110 THE USE OF FORSKOLIN AND ITS EFFECT ON IN VITRO-PRODUCED BRAHMAN-SIRED EMBRYOS SUBMITTED TO SLOW COOL FREEZING OR VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 214 ◽  
Author(s):  
J. H. Pryor ◽  
J. A. Trant ◽  
C. B. Ponchirolli-Schneider ◽  
C. R. Looney ◽  
C. R. Long ◽  
...  
Keyword(s):  
Statistical Significance ◽  
Survival Rates ◽  
Slow Cool ◽  
Blastocyst Stage ◽  
Developmental Differences ◽  
Treatment Groups ◽  
Student’S T ◽  
Blastocyst Hatching ◽  
Hatching Rates

The objective of this study was to further test the lipolytic effect of 10 μM forskolin on developmental differences between bovine in vitro produced (IVP) embryos submitted to slow cool (SC) freezing or vitrification (VT). Previously reported (phase I) IVP embryo hatching rates for control embryos (62%) were no different than 10 μM forskolin (67%; Pryoretal et al. 2009 Reprod. Fertil. Dev. 21, 163). For phase II: on Day 6 post-fertilization (IVF = Day 0), 207 Brahman-sired viable embryos were evenly divided and cultured for 24 h in G2.5 medium (Vitrolife, Englewood, CO, USA) with or devoid of 10 μL forskolin (Sigma, St. Louis, MO, USA). On Day 7, compact morula (CM, n = 31), blastocyst (BL, n = 76), and expanded BL (XBL, n = 100) were washed in Vigro Holding Plus medium (Bioniche, Pullman, WA, USA) and randomly allocated to 4 treatment groups; control SC (CSC; no treatment, n = 52), 10 μM forskolin SC (FSC; n = 55), control VT (CVT; n = 49), and 10 μM forskolin VT (FVT; n = 51). All embryos were packaged in sterile 0.25-mL plastic straws. The SC embryos were submitted to Vigro Ethylene Glycol Freeze Plus medium (Bioniche) for 5 min before freezing at 0.5°C/min from -6°C to -32°C and plunging in LN2. Embryos were vitrified using a bovine VT kit (Bioniche): VS1, 3 min; VS2, 45 s in 15 μL; diluent, in straw with VS2 separated by air columns, vitrified in LN2 vapor 1 cm from liquid for 1 to 15 min before plunging. The SC embryos were air thawed 5 s and placed in 30°C H2O bath for 10 s. The VT straws were air warmed 10 s and then in 35°C H2O for 20 s prior to shaking them down to mix columns. All embryos were cultured in G2.5 for 24-h survival and 48-h hatching rates. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student’s t. For mean survival rates, FSC was different than CSC but showed no difference between FVT and CVT (81.7 ± 0.09, 42.6 ± 0.09, 59.4 ± 0.09, 49.0 ± 0.10, respectively (P < 0.01). There were no statistical differences for hatching rates for combined embryo stages (58.2 ± 0.10, 37.8 ± 0.10, 34.4 ± 0.10, 28.1 ± 0.11 for FSC, FVT, CSC, and CVT, respectively; P > 0.07). However, when comparing hatching rates of only the blastocyst stage embryos (n = 176), FSC was superior to CSC and CVT but not different than FVT (74.8 ± 0.11, 29.5 ± 0.11, 29.1 ± 0.11, 48.5 ± 0.11, respectively; P < 0.01). In conclusion, FSC yielded significantly higher survival and blastocyst hatching rates than CSC, but there were no differences between CVT and FVT for survival and FVT for blastocyst hatching rates. These results indicate that the addition of 10 μM forskolin to culture 24 h prior to freezing 7 d IVP Brahman-sired embryos can increase survival and blastocyst hatching rates. The authors acknowledge support from the American Brahman Breeders Association.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

70 ONE-STEP CRYOPROTECTANT DILUTION FOLLOWING VITRIFICATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 182
Author(s):  
R. Morató ◽  
T. Mogas
Keyword(s):  
Embryo Transfer ◽  
Statistical Significance ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Dilution Method ◽  
Bovine Embryo ◽  
One Step

Although slow freezing continues to be the most widely used technique of cryopreservation for bovine in vivo- and in vitro-produced embryos, vitrification has been tested in different species with good results, especially when dealing with in vitro-produced embryos. Vitrification represents a minor expense in time and equipment associated with cryopreservation compared with conventional slow freezing. However, vitrification, which is the most common method for human embryo cryopreservation, has not been widely adopted by embryo-transfer practitioners for commercial use in cattle. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting, and it is difficult to perform in the field. The objective of this study was to develop a one-step dilution method suitable for one-step bovine embryo transfer using the cryotop vitrification method. Embryos produced in vitro by standard procedures were vitrified at the blastocyst stage at Day 7 post-insemination in a mixture of 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose using cryotop devices. Embryos were randomly assigned to 1 of 3 warming methods: (1) W3: warming was carried out following the cryotop method (1 M sucrose for 1 min, 0.5 M sucrose for 3 min, and 0 M sucrose for 6 min); (2) W1/0.5: embryos were warmed directly in 0.5 M sucrose for 3 min; and (3) W1/0: embryos were warmed directly in 0 M sucrose for 5 min. Survival rates were assessed in terms of blastocyst re-expansion, hatching, and hatched status at 3 and 24 h after warming. Data were analyzed using the statistical analysis systems package (SAS, v9.1). Data from at least 3 replicates were collected. Comparisons of vitrified–warmed blastocyst survival rates between groups were performed using the chi-squared test. The level of statistical significance was set at P < 0.05. When embryo survival was evaluated at 3 h postwarming, embryos warmed using the 3-step dilution protocol and those warmed directly in 0.5 M sucrose showed higher percentages of survival (W3: 89.8%, n = 98; W1/0.5: 87.5%, n = 64; P < 0.05) than those blastocysts that were warmed directly in 0 M sucrose (W1/0: 66.4%, n = 146). However, similar rates irrespective of the warming procedure were observed at 24 h postwarming (W3: 85.7%, W1/0.5: 88.2%, W1/0: 70.5%). Warmed in vitro-produced embryos exposed to W3 (47.6%) and W1/0.5 (35.6%) achieved higher percentages of embryos developing to the hatched blastocyst stage after 24 h of culture than those embryos warmed in W1/0 (20.4%; P < 0.05). Our results indicate that direct warming and dilution of cyotop-vitrified embryos in 0.5 M sucrose for 3 min may enable one-step bovine embryo transfer without requirement of a microscope or other laboratory equipment, simplifying the embryo-transfer procedure of vitrified embryos on farm at the same level of complexity as carrying out AI. Support came from Spanish MEC (RZ2010-00015-0-00; AGL2010-19069) and Generalitat de Catalunya (2009 SGR 621).

scholarly journals Cold atmospheric plasma coupled with air abrasion in liquid medium for the treatment of peri-implantitis model grown with a complex human biofilm: an in vitro study

2021 ◽  
Author(s):  
Wang Lai Hui ◽  
Vittoria Perrotti ◽  
Adriano Piattelli ◽  
Kostya (Ken) Ostrikov ◽  
Zhi Fang ◽  
...  
Keyword(s):  
Liquid Medium ◽  
Statistical Significance ◽  
Atmospheric Plasma ◽  
Air Abrasion ◽  
Implant Surface ◽  
Cold Atmospheric Plasma ◽  
Surface Features ◽  
Treatment Groups ◽  
Surface Decontamination

Abstract Objective Treatment of implants with peri-implantitis is often unsuccessful due to residual microbial biofilm hindering re-osseointegration. The aim of this study was to treat biofilm-grown titanium (Ti) implants with different modalities involving air abrasion (AA) and cold atmospheric plasma (CAP) to compare the effectiveness in surface decontamination and the alteration/preservation of surface topography. Materials and methods Saliva collected from a peri-implantitis patient was used to in vitro develop human biofilm over 35 implants with moderately rough surface. The implants were then mounted onto standardized acrylic blocks simulating peri-implantitis defects and treated with AA (erythritol powder), CAP in a liquid medium, or a combination (COM) of both modalities. The remaining biofilm was measured by crystal violet (CV). Surface features and roughness before and after treatment were assessed by scanning electron microscope (SEM). The data were statistically analyzed using Kruskal-Wallis followed by Tukey’s multiple comparison test. Results In the present peri-implantitis model, the human complex biofilm growth was successful as indicated by the statistical significance between the negative and positive controls. All the treatment groups resulted in a remarkable implant surface decontamination, with values very close to the negative control for AA and COM. Indeed, statistically significant differences in the comparison between the positive control vs. all the treatment groups were found. SEM analysis showed no post-treatment alterations on the implant surface in all the groups. Conclusions Decontamination with AA delivering erythritol with or without CAP in liquid medium demonstrated compelling efficacy in the removal of biofilm from implants. All the tested treatments did not cause qualitative alterations to the Ti surface features. No specific effects of the CAP were observed, although further studies are necessary to assess its potential as monotherapy with different settings or in combination with other decontamination procedures. Clinical relevance CAP is a promising option in the treatment of peri-implantitis because it has potential to improve the elimination of bacterial plaque from implant surfaces, in inaccessible pockets or during open-flap debridement, and should stimulate the process of the re-osseointegration of affected dental implants by not altering surface features and roughness.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann
Keyword(s):  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

1 ARGININE SUPPLEMENTATION IN VITRO INCREASES PORCINE EMBRYO DEVELOPMENT AND AFFECTSmRNA TRANSCRIPT EXPRESSION

2011 ◽  
Vol 23 (1) ◽  
pp. 107 ◽  
Author(s):  
B. K. Bauer ◽  
L. D. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Treatment Group ◽  
Mrna Expression ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Sequencing Analysis ◽  
Treatment Groups ◽  
Porcine Embryo ◽  
Arginine Supplementation

In vitro culture systems are suboptimal as compared to in vivo. Previous next-generation sequencing analysis of in vivo fertilized and in vitro cultured (IVC) or in vivo cultured (IVV) porcine blastocyst stage embryos identified an arginine transporter (SLC7A1) expressed 63 fold higher in IVC compared to IVV blastocysts (Bauer et al. 2010 Biol. Reprod. Epub ahead of print). Arginine catabolism may play important roles in placental and conceptus growth and development as it is a substrate for synthesis of nitric oxide synthase and polyamines. The objective of this study was to determine the effects arginine had on both embryo development and mRNA expression in in vitro fertilized embryos. Cumulus–oocyte complexes were matured for 44 h in M199 supplemented with EGF, FSH, and LH. Oocytes with a visible polar body (metaphase II) were selected and fertilized in modified Tris Buffered Medium for 5 h and then placed into one of 5 treatment groups (Porcine Zygote Medium 3 (PZM3) with 0 mM, 0.12 mM (current concentration of arginine in PZM3), 0.36 mM, 0.72 mM, or 1.69 mM arginine). Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25 μL drops of respective culture media and cultured to day 6 in 5% CO2, 5% O2, 90% N2 at 38.5°C. To determine the effect arginine had on development the percent of embryos that made it to the blastocyst stage for each treatment group were analysed using PROC GLM in SAS (SAS Institute, Cary, NC). A least significant difference post test comparison was completed to determine if significant differences existed between treatment groups (a,b,cP < 0.05). The percentage of cleaved embryos on Day 6 that developed to blastocyst was 57.2%b,c, 50.2%c, 67.3%a,b, 67.3%a,b, 70.4%a (N = 147, 163, 150, 120, and 134) in 0 mM, 0.12 mM, 0.36 mM, 0.72 mM, and 1.69 mM arginine, respectively. Real-time PCR was then completed to assess the affect arginine supplementation had on SLC7A1 mRNA expression. Three biological replicates, each containing 10 blastocyst pools to ensure enough starting material, were collected for each treatment group. RNA was isolated from each sample and 5 μL was linearly amplified (NuGEN Ovation Pico WTA System) so multiple genes could be compared and then purified using Bio-Rad MicroSpin Columns. Expression levels were calculated relative to the reference sample and the housekeeping gene, YWHAG. The ΔΔCT values were log-transformed and analysed using PROC GLM in SAS. The expression of SLC7A1 mRNA was decreased (P = 0.0006) compared to PZM3 in the 1.69 mM arginine group. These results illustrate the positive effects that additional arginine may be having on porcine embryo development during culture from the 2-cell to the blastocyst stage. Supplementing arginine to a final concentration of 1.69 mM during culture increases development of porcine embryos to blastocyst compared to PZM3 and also decreases the expression of SLC7A1. Evaluation of the transcriptional profile appears to be a good method of letting the embryo tell us what it needs for development, and in this case arginine. Funded by F21C.

Sign in / Sign up

Export Citation Format

Share Document