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>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<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.

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

113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 215 ◽  
Author(s):  
B. V. Sanches ◽  
B. D. O. Filho ◽  
J. H. F. Pontes ◽  
A. C. Basso ◽  
M. L. G. Meirinhos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Lipid Droplets ◽  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Genetic Material ◽  
Embryo Cryopreservation ◽  
Hatching Rate

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.

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.

130 In vitro embryo production using frozen semen from cloned and non-cloned Bos indicus bulls

2019 ◽  
Vol 31 (1) ◽  
pp. 190
Author(s):  
S. Romo ◽  
O. Sebastián ◽  
F. Guerrero ◽  
R. Romero ◽  
F. Muñoz ◽  
...  
Keyword(s):  
Bos Indicus ◽  
The State ◽  
Assisted Reproductive Techniques ◽  
Embryo Production ◽  
Humid Atmosphere ◽  
Frozen Semen ◽  
Significant Difference ◽  
Reproductive Techniques ◽  
In Vitro Embryo Production

Reproductive biotechnology has continued to evolve rapidly, allowing the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include the in vitro maturation and IVF of oocytes, sperm sexing and cloning. These modern assisted reproductive techniques can help produce offspring of desired genetic characteristics and of a pre-determined sex. However, studies of the bull’s contribution to in vitro reproductive performance are scarce in the Brahman breed. The aim of this study was to compare oocyte maturation and embryo production in vitro using frozen semen from 5 Brahman bulls (Bos indicus), cloned (n=1) and non-cloned (n=4), with characteristics and genetics of high commercial value. The age of the bulls at the time of semen collection and cryopreservation ranged from 2 to 7 years. The oocytes were obtained on 2 different dates (45 days between collections) using pooled oocytes collected by ovum pickup at random stages of the oestrous cycle, from a total of 15 Brahman donor cows. Oocytes were transported to a laboratory in the State of Chiapas, Mexico (Genemex Internacional). The oocytes were cultured in maturation medium for 24h. For IVF, conventional semen was used from one bull (B1) and his clone (B12), the grandson of B1 (B2), and 2 non-related bulls (B3 and B4). The gametes were co-incubated for 22h and afterward placed in medium for embryo development and cultured for 7 days in a humid atmosphere with 5% CO2 in air. Of the matured oocytes, 36/43 (84%), 16/32 (50%), 101/143 (70%), 46/67 (68%) and 53/65 (81%) were fertilized using semen from B1, B12, B2, B3 and B4, respectively. Of the fertilized oocytes, 15/30 (50%), 8/16 (50%), 45/101 (44%), 21/46 (45%) and 18/53 (34%) resulted in transferrable embryos, corresponding to semen from the same bulls, respectively. This would appear to be the first scientific report in Mexico about the use of semen from a cloned bull for in vitro embryo production. In IVF, similar results were observed between B1 and a non-related bull (B4). Similar results in transferrable embryos were observed between B1 and B12 but also similar to a related bull (B3) and a non-related bull (B4). A Fisher’s exact test of the IVF results comparing B1 and B12 found a significantly (P&lt;0.05) higher number of fertilized oocytes for B1. However, a significant difference was not found (P&gt;0.05) concerning the number of transferrable embryos produced by these two bulls. In conclusion, the Brahman bulls in this study differ in their contribution to IVF and embryo production. Further studies are required to determine the factors responsible for such effects, e.g. age differences or clone versus non-clone mosaicism. Results from this research contribute to the study and development of assisted reproductive techniques for increasing in vitro production efficiency in Zebu cattle. We thank the Rosales family, from El Herradero Ranch, in the State of Campeche, Mexico, for allowing the use of their cattle for this project.

135 IN VITRO DEVELOPMENT OF IN VIVO PRODUCED EMBRYOS FROZEN AT MORULA OR BLASTOCYST STAGES

2008 ◽  
Vol 20 (1) ◽  
pp. 148
Author(s):  
R. Sartori ◽  
G. M. Machado ◽  
M. M. Guardieiro ◽  
M. R. Bastos ◽  
L. Leme ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Zebu Cattle ◽  
Hatching Rate ◽  
Chi Square ◽  
Fluid Medium ◽  
Chi Square Test ◽  
Fresh Embryos

This study was designed to compare cryotolerance between morulae and blastocysts collected from superovulated heifers. Twenty pubertal beef heifers (10 Nelore and 10 crossbred Nelore � Simmental) were superovulated with 100 mg of FSHp (Folltropin-V, Bioniche, Ontario, Canada), and embryos were collected and evaluated 7 days after estrus. Grades 1 and 2 embryos (IETS) were divided into four groups: morulae cryopreserved (MC) in liquid nitrogen (n = 24); blastocysts cryopreserved (BC; n = 19); morulae fresh (MF; n = 23); and blastocysts fresh (BF; n = 18). For freezing, embryos were immersed in ethylene glycol (Ethylene Glycol Freeze Plus with 0.1 m sucrose, Bioniche, Pullman, WA, USA), and a standard protocol (cooling rate of –0.5�C/min) was used. Prior to in vitro culture, embryos were removed from nitrogen, kept at room temperature for 5 s, and put in a water bath at 30�C for 20 s. Within 5 h after recovery, thawed and fresh embryos were washed five times in holding solution (Holding Plus, Bioniche), transferred to synthetic oviduct fluid medium (SOF, Nutricell, Campinas, SP, Brazil), and cultured for 72 h. Embryos were evaluated at 48 and 72 h of culture. After the last evaluation, degenerate and non-hatched embryos were removed from culture, and the remaining embryos were measured by a graduated ocular coupled to the Motic Images Plus 2.0 program. Hatched blastocysts were kept in culture for an additional 48 h for post-hatching development assessment. For post-hatching culture PHD medium (Brand�o DO et al. 2005 Biol. Reprod. 71, 2048–2055) was added into each well, to have a final composition of 50% SOF and 50% SOF PHD. At 120 h of culture (48 h of PHD culture) only morphologically normal blastocysts were measured. Comparison among groups was performed by ANOVA or chi-square test. Data are presented as mean � SEM. After 48 h of culture, hatching rate (%) was significantly lower in cryopreserved (MC = 8.3 and BC = 21.5) than in fresh (MF = 56.5 and BF = 77.8) embryos (P < 0.05). However at 72 h, hatching rate was similar among BC (75.9), MF (78.3), and BF (88.9), being MC (41.7) still lower (P < 0.05). The diameter (µm) of hatched embryos after 72 h of culture was 272.8 � 27.1a (n = 8), 320.6 � 18.6ab (n = 14), 385.3 � 14.2c (n = 17), and 378.0 � 22.0bc (n = 16) for MC, BC, MF, and BF, respectively (a–cP < 0.05). After 120 h of culture, the diameter of MC (379.0 � 39.9; n = 8), although similar to BC (495.4 � 59.6; n = 10), was smaller than MF (509.1 � 36.5; n = 11) and BF (511.8 � 41.2; n = 14). The results of this study with zebu cattle suggest that morulae are less resistant to cryopreservation in liquid nitrogen than blastocysts. Moreover, frozen/thawed embryos, when put in culture, present a slower development compared with fresh embryos. Financial support from CNPq and FAPESP from Brazil.

170 POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED EITHER IN HOST CAPRINE REPRODUCTIVE TRACTS OR IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 193
Author(s):  
S. Menges ◽  
C. Bormann ◽  
B. Stroud ◽  
D. Kraemer ◽  
M. Westhusin ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Pregnancy Rate ◽  
Reproductive Tract ◽  
Animal Health ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
Culture Methods ◽  
Vitro Fertilization ◽  
Significant Difference

In vitro culture of bovine embryos is usually associated with poor pregnancy rate following cryopreservation. The objective of this study was to compare the post-thaw viability of in vitro-produced bovine zygotes, cultured in vitro or in the reproductive tract of a host goat. Cumulus-oocyte complexes were matured in vitro, and in vitro fertilization was carried out with frozen-thawed semen as per standard laboratory procedures. At 18-20 h post-fertilization, zygotes were stripped of remaining cumulus cells and randomly separated into culture treatments. In three replicates, a total of 606 embryos were surgically transferred 12 to 24 h post-ovulation to the oviducts of an estrous-synchronized goat (VIVO) and 550 embryos were cultured in G1.3 for 72 h and then moved to G2.3 medium for 96 h and in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (IVC). On Day 7, embryos were flushed from the excised tract with a 69.5% recovery rate or removed from culture. Embryos were classified according to IETS criteria with grades and stages recorded. All data were analyzed using the one-way analysis of variance and means were compared using Student's t-test. No differences were seen in the percentage of freezable quality embryos per total recovered between the two groups (34.3% vs. 32.3% for IVC and VIVO, respectively). However, there was a significant difference in the pre-freezing stage between the two culture groups (Stage 5.5 � 0.22 vs. Stage 4.8 � 0.26 for IVC and VIVO, respectively; P < 0.05), but no difference in the quality grade. All embryos greater than Stage 4, Grade 2 were frozen in groups of 5-10 in ethylene glycol with sucrose (Vigro Ethylene Glycol Freeze Plus; Bioniche Animal Health, Belleville, Ontario, Canada) in 0.25-mL straws. After thawing, embryo groups were washed, rehydrated, and incubated in G2.3 as above. Morphology was assessed by assigning grade and stage objectively at 24 h and 48 h post-thaw. Post-thaw viability in vitro was not different between groups (73.4% vs. 72.7% for IVC and VIVO, respectively). The average changes in morphology post-thaw from pre-freezing to 24 h and from 24 h to 48 h within each freezing group were determined. There was no significant difference in the mean change in stage (0.67 � 0.15 vs. 0.82 � 0.17 at 24 h and 0.31 � 0.09 vs. 0.37 � 0.10 at 48 h for IVC and VIVO, respectively) or grade (0.60 � 0.15 vs. 0.41 � 0.17 at 24 h and 0.03 � 0.06 vs. 0.14 � 0.07 at 48 h for IVC and VIVO, respectively) at either observation point. These results suggest that culture of in vitro-fertilized bovine embryos in the caprine reproductive tract did not alter post-thaw development or improve post thaw viability compared to in vitro cultured controls. However, morphological evaluation is too subjective to successfully predict pregnancy rate after transfer; therefore, further study is needed to determine if there are differences in pregnancy rates between these culture methods.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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

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.

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