217 KINETICS OF SPERM PENETRATION IS CORRELATED WITH IN VITRO FERTILITY OF BUFFALO (BUBALUS BUBALIS) BULLS

2009 ◽  
Vol 21 (1) ◽  
pp. 206 ◽  
Author(s):  
M. Rubessa ◽  
M. Di Fenza ◽  
E. Mariotti ◽  
S. Di Francesco ◽  
C. de Dilectis ◽  
...  
Keyword(s):  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
Optimal Time ◽  
High Fertility ◽  
Chi Square ◽  
Sperm Penetration ◽  
Blastocyst Rate ◽  
Kinetics Of ◽  
Time Point

It was previously demonstrated that the kinetics of early cleavage could be used to discriminate between bovine bulls with high and low field fertility (Ward F et al. 2001 Mol. Reprod. Dev. 60, 47–55). Marked differences exist in the kinetics of sperm penetration between bulls, and this may be a useful predictor of field fertility in cattle (Ward F et al. 2002 Theriogenology 57, 2105–2117). It is well known that the ability to fertilize oocytes in vitro and to sustain embryo development varies significantly among buffalo bulls. Therefore, the aim of this work was to evaluate whether the speed of oocyte penetration after IVF was correlated with the blastocyst rates obtainable with different bulls in buffalo species. In Experiment 1, in vitro-matured buffalo oocytes were co-incubated with MitoTracker-labeled spermatozoa (Ward F et al. 2002 Theriogenology 57, 2105–2117) from 6 different bulls, over 2 replicates. Oocytes were subsequently fixed every 3 h (up to 18 h) postinsemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight, and stained with 4′,6-diamidino-2-phenylindole for nuclei examination under a fluorescence microscope. In Experiment 2, in vitro-matured oocytes were fertilized with sperm from the same 6 bulls and were cultured to the blastocyst stage, over 4 replicates. Bulls were tested, collectively, on each batch of ovaries in both experiments. Differences in the percentages of monospermic penetration among bulls were analyzed by chi-square test. Correlation and multiple regression analyses were also carried out between the speed of penetration and blastocyst yields. Marked differences in the kinetics of sperm penetration were found among buffalo bulls, as shown in Table 1. Interestingly, a correlation was found between the blastocyst rate and the percentage of oocytes penetrated at 6 h (r = 0.71; P < 0.01), at 9 h (r = 0.65; P < 0.05), at 12 h (r = 0.77; P < 0.01), and at 18 h pi (r = 0.59; P < 0.05). Regression analysis showed that the optimal time of penetration for predicting the blastocyst rate was 12 h pi (R2 = 0.6). In conclusion, the kinetics of sperm penetration may be a useful marker to predict the in vitro-fertilizing ability of buffalo bulls. The great variability in the speed of oocyte penetration suggests inserting this assessment in the preliminary screening of bulls before their utilization in IVF programs. This may be helpful in selecting high-fertility bulls and identifying the optimal gamete co-incubation times for each bull used. Table 1.Percentage of oocytes penetrated at each time point (hpi, h postinsemination) by different bulls1

Morphokinetics and in vitro developmental potential of monopronucleated ICSI zygotes until the blastocyst stage

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 217-222
Author(s):  
Silvia Mateo ◽  
Francesca Vidal ◽  
Beatriz Carrasco ◽  
Ignacio Rodríguez ◽  
Buenaventura Coroleu ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Control Group ◽  
Study Group ◽  
Comprehensive Understanding ◽  
Developmental Potential ◽  
Morphokinetic Parameters ◽  
Developmental Capacity ◽  
Blastocyst Rate ◽  
Kinetics Of

SummaryThe aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

135 EXPRESSION OF APOPTOSIS-RELATED GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED THROUGH IN VITRO FERTILIZATION AND PARTHENOGENETIC ACTIVATION

2011 ◽  
Vol 23 (1) ◽  
pp. 172
Author(s):  
S. Saw ◽  
K. P. Singh ◽  
R. Kaushik ◽  
M. Muzaffar ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Culture Medium ◽  
Mammalian Species ◽  
Control Cell ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Immature Oocytes ◽  
Tissue Size

Apoptosis, a highly conserved evolutionary mechanism that allows an organism to tightly control cell numbers, tissue size, and protect itself from dangerous cells and unfavourable environments that threaten homeostasis, is generally directed by specific genes involved in the regulation of a series of pro-apoptotic (BAX) and anti-apoptotic (BCL-XL) proteins that are expressed during early development. All mammalian species show the highest level of spontaneous apoptotic processes at the blastocyst stage. These proteins prevent apoptosis by maintaining the cell survival by interfering with the release of cytochrome-C from mitochondria. In this study, immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 μg mL–1 porcine FSH for 24 h in a CO2 incubator (5% CO2, 90 to 95% relative humidity) at 38.5°C. The mature oocytes were used for IVF, and the cleaved embryos were cultured for 8 days in culture medium (CR2 medium containing 0.6% BSA and 10% FBS) for production of embryos at different stages. The parthenotes were produced with exposure of 7% ethanol, 6-dimethyl aminopurine and cultured for 8 days in culture medium. The total RNA was isolated from oocytes and embryos and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA), according to manufacturer protocol. The PCR cycle included heating to 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60 (BAX) and 62°C (BCL) for 30 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The amplified product of both genes were separated on agarose gel and densitometry data for band intensities were generated using AlphaDigiDocTM AD-1201 software under a WindowsTM environment and data analysed with the help of SYSTAT software. Relative abundance of BCL-XL transcripts in immature, mature oocytes and embryos produced through IVF (i.e. 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stage) were 25.33 ± 0.90, 12.67 ± 1.20, 37.67 ± 0.90, 30.67 ± 0.30, 23.67 ± 0.90, 18.33 ± 0.90, and 27.00 ± 1.20, respectively, whereas in parthenogenesis these values were 23.67 ± 0.88, 13.67 ± 1.20, 23.67 ± 1.20, 22.34 ± 0.88, 24.34 ± 0.88, 33.67 ± 0.88, and 45.34 ± 1.20, respectively. Relative abundance of BAX transcripts by IVF were 23.0 ± 0.60, 0.33 ± 0.10, 4.00 ± 0.60, 5.00 ± 0.60, 0.37 ± 0.06, 13.0 ± 0.66, and 56.7 ± 0.90; and by parthenonenesis were 22.3 ± 0.90, 0.13 ± 0.03, 13.67 ± 0.90, 14.0 ± 0.60, 15.33 ± 0.90, 64.67 ± 2.20, and 55.0 ± 2.10, respectively. In conclusion, the expression pattern of the apoptosis-related genes revealed that the incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages such as immature oocytes, morula, and blastocyst than the early cleavage stage embryos.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 317
Author(s):  
I. Venditto ◽  
E. Mariotti ◽  
L. Boccia ◽  
M. Rubessa ◽  
M. De Blasi ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Proteolytic Enzymes ◽  
Critical Role ◽  
Bubalus Bubalis ◽  
Positive Control ◽  
Negative Control ◽  
Chi Square ◽  
Chi Square Test ◽  
Response Trial

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.

80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage

2019 ◽  
Vol 31 (1) ◽  
pp. 165
Author(s):  
M. Poirier ◽  
D. Miskel ◽  
F. Rings ◽  
K. Schellander ◽  
M. Hoelker
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Coat Colour ◽  
Blastocyst Stage ◽  
Transcription Activator ◽  
Guide Rna ◽  
Blastocyst Rate ◽  
Copa Gene

Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.

The influence of sperm concentration, length of the gamete co-culture and the evolution of different sperm parameters on the in vitro fertilization of prepubertal goat oocytes

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 345-355 ◽  
Author(s):  
M.J. Palomo ◽  
T. Mogas ◽  
D. Izquierdo ◽  
M.T. Paramio
Keyword(s):  
Acrosome Reaction ◽  
Penetration Rate ◽  
Sperm Concentration ◽  
Semen Parameters ◽  
Oxygen Species ◽  
Vitro Fertilization ◽  
Rate Of Penetration ◽  
Sperm Penetration ◽  
Kinetics Of

SummaryThe aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 106 to 4 × 106 spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus–oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 106 sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8–12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12–14 h, with a concentration of 4 × 106 sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

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