Comparative Characteristic of the Methods of Isolation of Oocytes and Spermatozoa in Laboratory Animals (Review).

Over the past decade, there has been an increasing trend in the use of assisted reproductive technologies, which have significantly expanded the opportunities to overcome the problem of infertility. However, the problem of increasing the effectiveness of in vitro fertilization remains open. Isolation of germ cells from animals is a necessary process for various experimental studies. Animal germ cells can be used in experiments to study physical, chemical, genetic, immunological, and microbiological factors affecting reproduction efficiency and for the development of techniques that increase the effectiveness of in vitro fertilization. All of the above determines the relevance of studying existing methods of oocyte and sperm isolation for experimental in vitro studies. Here we discuss the existing methods of sperm and oocyte isolation from animals and their advantages and disadvantages, and also substantiate priority methods for use.

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall in vitro maturation efficiency and early embryonic stages.

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.

Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium.

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

In vitro exposure of porcine sperm to functionalized superparamagnetic nanoparticles.

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co-incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization-IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p > .05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p > .05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p > .05). In conclusion, in vitro exposure of boar sperm to OVGP1-functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP.

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

Goat production is affected by reproductive seasonality. In vitro 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 in vitro capacitation.

Why choose the rabbit to work in reproductive technology?

Rabbits have played a significant role in both livestock production and the advancement of reproductive scientific research. Their unique biological traits, including induced ovulation and a reproductive process that closely mirrors that of humans, have been pivotal in their use as a model. Moreover, their body size is perfectly aligned with the 3Rs principles: Replacement, Reduction, and Refinement. Consequently, techniques for gamete collection and embryo recovery, followed by their use in artificial insemination or embryo transfer, are characterized by being minimally invasive. However, refining in vitro fertilization and embryo culture techniques continues to present challenges. The incorporation of cutting-edge genomic editing tools, such as CRISPR/Cas9, has reestablished rabbits as essential models in genetic and biomedical research, driving scientific progress. This review aims to describe the most effective reproductive biotechnologies for both male and female rabbits and how these methodologies are in line with the 3Rs principles-Replacement, Reduction, and Refinement-highlighting their significance in conducting ethical research.

Enhancing felid conservation: Exploring the impact of in vitro culture media on domestic cat blastocyst production.

This study investigated the optimization of assisted reproductive techniques for wild felid conservation, focusing on in vitro procedures using the domestic cat as a model species. The research evaluated the impact of three different in vitro culture media on blastocyst formation. Oocytes and spermatozoa were collected and processed, followed by in vitro fertilization and culture. Results returned a similar blastocyst rate (ANOVA, p > .05), over 16% across all groups. While demonstrating the potential of these techniques, further investigations are warranted to evaluate embryo quality to refine optimal protocols and their applicability in felid conservation efforts.

Advanced assisted reproduction technologies in endangered mammalian species.

A new synergistic approach of classical conservation strategies combined with advanced assisted reproduction technologies (aART) allows for protection and rescue of endangered keystone species at the brink of extinction, which can help to safeguard complex ecosystems. Reproduction biology and management in mammal species is not only challenging in regards to their diverging sizes, anatomy, and often unknown physiology; it also requires customized training or chemical restraint protocols for safe handling. Besides these general challenges, there are several new assisted reproduction techniques (ART) specifically tailored to critically endangered mammals. The current portfolio of ART in these mammalian taxa is ranging from sexual cycle characterization and manipulation, semen collection and cryopreservation, artificial insemination, biobanking of living cells, oocyte collection, in vitro fertilization (IVF), and embryo production, embryo transfer as well as stem cell-derived in vitro gametogenesis for generating gametes in culture. The article covers advanced assisted reproduction technologies (aART), success and challenges, as well as ethical implications.

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

Cryopreservation of bovine oocytes and embryos is essential for long-term preservation and widespread distribution of genetic material, particularly in bovine in vitro 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 in vitro-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.

The Cryotop vitrification system is competent for the simultaneous cryopreservation of large numbers of pig in vitro-produced blastocysts.

This study aimed to compare the effectiveness, in terms of viability and quality post-warming, when vitrifying in vitro-produced (IVP) pig blastocysts with either a modified Cryotop (n = 161; 20 blastocysts/device) or the conventional Superfine Open Pulled Straw (SOPS; n=160; 5-6 blastocysts/device systems. Blastocyst viability, morphology, and apoptosis parameters were evaluated after 24 h of in vitro culture. The Cryotop system yields better results in terms of higher embryo viability and total cell numbers (p < .05) and lower apoptosis (p < .05) parameters than the SOPS procedure, defining a high effectiveness to simultaneously vitrify 20 pig IVP blastocysts at one time, thus increasing the yield of IVP blastocysts readily available for embryo transfer.

Effect of the addition of IGF-1 during in vitro culture on the embryonic development speed from different crossbreed bovine embryos.

Supplementation with insulin-like growth factor type 1 (IGF-1) during in vitro culture of bovine embryos has yielded mixed results, likely due to genetic variability among embryos. This work aimed to evaluate the effect of IGF-1 at two concentrations on the development speed embryos from primary F1 crossbreeds used in dual-purpose cattle farming in the Colombian low tropics. Specifically, we investigated the influence of IGF-1 and embryo breed on the blastocyst formation rate. Oocytes were sourced from non-pregnant cows: Bos taurus indicus (20 Brahman and 14 Gyr) and Bos taurus taurus (12 Holstein and 28 Romosinuano). Oocytes were fertilized with semen from specific bulls (Recoil for Holstein, Gabinete for Gyr, and UBER POI 1490 for Brahman). The resulting embryos from each crossbreed group were randomly distributed in three different cultured media with 50 ng/mL IGF-1, 100 ng/mL IGF-1, or no IGF-1 (control) for 7 days. Results showed that 50 ng/mL IGF-1 significantly increased embryo production by day 6 (25.9%±14.56%) compared to control (20.5%±11.84%) and 100 ng/mL IGF-1 (23.0%±9.54%) (p < 0.05). By day 7, both 50 ng/mL (42.6%±26.55%) and 100 ng/mL (49.7%±21.98%) IGF-1 groups exhibited significantly higher production rates compared to the control group (p < 0.001). The embryo breed also influenced development, with Gyr-Holstein (GxH) crossbreeds showing the highest production rates (p < 0.001). In conclusion, IGF-1 supplementation enhances in vitro embryo production, with the effect influenced by both breed and IGF-1 concentration. These findings suggest that breed-specific optimization of IGF-1 conditions is necessary to maximize embryonic development outcomes.

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 [150 mg/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.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

High and low performing sires differ in their contributions to early embryonic stress in the bovine.

Context Sires differ in their ability to produce viable blastocysts, yet our understanding of the cellular mechanisms regulated by the sire during early embryo development is limited. Aims The first aim was to characterise autophagy and reactive oxygen species (ROS) in embryos produced by high and low performing sires under normal and stress culture conditions. The second aim was to evaluate DNA damage and lipid peroxidation as mechanisms that may be impacted by increased cellular stress, specifically oxidative stress. Methods Embryos were produced using four high and four low performing sires based on their ability to produce embryos. Autophagy and ROS were measured throughout development. To evaluate oxidative stress response, autophagy, and ROS were measured in 2-6 cell embryos exposed to heat stress. To understand how cellular stress impacts development, DNA damage and lipid peroxidation were assessed. Key results Under normal conditions, embryos from low performing sires had increased ROS and autophagy. Under heat stress, embryos from low performing sires had increased ROS, yet those from high performing sires had increased autophagy. There was no difference in DNA damage or lipid peroxidation. Conclusions Results suggest that embryos from low performing sires may begin development under increased cellular stress, and autophagy potentially increases to mitigate the impacts of stress. Implications There is potential for improving embryonic competence through selection of sires with lower stress-related markers.

Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy.

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h.

Development of the world farm animal embryo industry over the past 30 years.

Embryo technologies have been used over the past three decades globally in most relevant farm species. From a sanitary perspective, embryos collected in vivo have long been recognized as the safest way to trade livestock germplasm, as long as the IETS washing and, if recommended, trypsin-decontaminating procedures are adopted. On the other hand, a number of questions have been raised about the safety of embryos produced in vitro, frequently inhibiting the international commerce of these embryos. In the major players of the world embryo industry, however, in vitro embryo production (IVEP) has become the technology of choice in cattle, and its adoption on a large-scale has caused the world embryo records to be scaled up exponentially. Nowadays, over a million cattle embryos are produced and transferred worldwide, of which approximately 80 % are in vitro produced. Moreover, the same trend is currently being observed in small ruminants and horses. In the present review, we describe the development of the world embryo industry over the past few decades and speculate on how this contributed to the apparent dichotomy between the perceived and the actual risk of transmission of infectious diseases by the transfer of in vitro produced embryos. Additionally, we discuss the future trends of the international market of livestock germplasm, in the light of the changes driven by emerging embryo technologies.

Disease risk of in vitro produced embryos: A review of current commercial practices in the context of international trade with emphasis on bovine embryos.

The ever-evolving procedures of the practice of in vitro production (IVP) of embryos has well outpaced the ability of governments and institutions to create standardized rules and regulations around the global trade of these reproductive products. There are several challenges related to identifying and quantifying disease transmission risk in IVP embryos, not the least of which is a lack of published information. The International Embryo Technology Society (IETS) Health and Safety Advisory Committee (HASAC) has identified and addressed several of the challenges related to the potential for disease transmission via IVP embryos. This review will outline the current sanitation practices recommended by the IETS for in vivo derived (IVD) embryos, as well as the current practices at most in vitro fertilization (IVF) labs and will present the case that IVP embryos are not only just as safe as IVD embryos from a disease transmission risk standpoint, but perhaps may even be safer than IVD embryos, as the oocyte, zygote, and embryo in the in vitro production system has no contact with the downstream reproductive anatomy (fimbria, isthmus, oviduct, or uterus) of the donor female.

The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility.

Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2 µg/ml) in "TALP-Sperm" and "TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.

Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.