303 EFFECT OF SEMEN BATCHES AFTER SEXING ON PRODUCTION EFFICIENCY OF BOVINE EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
T. L. G. Torregrossa ◽  
M. B. Fernandes ◽  
R. B. Prado ◽  
R. A. Vila ◽  
F. P. Elias ◽  
...  
Keyword(s):  
Production System ◽  
Production Efficiency ◽  
Animal Health ◽  
Personal Communication ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Significance Level ◽  
Sexed Semen ◽  
Developmental Rates

Within an in vitro production system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitro for 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1 FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1 LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitro for 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1 BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2 in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitro embryos according to the batch of frozen sexed semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF

97 IN VIVO AND IN VITRO EMBRYO PRODUCTION WITH Y-SEXED SORTED OR CONVENTIONAL SEMEN IN BEEF CATTLE

2014 ◽  
Vol 26 (1) ◽  
pp. 162
Author(s):  
H. Tribulo ◽  
J. Carcedo ◽  
R. Tribulo ◽  
J. Menajovsky ◽  
B. Bernal ◽  
...  
Keyword(s):  
Animal Health ◽  
T Test ◽  
High Fertility ◽  
In Vitro Production ◽  
Embryo Production ◽  
Sexed Semen ◽  
Chi Squared ◽  
In Vitro Embryo Production

An experiment was designed to evaluate in vivo and in vitro embryo production following the use of frozen–thawed conventional or Y-sexed semen from a Brangus bull with known high fertility. For in vivo embryo production, Brangus heifers (n = 12) were superovulated twice in a crossover design and inseminated with sexed or conventional semen. On Day 0, all heifers received an intravaginal progesterone device (DIB 1 g, Syntex S.A., Buenos Aires, Argentina) and 2.5 mg oestradiol benzoate and 50 mg progesterone (Progestar, Syntex S.A.) by intramuscular injection (IM). On Day 4, heifers were superstimulated with 200 mg of NIH-FSH-P1 Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada) in twice-daily decreasing doses over 4 days. In the a.m. and p.m. of Day 6, all heifers received PGF2a (Ciclase, Syntex) and DIBs were removed in the p.m.. In the a.m. of Day 8, heifers received 100 μg de Gonadolerin (Gonasyn, Syntex S.A.) and were randomly allocated to receive either one straw of conventional semen (24 × 106 sperm per dose) 12 and 24 h later or two straws of sexed semen (2.4 × 106 sperm per dose) 18 and 24 h after GnRH. Ova/embryos were collected nonsurgically on Day 15 and evaluated following IETS recommendations. Means were compared by t-test. Mean ( ± s.e.m.) number of ova/embryos, fertilized ova, and transferable embryos were 14.8 ± 2.7, 9.4 ± 1.8, and 7.1 ± 1.7 v. 16.8 ± 3.1, 9.9 ± 2.5, and 8.1 ± 2.0 for donors inseminated with conventional or sexed semen, respectively (P > 0.6). For in vitro production, oocytes were obtained from 50 ultrasound-guided follicle aspiration (OPU) sessions that was performed at random stages of the oestrous cycle and without superstimulation in 22 Brangus cows and heifers. Oocytes were classified and matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Semen samples from the same bull used for in vivo embryo production were selected using Percoll and capacitated in Fert medium and used at a final concentration of sperm/mL for nonsexed semen and 2 × 106 sperm mL–1 for sexed semen. After 16 h (sexed) or 18 h (conventional) in Fert medium, zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2 and saturated humidity for 7 days. The total number of oocytes matured and fertilized was 528 and 318 for conventional and sexed semen, respectively. Means were compared by t-test and proportions by chi-squared test. Mean (± s.e.m.) number of cleaved zygotes and blastocysts produced per OPU session did not differ between conventional (11.0 ± 1.4 and 7.1 ± 1.0) and sexed (8.7 ± 0.8 and 4.9 ± 0.7; P > 0.2) semen. However, the proportion of cleaved zygotes and blastocysts produced were significantly higher (P < 0.05) with conventional semen (61.2%; 329/538 and 39.4%; 212/538) than with sexed semen (54.4%; 173/318 and 30.8%; 98/318), respectively. In conclusion, comparable number of embryos can be obtained in vivo with sexed or conventional semen from a bull with proven high fertility. However, the proportion of blastocysts produced in vitro is likely to be reduced following the use of sexed as compared with conventional semen from the same bull.

33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor

2018 ◽  
Vol 30 (1) ◽  
pp. 156
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
B. C. Lee
Keyword(s):  
Calcium Ionophore ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Serum Progesterone ◽  
Significance Level ◽  
Chi Squared ◽  
Pregnancy And Delivery ◽  
Developmental Rates

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos. This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

264 EFFECT OF FROZEN SEMEN BATCH ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 289
Author(s):  
M. B. Fernandes ◽  
T. L. G. Torregrossa ◽  
R. B. Prado ◽  
R. A. Vila ◽  
F. P. Elias ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Development Rate ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Significance Level ◽  
Frozen Semen ◽  
Vitro Production

Within an in vitro production controlled system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effect are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation, according to the frozen semen batch used in the in vitro fertilization, identified with the date of ejaculate and its freezing. In an embryo commercial production system, over 12 months, 10 619 viable oocytes were obtained by ultrasound-guided follicular aspiration from 642 Nelore cows (Bos indicus). The oocytes were matured in vitro for 24 h in TCM-199 supplemented with 0.5 μg mL-1 FSH, 50 μg mL-1 LH, and 10% fetal bovine serum. They were then inseminated for 18 hours in IVF-TALP medium, using the semen from 4 bulls (A to D) subdivided into 4 frozen batches (I to IV) and selected by 45/90% Percoll gradient. Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium drops (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) for 163 h at 39°C in a humidified atmosphere of 5% CO2 in air. The oocyte distribution, the total number of blastocysts, and the embryo development rate by each bull and respective batch are described in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is a difference between the used batches of each bull regarding the development rate of blastocysts 163 h after IVF Therefore, there is intra-bull variation in the ability to develop in vitro embryos according to the batch of frozen semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

296 EFFECT OF GROWTH HORMONE ON EMBRYO DEVELOPMENT AND ULTRASTRUCTURE OF BOS TAURUS IVP BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
L. M. C. Pegoraro ◽  
M. N. Dode ◽  
C. F. Weissheimer ◽  
F. G. Leivas ◽  
A. Vieira ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Culture Medium ◽  
Production Systems ◽  
Bos Taurus ◽  
In Vitro Production ◽  
Assisted Reproductive Technique ◽  
Development Rates ◽  
Developmental Rates ◽  
Chi Square Analysis

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.

221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 209
Author(s):  
S. Miyashita ◽  
K. Miyata ◽  
C. Tachibana ◽  
Y. Inaba ◽  
H. Koyama ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Large Mass ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Stage Of Development ◽  
The Mean ◽  
Vitro Production

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

282 DIFFERENT CONCENTRATIONS OF FORSKOLIN FOR MEIOSIS BLOCK AND TO IMPROVE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 230
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Crocomo ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Least Squares ◽  
Control Group ◽  
In Vitro Production ◽  
Intact Cells ◽  
Significance Level ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Group Control

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P < 0.05). We conclude that all the experimental groups reached the stage of M II after the addition of forskolin and the highest concentration of forskolin caused cellular degeneration without harming embryo production on the seventh day.

175 Nobiletin enhances the quality of in vitro-matured bovine oocytes and blastocysts by altering the transcription of key developmental genes

2019 ◽  
Vol 31 (1) ◽  
pp. 212
Author(s):  
Y. N. Cajas ◽  
K. Cañón-Beltrán ◽  
M. E. González ◽  
P. Ramos-Ibeas ◽  
A. Gutierrez-Adán ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Statistical Significance ◽  
Housekeeping Genes ◽  
Cumulus Cells ◽  
Cell Junction ◽  
In Vitro Production

One of the problems associated with in vitro production of embryos in bovine is the increase in reactive oxygen species (ROS), which leads to cell alterations and death. Nobiletin is a polymethoxyflavone isolated from citrus fruits with various beneficial effects on cell cycle regulation and inhibition of ROS production. In a preliminary study, we demonstrated that supplementation of 25 or 50 µM nobiletin to the in vitro maturation (IVM) medium reduces oxidative stress and improves oocyte nuclear and cytoplasmic maturation and embryo development. Thus, in this study, we aimed to evaluate the antioxidant activity of nobiletin during IVM on bovine matured oocytes, their cumulus cells (CC), and blastocysts by quantitative changes of gene expression. Immature cumulus oocytes complexes (COC) were aspirated from ovaries of slaughtered heifers. Selected COC underwent IVM in TCM-199+10% FCS and 10ng mL−1 epidermal growth factor (EGF; Control) supplemented with 25 µM (Nob25) or 50 µM (Nob50) nobiletin (MedChemExpress, Monmouth Junction, NJ, USA) or 0.001% dimethyl sulfoxide (DMSO control), a vehicle for nobiletin dilution, in 5% CO2 in air at 38.5°C. After 24h, 50 matured oocytes/group and their CC were snap-frozen in LN2 for gene expression analysis. The remaining oocytes were fertilized (Day 0) and cultured in vitro. Blastocysts (Day 7; n=50/group) were snap-frozen in LN2 for gene expression analysis (5 replicates). The mRNA abundance of candidate genes related with oxidative stress (SOD2, CYP51); apoptosis (BAX); quality (BMP15, BMP7, CLIC1, MAPK1, ABCB1); and cell junction (GJA1) was measured by quantitative PCR; H2AFZ and 18S rRNA were used as housekeeping genes. Statistical significance was assessed by one-way ANOVA. Supplementation of IVM medium with Nob25 or Nob50 produced changes in the expression levels of genes related to oxidative stress and apoptosis during IVM compared with controls. SOD2 and CYP51 were down-regulated in oocytes and CC (P&lt;0.05) but not in blastocysts, whereas BAX was down-regulated only in CC (P&lt;0.05). Nobiletin supplementation in IVM increased the expression of MAPK1 in oocytes and blastocysts (P&lt;0.05); however, no differences were observed in CC. BMP15 for oocytes and their CC and GJA1 for CC were up-regulated in Nob25 and Nob50 groups compared with controls (P&lt;0.05). The relative abundance of CLIC1 decreased in blastocysts from both nobiletin groups compared with controls (P&lt;0.05). No significant differences in the expression in ABCB1 and BMP7 were detected. In conclusion, our results suggest that supplementation of 25 or 50 µM nobiletin to the IVM medium reduces oxidative stress in oocytes and CC, decreases CC apoptosis, and provokes positive changes in the expression of genes related to oocyte and embryo quality. This research was supported by Spanish MINECO (AGL2015-70140-R and AGL2015-66145-R). Y. N. Cajas was supported by a grant from SENESCYT-Ecuador.

A review of the efficacy of mycotoxin detoxifying agents used in feed in light of changing global environment and legislation

2016 ◽  
Vol 9 (3) ◽  
pp. 419-433 ◽  
Author(s):  
E. Wielogórska ◽  
S. MacDonald ◽  
C.T. Elliott
Keyword(s):  
Agricultural Production ◽  
Production Efficiency ◽  
Scientific Literature ◽  
Animal Feed ◽  
Animal Health ◽  
Global Environment ◽  
Low Levels ◽  
Feed Samples

In the recent years, mycotoxins have undoubtedly gained a keen interest of the scientific community studying food safety. The main reason is their profound impact on both human and animal health. International surveys reveal a low percentage of feed samples being contaminated above permitted/guideline levels, developed to protect consumers of animal derived products. However, the deleterious impact of feed co-contaminated at low levels with numerous both known and regulated as well as novel mycotoxins on producing animals has been described. Associated effects on agro-economics world-wide include substantial pecuniary losses which are borne by the society as a whole. Even though good agronomic practice is thought to be the most effective way of preventing animal feed contamination, the EC have recognised the need to introduce an additional means of management of feed already contaminated with low-levels of mycotoxins to alleviate detrimental effects on agricultural production efficiency. This review discusses types of feed detoxifying agents described in scientific literature, their reported efficacy in both in vitro and in vivo systems, and comparison with available commercial formulations in the light of increasing knowledge regarding mycotoxin prevalence in the changing global environment.

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