarian Tissue ◽  
Testicular Tissue ◽  
Mature Oocyte ◽  
Gonadotropin Releasing Hormone ◽  
Oocyte Cryopreservation ◽  
Ovarian Tissue Cryopreservation ◽  
Embryo Cryopreservation ◽  
Gonadotropin Releasing Hormone Agonist ◽  
Tissue Cryopreservation

Fertility preservation has becoming increasingly important for patients at risk for gonadal failure, including those needing treatment for cancer or autoimmune conditions, genetic conditions that predispose to gonadal insufficiency, and age-related fertility decline. Embryo cryopreservation and mature oocyte cryopreservation are the standards for fertility preservation in postpubertal women. Ovarian tissue cryopreservation and gonadotropin-releasing hormone agonist use for ovarian suppression are experimental methods that may be offered to patients for whom embryo and/or mature oocyte cryopreservation are not applicable. The cryopreservation of spermatozoa is the standard for fertility preservation in postpubertal males, but testicular tissue cryopreservation may be offered to prepubertal males.   This review contains 10 figures, 6 tables and 53 references Key words: controlled ovarian stimulation, embryo cryopreservation, gonadotropin-releasing hormone agonist, in vitro maturation, oocyte cryopreservation, ovarian tissue cryopreservation, sperm extraction, testicular tissue cryopreservation  

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.

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH

2009 ◽  
Vol 46 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Andy Michael Laughlin ◽  
Thomas H. Welsh ◽  
Charles C. Love ◽  
Dickson D. Varner ◽  
Alan R. Parrish ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Testicular Tissue

Female fertility preservation strategies: cryopreservation and ovarian tissuein vitroculture, current state of the art and future perspectives

Zygote ◽  
2016 ◽  
Vol 24 (5) ◽  
pp. 635-653 ◽  
Author(s):  
M.A. Filatov ◽  
Y.V. Khramova ◽  
M.V. Kiseleva ◽  
I.V. Malinova ◽  
E.V. Komarova ◽  
...  
Keyword(s):  
Fertility Preservation ◽  
State Of The Art ◽  
Ovarian Tissue ◽  
Female Fertility ◽  
Ovarian Tissue Cryopreservation ◽  
Future Perspectives ◽  
Current State ◽  
Female Fertility Preservation ◽  
Tissue Cryopreservation

SummaryIn the present review, the main strategies of female fertility preservation are covered. Procedures of fertility preservation are necessary for women who suffer from diseases whose treatment requires the use of aggressive therapies, such as chemotherapy and radiotherapy. These kinds of therapy negatively influence the health of gametes and their progenitors. The most commonly used method of female fertility preservation is ovarian tissue cryopreservation, followed by the retransplantation of thawed tissue. Another approach to female fertility preservation that has been actively developed lately is the ovarian tissuein vitroculture. The principal methods, advantages and drawbacks of these two strategies are discussed in this article.

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