52 VITRIFICATION OF DAY-8 EQUINE EXPANDED BLASTOCYST: AN IN VITRO COMPARISON OF DIRECT VERSUS INDIRECT MECHANICAL INTRODUCTION OF CRYOPROTECTANT

2014 ◽  
Vol 26 (1) ◽  
pp. 140
Author(s):  
F. A. Diaz ◽  
D. L. Paccamonti ◽  
K. R. Bondioli ◽  
G. T. Gentry

The cryopreservation of equine expanded blastocysts (>300 μm) has been largely unsuccessful primarily due to the low permeability of the embryo to cryoprotectants. This low permeability has been attributed to the acellular glycoprotein capsule that develops when an embryo approaches approximately 300 μm in diameter. Mechanical alternatives may provide a means to overcome the capsule barrier and the relative large embryo size to successfully cryopreserve equine embryos. The objective of this experiment was to compare re-expansion rates of vitrified equine expanded blastocysts following either direct or indirect mechanical introduction of cryoprotectants using a coaxial microinjection system (Dracula pipette). Twenty-six Day-8 expanded blastocysts were subjected to capsule puncture, cryoprotectant injection, and blastocoele fluid extraction (direct treatment) or capsule puncture and blastocoele fluid extraction (indirect treatment) before cryopreservation. The Dracula pipette incorporates the injection pipette within the holding pipette, facilitating aspiration of blastocoele fluid or injection of cryoprotectant in a single unit. A standard vitrification protocol using a final concentration of 3.4 M glycerol and 4.6 M ethylene glycol at cryopreservation was used. Vitrified embryo re-expansion was assessed following in vitro culture at 24, 48, and 72 h post-warming. Differences across treatments were analysed using the Student's t-test for re-expansion and the chi-squared test of independence for capsule loss. Pre-vitrification embryo mean diameter (mean ± standard error) for direct and indirect treatment groups were not different, 979 ± 85.6 μm and 912 ± 101.4 μm, respectively (P = 0.62). Post-vitrification embryo mean diameters were not different for the direct and indirect treatments (688 ± 63 and 662 ± 75 μm, respectively; P = 0.79). Following 72 h of in vitro culture, there was no difference in mean embryo diameter (1813.16 ± 209 μm v. 1383.88 ± 198 μm; P = 0.21), or re-expansion rates (69 v. 69%) for direct and indirect treatment groups, respectively. However, partial or total capsule loss was 69% (9/13) for direct treatment embryos compared with 30% (4/13) for indirect treatment embryos (P = 0.049). Results from this experiment demonstrate that capsule puncture and blastocoele fluid extraction before vitrification resulted in high re-expansion rates of Day-8 equine expanded blastocysts after warming. More importantly, the relatively large percentage of capsule failure when directly introducing cryoprotectant into the embryo may interfere with maternal recognition of pregnancy following embryo transfer. Nonetheless, based on the embryonic re-expansion rate of vitrified equine embryos following the indirect technique, we anticipate that a relatively high pregnancy rate can be obtained if this technique is used.

2020 ◽  
Vol 21 (12) ◽  
Author(s):  
Erma Prihastanti ◽  
ENDAH D. HASTUTI ◽  
SRI W.A. SUEDY

Abstract. Prihastanti E, Hastuti ED, Suedy SWA. 2020. Short Communication: Comparing the growth of stem explants between Citrus reticulata var. Tawangmangu and C. reticulata var. Garut using in vitro culture methods. Biodiversitas 21: 5845-5849. Several efforts have been made to preserve Citrus reticulata var. Tawangmangu and Citrus reticulata var. Garut as indigenous Indonesian mandarin cultivars, including in vitro tissue culture methods. This study aimed to determine growth differences of the stem explants of C. reticulata var. Tawangmangu and C. reticulata var. Garut,which planted on the same Murashige and Skoog (MS) media. The treatment groups were derived from different explants, grown in 4 separate culture bottles for 35 days at 25°C. The observed parameters included the percentage of explants indicating callus development browned-colored explants, and the contaminated explants. Among C. reticulata var. Tawangmangu explants, 23.53% indicated callus development, 29.42% were browned-colored explants, and 0% indicated contamination. In contrast, among the C. reticulata var. Garut explants, 0% indicated callus development, 7.14% brown-colored, and 7.14% indicated contamination. The stems explants from C. reticulata var. Tawangmangu showed a tendency to develop calluses, but the explants of C. reticulata var. Garut was able to support the growth of shoots. C. reticulata var. Tawangmangu and Garut mandarin stem explants showed differences of shoot growth because physiological conditions varied according to the variety.


2016 ◽  
Vol 28 (2) ◽  
pp. 139
Author(s):  
C. Buemo ◽  
A. Gambini ◽  
L. Moro ◽  
R. F. Y. Martin ◽  
D. Salamone

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.


2010 ◽  
Vol 22 (1) ◽  
pp. 214 ◽  
Author(s):  
J. H. Pryor ◽  
J. A. Trant ◽  
C. B. Ponchirolli-Schneider ◽  
C. R. Looney ◽  
C. R. Long ◽  
...  

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.


2017 ◽  
Vol 29 (1) ◽  
pp. 137
Author(s):  
A. Moresco ◽  
H. L. Bateman ◽  
J. Newsom ◽  
W. F. Swanson

Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN2 vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN2. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 M sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN2. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P > 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P > 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P > 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P < 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids. This study was funded by the Institute of Museums and Library Services.


2018 ◽  
Vol 30 (8) ◽  
pp. 1055 ◽  
Author(s):  
N. J. Donfack ◽  
K. A. Alves ◽  
B. G. Alves ◽  
R. M. P. Rocha ◽  
J. B. Bruno ◽  
...  

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.


2020 ◽  
Author(s):  
Jessie Wang ◽  
Abhinav Dey ◽  
Adam Kramer ◽  
Yuan Miao ◽  
Juan Liu ◽  
...  

AbstractPurposeTo determine the efficacy of nanoparticle-encapsulated FL2 siRNA (FL2-NPsi), a novel therapeutic agent targeting the Fidgetin-like 2 (FL2) gene, for the treatment of corneal alkaline chemical injury.MethodsEighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 μM, 10 μM, 20 μM) and FL2-NPsi (1 μM, 10 μM, 20 μM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical (N=10 per group), histopathologic (N=6 per group), and immunohistochemical (N=4 per group) analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro qPCR. Toxicity assays were performed to assess for apoptosis (TUNEL assay, N=3 per group) and nerve damage (whole mount immunochemical staining, N=2 per group). Statistical analyses were performed using student’s t-test and ANOVA.ResultsCompared to controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 μM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization (p=0.0003 at Day 4 and p<0.0001 at Day 8) as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p=0.917).ConclusionDose-response studies found both 10 and 20 μM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries.


2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
M. Takayama ◽  
S. Sato ◽  
Y. Nishimura ◽  
K. Imai ◽  
O. Dochi

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.


Author(s):  
Prathima S. ◽  
Savitha C. ◽  
Tejeswini KK ◽  
A GS

Background: The aim of this study was to compare the low dose of vaginal misoprostol and dinoprostone gel for the induction of labour at term of pregnancy.Methods: Retrospective comparative study was undertaken on 300 subjects with 2 parallel treatment groups. Data were analyzed using Graphpad Instat 3 McIntosh software by Student’s t test, Mann–Whitney U test, the Chi-squared test or fisher’s exact test.Results: Comparatively narrow spectrum low cost cefotaxime is as effective as more expensive commonly used amoxicillin-clavulanic acid with no significant difference of infectious morbidity.Conclusions: Less costly cefotaxime should be preferred compared to more costly triple drug regimen for prophylaxis at caesarean section.


Author(s):  
M. John Hicks

Acid-etching of enamel surfaces has been performed routinely to bond adhesive resin materials to sound dental enamel as a caries-preventive measure. The effect of fluoride pretreatment on acid-etching of enamel has been reported to produce inconsistent and unsatisfactory etching patterns. The failure to obtain an adequate etch has been postulated to be due to fluoride precipitation products deposited on the enamel surface. The purpose of this study was to evaluate the effects of fluoride pretreatment on acid-etching of carieslike lesions of human dental enamel.Caries-like lesions of enamel were created in vitro on human molar and premolar teeth. The teeth were divided into two fluoride treatment groups. The specimens were exposed for 4 minutes to either a 2% Sodium Fluoride (NaF) solution or a 10% Stannous Fluoride (SnF2) solution. The specimens were then washed in deionized-distilled water. Each tooth was sectioned into four test regions. This was carried out to compare the effects of various time exposures (0 to 2 minutes) and differing concentrations (10 to 60% w/w) of phosphoric acid (H3PO4) on etching of caries-like lesions. Standard preparation techniques for SEM were performed on the specimens.


Planta Medica ◽  
2010 ◽  
Vol 76 (12) ◽  
Author(s):  
K Graikou ◽  
H Damianakos ◽  
K Syklowska-Baranek ◽  
A Pietrosiuk ◽  
M Jeziorek ◽  
...  

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