In vitro fertilization and development of mouse follicular oocytes matured in TYH medium supplemented with FSH and/or 5% fetal calf serum.

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

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

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab68 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab213 ◽

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.)

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Evaluation of the sperm separability of blanc-blue-belge bull by swim-up method and in vitro embryo production with hybrid Zebu bovine oocyte in Vietnam

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/18/1/15265 ◽

2020 ◽

Vol 18 (1) ◽

pp. 59-66

Author(s):

Nguyen Huu Duc ◽

Pham Thu Giang ◽

Tran Thi Binh Nguyen ◽

Bui Dai Phong

Keyword(s):

In Vitro Fertilization ◽

Calf Serum ◽

Basic Medium ◽

Embryo Production ◽

Vitro Fertilization ◽

Bovine Sperm ◽

Maturation Medium ◽

In Vitro Embryo Production ◽

The Right

The objective of this study was to determine the right conditions for the separation of Blanc-Blue-Belge bovine sperm (BBB) by swim-up mothed; determine the maturity of hybrid Zebu bovine eggs; and culture of embryos after in vitro fertilization. After 60-80 minutes of swim-up in CAP-05, BBB bovine sperms were healthy, straight movement and separated with a concentration of 106 sperm/ml. Hybrid Zebu bovine eggs developed and matured in the maturation medium with the basic medium TCM-199 supplemented with 10% calf serum, FSH (0.75 µg / ml), LH (0.15 µg / ml) and Estradiol (2.5 µg / ml), the results showed that the IVM-08 medium had significantly higher maturation rates than IVM-03, the proportion of mature eggs reached 71,11% compared to 51.69%, respectively (P <0.01). In vitro fertilization of hybrid Zebu bovine egg in IVF-08 medium. In vitro fertilized embryos (BBB x hybrid Zebu) developed from bovine sperms separated by the swim-up method achieved a better rate of morula-blastocysts in IVC-09 than IVC-06 medium, 21.68% compared to 8.56%, respectively (P <0.01). The conclusion was that the suitable conditions for BBB bovine sperm separation and in vitro embryo production (BBB x hybrid Zebu) were determined. This is the premise to create bovine semen, BBB bovine embryos with defined gender.

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183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab183 ◽

2014 ◽

Vol 26 (1) ◽

pp. 206 ◽

Cited By ~ 1

Author(s):

S. Chastant-Maillard ◽

K. Reynaud ◽

S. Thoumire ◽

M. Chebrout

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Fetal Calf Serum ◽

Selection Process ◽

Calf Serum ◽

Germinal Vesicle ◽

Sperm Head ◽

Cell Stage ◽

Vitro Fertilization

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).

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208 TIME LAPSE CINEMATOGRAPHIC ANALYSIS OF CLEAVAGE AND BLASTULATION IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab208 ◽

2009 ◽

Vol 21 (1) ◽

pp. 202

Author(s):

K. Imai ◽

T. Somfai ◽

Y. Inaba ◽

Y. Aikawa ◽

M. Ohtake ◽

...

Keyword(s):

In Vitro Fertilization ◽

Calf Serum ◽

Development Program ◽

Time Lapse ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Blastocyst Development ◽

Vitro Fertilization ◽

Cell Divisions

Since the 1980s, several different bovine in vitro embryo production systems have been developed, and more than 291 000 embryos have been transferred throughout the world (Thibier M 2007 IETS Newsletter 25(4), 15–20). However, we have limited knowledge about the cleavage pattern of the first, second, and third cell divisions and the developmental activities of embryos during in vitro culture (IVC). The present study was conducted to determine the developmental activities of bovine embryos obtained by ovum pickup (OPU), in vitro maturation (IVM), and in vitro fertilization (IVF). We analyzed embryonic development by time-lapse cinematography (TLC). A total of 92 cumulus–oocyte complexes were collected by OPU from Japanese Black cows and were subjected to IVM and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52(Suppl.), S19–S29). Inseminated oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 in air at 38.5°C. Kinetics of embryo development were measured by TLC for 168 h after IVF by using a Cultured Cell Monitoring System (CCM–M1.4ZS, Astec, Fukuoka, Japan). A total of 672 photographs of the embryos were taken (1 photograph every 15 min) during IVC. Image stacks were analyzed by the CCM–M1.4 software. Timing of the first, second, and third cell divisions, blastulation, and embryonic contractions were recorded. The results are reported as time (h) passed after insemination. In total, 75 (81.5%) embryos cleaved and 61 (66.3%) embryos developed to the blastocyst stage. The first, second, and third cell divisions in these viable embryos occurred at 24.0 ± 0.5, 32.1 ± 0.2, and 39.4 ± 0.4 h (mean ± SE) after IVF, respectively. On the other hand, in nonviable embryos (those that failed to develop to the blastocyst stage; n = 14), these cell divisions occurred at 29.5 ± 2.2, 41.3 ± 3.3, and 57.2 ± 7.6 h after IVF, respectively. There tended to be a difference (P = 0.06; paired t-test) in the timing of the first cell division between viable and nonviable embryos. Blastulation of embryos began at 114.4 ± 1.1 h, embryos developed to the blastocyst stage at 127.3 ± 1.4 h, and blastocysts began to expand at 138.4 ± 1.7 h after IVF, respectively. During blastocyst development, embryonic contractions (shrinkage attributable to the rupture of the blastocoele) and tight-shrinkage (shrinking of the embryo to less than 70% of its surface area) were observed in all embryos. The mean numbers of contractions and tight-shrinkages in blastocysts were 5.3 ± 2.7 and 2.1 ± 1.0 times, respectively. The frequency of contractions from the beginning of blastulation to the blastocyst stage was significantly lower (P < 0.01) than after the blastocyst stage. It took 6.9 ± 4.6 h for the embryos to re-expand after the tight-shrinkages. These results indicate that viable in vitro-produced embryos can be selected at early stages by TLC. Further studies are necessary to clarify the importance of the pulsating activity in OPU–IVF embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

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Meiotic Status Does Not Affect the Vitrification Effectiveness of Domestic Cat Oocytes

Animals ◽

10.3390/ani10081371 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1371

Author(s):

Natalia Sowińska ◽

Jennifer Zahmel ◽

Wojciech Niżański ◽

Romy Hribal ◽

Lorena Fernandez-Gonzalez ◽

...

Keyword(s):

In Vitro Maturation ◽

Fetal Calf Serum ◽

Calf Serum ◽

Toxicity Assessment ◽

Domestic Cat ◽

Developmental Potential ◽

Vitro Fertilization ◽

Negative Effect ◽

Meiotic Competence

Cryopreservation is important for animal fertility and biodiversity. Unfortunately, cryopreservation of feline oocytes is still an experimental technique. The aims of this study were to analyze the potential toxicity of the cryoprotectants in the vitrification solution (VS) on cat oocytes and to investigate whether the meiotic status of oocytes influences their developmental potential after vitrification. Two experiments were conducted with the VS composed of 20% ethylene glycol, 20% dimethyl sulfoxide, 20% fetal calf serum, 1.5 M trehalose, and 10% Ficoll PM-70: (1) toxicity assessment of the VS on immature cumulus oocyte complexes (COCs), and subsequently in vitro maturation (IVM) and in vitro fertilization; (2) assessment of the influence of the meiotic status on vitrification effectiveness, where immature and in vitro matured COCs were vitrified on the Cryotop. After rewarming, vitrified oocytes were subjected to IVM (immature) and intracytoplasmic sperm injection (ICSI) with fresh epididymal sperm. The toxicity test revealed no negative effect of oocyte exposure to the applied VS on their developmental potential (p > 0.05). Although the vitrification procedure itself significantly reduced the meiotic competence of oocytes, their meiotic status before vitrification (immature vs. in vitro matured) did not influence fertilization and morula rates. The only parameter affected by vitrification was the rate of oocytes suitable for ICSI, which was significantly lower for immature oocytes. Regardless of the meiotic status of vitrified oocytes, morphologically normal morulae were obtained. Moreover, the two meiotic stages examined are suitable for vitrification, with mature oocytes being a better choice when a well-equipped laboratory is available.

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