234 HORMONAL FOLLICLE STIMULATION IN HOLSTEIN COWS FOR IN VITRO EMBRYO PRODUCTION USING SPERM SORTED BY FLOW CYTOMETRY

2016 ◽  
Vol 28 (2) ◽  
pp. 248 ◽  
Author(s):  
L. Ferré ◽  
Y. Bogliotti ◽  
J. Chitwood ◽  
M. Kjelland ◽  
P. Ross
Keyword(s):  
Transvaginal Ultrasound ◽  
Prostaglandin F2α ◽  
Holstein Cows ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
Group 2 ◽  
Group 1

Transvaginal ultrasound needle-guided ovum pick-up (OPU) and in vitro embryo production (IVP) offer a reliable alternative to conventional embryo transfer to produce offspring. The success of OPU/IVP greatly depends on the number and quality of retrieved oocytes. The aim of this study was to compare OPU/IVP performance from stimulated Holstein cows. Holstein (Bos taurus) >8-year-old pluriparous open dry cows (n = 28) were used for OPU as oocyte donors. Follicular waves in all groups were synchronized by gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF), and CIDR administrated on Day 0, followed by stimulation treatments 48 h later. No pre-synch was used. Total hormone dosage were administrated as follows: Group 1: pFSH = 180 mg (Folltropin, Bioniche, Belleville, ON, Canada; n = 7), Group 2: pFSH/LH = 500 IU (Pluset, Calier, Barcelona, Spain; n = 7), Group 3: eCG = 1500 IU (eCG, Biogénesis-Bagó, Buenos Aires, Argentina; n = 7) and Group 4: Control (n = 7), no stimulation. All injections were performed intramuscularly (i.m.) twice a day, during three days. OPU was performed 48 (Group 1) or 24 h (Group 2 and 3) after the last injection. The control group received saline solution i.m. Follicles were classified according to diameter in 4 categories: small (2–5 mm); medium (6–9 mm); large (10–14 mm) and extra large (>15 mm). A Mindray DP-30 Vet (Mindray Medical, Shenzhen, China) was equipped with a micro-convex transducer 5.0- to 8.5-MHz probe along with a disposable 21G needle. The OPU flow rate was 15 mL min–1. Retrieved oocytes were classified according to IETS guidelines as viable (grade 1 + 2) and non-viable (grade 3 + 4). The IVP protocol was according to that in Reprod. Fertil. Devel. (2004, 16, 253). Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient (PureSperm, Nidacon, Mölndal, Sweden) and diluted to 1 × 106 sperm mL–1. ANOVA was used for comparisons of mean values and a chi-squared test was used for proportions. Results are presented in the Table 1. In conclusion, pFSH stimulation before ovum pick-up in Holstein cows increased the number of collected and viable oocytes, cleavage, embryo development, and hatching rates in comparison to other follicle stimulation hormones and non-stimulation. A cost-benefit analysis of these methods could be valuable in order to inform whether or not a stimulation protocol is necessary for a commercial IVP operation. Table 1.Numbers of follicles, collected and viable oocytes, cleavage rate, blastocysts and hatching rate

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

2019 ◽  
Vol 31 (1) ◽  
pp. 142
Author(s):  
E. V. Kornienko ◽  
A. B. Romanova ◽  
M. A. Ikonopistseva ◽  
G. P. Malenko
Keyword(s):  
Ethylene Glycol ◽  
Fertilization Rate ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Hollow Fibres ◽  
Group 2 ◽  
Bovine Oocytes ◽  
Group 1

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P<0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P<0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

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 Comparison of different irrigation and agitation methods for the removal of two types of calcium hydroxide medicaments from the root canal wall: An in-vitro study

2017 ◽  
Vol 90 (3) ◽  
pp. 327-332 ◽  
Author(s):  
Deepak Singh Kirar ◽  
Pradeep Jain ◽  
Pallav Patni
Keyword(s):  
Calcium Hydroxide ◽  
Root Canal ◽  
Positive Control ◽  
Positive Control Group ◽  
Control Group ◽  
Passive Ultrasonic Irrigation ◽  
Ultrasonic Irrigation ◽  
Group 2 ◽  
Group 1

Background and aim: Comparison of different irrigation and agitation methods for the removal of two types of calcium hydroxide medicaments from the root canal walls.Methods: Fifty extracted single rooted teeth were selected for this study. After decoronation, the root canals of these teeth were prepared to the size F3 (30 no.) using rotary ProTaper file system. These samples were randomly divided into four groups. Group 1 (n=20) were filled completely with water based calcium hydroxide (CH), Group 2 (n=20) were filled with oil based CH using lentulo spiral, Group 3 (n=5) - the positive control group received the CH as intracanal medication, but no subsequent removal, Group 4 (n=5) - the negative control did not receive CH placement. Further on, Group 1 and Group 2 were divided into four sub-groups (n=5). In sub-group A we performed conventional syringe irrigation with side-vented needle sub-group B) manual dynamic agitation, sub-group C sonic agitation using endoactivator, sub-group D passive ultrasonic irrigation (PUI). Roots were split longitudinally into mesial and distal halves. Digital images of the root canal walls were acquired by a Dental Operating Microscope (DOM) and assessed by using a scoring criteria at different thirds (coronal, middle and apical) of the root canal as follows: score 1, score 2, score 3, and score 4. Data were analyzed applying one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests at a 95% confidence interval (P < 0.05).Results: Statistically significant differences were not found between the experimental groups and the negative group in any one third of the root canal (P>0.05). However, a difference did exist between the experimental groups and the positive control group (P<0.05). None of the experimental groups totally removed CH substances from root canal walls.Conclusion: Among all experimental groups, removal of CH was best achieved by sonic agitation using endoactivator followed by passive ultrasonic irrigation (PUI), manual dynamic agitation and conventional syringe irrigation with side-vented needle.

scholarly journals Effect of Different Irrigation Solutions on the Diffusion of MTA Cement into the Root Canal Dentin

Materials ◽  
2020 ◽  
Vol 13 (23) ◽  
pp. 5472
Author(s):  
José Pedro Martinho ◽  
Sara França ◽  
Siri Paulo ◽  
Anabela Baptista Paula ◽  
Ana Sofia Coelho ◽  
...  
Keyword(s):  
Root Canal ◽  
Ethylenediaminetetraacetic Acid ◽  
Treatment Success ◽  
Confocal Laser ◽  
Control Group ◽  
Middle Section ◽  
Group 2 ◽  
Root Canal Dentin ◽  
Group 1

(1) Aim: This study aims to analyze the in vitro infiltration of a silicate root canal sealer into dentinal tubules after using different endodontic irrigating solutions. (2) Methods: Twenty-nine teeth with single roots were separated into three groups according to the final irrigation protocol: G1 n = 10) = 17% EDTA (ethylenediaminetetraacetic acid) + 3.0% sodium hypochlorite (NaOCl), G2 (n = 10) = 17% EDTA + 2.0% chlorhexidine and G3 (Control group, n = 9) = 17% EDTA + saline solution. Root canals were filled using cold lateral compaction technique with MTA Fillapex sealer and gutta-percha. The sealer was labeled with rhodamine B. The teeth were segmented at the middle and third apical sections, which were visualized using 10× confocal laser microscopy to determine the sealer penetration percentage. (3) Results: In the apical section, no statistically significant differences were found between the groups regarding sealer penetration. In the middle section, Group 1 obtained the highest percentage, and Group 2 the lowest (p = 0.004). Group 1 also presented statistically significant differences in the Control Group (p = 0.031) and had close sealer penetration values. Meanwhile, the Control Group (p = 0.023) and Group 2 (p = 0.029) revealed a significant decrease of sealer penetration between the apical and middle sections. (4) Conclusion: The obtained results support that final irrigation with NaOCl promoted similar sealer penetration in the apical and middle sections. On the other hand, a significant decrease in the sealer penetration of the middle section was observed for the chlorhexidine and saline groups. Compared to other irrigant solutions, NaOCl promotes more uniform sealer penetration, which can correlate with better sealing and, consequently, higher endodontic treatment success.

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

266 OVUM PICKUP AND IN VITRO EMBRYO PRODUCTION DURING THE SUMMER: DIFFERENCES BETWEEN HOLSTEIN HEIFERS, HIGH-PRODUCING HOLSTEIN COWS IN PEAK LACTATION, AND REPEAT-BREEDER HOLSTEIN COWS

2010 ◽  
Vol 22 (1) ◽  
pp. 290 ◽  
Author(s):  
R. M. Ferreira ◽  
H. Ayres ◽  
M. L. Ferraz ◽  
A. B. Araújo ◽  
M. R. Chiaratti ◽  
...  
Keyword(s):  
Quality Control ◽  
Heat Stress ◽  
Estradiol Benzoate ◽  
Low Fertility ◽  
Holstein Cows ◽  
Embryo Production ◽  
Bovine Reproduction ◽  
In Vitro Embryo Production ◽  
Peak Lactation

The association of ovum pickup (OPU) and in vitro embryo production (IVP) has been widely used to improve bovine reproduction. However, previous reports have indicated the occurrence of low fertility associated with summer heat stress. In the present work, we hypothesized that different categories of Holstein cattle [heifers (H), high-producing cows in peak lactation (PL), and repeat-breeders (RB)] would respond differently to OPU and IVP during the summer, because of their different metabolisms. This experiment was conducted on 2 commercial dairy farms in southeast Brazil in summer 2009. Cattle (n = 36/category) started a protocol to synchronize follicular wave emergence: 2 mg of estradiol benzoate (Sincrodiol®, OuroFino, Minas Gerais, Brazil) + 50 mg of progesterone (OuroFino) + 150 μg of D-cloprostenol (Sincrocio®, OuroFino) i.m. + a norgestomet ear implant (Crestar®, Intervet, São Paulo, Brazil) on Day 0, implant removal and OPU on Day 5. Respiration rate (RR), rectal temperature (RT), and cutaneous temperature (CT) were recorded on Day 0. Semen from a single Holstein bull previously tested was used in IVP, and oocytes from slaughterhouse were submitted to IVP as a quality control. Statistical analyses were done using PROC GLIMMIX of SAS (SAS Institute, Cary, NC, USA). The average and maximum environmental temperature and humidity were 30 and 39.8°C and 61 and 88%. Heifers were on average 15.7 months old; PL and RB cows had 112.8 ± 5.4a v. 422.8 ± 27.6b days in milk, milk production of 32.8 ± 0.9a v. 21.7 ± 1.1b kg, and number of insemination of 0.9 ± 0.1a v. 6.8 ± 0.3b; P < 0.0001; mean ± SE). Heifers and cows had different RR (H = 11.2 ± 0.3a, PL = 18.2 ± 0.6b, RB= 18.5 ± 0.4b breaths/min; P < 0.0001), RT (H = 38.7 ± 0.1a, PL = 39.56 ± 0.2b, RB = 39.32 ± 0.1b; P < 0.0001), and CT (H = 31.3 ± 0.2a, PL = 33.2 ± 0.3b, RB = 32.9 ± 0.3b; P < 0.0001). At OPU, heifers had greater number of follicles than PL cows, but they were similar to RB cows (H= 18.5 ± 1.9a, PL = 12.4 ± 1.1b, RB = 17.2 ± 2.0a; P = 0.04). Heifers had also greater number of oocytes (H = 9.6 ± 1.6a, PL = 5.0 ± 0.9b, RB = 8.8 ± 13ab; P = 0.03) and viable oocytes (H = 7.6 ± 1.5a, PL = 3.6 ± 0.8b, RB = 6.8 ± 1.2ab; P = 0.05) recovered from OPU than PL cows and similar to RB cows. However, at IVP, heifers had greater rates than both other categories (cleavage at Day 3: H = 47.8%a, PL = 31.1%b, RB = 35.4%b, P = 0.008; blastocyst at Day 7: H = 21.0%a, PL = 4.1%b, RB = 3.8%b, P < 0.0001) and more grade I embryos (H = 1.3 ± 0.4a, PL = 0.3 ± 0.2b, RB = 0.5 ± 0.2b, P = 0.04). The quality control had 80.7% cleavage and 45.4% blastocyst rates. The differences found among heifers and cows are probably related to their metabolism under heat stress, compromising oocyte number and quality. Also, although RB had similar number of viable oocytes than heifers, these oocytes are probably compromised, leading to poorer results at IVP, as observed. Fazendas Santa Rita e Campestre, Vida Reprodutiva, LMMD, SAMVET, VITROGEN, OuroFino Saúde Animal, FAPESP (proc09/00938-3).

scholarly journals Superstimulation prior to the ovum pick-up to improve in vitro embryo production in lactating and non-lactating Holstein cows

2014 ◽  
Vol 82 (2) ◽  
pp. 318-324 ◽  
Author(s):  
L.M. Vieira ◽  
C.A. Rodrigues ◽  
A. Castro Netto ◽  
B.M. Guerreiro ◽  
C.R.A. Silveira ◽  
...  
Keyword(s):  
Holstein Cows ◽  
Embryo Production ◽  
In Vitro Embryo Production

scholarly journals Effect of Different Finishing and Polishing Methods for Color Stability of the E-Max Dental Ceramics: In vitro Study

2021 ◽  
Vol 14 (4) ◽  
pp. 1508-1513
Author(s):  
Ibraheem F Alshiddi
Keyword(s):  
Surface Brightness ◽  
Color Stability ◽  
Color Measurement ◽  
Control Group ◽  
Group 4 ◽  
Significant Difference ◽  
Group 3 ◽  
Group 2 ◽  
Group 1

In order to assess the influence of finishing and polishing on the surface brightness and color stability of the ceramic veneer, fifty specimens were fabricated with 10 mm diameter and 2 mm thickness using IPS E-Max Ceramic. After glazing, 10 specimens were untouched as control group, and the other 40 specimens were abraded using 125µm diamond bur to create surface roughness. Forty specimens were divided into four groups (n=10), in group 1: specimens were finished using diamond point, in group 2 specimens’ surface was polished with a polishing kit, Group 3: Each specimen surface was polished with the polishing kit as in protocol 2 and was polished a polishing past and group 4 Each specimen was glazed by heating at 621℃ for 3 minutes followed by a temperature increase of 83℃/min up to 918℃ for 30 seconds. Color measurement was performed using spectrophotometer. Color stability data were analyzed using two-way ANOVA and Tukey’s HSD test (α=0.05). For Ra values, paired-samples t-tests were used to analyze the data and compare groups. The change in L and E showed a significant difference among the study groups; (group 1, group 2, group 3 and group 4) with respect to three variables L, a and b. A significant difference was noted when compared each group with the control; however, only group 2 showed a significant difference from group 4; the remaining groups demonstrated similar findings for all three variables. The study displayed a significant impact of the finishing and polishing technique on the surface brightness and color stability of ceramic restoration. However, it was evident that combination of two or three polishing techniques which includes polish kit and glaze enhances the surface finish and adds color stability by alternating the yellow – blue axis (increase in b) and red- green axis (decrease in a).

scholarly journals Transvaginal ultrasound-guided oocyte aspiration for production of embryos <i>in vitro</i>

2002 ◽  
Vol 45 (1) ◽  
pp. 99-108
Author(s):  
J. A. Carter ◽  
S. Bellow ◽  
M. Meintjes ◽  
O. Perez ◽  
E. Ferguson ◽  
...  
Keyword(s):  
Transvaginal Ultrasound ◽  
Farm Animals ◽  
Embryo Research ◽  
Ultrasound Guided ◽  
Cyclic Activity ◽  
Embryo Production ◽  
Early Postpartum ◽  
In Vitro Embryo Production ◽  
Oocyte Aspiration

Abstract. reproductive potential in genetically valuable animals (BEAL et al., 1992). Now that repeatable oocyte retrieval methods are being fine-tuned, it is likely these procedures will become routinely used to obtain oocytes for further gamete and embryo research and also by seedstock producers for in vitro embryo production from farm animals in the commercial sector. The use of transvaginal ultrasound-guided oocyte aspiration and IVF procedure does offer an alternative to cattle producers who have genetically valuable cows that for some reason are unable to produce viable embryos through standard embryo collection procedures. This technology can be used on oocytes harvested from older ovulating or nonovulating cows, females with physical injuries (e.g., fractured leg) and problem cows having an abnormal cervix. Good success has been reported using IVF procedures on oocytes obtained from supplemental follicles of cows with cystic ovarian disease. With IVF the potential exists for more embryos to be produced in a shorter period of time, since the procedure can be repeated on the same cow 3 to 4 times or more a month. At this station, we are harvesting oocytes from early postpartum (< 40 days) beef and dairy cattle, before the female begins cyclic activity. The approach allows the opportunity to produce one or more extra calves from the cow before she is mated for a natural pregnancy. Currently, transvaginal ultrasound-guided oocyte aspiration is now being used to harvest valuable oocytes from minor farm animal breeds, from domestic females representing rare bloodlines, clinically infertile females and reproductively senescent cows. Research continues to find applications for this technology, including harvesting oocytes from young prepubertal heifers and early postpartum beef cows for in vitro embryo production. The use of ultrasound-guided oocyte aspiration should not be overlooked to obtain oocytes for in vitro embryo production and to aid in germplasm preservation of endangered exotic species.

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