G. M. Machado

Effect of Percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos.

The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1-4 mL of Percoll, centrifuged for 20 min at 700 g; T2-800 microL of Percoll, centrifuged for 20 min at 700 g; and T3-800 microL of Percoll, centrifuged for 5 min at 5,000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P<0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P>0.05), but were affected by sire (P<0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production.

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.

Methylation status in the intragenic differentially methylated region of the IGF2 locus in Bos taurus indicus oocytes with different developmental competencies

Oocyte quality is one of the most important aspects of in vitro embryo development. Extensive epigenetic programming must occur during oocyte growth and maturation. A specific DNA methylation pattern of the imprinted genes must be established on differentially methylated regions (DMR). The insulin-like growth factor 2 (IGF2) gene is an important growth factor, and it is imprinted in several mammalian species. The aim of this study was to evaluate the methylation pattern on the DMR of the last exon of IGF2 in immature and mature bovine oocytes with different developmental competencies. Mature oocytes from large follicles were less methylated (28.93%) than immature oocytes from large follicles (77.38% P = 0.002), and there was also a tendency towards lower methylation in mature oocytes from large follicles (28.93%) compared with mature oocytes from small follicles (52.58% P = 0.07). Immature oocytes from small and large follicles showed 53.85% (7/13) and 91.66% (11/12) hypermethylated sequences, respectively, whereas mature oocytes from small and large follicles showed 61.11% (11/18) and 40% (4/10), respectively. The hypomethylation pattern in mature oocytes from large follicles may be related to the higher competence of these oocytes. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a molecular marker for oocyte competence in cattle and as a model for studies in other species.

Post‐hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro

This study evaluates the post‐hatching development of in vitro‐produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post‐hatching development (PHD) system. As a control group, in vivo‐produced (IVV), Day‐7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day‐7 and Day‐14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day‐14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day‐7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day‐14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day‐7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus. Mol. Reprod. Dev. 80: 936–947, 2013.

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre‐implantation bovine embryo development by characterizing the allele‐specific expression pattern of the X chromosome‐linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4‐, 8‐ to 16‐cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT‐PCR‐RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele‐specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4‐, 8‐ to 16‐cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele‐specific expression of an X‐linked gene that is subject to XCI in in vitro bovine embryos from the 4‐cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. 77: 615–621, 2010.

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: Assessment of morphological, molecular and functional patterns ☆

This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.

Morphology, sex ratio and gene expression of Day 14 in vivo and in vitro bovine embryos

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo- and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down- and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality.

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation.

PurposeThe aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1–3 mm and  ≥ 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.MethodsGenomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.ResultsThere were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 ± 14.11 % and 82.93±5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 ± 10.40 % and 79.64 ± 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 ± 12.01 % and 81.19 ± 10.15 %.ConclusionsIn conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.

135 In vitro development of in vivo produced embryos frozen at morula or blastocyst stages

This study was designed to compare cryotolerance between morulae and blastocysts collected from superovulated heifers. Twenty pubertal beef heifers (10 Nelore and 10 crossbred Nelore × Simmental) were superovulated with 100 mg of FSHp (Folltropin-V, Bioniche, Ontario, Canada), and embryos were collected and evaluated 7 days after estrus. Grades 1 and 2 embryos (IETS) were divided into four groups: morulae cryopreserved (MC) in liquid nitrogen (n = 24); blastocysts cryopreserved (BC; n = 19); morulae fresh (MF; n = 23); and blastocysts fresh (BF; n = 18). For freezing, embryos were immersed in ethylene glycol (Ethylene Glycol Freeze Plus with 0.1 m sucrose, Bioniche, Pullman, WA, USA), and a standard protocol (cooling rate of –0.5°C/min) was used. Prior to in vitro culture, embryos were removed from nitrogen, kept at room temperature for 5 s, and put in a water bath at 30°C for 20 s. Within 5 h after recovery, thawed and fresh embryos were washed five times in holding solution (Holding Plus, Bioniche), transferred to synthetic oviduct fluid medium (SOF, Nutricell, Campinas, SP, Brazil), and cultured for 72 h. Embryos were evaluated at 48 and 72 h of culture. After the last evaluation, degenerate and non-hatched embryos were removed from culture, and the remaining embryos were measured by a graduated ocular coupled to the Motic Images Plus 2.0 program. Hatched blastocysts were kept in culture for an additional 48 h for post-hatching development assessment. For post-hatching culture PHD medium (Brandao DO et al. 2005 Biol. Reprod. 71, 2048–2055) was added into each well, to have a final composition of 50% SOF and 50% SOF PHD. At 120 h of culture (48 h of PHD culture) only morphologically normal blastocysts were measured. Comparison among groups was performed by ANOVA or chi-square test. Data are presented as mean ± SEM. After 48 h of culture, hatching rate (%) was significantly lower in cryopreserved (MC = 8.3 and BC = 21.5) than in fresh (MF = 56.5 and BF = 77.8) embryos (P < 0.05). However at 72 h, hatching rate was similar among BC (75.9), MF (78.3), and BF (88.9), being MC (41.7) still lower (P < 0.05). The diameter (µm) of hatched embryos after 72 h of culture was 272.8 ± 27.1a (n = 8), 320.6 ± 18.6ab (n = 14), 385.3 ± 14.2c (n = 17), and 378.0 ± 22.0bc (n = 16) for MC, BC, MF, and BF, respectively (a–cP < 0.05). After 120 h of culture, the diameter of MC (379.0 ± 39.9; n = 8), although similar to BC (495.4 ± 59.6; n = 10), was smaller than MF (509.1 ± 36.5; n = 11) and BF (511.8 ± 41.2; n = 14). The results of this study with zebu cattle suggest that morulae are less resistant to cryopreservation in liquid nitrogen than blastocysts. Moreover, frozen/thawed embryos, when put in culture, present a slower development compared with fresh embryos. Financial support from CNPq and FAPESP from Brazil.