Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius)

2005 ◽  
Vol 17 (5) ◽  
pp. 523 ◽  
Author(s):  
J. A. Skidmore ◽  
M. Billah ◽  
N. M. Loskutoff
Keyword(s):  
Polyethylene Glycol ◽  
Survival Rate ◽  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Survival Rates ◽  
Camelus Dromedarius ◽  
Dromedary Camel ◽  
Vitrification Solution

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 m sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 m sucrose + 0.375 m glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

scholarly journals Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

2016 ◽  
Vol 36 (3) ◽  
pp. 209-215 ◽  
Author(s):  
Carolina R. Jimenez ◽  
Jurandy M. Penitente-Filho ◽  
Ciro A.A. Torres ◽  
Amanda M. Medeiros ◽  
Leandro S. Silva
Keyword(s):  
Liquid Nitrogen ◽  
Trypan Blue ◽  
Ovarian Tissue ◽  
Survival Rates ◽  
Histological Evaluation ◽  
Minimal Essential Medium ◽  
Preantral Follicles ◽  
Vitrification Solution

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Beebe ◽  
S. McIlfatrick ◽  
R. Ashman ◽  
M. Nottle
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Direct Contact ◽  
Survival Rates ◽  
Genetic Material ◽  
Basic Medium ◽  
Embryo Cryopreservation ◽  
Fetal Bovine ◽  
And Storage

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5�C in an humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean � SEM; 109 � 6 and 112 � 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving embryos.

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
P. Rodriguez Villamil ◽  
F. Ongaratto ◽  
M. Fernandez Taranco ◽  
G. A. Bó
Keyword(s):  
Ethylene Glycol ◽  
Solid Phase ◽  
Animal Health ◽  
Survival Rates ◽  
Control Group ◽  
Cresyl Blue ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

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

scholarly journals 86COMPARISON OF TWO VITRIFICATION PROTOCOLS FOR CROSSBRED BOS INDICUS×BOS TAURUS IN VITRO-PRODUCED EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 164 ◽  
Author(s):  
L.S.A. Camargo ◽  
R.S. Oliveira ◽  
J.H.M. Viana ◽  
W.F. Sá ◽  
A.M. Ferreira ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Dairy Herds ◽  
Hatching Rate ◽  
Humid Atmosphere ◽  
Significant Difference ◽  
Vitrification Solution

Dairy herds in tropical countries are often maintained as crossbred B. indicus×B. taurus hybrids to take advantage of heterosis, such as resistance to heat stress. Creating crossbred B. indicus×B. taurus embryos by in vitro methods may offer a means of rapidly improving tropical dairy herds, especially if the embryos can be cryopreserved. The aim of this study was to compare the viability of in vitro-produced crossbred B. indicus×B. taurus embryos (1/2, 3/4) using two vitrification solutions and equilibration/dilution temperatures. Cumulus-oocyte complexes were aspirated from purebred B. indicus and crossbred (B. indicus×B. taurus hybrid) ovaries, matured in vitro, and fertilized with spermatozoa collected from a Holstein bull. Presumptive zygotes were co-cultured in CR2aa medium with cumulus cells, in a humid atmosphere of 5% CO2-air at 38.8°C. On day 7 of co-culture, embryos were assessed and classified as good or excellent, and those at the appropriate developmental stage were vitrified using one of two vitrification solutions, a mixture of either glycerol/ethylene glycol (GE) or dimethylsulphoxide/ethylene glycol (DE). Embryos (n=34) assigned to GE vitrification were equilibrated in a medium of PBS+20% FCS (HM1) containing 10% v/v G for 5min, followed by 10% v/v G+20% v/v E for 5min., and then transferred to a vitrification solution of 25% v/v G+25% v/v E in HM1 for 30s. The embryos were immediately aspirated into an Open Pulled Straw (OPS) and plunged into liquid nitrogen. Embryos vitrified in GE were warmed by immersing the OPS in HM1 containing 1M sucrose for 1min (37°C), then stepwise diluted in fresh HM1 containing 1M, 0.5M, and 0.25M sucrose for 5min; and finally washed in HM1. Stepwise equilibration and dilution of GE embryos was at 20°C. Embryos (n=43) assigned to DE vitrification were equilibrated in a medium of PBS+5% FCS (HM2) containing 10% v/v D+10% v/v E for 1min, and then transferred to a vitrification solution of 20% v/v D+20% v/v E in HM2 for 30s. The embryos were immediately aspirated into an Open Pulled Straw (OPS) and plunged into liquid nitrogen. Embryos vitrified in DE were warmed by immersing the OPS in HM2 containing 0.25M sucrose for 1min (39°C), then stepwise diluted in fresh HM2 containing 0.25M and 0.15M sucrose for 5min, and finally washed in HM2. Stepwise equilibration and dilution of DE embryos was at 39°C. Diluted embryos from both groups and untreated control embryos (n=49) were cultured in TCM-199 with monolayer granulosa cells for 72h in conditions described above. Blastocyst re-expansion and hatching was assessed and analyzed by chi-square test. Overall, 67% of the thawed embryos were expanded blastocysts (remainder were blastocysts) and 56% were excellent quality (remainder were good). No significant difference (P&gt;0.05) was found between the rates of blastocyst re-expansion and hatching for the GE and DE vitrification procedures (respectively, 59 and 79%, and 41 and 58%). However the hatching rate of control embryos (77%) was significantly higher than that of vitrified embryos (P&lt;0.05). These results indicate that both vitrification procedures are promising for the cryopreservation of crossbred B. indicus×B. taurus in vitro-produced embryos. Supported by CNPq.

scholarly journals Cryopreservation of Limnoperna fortunei (golden mussel) sperm with polyethylene glycol

2018 ◽  
Author(s):  
Milica Markovic ◽  
Juliana Alves Americo ◽  
Inês Julia Ribas Wajsenzon ◽  
Yasmin Rodrigues da Cunha ◽  
Thaísa Vieira Santos de Souza ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Survival Rate ◽  
Ethylene Glycol ◽  
Limnoperna Fortunei ◽  
Polyethylene Glycol 4000 ◽  
Cryoprotective Agents ◽  
Viable Sperm ◽  
Golden Mussel ◽  
And Storage ◽  
Cryopreservation Protocol

Background. Insufficient quantities of freshly harvested Limnoperna fortunei gametes and embryos constrain reproduction research in the laboratory. Cryopreservation would allow the accumulation and storage of gametes when they are available. The lack of available cryopreservation protocol for Limnoperna fortunei in the literature led our research group to undertake a study to establish which cryoprotective agents can be might be most useful for cryopreservation of this species’ sperm. Methods. 2%, 5%, and 10% concentration of ethylene glycol, dimethyl sulfoxide, glycerol, and polyethylene glycol 4000 as well as 0.2 M glucose, sucrose, and trehalose (mixed into the 10% concentration of the aforementioned agents) were tested in a 1:1 ratio with sperm, in 0.25 ml straws, frozen in liquid nitrogen. Results. After 48 hours the best survival rate was in the samples with 10% polyethylene glycol 4000, 36.1%, which also resulted in viable sperm after 7 and 15 days.

scholarly journals Cryopreservation of Limnoperna fortunei (golden mussel) sperm with polyethylene glycol

2018 ◽  
Author(s):  
Milica Markovic ◽  
Juliana Alves Americo ◽  
Inês Julia Ribas Wajsenzon ◽  
Yasmin Rodrigues da Cunha ◽  
Thaísa Vieira Santos de Souza ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Survival Rate ◽  
Ethylene Glycol ◽  
Limnoperna Fortunei ◽  
Polyethylene Glycol 4000 ◽  
Cryoprotective Agents ◽  
Viable Sperm ◽  
Golden Mussel ◽  
And Storage ◽  
Cryopreservation Protocol

Background. Insufficient quantities of freshly harvested Limnoperna fortunei gametes and embryos constrain reproduction research in the laboratory. Cryopreservation would allow the accumulation and storage of gametes when they are available. The lack of available cryopreservation protocol for Limnoperna fortunei in the literature led our research group to undertake a study to establish which cryoprotective agents can be might be most useful for cryopreservation of this species’ sperm. Methods. 2%, 5%, and 10% concentration of ethylene glycol, dimethyl sulfoxide, glycerol, and polyethylene glycol 4000 as well as 0.2 M glucose, sucrose, and trehalose (mixed into the 10% concentration of the aforementioned agents) were tested in a 1:1 ratio with sperm, in 0.25 ml straws, frozen in liquid nitrogen. Results. After 48 hours the best survival rate was in the samples with 10% polyethylene glycol 4000, 36.1%, which also resulted in viable sperm after 7 and 15 days.

Vitrication of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives

1997 ◽  
Vol 9 (7) ◽  
pp. 741 ◽  
Author(s):  
S. Saha ◽  
T. Suzuki
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Different Ages ◽  
One Step ◽  
Vitrification Solution ◽  
Hatching Rates

The effect of embryo age on development and ratio of live : dead cells after vitrication and warming was examined. One-step and three-step addition of cryoprotectants in vitrification solution (40% ethylene glycol, 0·3 M trehalose and 12% polyvinylpyrrolidone) were compared usingin vitro produced (IVP) bovine blastocysts and expanded blastocysts. Rates of development and hatching were 74 ·2% and 41· 9% for Day 7, 57·8% and 23· 8% for Day 8, 33· 7% and 6·1% for Day 9 embryos with one-step addition. In three-step addition, those rates were 86·2% and 77·3% for Day 7, 72·3% and 39·0% for Day 8, 47·3% and 10·5% for Day 9 embryos. Day 7 embryos showed highest (P < 0·01) development and hatching rates with one exception. Hatching rate of Day 7 embryos with three-step addition was higher (P < 0·01) than with one-step addition. The ratio of live : dead cells differed between one-step (94%) and three-step (97%) additions for Day 7 embryos (P < 0· 05). The results indicate the higher resistance of younger IVP bovine embryos against vitrication and the potential for three-step addition of cryoprotectants to yield a higher survival rate after warming than with one-step addition.

scholarly journals Cryopreservation of Limnoperna fortunei (golden mussel) sperm with polyethylene glycol

2018 ◽  
Author(s):  
Milica Markovic ◽  
Juliana Alves Americo ◽  
Inês Julia Ribas Wajsenzon ◽  
Yasmin Rodrigues da Cunha ◽  
Thaísa Vieira Santos de Souza ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Survival Rate ◽  
Ethylene Glycol ◽  
Limnoperna Fortunei ◽  
Polyethylene Glycol 4000 ◽  
Cryoprotective Agents ◽  
Viable Sperm ◽  
Golden Mussel ◽  
And Storage ◽  
Cryopreservation Protocol

Background. Insufficient quantities of freshly harvested Limnoperna fortunei gametes and embryos constrain reproduction research in the laboratory. Cryopreservation would allow the accumulation and storage of gametes when they are available. The lack of available cryopreservation protocol for Limnoperna fortunei in the literature led our research group to undertake a study to establish which cryoprotective agents can be might be most useful for cryopreservation of this species’ sperm. Methods. 2%, 5%, and 10% concentration of ethylene glycol, dimethyl sulfoxide, glycerol, and polyethylene glycol 4000 as well as 0.2 M glucose, sucrose, and trehalose (mixed into the 10% concentration of the aforementioned agents) were tested in a 1:1 ratio with sperm, in 0.25 ml straws, frozen in liquid nitrogen. Results. After 48 hours the best survival rate was in the samples with 10% polyethylene glycol 4000, 36.1%, which also resulted in viable sperm after 7 and 15 days.

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