Sheep Oocytes Research Articles

Strip Pulled Straw: Cost Effective Alternative Cryodevice for the Vitrification of Oocytes

BACKGROUND: Increased cooling and warming rates by using a suitable cryodevice allows the use of lower cryoprotectant concentration and reduces cryoinjuries as a result of the rapid passage through the 'dangerous' temperature zone. OBJECTIVE: To evaluate the effectiveness of newly customized strip pulled straw (SPS) with respect to post warming quality, viability, and in vitro maturation for immature oocytes post-vitrification of. MATERIALS AND METHODS: SPS was prepared using conventional French mini straw to combine the merits of OPS and the Cryotop system. Immature sheep oocytes were treated in 15% EG + 15% DMSO, loaded on SPS and plunged into liquid nitrogen (LN). Post thaw quality, viability, and maturation rates of oocytes were determined after 1 week storage in LN. RESULTS: SPS achieved a post-thaw morphological survival of 90.9% with 9.0% morphological defects, 96.4% viability and 51% in vitro maturation. In comparison to OPS, SPS had higher post thaw survival (86.5% vs 67.9%) and maturation rate (57.7% vs 50.5%) with lower morphological defects (13.5% vs 32.1%). Cumulus cell loss was the highest among morphological abnormalities of post warm oocytes in SPS (40.9%) and OPS (44.1%). The data showed acceptable post thaw survival, viability and in vitro maturation rate of immature ovine oocytes using SPS as compared to traditional OPS. CONCLUSION: SPS can be used as a cost effective alternative device for oocyte vitrification.

Zinc supplementation promotes oocyte maturation and subsequent embryonic development in sheep

Zinc plays a crucial role in the growth and reproductive functions of animals. Despite the positive effects of zinc that have been reported in oocytes of cows, pigs, yaks, and other animals, the influence of zinc on sheep is little known. To investigate the effect of zinc on the in vitro maturation of sheep oocytes and subsequent parthenogenesis-activated embryonic development, we added different concentrations of zinc sulfate to the in vitro maturation (IVM) culture medium. The IVM culture medium with zinc improved the maturation of sheep oocytes and the subsequent blastocyst rate after parthenogenesis activation. Notably, it also enhanced the level of glutathione and mitochondrial activity while reducing levels of reactive oxygen species. Thus, zinc addition to the IVM medium improved the quality of oocytes with a positive effect on the subsequent development of oocytes and embryos.

EFFECTIVENESS OF OVINE FOLLICULAR FLUID AND EWE AGE ON IN VITRO NUCLEAR MATURATION AND EMBRYO EVOLUTION OF AWASSI EWES

Two experiments were conducted to study the efficiency of the ovine follicular fluid (OFF) and the age of ewe on in vitro nuclear maturation of Awassi sheep oocytes (Experiment 1) and the subsequent stages of early embryos and their quality (Experiment 2). The OFF has been added to the basic maturation medium (TCM-199) with three different levels (A: 0% (Control group), B:15%, C:25% and D:50%) while the ages of the ewes were determined into two main groups (> 2 and 1-2 year). In the first experiment, across the two age groups, matured oocytes in C solution achieved the highest (p=0.006) rates of metaphase-II (85.70 and 82.35 % respectively). In the second experiment, oocytes belonging to > 2 and 1-2-year ewe age groups (C Solution) achieved the highest (p=0.001) rates of fertilization (23.17 and 30.73% respectively). Similar trend applies to the cleavage stage, the rates reached 35.14 and 41.27% respectively (p=0.01). No significant differences were noticed at the different stages of cleavage (2-4 cell, morula, and blastocyst). The resulting embryos of B (> 2-year ewe age group) and C solution (1-2-year age group) achieved the highest (p=0.04) rates of Type1 embryos (69.56 and 76.92% respectively). A significant difference was noticed in type2 embryos (p=0.02), embryos of the first and second age groups (Solution D) achieved the highest rates (46.15 and 33.33% respectively). It can be concluded that the adding of 25% OFF to the maturation media led to significant improvement in the rates of maturation, cleavage and type1 embryos of sheep oocytes.

Effect of addition of follicular fluid, oviductal or granulosa cell co-culture on In vitro cleavage and embryonic development in sheep oocytes

Effect of addition of follicular fluid, oviductal or granulosa cell co-culture on In vitro cleavage and embryonic development in sheep oocytes

MRNA 3ʹ -UTR-mediate translational control through PAS and CPE in sheep oocyte

In oocytes, the cytoplasmic polyadenylation and maternal mRNAs translation is regulated by cis-elements, including polyadenylation signal (PAS) and cytoplasmic polyadenylation element (CPE) in 3ʹ-UTR. Recent studies illustrate non-canonical polyadenylation mechanisms of translational regulation in mouse oocytes, which is different from that in Xenopus oocytes. However, it is still unclear if this regulation in rodent oocytes functions in the domestic animal oocyte. Here, by using sheep as an animal model, we cloned the 3ʹ-UTRs of Cpeb1 or Btg4 and ligated it into the pRK5-Flag-Gfp vector. Variant numbers and positions of PASs and CPEs within the 3ʹ-UTRs were constructed to detect their effects on translational control. After in vitro-transcription and microinjection into sheep fully grown germinal vesicle stage oocytes, the expression efficiency of mRNAs was detected by the GFP and flag expression. Our results show that: (i) PAS located at the proximal end of 3ʹ-UTR can mediate the translation of the maternal mRNAs, as long as they locate far from CPEs; (ii) The proximal PAS has higher efficiency in regulating transcription than the distal one; (iii) increase of PAS number can promote the translational activity more efficiently; (iv) a single CPE located close to PAS (<50 bp) in 3ʹ-UTRs of Cpeb1 or Btg4 could partially repress translation. In 3ʹ-UTRs of Btg4, two CPEs have a higher inhibitory effect, and three CPEs can completely inhibit mRNA translation. These results confirm the existence of the non-canonical mechanism in domestic animal oocytes.

Evaluation of the Maturity and Gene Expression of Sheep Oocytes and Embryos Cultured in Media Supplemented with Marjoram (Origanum vulgare) Extract.

This study aimed to evaluate the effects of marjoram extract on oocyte maturation and gene expression in sheep oocytes and embryos. The first experiment studied the effect of the extract as an antioxidant to improve the in vitro maturation media used for sheep oocytes; the oocytes were matured in a TCM199 medium supplemented with 1 or 10 µg/mL of marjoram extract or the control, 0 µg, for 24 hr. Then, the maturation was estimated, and the gene expression was measured by using qPCR. The second experiment studied the effect of the extract on the development of sheep embryos produced in vitro; the fertilized oocytes were cultured in a SOF medium supplemented with 1 or 10 µg/mL of marjoram extract or the control, 0 µg, for 7 days. Then, the gene expression was measured using qPCR. The results showed that the marjoram extract did not improve nuclear maturation or the blastocyst rate. There was a significant increase in the level of GDF-9 gene expression in mature oocytes in the treatment groups. An increase in the expression of BCL-2 and EGR-1 genes was observed for the blastocysts in the 10 µg/mL group. We concluded that the marjoram extract did not improve nuclear maturation, but it did affect the expression of some genes in sheep oocytes and embryos.

HDAC1 in the Ovarian Granulosa Cells of Tan Sheep Improves Cumulus Cell Expansion and Oocyte Maturation Independently of the EGF-like Growth Factors.

Simple SummaryThis study aimed to explore the possible role of histone deacetylase 1 (HDAC1), an epigenetic modifying enzyme, in the improvement of oocyte maturation recovered from the Tan sheep of China. We supplemented a specific inhibitor of HDAC1 to in vitro-cultured cumulus oocyte complexes (COCs). The results showed that the cumulus cell expansion and oocyte maturation ratio were impaired, while the production of the epidermal growth factor (EGF) was only slightly changed, despite the fact that the pan-acetylation levels of the histones were significantly elevated. This study implies that HDAC1 may participate in the maturation of oocytes in Tan sheep independently of the classic gonadotrophin-induction pathway.Previous studies have shown that some of the histone deacetylases (HDACs) play diverse roles in the regulation of ovarian somatic cell development, oocyte maturation and early embryonic development in different species including sheep. This study aimed to clarify whether HDAC1 also played pivotal roles in regulating oocyte maturation in Tan sheep. The results showed that HDAC1 was expressed in the nuclei of both the granulosa cells and oocytes of the growing follicles in the Tan sheep’s ovaries. However, the level of HDAC1 was unaffected by luteinizing hormone (LH) induction in cultured granulosa cells. Meanwhile, the specific inhibition of HDAC1 using pyroxamide did not induce significant changes in the expression levels of EGF-like growth factors in vitro, whereas both the cumulus expansion and oocyte maturation of the cultured cumulus oocyte complexes (COCs) were significantly inhibited by pyroxamide. Additionally, the numbers of histone acetylation sites (H4K5, H4K12, H3K14 and H3K9) in ovarian granulosa cells were significantly increased. In conclusion, a constant expression of HDAC1 in the growing follicles of Tan sheep may be pivotal for supporting oocyte growth and maturation, although its action may not be closely correlated with LH induction, nor does it directly affect the expression of the EGF-like factors. Our study implies that there may exist diverse functions of the respective HDACs in modulating female reproduction in sheep.

Effect of Ovarian Storage Temperature and Time on Post Thaw Viability and Maturation Rate of Vitrified Immature Oocytes in Sheep

BACKGROUND: Vitrification of oocytes as a method of cryopreservation is quite successful, although it is still being standardized because of structural and molecular sensitivity of oocytes to the cooling and freezing process. OBJECTIVE: To investigate the effect of ovarian storage temperature and time on post thaw viability and maturation rate of vitrified immature oocytes in sheep. MATERIALS AND METHODS: The work consisted of oocyte collection from ovaries of abattoir sheep stored at various temperature (0 °C , 4 °C and 25 °C) and time (0 h, 6 h, 12 h and 24 h) combinations and post thaw viability and in vitro maturation rate evaluation. Vitrification was done in 30% vitrification solution, using ethylene glycol and DMSO, with post vitrification evaluation after 1 week 's storage. RESULTS: Significantly higher post thaw viability was observed after storage at 0 °C for 6 h (95.3%) followed by 12 h (85%), with lowest value at 24 h (66.7%). However at 4 °C and 25 °C, values were nonsignificantly higher after 6 h (96.5 and 100% respectively) followed by 12 h (93 and 100%), with significantly lower values after 24 h (85.7 and 90.7%). At storage temperatures of 25 °C and 4 °C, a significantly higher percentage of mature oocytes was observed after 6 h (40 and 39.1%), 12 h (37.3 and 38.1%) and 24 h (34.6 and 36.4%) storage times compared to that at 0 °C (20.3% at 6 h, 14.2% at 12 h and only 13.8% at 24 h). However, at all storage temperatures, there was a tendency for the level of mature oocytes to decrease with storage time, and the levels were significantly lower than the control. CONCLUSION : Acceptable post thaw viability and in vitro maturation rates for oocytes is maintained up to 24 h in ovaries stored at 4 °C and 25 °C compared to at 0 °C, and these conditions may be used for the storage of ovaries meant for oocyte preservation.

Maturation rate of sheep oocytes cultured in suboptimal environment using CO2 exhaled by human and glutathione addition

This study aimed to identify the effect of the use of CO2 exhaled by human (CEbH) in in vitro maturation (IVM) with the addition of Glutathione also known as GSH on the oocytes maturation rate. It is known that CO2 derived from human respiration can be used as alternative support for IVM of oo-cytes. However, this method is categorized as using a suboptimal culture environment, and it results in increased production of reactive oxidative species (ROS). Thus, the addition of the antioxidant GSH is needed. In total, 273 oocytes were cultured in three groups: 5% CO2 (control) and CEbH with 0 mM and 1 mM of GSH addition, respectively. The results showed that the control group produced a better maturation rate (p &lt; 0.05) compared to the remaining two groups (65.92% vs. 24.44% and 30.61%), which used CEbH. Statistically, 0 mM GSH vs. 1 mM GSH did not show any significant differences (p &gt; 0.05). However, 1 mM GSH led to more mature oocytes (24.44% vs 30.61%) compared to the group without GSH. In conclusion, IVM using CEbH supports oocyte maturation, and the addition of GSH can improve the maturation rate in a suboptimal culture environment.

PLCζ can stably regulate Ca2+ fluctuations in early embryo

Phospholipase C zeta (PLCζ) is an important inducer of Ca2+ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca2+ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca2+ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca2+ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca2+ in early embryos at different stages, Ca2+ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca2+ fluctuations differed. PLCζ resulted in Ca2+ peaks that appeared at the cleavage and morula stages of early embryos, and Ca2+ returned to normal levels at the morula stage. However, the Ca2+ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca2+ fluctuations at different stages during early embryonic development.

Serum supplementation during in vitro fertilization of sheep oocytes influences blastocyst quality through the differential abundance of mRNA transcripts.

Incubation with estrous sheep serum (ESS) is required to induce in vitro capacitation of spermatozoa during in vitro fertilization of small ruminants. However, the effect of adding different serum concentrations in the fertilization media on the quality of resulting blastocysts has not yet been studied. Here, 298 sheep oocytes were co-incubated with capacitated spermatozoa with either 10% or 2% ESS. There were no differences between treatments in cleavage (10% ESS: 63.81 ± 5.87% and 2% ESS: 45.31 ± 5.87%) and blastocyst rates (10% ESS: 20.83 ± 2.12% and 2% ESS: 15.93 ± 2.12%). Nonetheless, in vitro-produced blastocysts from the 10% ESS treatment showed a higher transcript abundance of mRNAs involved in apoptosis (ITM2B and BCL2), antioxidant defence (GPX1) and growth-related imprinting (IGF2R). Our data suggest that ESS supplementation during in vitro fertilization can influence the quality of sheep embryos at later stages of development by increasing the transcription of developmentally important genes.

Exogenous Melatonin Directly and Indirectly Influences Sheep Oocytes.

Understanding whether and how melatonin (MT) may impact sheep oocyte development competence is central to our ability to predict how sheep oocytes will respond to artificially regulated estrus. Implanting MT can make sheep enter estrus during the non-breeding season. One study found that the blastocyst rate increased under MT treatment, while another found that the blastocyst rate decreased. Therefore, we conducted a meta-analysis of MT directly and indirectly influencing sheep oocytes. A total of 433 articles were collected from which 20 articles and 34 treatments were finally selected. A method for estimating the default value was established for the litter size analysis. We found that exogenous MT add into in vitro maturation medium was positively related to the blastocyst rate in the lab. However, subcutaneous implanting MT did not affect the in vivo ovulation rate, fertilization rate, blastocyst rate, or pregnancy rate at farm. MT did not affect the in vitro cleavage rate. However, MT improved the in vivo cleavage rate. We hypothesized that implanted MT could increase the concentration of MT in oviduct fluid in vivo, and also that in vitro MT could increase the early cleavage rate of sheep zygotes without affecting the total cleavage rate. In the analysis of oocyte apoptosis caused by injury, the results suggested that pyroptosis would be more suitable for further research. MT produces responses in all body organs, and thus implanting of MT during non-breeding seasons should consider the effect on animal welfare.

SIRT2 Is Critical for Sheep Oocyte Maturation through Regulating Function of Surrounding Granulosa Cells.

Oocyte in vitro maturation is crucial for in vitro embryo production technology, which provides oocytes resources for in vitro fertilization and somatic cell nuclear transfer. Previous studies proved that SIRT2, a member of the sirtuin family, plays a role in oocyte meiosis, but its role in sheep oocyte maturation and its regulating mechanism remains unknown. Firstly, we confirmed the role of Sirt2 in sheep oocytes maturation by supplementation of SIRT2 inhibitor and activator. To further explore the specific mechanism, we performed knockdown of Sirt2 in granulosa cells and then cocultured them with oocytes. Moreover, we determined the effects of Sirt2 on granulosa cell oxidative apoptosis, cell migration, and diffusion, and examined its effects on granulosa cell mitochondrial function, mitophagy, and steroid hormone levels. The results showed that supplementation of SIRT2 inhibitor decreased the oocytes maturation rate (69.28% ± 1.28 vs. 45.74% ± 4.74, p < 0.05), while resveratrol, a SIRT2 activator, increased its maturation rate (67.44% ± 1.68 vs. 78.52 ± 1.28, p < 0.05). Knockdown of Sirt2 in sheep granulosa cells also reduced the oocytes maturation rate (47.98% ± 1.43 vs. 33.60% ± 1.77, p < 0.05), and led to decreased cell migration and expansion ability, oxidative apoptosis, abnormal mitochondrial gene expression, decreased mitochondrial membrane potential and ATP level, and increased mitophagy level. Overexpression of Sirt2 improved mitochondrial membrane potential and ATP level and improved mitochondrial function. Furthermore, we found that Sirt2 knockdown in granulosa cells promotes the secretion of P4 through regulating p-ERK1/2. In conclusion the present study showed that SIRT2 is critical for sheep oocyte maturation through regulating the function of ovarian granulosa cells, especially affecting its mitochondrial function.

The Effect of Ghrelin on the Maturation of Sheep Oocytes and Early Embryonic Development In Vitro.

Simple SummaryDifferent gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their changes, and 200 ng/mL ghrelin was found to be the optimal concentration. The RNA-seq analysis showed that the Cell cycle signaling pathway was enriched. The results suggest that adding ghrelin shortens the duration of IVM of sheep oocytes and hinders early embryonic development.In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 μM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 μM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 μM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 μM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 μM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.

High pressure frozen oocytes have improved ultrastructure but reduced cleavage rates compared to conventionally fixed or vitrified oocytes.

Live birth rates are lower for cryopreserved oocytes than for fresh IVF cycles, indicating a need for improved methodologies. The aim of this study was to determine if high pressure freezing (HPF) could improve both ultrastructural preservation and cryopreserved oocyte quality when compared to conventional fixation and vitrification methods. Sheep oocytes and embryos were prepared by HPF or vitrification, with or without cryoprotectants. Frozen oocytes were prepared for transmission electron microscopy or warmed, in vitro fertilised and the recovery and cleavage rates recorded. Blastocyst rates were similar between fresh, HPF and vitrified embryos. HPF oocytes had improved ultrastructure compared to conventional fixation or vitrification, but had poorer survival and cleavage rates compared to vitrified oocytes. Freeze-substitution of cryopreserved oocytes and transmission electron microscopy demonstrated disruption of the oocyte ultrastructure in the presence of cryoprotectants. Superior preservation of ultrastructure was observed in HPF oocytes compared to vitrification or conventional fixation methods. In the presence of CP, both embryos and oocytes could survive HPF and warming but oocytes had reduced development. The HPF method has potential to be developed and lead to improved oocyte and embryo cryopreservation and outcomes for assisted reproduction.

THE EFFECTIVITY OF CYSTEAMINE SUPPLEMENTATION ON IMPROVING THE IN VITRO FERTILIZATION RATE OF SHEEP OOCYTES

The purpose of this study was to evaluate the effectiveness of cysteamine supplementation on the maturation medium and/or in vitro fertilization medium with regards to improving the normal fertilization rate of sheep oocytes, which are characterized by the formation of two pronuclei. Grade A and B oocytes were matured in medium-199 with 0.3% bovine serum albumin (BSA), 10 IU/mL follicle stimulating hormone (FSH), 10 IU/mL human chorionic gonadotrophin (hCG), and 50 μg/mL gentamicin added for 24 hours in a 5% CO2 incubator at 39° C. The treatment group was divided into the following groups: a control group with no cysteamine (P1), a group with 100 μM cysteamine in the maturation medium (P2), a group with 100 μM cysteamine in the fertilization medium (P3), and a group with 100 μM cysteamine both in the maturation and fertilization medium (P4). The fertilization was carried out by incubating sperm-oocytes for 12 hours and then staining them with 1% aceto-orcein to observe the formation of a pronucleus. Normal fertilization rates obtained by each treatment group were 56.5% (P1), 57.1% (P2), 57.8% (P3), and 59.9% (P4) with no significant difference (P0.05) between groups. It was concluded that 100 μM cysteamine supplementation in both the maturation medium and fertilization medium was not able to increase the normal fertilization rate of sheep oocytes.

Effects of Epidermal Growth Factor and Myo-Inositol on Nuclear Configuration and Subsequent Embryonic Development of Sheep Oocytes

Growth factors and vitamin-like substances have great positive importance in most biological interactions in the cellular level. The addition of these elements in the culture media will increase the yield of the resulting embryos and improve quality. We examined the effects of epidermal growth factor (EGF) and myo-inositol (MI) on meiotic maturation and yields of blastocyst of Awassi sheep oocyte across two experiments. The oocytes obtained were subjected into three treatments: A (without EGF nor MI), B (10 ng/ml EGF + 20 mmol/l MI) and C (50 ng/ml EGF +40 mmol/l MI). Oocytes were then cultured in Ham's F-10 medium supplemented with 5% (v: v) fetal calf serum and 40 ng/ml follicle - stimulating hormone. In the first experiment, during the 27-h culture period, the oocytes were assessed for germinal vesicle break down, metaphase-I and metaphase-II stages across three-time intervals (9, 21 and 27-h). Results of the experiment showed that EGF and MI enhanced the rates of germinal vesicle break down phase (1.53%; 27-h interval; lowest value), metaphase-I (33.87%; 21-h interval) and metaphase-II (89.23%; 27-h interval). In the second experiment, the oocytes incubated in treatment B achieved the highest rates of cleavage (81.96%), 2-8 cell (62.35%) and blastocyst (45.09%). It is concluded from the present study that incubating sheep oocytes in culture media containing a cocktail of EGF (10 ng/ml) and MI (20 mmol/l) significantly improves the rates of metaphase-II, fertilization and blastocyst rates.

Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.