scholarly journals 331INSEMINATION OF OOCYTES WITH AGED SEMEN ALTERS SEX RATIO OF BOVINE EMBRYOS PRODUCED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 285
Author(s):  
J.L. Edwards ◽  
P. Bredbacka ◽  
A.M. Saxton ◽  
F.N. Schrick
Keyword(s):  
Sex Ratio ◽  
Sperm Motility ◽  
Block Design ◽  
Water Bath ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Frozen Semen ◽  
Effects Of Aging

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P<0.05) by Fisher’s Exact test. Bull or replicate variation was noted but the direction of aging for increasing proportion of females was consistent. Preliminary observations suggest that biological differences between X- and Y-bearing sperm may exist such that alternative strategies for altering sex ratio in livestock species may be possible.

scholarly journals Effect of Ram Breed on the Efficiency of in vitroDevelopment of Sheep Embryos

2017 ◽  
Vol 14 (4) ◽  
pp. 1309-1313
Author(s):  
Y. Al-Anazi ◽  
M. G. Al-Mutary ◽  
M. M. Alfuraiji ◽  
M. Al-Ghadi ◽  
A. R. Al-himaidi ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Cell Stage ◽  
In Vitro Development ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Stage 4 ◽  
Before And After ◽  
Vitro Development ◽  
Fresh Semen

The aim of this work was to investigate the impacts of ram breed on in vitro embryo development from fresh or frozen semen. Semen was collected from Najdi and Naimi rams and frozen; the mass and progressive motility of the spermwere assessed in each trial before and after freezing. Then, 970 oocytes in six replicates were fertilized with fresh and frozen semen in vitro. Different stages of sheep embryos were recorded. There were no significant differences in mass and progressive sperm motility of fresh or frozen ram semen between Najdi and Naimi,but there were significant differences between frozen and fresh semen within each breed. Our results showed significant (P<0.05) differences in 2-cell stage, 4-cell stage, 8-cell stage, morula, fragmented embryos, cleavage and blastocyst rates in the frozen semen group compared to fresh semen group in both breeds. In addition, significant (P<0.05) differencesbetween the two breeds were shown in 8-cell and16-cell embryonic stages.In conclusion, there were slight breed effects on the efficiency of in vitro development of sheep embryos.

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 146
Author(s):  
K. E. Brooks ◽  
B. L. Daughtry ◽  
S. S. Fei ◽  
M. Y. Yan ◽  
B. Davis ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Rna Sequencing ◽  
Live Cell ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage

Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

89 Influence of sperm preincubation on development and sex ratio of in vitro-produced bovine embryos

2022 ◽  
Vol 34 (2) ◽  
pp. 281
Author(s):  
A. Fries ◽  
B. Zimmer ◽  
B. Rabenau ◽  
F. Kotarski ◽  
C. Wrenzycki
Keyword(s):  
Sex Ratio ◽  
Bovine Embryos

scholarly journals 183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 213 ◽  
Author(s):  
J. Small ◽  
M. Colazo ◽  
D. Ambrose ◽  
R. Mapletoft ◽  
J. Reeb ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Animal Health ◽  
Beef Cows ◽  
Water Bath ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Frozen Embryos

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

scholarly journals Effect of Linoleic Acid-Albumin on Post-Thaw Survival of in vitro-Produced Bovine Embryos at the 16-Cell Stage.

2000 ◽  
Vol 62 (4) ◽  
pp. 465-467 ◽  
Author(s):  
eiichiro KTOMINAGA ◽  
Yukako HAMADA ◽  
Tsuyoshi YABUUE ◽  
Tetsushi ARIYOSHI
Keyword(s):  
Linoleic Acid ◽  
Bovine Embryos ◽  
Cell Stage

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

264 TESTOSTERONE IN THE OOCYTE CULTURE DOES NOT ALTER SEX-RATIO OF IN VITRO PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 229
Author(s):  
C. Díez ◽  
P. Bermejo-Alvarez ◽  
A. Gutiérrez-Adan ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Bovine Embryos ◽  
Experimental Conditions ◽  
Vitro Fertilization ◽  
Grant Support ◽  
High Concentrations

The production of sex-known offspring is a main objective in reproductive biotechnology. It has been reported that bovine ova developed in follicles with high concentrations of testosterone in vivo yielded significantly more male embryos in vitro (Grant V et al. 2008 Biol. Reprod. 78, 812–815). In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in fully in vitro conditions. Immature bovine cumulus–oocyte complexes (COCs; n = 750) from slaughterhouse ovaries were cultured in 199 HNaCO3 with polyvinyl alcohol (PVA) 0.1 mg mL–1 as a basic medium. Culture was made in two steps, a 24 h meiotic arrest (roscovitine 25 μm), and a subsequent in vitro maturation period with FSH-LH for 24 h. Testosterone (T-86500, Sigma-Aldrich, St. Louis, MO, USA) was added throughout the entire oocyte culture at 0, 30, 300, and 1500 nm. After in vitro fertilization (Day 0), zygotes were freed of cumulus cells by pipetting, and subsequently cultured in SOF + 6 g L–1 BSA up to Day 3. At this time, embryo development was recorded, and all embryos having 3 or more cells were treated with pronase to remove the zona pellucida. Zona-free embryos were washed in PBS containing PVA 0.1 mg mL–1 and individually frozen at –80°C until sex analysis by PCR (Bermejo-Alvarez P et al. 2008 Biol. Reprod. doi:10.1095/biolreprod.108.070169). A total of 252 embryos from 5 replicates were sexed. Data for development and sex-ratio are presented as % LSM ± SD. There were no interactions between testosterone treatment, embryonic sex, and embryonic stage analyzed. Testosterone did not affect development rates (P > 0.05) at any stage: cleavage (47.8 ± 6.8, 56.5 ± 6.8; 50.9 ± 6.8; 62.2 ± 6.8), 3 to 4 cells (40.6 ± 5.2, 45.8 ± 5.2; 37.8 ± 5.2; 47.7 ± 5.2) and >5 cells rates (24.5 ± 4; 27.3 ± 4; 21.3 ± 4; 25.3 ± 4) for 0, 30, 300, and 1500 nm testosterone, respectively. Cumulative percentages of male embryos were as follows: 53 ± 8 (n = 56), 42.6 ± 8 (n = 52), 53.6 ± 6 (n = 81) and 57.6 ± 8 (n = 63) for 0, 30, 300, and 1500 nm groups respectively (P > 0.05). These results show that the testosterone effects on oocyte ability to select Y-chromosome bearing spermatozoa are not reproducible in vitro under the present experimental conditions. Grant support: MEC, project AGL2008-01530; RTA2008-0082; M. Muoz is supported by FICYT.

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