68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

86 EFFECT OF CRYOPROTECTANT EXPOSURE, VITRIFICATION, AND WARMING TIME OF BOVINE CUMULUS OOCYTE COMPLEXES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
R. J. Mapletoft ◽  
M. Anzar
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Time Interval ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Vitro Fertilization ◽  
Vitrification Solution ◽  
High Concentrations

Despite the importance of cryoprotectants for avoiding ice crystal formation, the high concentrations required for vitrification may be toxic to bovine oocytes. During warming (thawing), the removal of permeating cryoprotectants from cells can lead to osmotic injury, and the most appropriate time interval for warming and cryoprotectant removal from vitrified oocytes is currently uncertain. The present study aimed to evaluate the effect of cryoprotectant exposure, vitrification, and warming time of bovine cumulus oocyte complexes (COC) on fertilization and ability to develop as embryos in vitro. Follicles <8 mm in diameter were aspirated from slaughterhouse-derived bovine ovaries. Cumulus oocyte complexes with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed 3 times in Dulbecco’s PBS + 5% newborn calf serum (CS), and randomly divided into 4 groups: 1) control group: no treatment; 2) VS1 group: COC were exposed to vitrification solution 1 [VS1: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in TCM-199 + 20% CS] for 5 min; 3) VS1+VS2 group: COC were exposed to VS1 for 5 min followed by vitrification solution 2 (VS2: 15% EG, 15% DMSO, and 0.5 M sucrose in TCM-199 + 20% CS) for 30 s; and 4) vitrified group: COC were exposed to VS1 and VS2, and then vitrified in liquid nitrogen using cryotops. The COC in VS1, VS1+VS2, and vitrified groups were exposed to a warming solution (0.5 M sucrose in TCM-199) for 1 or 5 min. The COC from all groups were in vitro matured (IVM) for 22 h in TCM-199 containing 5% CS, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin at 38.5°C, 5% CO2, and high humidity, incubated with frozen–thawed sperm in Brackett-Oliphant capacitating medium for 18 h, and the presumptive zygotes were cultured in Charles Rosenkrans 1 amino acids (CR1aa) + 5% CS for 9 days. Data were analysed using Proc Glimmix in SAS® 9.2 (SAS Institute Inc., Cary, NC, USA). Cleavage and blastocyst rates in the vitrified group (25 and 2%, respectively) were significantly lower (P < 0.0001) than in control (75 and 27%), VS1 (68 and 19%), or VS1+VS2 (63 and 22%) groups. Cleavage and blastocyst rates did not differ among non-vitrified groups (P > 0.05). In VS1, VS1+VS2, and vitrified groups, warming time had no effect on cleavage or blastocyst rates (P > 0.05). In conclusion, although cryoprotectant exposure and warming times had no apparent adverse effect, vitrification of bovine COC drastically reduced cleavage and blastocyst rates. Further studies are required to understand how vitrification of bovine COC affects subsequent fertilization and embryo development. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals 213EFFECTS OF TIME OF MATURATION AND SHEEP SERUM ON IN VITRO FERTILIZATION IN THE ENDANGERED ELD'S DEER (CERVUS ELDI THAMIN) OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 228
Author(s):  
B. Siriaroonrat ◽  
P. Comizzoli ◽  
N. Songsasen ◽  
R.E. Spindler ◽  
S.L. Monfort ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Calf Serum ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Fertilization Success ◽  
Estrous Cycles ◽  
Vitro Fertilization ◽  
Offspring Production ◽  
Oviduct Fluid

The Eld’s deer, native to Southeast Asia, is threatened with extinction. Although artificial insemination is effective for offspring production, in vitro fertilization (IVF) would be more useful for rapidly disseminating genetic material from valuable founders. The objectives of this study were to: 1) determine if oocytes recovered from exogenous gonadotropin-treated hinds require additional in vitro maturation;; and 2) assess if fertilization is enhanced by supplementing Deer Synthetic Oviduct Fluid (DSOF;; Berg DK et al., 2003 Theriogenology 59, 189–205) with 1-day postestrus sheep serum (SS). Estrous cycles in Eld’s deer hinds (n=10) were synchronized with PGF2α analog (Lutalyse™, 500mg), followed by a 14-day intravaginal CIDR-G insertion;; ovine FSH (Ovagen™; 0.05 unit×8 injections) was administered at 12-h intervals beginning 84h before CIDR-removal. COCs (n=160) were retrieved laparoscopically 40–46h post-CIDR-removal and either fixed or matured in vitro (for 12h v. 24h) in TCM-199 (Earle’s salt) supplemented with 0.33mM pyruvate, 2mM glutamine, 100IUmL−1 penicillin, 100μgmL−1 streptomycin, 10% fetal calf serum, 5μgmL−1 FSH and LH and 1μgmL−1 E2 (5% CO2, 38.5°C). After 12- or 24-h IVM, cumulus cells were partially removed and oocytes (n=110) fertilized in DSOF with pooled frozen-thawed sperm (3 males;; 2×106 motile sperm mL−1), in the absence or presence of SS (20%, v/v). Additional oocytes (n=18) were used for parthenogenetic control. At 20-h postinsemination, presumptive zygotes were fixed and stained (Hoechst 33342) to assess fertilization success (presence of two pronuclei). Data were analyzed by ANOVA. Overall, 16.0±2.6 (mean±SEM) COCs were recovered/female. The majority of COCs were of excellent quality (grade I; 67.7±3.8%). At time of aspiration, 85% of the oocytes (n=11/13) were in metaphase I stage, 7.5% in telophase and 7.5% degenerate. No parthenogenic activation was observed. Likewise, no polyspermy was observed in any treatment. Fertilization was higher (P&lt;0.05) in oocytes matured for 24h and fertilized in the absence (64.4±3.1%) compared to presence (26.9±11.2%) of SS. In the absence of SS, a higher (P&lt;0.05) proportion of oocytes were fertilized after 24h (64.4±3.1%) compared to 12h (27.1±9.0%) IVM. There was no effect (P&gt;0.05) of SS on fertilization among oocytes subjected to 12-h IVM (27.1±9.0% v. 12.5±9.5%). When SS was present during fertilization, no difference (P&gt;0.05) was observed among oocytes matured for 12 or 24h. Results demonstrate that: 1) Eld’s deer oocytes require an additional 24-h IVM to complete maturation;; 2) DSOF supports sperm-oocyte interaction;; and 3) SS is not essential for successful fertilization. (Supported by Morris Animal Foundation.)

scholarly journals EFFECT OF VAGINAL SILDENAFIL ON IN VITRO FERTILIZATION SUCCESS RATES IN WOMEN WITH PREVIOUS FAILED IN VITRO FERTILIZATION ATTEMPTS

2018 ◽  
Vol 11 (6) ◽  
pp. 486 ◽  
Author(s):  
Ensieh Shahrokh Tehraninejad ◽  
Noushin Khazei ◽  
Elnaz Ayati ◽  
Ali Movafegh ◽  
Omid Azimaraghi
Keyword(s):  
Clinical Trial ◽  
In Vitro Fertilization ◽  
Endometrial Thickness ◽  
Pregnancy Rates ◽  
Control Group ◽  
Vitro Fertilization ◽  
Group A ◽  
The Mean ◽  
Group B

Objectives: Endometrial thickness of <9 mm is a predictor of in vitro fertilization (IVF) failure, although neither pregnancy rates nor the pregnancy outcomes are dependent on the endometrial thickness alone. The impact that uterine artery blood flow has on endometrial growth is dependent on nitric oxide which concentrations could be altered by halting a cyclic guanosine monophosphate-mediated pathway with a phosphodiesterase type 5 selective inhibitor such as sildenafil.Methods: In this clinical trial, 72 patients aged below 45 years which have had at least two earlier failed IVF attempts were randomly split into two groups each consisting of 36 patients. Both groups were started on a long IVF protocol. The case group was also administered 100 mg vaginal sildenafil suppositories daily, starting on day 3 of menstruation which was continued until human chorionic gonadotropin administration. Endometrial thickness was measured using ultrasonography in both groups plus pregnancy rates were assessed in both groups.Results: The mean age of the patients in Group A who received sildenafil; in this clinical trial, 72 patients aged below 45 years which have had at least two previous failed IVF attempts were randomly split into two groups each consisting of 36 patients was 33.8±4.8 in contrast to Group B (control group) with the mean age of 33.8±4.8. Mean endometrial thickness of 8.6±0.1 mm was recorded in Group B compared to 9.0±0.7 mm in Group A (p=0.03). Of all the 36 participants who received sildenafil citrate during the IVF cycle, 12 (33.3%) patients had successful pregnancies while 24 (66.7%) failed to get pregnant. In the control group, out of the 36 participants, 10 (27.8%) patients got pregnant while 26 (72.2%) failed the cycle (p=0.9).Conclusion: This study showed that although using vaginal sildenafil during the IVF cycle does improve endometrial thickness before implantation, this does not necessarily lead to higher pregnancy rates.

Levels interleukine-4 and interleukin-17 (IL-4, IL-17) of blood in patients with habitual recurrent pregnancy loss, which occurred in a loop in vitro fertilization

2016 ◽  
pp. 137-139
Author(s):  
K.P. Golovatyuk ◽  
Keyword(s):  
In Vitro Fertilization ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Recurrent Miscarriage ◽  
Interleukin 4 ◽  
Control Group ◽  
Interleukin 17 ◽  
Vitro Fertilization ◽  
Blood Mononuclear Cells

The objective: was to investigate the levels of cytokines IL-4 and IL-17 in serum and conditioned medium cultures of blood mononuclear cells (MNC) and evaluation association between their products and miscarriage, which occurred in IVF cycles. Patients and methods. We observed 240 patients with recurrent miscarriage, came in IVF cycles, and 100 apparently healthy fertile women in the control group. The concentrations of IL-4 and IL-17 in serum and conditioned medium of MNC cultures were determined. Results. The levels of IL-4 in the serum and conditioned medium in spontaneous and stimulated mitogen secretion was not significantly different from those in the control group, whereas IL-17 levels were higher than those in the control group serum, in conditioned media of stimulated and non-stimulated MNCs. Conclusion. Disregulation of activity of circulating blood mononuclear cells in women with recurrent miscarriage that followed IVF, is accompanied by increased secretion of IL-17 and almost constant production of IL-4 on the back of high stimulation index of production of these cytokines. Key words: in vitro fertilization, miscarriage, interleukin-4, interleukin-17, serum stimulated and non-stimulated mononuclear blood.

scholarly journals A Brief Incubation of Cumulus-Enclosed Mouse Eggs in a Calcium-Free Medium Containing a High Concentration of Calcium-Chelator Markedly Improves Preimplantation Development

2020 ◽  
Vol 17 (10) ◽  
pp. 3505
Author(s):  
Valeria Merico ◽  
Silvia Garagna ◽  
Maurizio Zuccotti
Keyword(s):  
In Vitro Fertilization ◽  
Mammalian Species ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Preimplantation Development ◽  
High Concentration ◽  
Vitro Fertilization ◽  
Positive Effects

The presence of cumulus cells (CCs) surrounding ovulated eggs is beneficial to in vitro fertilization and preimplantation development outcomes in several mammalian species. In the mouse, this contribution has a negligible effect on the fertilization rate; however, it is not yet clear whether it has positive effects on preimplantation development. Here, we compared the rates of in vitro fertilization and preimplantation development of ovulated B6C3F1 CC-enclosed vs. CC-free eggs, the latter obtained either after a 5 min treatment in M2 medium containing hyaluronidase or after 5–25 min in M2 medium supplemented with 34.2 mM EDTA (M2-EDTA). We found that, although the maintenance of CCs around ovulated eggs does not increment their developmental rate to blastocyst, the quality of the latter is significantly enhanced. Most importantly, for the first time, we describe a further quantitative and qualitative improvement, on preimplantation development, when CC-enclosed eggs are isolated from the oviducts in M2-EDTA and left in this medium for a total of 5 min prior to sperm insemination. Altogether, our results establish an important advancement in mouse IVF procedures that would be now interesting to test on other mammalian species.

Salivary oestradiol and progesterone after in vitro fertilization and embryo transfer using different luteal support regimens

1990 ◽  
Vol 2 (4) ◽  
pp. 351 ◽  
Author(s):  
YF Wong ◽  
EP Loong ◽  
KR Mao ◽  
PP Tam ◽  
NS Panesar ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Transfer ◽  
Luteal Phase ◽  
Biologically Active ◽  
Luteal Support ◽  
Vitro Fertilization ◽  
Group A ◽  
Group B ◽  
Ivf Et

Salivary oestradiol (E2) and progesterone (P) levels have been shown to reflect the biologically active fractions in the serum. The luteal-phase status of stimulated cycles was investigated after in vitro fertilization and embryo transfer (IVF-ET). Thirty patients were randomly allocated to one of three luteal therapy groups: group A had no support, group B had intramuscular P and group C had intramuscular P and human chorionic gonadotrophin (hCG). One pregnancy was achieved in group A, two in group B and three in group C. Significant correlations between salivary and serum levels of E2 and of P in matched samples during luteal phase were found. Salivary E2 levels from luteal day 8 through day 14 and P levels from day 3 through day 14 were significantly higher in the pregnant than in the nonpregnant cycles. Among the nonpregnant cycles, salivary E2 and P levels were significantly higher in group C than in group A or B. These findings suggest that, in stimulated cycles for IVF-ET, determination of salivary E2 and P levels may be used as reliable alternatives to serum concentrations for assessing the luteal phase. Also, the additional hCG has an enhanced luteotrophic effect, as reflected by the higher salivary E2 and P levels, which may lead to a better pregnancy rate.

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