109 INTRAFOLLICULAR GLUCOSE CONCENTRATION HAS AN INFLUENCE ON THE SEX OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 160
Author(s):  
E. Abele ◽  
H. Stinshoff ◽  
A. Hanstedt ◽  
S. Wilkening ◽  
S. Meinecke-Tillmann ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Follicular Fluid ◽  
Glucose Concentration ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Bovine Embryos ◽  
Developmental Rates ◽  
Group 2 ◽  
Group 1

Several factors have been shown to alter the sex ratio of bovine embryos generated in vitro, i.e. the maturity of the oocyte at the time of insemination, the duration of sperm-oocyte co-incubation and the culture conditions after in vitro fertilization. It has been shown that the presence of glucose during in vitro culture reduced the development of female embryos to the blastocyst stage compared with controls cultured in the absence of glucose. The sex ratio of bovine embryos has also been linked with changes in the composition of the follicular fluid in which the oocyte undergoes growth and maturation, i.e. the intrafollicular testosterone concentration. However, no information is available regarding the effect of intrafollicular glucose concentration on the sex ratio of embryos after in vitro production (IVP). The purpose of this study was to determine whether different glucose concentrations in the follicular fluid at the time of cumulus–oocyte complex (COC) collection have an effect on the sex ratio of the resulting blastocysts after IVP. Ovaries from a local abattoir were transported to the laboratory within 2 h of slaughter. Follicles (3–8 mm) were individually dissected and the glucose concentration of each follicle was measured using a blood glucose monitoring system (Freestyle Freedom Lite, Abbott, Germany). Based on a glucose concentration, COC [low glucose: <1.1 mM (group 1) and high glucose: >1.1 mM (group 2)] were pooled in groups and used for blastocyst production employing standard protocols for IVP. Developmental rates were recorded at Day 3 (cleavage) and Day 7/8 (blastocyst stage). Total cell number of blastocysts was determined after Hoechst staining. Sex of the embryos was analysed via PCR using bovine X- and Y-chromosome specific primers. Developmental rates for COC stemming from follicles with different glucose concentrations did not show significant differences (P > 0.05) compared to each other [Cleavage rate: group 1: 81.8 ± 4.7% (93/117); group 2: 79.3 ± 4.9% (94/123); blastocyst rate: group 1: 35.6 ± 5.2% (38/117); group 2: 31.6 ± 5.2% (38/123)]. Total cell numbers were similar in embryos of both groups [Group 1: 117.7 ± 8.1 (n = 18); group 2: 117.2 ± 6.4 (n = 18)]. The overall sex ratio significantly differed (P < 0.05) from 1:1 in favour of females in both groups [Group 1: 85 v. 15% (n = 20); group 2: 63.6 v. 36.4% (n = 22)]. No significant difference (P > 0.05) in the overall sex ratio was detected in blastocysts produced under standard IVP conditions employed in the laboratory [without measurement of follicular glucose concentration, 55.0 v. 45.0%, (n = 20)]. In conclusion, under the conditions used in the present study, the intrafollicular glucose concentration from which the immature COC was collected affects the sex of the resulting embryo after IVP, favouring females. Further studies are needed to confirm these findings in living cows using the ovum pickup technique.

147 QUALITY OF PORCINE EMBRYOS CULTURED WITH HYALURONAN

2010 ◽  
Vol 22 (1) ◽  
pp. 232
Author(s):  
B. Gajda ◽  
I. Grad ◽  
E. van der Tuin ◽  
Z. Smorag
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Apoptotic Index ◽  
Total Cell ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Group 2 ◽  
Group 1

Hyaluronan (HA) is a high molecular weight polysaccharide found in the mammalian follicular, oviduct, and uterine fluids. When HA is added in maturation and culture media, it improves the developmental potential of bovine (Stojkovic M et al. 2002 Reproduction 124, 141-153; Palasz AT et al. 2008 Zygote 16, 39-47), and porcine oocytes (Sato E et al. 1990 Mol. Reprod. Dev. 26, 391-397) and embryos (Miyano T et al. 1994 Theriogenology 41, 1299-1305). Physiological concentration of HA in follicular, oviductal, and uterine fluids of pigs range from 0.04 to 1.83 mg mL-1 (Kano K et al. 1998 Biol. Reprod. 58, 1226-1232). The aim of the present study was to investigate the effect of different concentrations of HA on the development and quality of cultured porcine embryos. Zygotes from superovulated pigs were cultured in vitro in NCSU-23 medium supplemented with BSA and 0 mg mL-1 (control group), 0.25 mg mL-1 (Exp. Group 1), and 0.5 mg mL-1 (Exp. Group 2) of HA (Animal Pharma BV). Experiments were replicated 3 times with 30 to 40 embryos per each treatment group. Embryos were cultured up to the blastocyst stage at 39°C in an atmosphere of 5% CO2 in air, in 4-well plastic dishes, which contained approximately 0.8 mL of the NCSU-23 medium. Embryo quality criteria were cleavage (on Day 2 after in vitro culture), morula (on Day 4) and blastocyst (on Days 6 to 8) rates, total cell number per blastocyst, and degree of apoptosis (on Day 7) assessed by TUNEL method. Results were analyzed by ANOVA test. There was no difference in percentage of cleaved embryos between control and treated Group 1 and 2.The proportion of embryos developed to the morula and blastocyst stage was 80.0 and 60.0% for Group 1 (0.25 mg of HA), 73.7 and 44.7% for Group 2 (0.5 mg of HA), and 73.4 and 46.7% for control, respectively (difference NS). Supplementation with HA did not increase the cell number of the blastocysts but significantly reduced number of apoptotic nuclei from 2.0 for control to 0.7 (P < 0.01) and 0.6 (P < 0.01) for Group 1 and 2, respectively, and apoptotic index from 9.70 for control to 3.01 (P < 0.05) and 1.95 (P < 0.05) for Group 1 and 2, respectively. These results indicate that supplementation of culture medium NCSU-23 with HA improves the quality (assessed by apoptotic index) of pig embryos but does not increase the total cell number in pig blastocysts as reported by Kim HS et al. 2005 (Theriogenology 63, 1167-1180). However, further research to test the HA’s effect on cryopreservation of in vitro and in vivo produced pig embryos are needed.

35 IN VITRO BOVINE EMBRYO DEVELOPMENT AFTER NUCLEAR TRANSFER BY HANDMADE CLONING USING A MODIFIED WOW CULTURE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 126 ◽  
Author(s):  
C. Feltrin ◽  
F. Forell ◽  
L. dos Santos ◽  
J. L. Rodrigues
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Inner Diameter ◽  
Group 2 ◽  
Group 1 ◽  
Handmade Cloning

The effect of the microenvironment on embryo development during in vitro culture of zona-free embryos after nuclear transfer is still unclear. The aim of this experiment was to determine the effect of the dimensions of the well (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) culture system on the in vitro development of handmade cloned bovine embryos to the blastocyst stage. Appropriately ground steel needles were pressed slightly by hand to the bottom of the well of a polystyrene four-well dish (176740, Nunc, Life Technologies AS, Roskilde, Denmark). Embryos were produced by the handmade cloning (HMC) technique (Vajta et al. 2003 Biol. Reprod. 68, 571-578) with modifications, using primary cultures of skin fibroblast cells from an adult cow as nuclear donors. Cumulus-oocyte complexes were in vitro-matured in M-199 supplemented with 10% estrous cow serum (ECS), FSH, hCG, and estradiol (E2) for 17 h. After maturation, cumulus cells were removed by pipetting. Following zona pellucida removal in 0.5% protease (Sigma, Brazil), zona-free oocytes were incubated for 15 min in 5 mg/mL cytochalasin B (Sigma) and subsequently hand-bisected and screened for nuclear material under UV light after incubation in 10 mg/mL bisbenzimide (Hoechst 33342). Next, two enucleated halves and one donor cell were aggregated after a quick exposure to phytohemagglutinin (PHA) and subsequently fused by two electrical DC pulses of 1 kV/cm for 20 �s, in a BTX 453 chamber coupled to an ECM 2001 Electro Cell Manipulator System (BTX, Inc., San Diego, CA, USA), with additional exposure to brief pre- and post-fusion AC pulses of 15 V. Reconstructed embryos were chemically activated in 5 mM ionomycin (Sigma) for 5 min, followed by 2 mM 6-DMAP (Sigma) for 2.5 h. Finally, activated reconstructed cloned embryos were in vitro-cultured in one of two WOW culture systems (larger vs. smaller micro-wells) in 4-well plates containing 400 mL modified SOF medium supplemented with 10% ECS, under mineral oil, at 5% CO2, 5% O2 and 90% N2, and 39�C for 7 days. In Group 1 (large-size micro-well), embryos were cultured in individual cylindrical micro-wells with an inner diameter and depth of approximately 280 and 250 mm, respectively, whereas in Group 2 (small size micro-well), embryos were cultured in individual conical micro-wells with approximately 130 mm inner diameter and 150 mm depth. Data analysis was performed by the chi-square test. After four replicates, cleavage rates were significantly higher (P < 0.05) in Group 2 (51/63, 80.9%) than in Group 1 (43/67, 64.1%). Embryo development to the blastocyst stage was also greater (P < 0.05) in the small micro-wells (16/63, 25.3%) than in the large ones (8/67, 11.9%). In summary, these results show a significant increase in cleavage and blastocyst developmental rates in handmade cloned embryos cultured in a modified WOW system using individual small size micro-wells, suggesting that a small, tighter micro-well provides favorable in vitro conditions for embryo development.

315 USE OF PERIFOLLICULAR BLOOD FLOW TO PREDICT THE DEVELOPMENTAL COMPETENCE OF BOVINE CUMULUS-OOCYTE COMPLEXES COLLECTED DURING REPEATED OVUM PICKUP SESSIONS ONCE OR TWICE WEEKLY

2009 ◽  
Vol 21 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Hanstedt ◽  
K. Höffmann ◽  
Ä Honnens ◽  
H. Bollwein ◽  
C. Wrenzycki
Keyword(s):  
Blood Flow ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Small Individual ◽  
Color Flow ◽  
Color Flow Imaging ◽  
Significant Difference ◽  
Developmental Rates ◽  
Group 2 ◽  
Group 1

On average, only 20% of the cumulus–oocyte complexes (COC) develop to the blastocyst stage (Merton et al. 2003 Theriogenology 59, 651–674). An increase in the blood supply to individual follicles appears to be associated with follicular growth rates, whereas a reduction seems to be closely related to follicular atresia (Acosta et al. 2003 Reproduction 125, 759–767). The purpose of this study was to determine whether qualitative perifollicular blood flow changes can be used to predict the developmental competence of COC collected during repeated ovum pickup (OPU) sessions once or twice weekly. Lactating Holstein cows (n = 20) were used as oocyte donors. After dominant follicle removal, OPU was performed twice (group 1, for 3 weeks) or once (group 2, for six weeks) weekly employing a 7.5-MHz transducer (GE 8C-RS) of an ultrasound scanner (GE Logiq Book). Follicle size and Doppler characteristics were recorded by transvaginal ultrasonography just before COC collection using color flow imaging. Owing for technical limitations for measurement of blood flow in small individual follicles, only the presence or absence of blood flow was assessed for each follicle. When a clearly visible blue or red spot (blood flow) was detected in the follicle wall, it was considered as a follicle with detectable blood flow. Follicles with or without detectable blood flow from each individual cow were aspirated separately. After morphological classification of COC, standard protocols for IVP were used for embryo production (Wrenzycki et al. 2001 Biol. Reprod. 65, 323–331). Cleavage and blastocyst rates were recorded at Day 3 and Day 8, respectively. In total, 464 (246 with and 218 without detectable blood flow) and 243 (125 with and 118 without detectable blood flow) follicles ≥3 mm were aspirated in group 1 and group 2, respectively. Morphology of the COC was similar in all groups. Developmental rates for COC stemming from follicles with or without detectable blood flow in group 1 did not show differences for cleavage rates, 54.0% (34/63) and 56.7% (45/81), and for blastocyst rates, 25.4% (16/63) and 22.2% (18/83), respectively. In group 2, the cleavage rates were also similar for COC originating from follicles with and without detectable blood flow, 54.3% (25/46) and 51.5% (34/66). However, developmental rates up to the blastocyst stage did show a significant difference, 23.9% (11/46) and 15.2% (10/66) for COC aspirated from follicles with or without detectable blood flow (P ≤ 0.05). These results show that using COC originating from follicles with detectable perifollicular blood flow collected once weekly may have a higher developmental competence compared to those from follicle without detectable blood flow. Within the detection limits of this study, differences in perifollicular blood flow during repeated OPU sessions once weekly were predictive of oocyte competence. Ruthe Research Farm, Germany, for providing the animals; Masterrind GmbH, Germany, for donation of the semen; and the HW Schaumann Stiftung for financial support.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

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 258 APOPTOSIS IN FAST- AND SLOW-CLEAVING BOVINE EMBRYOS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 258
Author(s):  
L. Vandaele ◽  
B. Mateusen ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Univariate Analysis ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Tunel Staining ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Random Factor ◽  
Quality Marker

Fast-cleaving embryos have significantly higher chances of reaching advanced developmental stages, and hatched blastocysts developed from fast-cleaving embryos have higher total cell and inner cell mass cell numbers. Few data are available on the prevalence of apoptosis in blastocysts resulting from fast-cleaving bovine embryos. Therefore, it was the aim of this study to search for a correlation between this early quality marker (timing of first cleavage) and a later quality marker (apoptosis in blastocyst stage). A total of 836 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h after fertilization and cultured in 50 μL droplets of modified SOF medium without serum at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 30 h, 36 h and 48 h post-fertilization (PF), zygotes that had developed to the 2-cell stage or beyond were placed in new droplets and cultured in separate groups. In each of the 3 replicates a control group of 100 presumed zygotes was cultured in SOF medium without manipulation. After 24 h of culture, the SOF medium was supplemented with fetal calf serum (FCS) up to 10%. Blastocysts were evaluated at Days 7 and 8, and fixed in 4% paraformaldehyde. After TUNEL staining, total cell number and TUNEL-positive cells were counted for each group. An univariate analysis of variance was used with % apoptotic cells as the dependent variable, timing of first cleavage as the fixed factor, and replicate as the random factor (mixed model ANOVA). The mean cumulative blastocyst yields in the control group were 20.9% and 27.0% at Days 7 and 8, respectively, which were not different from the total yields from manipulated embryos (Table 1). The cumulative cleavage rates for manipulated embryos were 44.9, 65.1, and 85.7% at 30, 36, and 48 h PF, respectively. The blastocyst yields at Days 7 and 8 PF were declining for embryos which cleaved later, with a distinct drop (P < 0.05) between the 36-h and 48-h groups. The percentage of apoptotic cells in Day 7 blastocysts was significantly lower in the 30-h compared with the 36-h and 48-h groups (P < 0.05). Within the 30-h and 36-h groups, Day 8 blastocysts had significantly more apoptotic cells than Day 7 blastocysts (P < 0.05), but no differences could be detected between groups at day 8. In conclusion, this experiment has confirmed the increased chances of early-cleaving embryos to reach the blastocyst stage. The higher percentages of apoptosis in late cleaving embryos at Day 7 and in all blastocysts at day 8 as determined by TUNEL staining could be an indication of lower blastocyst quality. Table 1. Blastocyst yield (number and percentage) and percentage of apoptotic cells (APC) at Days 7 and 8 PF

scholarly journals In Vitro Embryo Production and Transfer of Bubaline Embryos using Oocytes Derived from Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA)

2014 ◽  
Vol 2 (2) ◽  
pp. 180-184
Author(s):  
FP Aquino ◽  
Eufrocina P. Atabay
Keyword(s):  
Transvaginal Ultrasound ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Follicular Aspiration ◽  
Group 2 ◽  
Group 1 ◽  
Donor Cows

Transvaginal ultrasound-guided follicular aspiration (TUFA) has become a popular tool for embryo production in vitro due to its high degree of repeatability in terms of recovering oocytes from live animals. In Study 1, the quantity and quality of oocytes from Bulgarian Murrah buffalo cows (n=10) of varying ages (Group 1, 8-12; and Group 2, 13-17 years) were assessed. Group 1 buffalo donor cows yielded significantly higher (P<0.05) number of oocytes vs Group 2 buffalo donor cows (71 vs 29 oocytes, respectively), though in terms of oocyte quality, no difference was observed. In Study 2, oocytes collected (n=100) in Study 1 were matured, fertilized in vitro and the resulting zygotes were cultured which developed to blastocyst stage embryos. The maturation, fertilization and blastocyst development rates obtained were 53.0%, 40.0% and 32.5%, respectively. In Study 3, the viability of resulting blastocyst stage embryos was determined by transferring to recipient cows. Of 10 recipients 1 got pregnant and delivered a 35 kg male calf after 310 days gestation period. Overall, the results of the studies conducted demonstrated the potential of TUFA technology in the in vitro production of embryos which eventually could be used in the production of live offspring.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10369Int J Appl Sci Biotechnol, Vol. 2(2): 180-184 

scholarly journals Effect of Splinting in Accuracy of Two Implant Impression Techniques

2014 ◽  
Vol 40 (6) ◽  
pp. 633-639 ◽  
Author(s):  
Erica Dorigatti de Avila ◽  
Fernanda de Matos Moraes ◽  
Sabrina Maria Castanharo ◽  
Marcelo Antonialli Del'Acqua ◽  
Francisco de Assis Mollo
Keyword(s):  
Confidence Interval ◽  
Analysis Of Variance ◽  
Impression Material ◽  
Titanium Screw ◽  
Standard Preparation ◽  
Implant Abutment ◽  
Interface Gaps ◽  
Group 2 ◽  
Group 1

Because there is no consensus in the literature about the need for a splint between copings, the aim of this study was to evaluate, in vitro, the accuracy of 2 impression techniques for implant-supported prostheses. A master cast was fabricated with four parallel implant abutment analogs and a passive framework. Two groups with 5 casts each were formed: Group 1 (squared impression copings with no splint: S) and Group 2 (splinted squared impression copings, using metal drill burs and Pattern resin: SS). The impression material used was polyvinyl siloxane with open trays for standard preparation of the casts. For each cast, the framework was positioned, and a titanium screw was tightened with 10 N·cm torque in analog A, after which measurements of the abutment-framework interface gaps were performed at analogs C and D. This process was repeated for analog D. These measurements were analyzed using software. A one-way analysis of variance (ANOVA) with a confidence interval of 95% was used to analyze the data. Significant differences were detected between S and SS in relation to the master cast (P ≤ 0.05). The median values of the abutment-framework interface gaps were as follows: master cast: 39.64 μm; squared impression copings with no splint: 205.86 μm; splinted squared impression copings: 99.19 μm. Under the limitations of this study, the technique presented for Group 2 produces better results compared with the technique used for Group 1.

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