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<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<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>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>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.)

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.

The relation between ovarian reserve and In-vitro fertilization success

2016 ◽  
Vol 1 (1) ◽  
pp. 8-15 ◽  
Author(s):  
Prashant Purohit ◽  
◽  
Mike Savvas ◽  
Keyword(s):  
In Vitro Fertilization ◽  
Ovarian Reserve ◽  
Fertilization Success ◽  
Vitro Fertilization

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.

scholarly journals A Potential Relationship Between Estrogen Receptors Polymorphisms, Sperm Function and in vitro Fertilization Success: A Preliminary Study*

2021 ◽  
Vol 75 ◽  
pp. 304-317
Author(s):  
Joanna Talarczyk-Desole ◽  
Mirosław Andrusiewicz ◽  
Małgorzata Chmielewska ◽  
Anna Berger ◽  
Leszek Pawelczyk ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Estrogen Receptors ◽  
Strong Association ◽  
Fertilization Success ◽  
World Health ◽  
Semen Parameters ◽  
Sperm Function ◽  
Allele Distribution ◽  
Vitro Fertilization

Background: Estrogen receptor 1 (ESR1) and 2 (ESR2) play an important role in regulating fertility in the human reproductive system. Polymorphisms of these receptor genes have been implicated in male infertility in both Chinese and Caucasian populations. However, studies have produced inconsistent results. Spermatozoa defects that result in conception deficiencies could be related to estrogens, their receptors, or genes involved in estrogen-related pathways. This study aims to explore the potential association between the ESR1 and the ESR2 polymorphisms in relation to semen parameters of Caucasian males as well as fertilization success. Materials/Methods: A total of 116 males were included in this study. Forty couples underwent conventional in vitro fertilization, while 76 couples were treated by intracytoplasmic sperm injection. Standard semen analyses were performed according to the World Health Organization criteria. Polymerase chain reaction and restriction fragment length polymorphisms were used to determine genotype and allele distributions. Results: A strong association between the ESR1 rs2234693 recognized by PvuII enzyme, genotype/allele distribution and fertilization success was shown. The T allele occurrence was significantly lower in the case of fertilization failure (p = 0.02). Additionally, the TT genotype was absent in the same group (p=0.02). In the case of the remaining analyzed polymorphisms, little to no interdependence of genotype/allele distribution and fertilization success was noted. Conclusions: Apart from ESR1 rs2234693, the study failed to demonstrate that fertilization success was associated with the selected polymorphisms. In most cases, we did not discover a relationship between both estrogen receptors polymorphisms and sperm function.

scholarly journals Features of the preparation of biological material for genome editing in cattle

2019 ◽  
Vol 191 (12) ◽  
pp. 40-44
Author(s):  
A. Barkova ◽  
M. Modorov ◽  
G. Isaeva ◽  
A. Krivonogova
Keyword(s):  
Genome Editing ◽  
Biological Material ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Expected Time ◽  
Donor Material ◽  
Material Transportation

Abstract. To carry out genome editing in cattle, an effective and well-functioning system for obtaining gametes, fertilizing eggs and their cryopreservation is necessary. Aim of the work: review and research of present-day existing methods of obtaining, insemination and cryopreservation of donor material, in order to provide genome editing in cows. Methods and materials. The work is completed according to the theme No. 0532-2019-0001 “Development of complex technology of marker-based genome selection of agricultural animals” within State Order of Ministry of Education and Science of the Russian Federation. The analysis of open scientific literature on the issues of in vitro fertilization in animals, cryopreservation of oocytes and embryons, sperm preparation and methods of insemination of cows’ oocytes, and cryopreservation of oocytes and embryons of animals is done. Features of the preparation of biological material of cattle for genome editing by microinjection into ooplasm are described. Results of research and duscussion. At present time there are two ways to obtain donor material from cattle: from live animals and taking ovaries after slaughtering cows. Material transportation is carried out at a temperature of 30–37 °C depending on the distance to the laboratory and expected time period of transportation. Oocyte-cumulus complexes can be removed by ovarian dissection and aspiration of visible follicles. In both cases, immature eggs are predominantly obtained. Subsequent ripening is carried out in vitro using special media in a CO2 incubator. The culture medium for oocyte maturation should contain hormones that mimic the peak of LH (luteinizing hormone), which occurs in vivo during the maturation of oocytes before ovulation. To accumulate a certain number of eggs at the stage of MII, it is recommended to carry out their cryopreservation by the method of vitrification, having previously released the oocyte from the cumulus cells. After thawing, oocytes need to be incubated for 2–3 hours 38.5 °C in 5–6.5% CO2 to restore the spindle. In order to make editing more effective, the introduction of genetic material is recommended to be carried out in parallel with the fertilization method “icsi”. In humans, mice and rabbits, an injection of sperm into the cytoplasm is sufficient to activate the oocyte, however, in cattle, just micro-injection of the sperm is not enough and often the male pronucleus does not form. To solve the problem, various methods are used, including freezing-thawing of sperm, resulting in damage of a membrane, or addition of heparin-glutathione into the medium that increases decondensation of the sperm DNA.

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