In Vitro Embryo Production Research Articles

OPA1 deficiency induces mitophagy through PINK1/Parkin pathway during bovine oocytes maturation

In vitro embryo production (IVP) technology has been increasingly applied to beef cattle breeding. In vitro maturation (IVM) technology is the basis of IVP. However, the quality of in vitro-generated mature oocytes is still poor. Mitochondria are the energy factories of oocytes, so they are crucial for oocyte quality. OPA1 is a protein located on the mitochondrial inner membrane, and its main function is to mediate mitochondrial inner membrane fusion. This work demonstrated that OPA1 is expressed at different stages of meiosis in bovine oocytes. The inhibition of OPA1 activity resulted in a reduced rate of first polar body excretion from bovine oocytes and disruption of the spindle structure. OPA1 deficiency impacted mitochondria by leading to mitochondrial dysfunction, promoting mitochondrial fission, and inducing mitophagy through the PINK1/Parkin pathway. Taken together, our findings suggest that OPA1 is essential for bovine oocyte maturation and that OPA1 deficiency leads to mitochondrial dysfunction and promotes mitochondrial fission as well as mitophagy.

Effect of Hormones, Season and Lactation on Follicular Size, Oocyte Competency and Embryo Production in Sahiwal Cattle

Background: Transvaginal ultrasound-guided ovum pick-up (OPU) and in vitro embryo production (IVEP) techniques are widely used for enhancing reproductive efficiency. The number and quality of embryos have been reported to be influenced by various factors like source of hormones, season, lactation in cattle. Methods: Twenty-one multiparous, normal cyclic Sahiwal cows (aged: 6-8 years; lactating: n-11 and non-lactating: n-10) were selected randomly and the experiment was conducted during hot (April to September) and cold (October to March) seasons. All the donor cows were administered 10 µg GnRH at random stage of oestrous cycle followed by FSH (Folltropin-V™ or Stimufol) at the rate of 100, 60 and 40 mg after 48, 60 and 72 h of GnRH administration. After 24 hours of coasting period, follicular aspiration was performed by stabilizing the targeted follicles with the help of ultrasonography. The procedures of COCs grading, in vitro maturation, fertilization, followed by culture were performed as per the standard protocols. Results: The hormonal treatment was effective in improving the oocytes’ developmental competency and in vitro embryo production; with no statistical differences between the two sources. The follicle size distribution revealed a lower (P less than 0.05) medium-sized and small-sized follicular proportion during the hot season with a higher average count of dominant (large-sized) follicles. While, good quality (A+B+C) oocytes, matured and fertilized oocytes were significantly higher (P less than 0.05) in non-lactating donors, especially among cool-season collections.

Injectable progesterone in 13-month-old prepubertal Nellore heifers: effects on in vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of injectable progesterone administration before ovum pick-up (OPU) on in vitro embryo production (IVEP) and pregnancy rate in prepubertal Nellore heifers. Twenty-three Nellore females, 13.04 ± 0.8 months of age, were randomly assigned to two groups: control group (no progesterone application) and progesterone group (intramuscular administration of 1 mL [150mg/mL] progesterone 7 days before OPU). The second OPU was performed after 28 days (crossover between groups). After OPU, oocytes were selected for IVEP and a sample was fixed in 4% paraformaldehyde. Viable oocytes were subjected to immunofluorescence staining for IGFBP2 and caspase-3. For each oocyte, one image was generated per channel (A555 and A488 filters) and analyzed with the ImageJ program for the quantification of fluorescence intensity (in pixels). The produced embryos were transferred to multiparous Nellore cows and pregnancy was diagnosed by ultrasound 30 days after transfer. Data were analyzed using the MIXED and GLIMMIX procedure of SAS®. Treatment with injectable progesterone had no effect on the number or size of the antral follicles of heifers. No differences between the control and progesterone groups were observed for total number of aspirated oocytes (P = 0.78), viable oocytes (P = 0.80), cleavage rate (P = 0.60), or blastocyst rate (P = 0.56). There were no differences in pixel intensity between the two groups, nor in the levels of IGFBP2 or caspase-3. However, a difference was observed in the pregnancy rate of embryos produced from heifers of the progesterone group (42.1%; 32/76) when compared to the rate of embryos produced from control heifers (28.4%; 23/81) (P = 0.02). The use of injectable progesterone before OPU in prepubertal Nellore heifers does not improve IVEP but increases the pregnancy rate after transfer.

In vitro maturation of bovine oocytes: the role of ROS, oxidative stress, and the impact of natural antioxidant supplementation

According to available data in the literature, Brazil has one of the largest cattle herds in the world and is one of the leading embryo producers, with a clear predominance of embryos produced in vitro. These data influence the ongoing pursuit of optimizing the results obtained in IVEP (in vitro embryo production). In this technique, in vitro maturation (IVM) is such a crucial stage that it justifies studies involving components that, when added to the media, can lead to improved cleavage rates and blastocyst development. It is well known that oocyte quality is influenced by oxidative stress, and in this context, the inclusion of antioxidants in the IVM medium may enhance these outcomes. Thus, the present study aimed to review oocyte maturation, the impact of oxidative stress, and the addition of antioxidants, such as Moringa oleifera, to the IVM media.

Methodological approaches in vitrification: Enhancing viability of bovine oocytes and invitro-produced embryos.

Cryopreservation of bovine oocytes and embryos is essential for long-term preservation and widespread distribution of genetic material, particularly in bovine invitro embryo production, which has witnessed substantial growth in the past decade due to advancements in reproductive biotechnologies. Among current cryopreservation methods, vitrification has emerged as the preferred cryopreservation technique over slow freezing for preserving oocytes and invitro-produced (IVP) embryos, as it effectively addresses membrane chilling injury and ice crystal formation. Nonetheless, challenges remain and a simple and robust vitrification protocol that guarantees the efficiency and viability after warming has not yet been developed. Furthermore, although slow cooling can easily be adapted for direct transfer, an easier and more practical vitrification protocol for IVP embryos is required to allow the transfer of IVP embryos on farms using in-straw dilution. In addition, the susceptibility of bovine oocytes and embryos to cryoinjuries highlights the need for novel strategies to improve their cryotolerance. This manuscript examines various methodological approaches for increasing the viability of bovine oocytes and IVP embryos during vitrification. Strategies such as modifying lipid content or mitigating oxidative damage have shown promise in improving cryotolerance. Additionally, mathematical modelling of oocyte and embryo membrane permeability has facilitated the rational design of cryopreservation protocols, optimizing the exposure time and concentration of cryoprotectants to reduce cytotoxicity.

Efficient Method for Liposome-Based Gene Transfer in Cattle Embryos.

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.

Impact of semen cryopreservation season on invitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. Invitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo invitro capacitation.

A new hormonal protocol supports early development of invitro-produced embryos after transfer to anoestrus mares.

The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 67% showed evident intrauterine development. Hence, ICSI-derived embryos can be successfully transferred to primed anoestrus mares, but more studies are required to ensure further embryo development and foaling.

In Vitro Embryo Production: Overview

Reproductive biotechnologies, also known as assisted reproductive technologies (ART), have undergone significant development over time, reaching a remarkable level of evolution in the 20th century. Currently, several biotechnologies stand out, among which Artificial Insemination (AI), widely spread globally, Embryo Production (EP) both in vivo and in vitro for embryo transfer, Intracytoplasmic Sperm Injection (ICSI), sexing, among other innovations. In Vitro Production (IVP) of embryos has emerged as the biotechnology that has experienced the greatest development and evolution in recent decades, covering various fields such as research in assisted human reproduction, obtaining transgenic animals for biomedicine, preservation of endangered species and conservation of animals of high genetic value. This research aims to analyze the relevance of IVP, as well as the fundamental elements that make up this complex process. IVP of embryos involves several stages, from oocyte procurement, oocyte selection and in vitro maturation, sperm preparation, in vitro fertilization, to the culture of the resulting zygotes to blastocysts. These blastocysts, once transferable to recipients, culminate in the birth of a new being. However, this process has both advantages and disadvantages, which motivates continuous innovation in the field.

Impact of sperm sex sorting on sperm quality and in vitro embryo production in bovine

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen – CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality.

Utilization of Assisted Reproductive Technologies in Breeding Auliekol Cattle: A Comparative Study.

This study evaluates the utilization of in vitro embryo production (IVEP) technology for the conservation and breeding of the Auliekol cattle breed, a primary beef breed in Kazakhstan facing population decline due to the cessation of breeding programs and the incursion of transboundary diseases. We assessed the effect of consecutive ovum pick-up (OPU) procedures on oocyte yield and embryo production in Auliekol and Aberdeen Angus cows. A total of 2232 and 3659 oocytes were aspirated from Auliekol and Aberdeen Angus donors, respectively, with significantly higher yields and embryo production observed in Aberdeen Angus cows. The application of a meiotic block using Butyrolactone I (BLI) and subsequent in vitro fertilization (IVF) protocols was employed, with embryo development monitored up to the morula/blastocyst stage. Results indicated that Auliekol cows exhibited lower oocyte recovery, cleavage, and blastocyst rates compared to Aberdeen Angus cows, likely due to genetic characteristics. Despite the challenges, IVEP presents a valuable tool for the preservation and future propagation of the Auliekol breed, highlighting the need for further research to enhance reproductive outcomes and conservation strategies.

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P &amp;gt; 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

Effect of Two Different Sperm Selection Methods on Boar Sperm Parameters and In Vitro Fertilisation Outcomes.

Porcine in vitro embryo production (IVP) protocols have conventionally used density gradient selection (DGS) by centrifugation to prepare sperm samples and achieve successful fertilisation. However, the possible toxicity of the solutions used and the potential damage caused by the centrifugation step may have a negative effect on the quality of the sample. Microfluidic chip-based sperm (MCS) sorting has been proposed as an alternative technique for the selection of high-quality sperm with the purpose of improving reproductive outcomes in IVF. This device does not require centrifugation or any toxic solution to prepare the sample for fertilisation. The sample is not subjected to unnecessary stress, and the process is less operator-dependent. In this study, we compared the sperm parameters of unselected extender-diluted boar semen samples with selected samples using DGS and MCS methods. The results show an expected reduction in sperm concentration after both methods. All the groups were significantly different from one another, with MCS being the group with the lowest concentration. Though the three groups had a similar overall motility, significant differences were found in progressive motility when comparing the unselected group (control, 19.5 ± 1.4%) with DGS and MCS. Progressive motility in DGS was also significantly higher than in MCS (65.2 ± 4.9% and 45.7% ± 5.3, respectively). However, MCS selection resulted in enriched sperm samples with a significantly lower proportion of morphologically abnormal sperm compared to DGS. After fertilisation, no statistical differences were found between the two methods for embryological parameters such as cleavage rates, blastulation rates, and embryo quality. The number of cells in blastocysts derived from MCS was significantly greater than those derived from DGS sperm. Thus, we demonstrate that MCS is at least as good as the standard DGS for most measures. As a more gentle and reproducible approach for sperm selection, however, it could improve consistency and improve IVP outcomes as mediated by a greater proportion of morphologically normal sperm and manifested by a higher cell count in blastocysts.

Successful ultrasound-guided ovum pick-up (OPU) and subsequent in vitro embryo production in a domestic cat

Ovum Pick Up (OPU) is a minimally invasive technique widely used in cattle and mares for oocyte retrieval, involving ultrasound-guided puncture of ovarian follicles. It has been demonstrated that this technique is safe for its repeated use in the same female without affecting her reproductive health, allowing for the retrieval of oocytes in individuals regardless of their reproductive status. The oocytes obtained through OPU can subsequently be used for in vitro embryo production (IVP) using assisted reproductive techniques (ARTs) or be cryopreserved in biobanks for their future use. Traditionally, the minimally invasive technique of choice performed in vivo in domestic and wild felines was LOPU (laparoscopic-guided ovum pick up). The present study was designed to explore if ultrasound-guided OPU in the domestic cat is safe and effective. In an initial series of ex vivo experiments (n = 92 ovaries, n = 434 oocytes), the effect of different aspiration pressures for oocyte collection was explored. These experiments identified 43 mmHg as the optimal aspiration pressure, resulting in the highest recovery rate and a favorable maturation and blastocyst rate. Subsequently, 16 grade I and II oocytes were retrieved by OPU and 101 oocytes were retrieved following ovariectomy and slicing. Sixteen oocytes obtained with each technique were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). A total of 14 presumptive zygotes were selected for in vitro culture (IVC) from each group (OPU and slicing), obtaining a cleavage rate of 57.1 % and 64.2 %, a morula rate of 28.5 % in both groups, and a blastocyst rate of 7.14 % and 14.2 % respectively. The hormonal stimulation protocol was well-tolerated, with no adverse effects observed. Moreover, no complications arose during the ovariectomy performed post-OPU. The use of this technique in domestic cats represents a significant step forward in terms of safety, replicability, and invasiveness, serving as a valuable model for its application in wild felids species. Additional research involving a greater number of animals is required to validate these encouraging findings.

PAPP-A enhances the antioxidative effects of IGF-1 during bovine in vitro embryo production

We investigated whether exogenous pregnancy-associated plasma protein-A (PAPP-A) enhances the antioxidant role of insulin-like growth factor-1 (IGF-1) in bovine in vitro embryo production (IVP). We performed standard in vitro maturation (IVM) and in vitro culture (IVC) or added menadione to promote an oxidative stressed microenvironment and evaluated the antioxidant effect of IGF-1 alone or in combination with PAPP-A (IGF-1/PAPP-A). In IVM, the treatments did not affect oocyte nuclear development, total GSH content, cumulus cell gene expression, and blastocyst yield. Nevertheless, IGF-1/PAPP-A treatment prevented an increase in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels. In IVC, the treatments did not affect the total GSH content on blastocysts and IVC media, but IGF-1 and IGF-1/PAPP-A treatments increased blastocyst yield compared to the menadione group. In addition, IGF-1/PAPP-A treatment had lower ROS levels and regulated genes related to embryonic quality compared to the control and menadione groups. Overall, we showed that PAPP-A could enhance the antioxidant role of IGF-1 during IVP in cattle by avoiding higher ROS levels in oocytes and blastocysts and modulating the transcriptional abundance of genes involved in oxidative protection and embryonic quality.

Docosahexaenoic acid supplementation during porcine oocyte in vitro maturation improves oocyte quality and embryonic development by enhancing the homeostasis of energy metabolism

Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 μM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 μM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 μM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.

Analysis of physical traits, clinical parameters, and energy metabolism of invivo- and invitro-derived Flemish newborn calves during the first day of life.

Studies investigating physiological deviations from normality in newborn calves derived from invitro fertilization procedures remain important for the understanding of factors that reduce calf survival after birth. The aim of this study was to investigate parameters affecting health and welfare of newborn Flemish calves derived from invitro embryo production (IVP) in the first hours of life in comparison to invivo-derived calves. Physical traits of newborn calves and fetal membranes (FM) were recorded soon after birth. Newborn venous blood samples were collected at several time points within the first 24 h of life for analyses of energy substrates, electrolytes, blood gases, acid-base balance, blood chemistry, and haematology. A liver biopsy was taken within the first hour after birth for analysis of gene expression of key enzymes of the fructolytic and glycolytic pathways. Newborn IVP calves were heavier and larger at birth, which was associated with heavier FM. At several time points during the first 24 h of life, IVP-derived calves had altered rectal temperature, blood gases, electrolyte concentrations, blood parameters for liver, kidney and muscle function, and acid-base balance, plasma lipid metabolism, and hemogram parameters. The relative mRNA abundances for triokinase and lactate dehydrogenase-B were greater in IVP calves. In summary, IVP-derived newborn calves were at higher risk of clinical problems after birth, which was markedly greater in heavier and larger calves. Such animals take longer to adapt to extrauterine life and should receive a special attention during the immediate neonatal period.

The Simulated Physiological Oocyte Maturation (SPOM) System Enhances Cytoplasmic Maturation and Oocyte Competence in Cattle.

In vitro embryo production is a widely applied technique that allows the expansion of genetics and accelerated breeding programs. However, in cattle, this technique still needs improvement in order to reach quality and pregnancy rates comparable to in vivo-derived embryos. One of the limitations of this technique is related to in vitro maturation, where a heterogeneous population of oocytes is harvested from follicles and cultured in vitro in the presence of gonadotropic hormones to induce maturation. As a result, oocytes with different degrees of competence are obtained, resulting in a decrease in the quality and quantity of embryos obtained. A novel system based on the use of cyclic adenosine monophosphate (cAMP) modulators was developed to enhance bovine oocyte competence, although controversial results were obtained depending on the in vitro embryo production (IVP) system used in each laboratory. Thus, in the present work, we employed a reported cAMP protocol named Simulated Physiological Oocyte Maturation (SPOM) under our IVP system and analysed its effect on cytoplasmic maturation by measuring levels of stress-related genes and evaluating the activity and distribution of mitochondria as a marker for cytoplasmic maturation Moreover, we studied the effect of the cAMP treatment on nuclear maturation, cleavage, and blastocyst formation. Finally, we assessed the embryo quality by determining the hatching rates, total cell number per blastocyst, cryopreservation tolerance, and embryo implantation. We found that maturing oocytes in the presence of cAMP modulators did not affect nuclear maturation, although they changed the dynamic pattern of mitochondrial activity along maturation. Additionally, we found that oocytes subjected to cAMP modulators significantly improved blastocyst formation (15.5% vs. 22.2%, p < 0.05). Blastocysts derived from cAMP-treated oocytes did not improve cryopreservation tolerance but showed an increased hatching rate, a higher total cell number per blastocyst and, when transferred to hormonally synchronised recipients, produced pregnancies. These results reflect that the use of cAMP modulators during IVM results in competent oocytes that, after fertilisation, can develop in more blastocysts with a better quality than standard IVM conditions.

Role of Follicular Size on Oocyte Recovery Rate and in Vitro Embryo Production Stages in Local Iraqi Sheep

In vitro embryo production (IVEP) encompasses a series of three basic steps, namely in vitro maturation (IVM), in vitro fertilisation (IVF), and in vitro embryo culture. IVEP approach has shown to be a valuable tool in advancing our comprehension of early mammalian development and expediting genetic enhancement in livestock. The aim of this research was to compare the impact of small and large follicles on the total number of oocytes recovered through the IVEP process. Therefore, 305 Ewe genitalia were collected at the Al-Fallujah abattoir in the Al-Anbar province. The follicles were identified, and their diameter was measured using an automated vernier calliper. Subsequently, the oocytes were classified into three distinct groups, namely excellent (A), acceptable (B), and poor (C). The results of the maturation rate of large follicles were 43.9% (51/116) and small follicles 30.7% (20/65). There was a statistical difference in maturation rate (p&lt;0.05). The fertilization rate of matured oocytes collected from large follicles was 60.7% (31/51) and 50% (10/20) from small follicles. There was no statistical difference. The results of Blastocyst production rate. showed a higher percentage 58.06 (18/31) of Blastocyst produced from large follicles as compared a percentage of 30% (3/10) of Blastocyst production from small follicles. There was a statistical difference (P&lt;0.05). It was concluded from the current study that the size of the follicle has an effect on IVM, IVF and IVC.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.