Background: Mitochondrial morphological homeostasis is essential for energy production and developmental competence in preimplantation embryos. While epigenetic modifications, particularly histone acetylation, are primarily recognized as regulators of gene transcription, accumulating evidence suggests that acetylation-dependent signaling also governs mitochondrial biogenesis and quality control. However, how PCAF-mediated acetyltransferase activity specifically modulates mitochondrial morphology during early embryonic development remains unknown. Methods: Porcine parthenogenetic embryos were cultured for seven days in PZM-5 medium. Garcinol (5 μM) was applied from the 4-cell stage to inhibit PCAF activity, and mitochondrial ultrastructure was assessed by transmission electron microscopy using a Bio-HVEM system. Results: Ultrastructural analysis of porcine blastocysts revealed morphologically diverse mitochondrial populations, encompassing spherical, hooded, cap-shaped, ring-shaped, horseshoe-shaped, elongated, and shell-like configurations. Mitochondrial fusion predominantly occurred in a side-to-side orientation, with tip-to-tip and side-to-tip contacts observed less frequently, and these combinatorial fusion states collectively contributed to the morphological heterogeneity of the organelle pool. PCAF inhibition disrupted this dynamic equilibrium, inducing aberrant fusion events and the formation of hyperfused clusters containing three or more organelles. Concurrently, mitochondria-derived vesicles, mitochondrial multivesicular body-like structures, and mitophagy-associated autophagy were increased throughout the cytoplasm, reflecting quality-control responses to mitochondrial damage. Conclusions: Collectively, these findings highlight that PCAF-mediated acetyltransferase activity is required for mitochondrial morphostasis, and that its suppression disrupts the fission-fusion balance while activating compensatory quality control responses. These results provide new insight into how epigenetic acetylation signaling is functionally coupled to mitochondrial structural integrity during early embryonic development.
Background: Canine mammary tumors are one of the most common tumors in female dogs, and the development of effective therapeutic agents is a critical clinical challenge. Chrysin, a natural flavonoid compound, has been reported to exhibit anticancer activity in various human cancers through the regulation of cell proliferation and apoptosis. However, the anticancer effects of chrysin on canine mammary tumors have not yet been fully elucidated. Therefore, this study investigated the antiproliferative and apoptosis-inducing effects of chrysin on CMT-U27 cells derived from canine mammary tumors. Methods: Antiproliferative activity was confirmed by treating CMT-U27 cells with chrysin at concentrations of 0-10 μM and evaluating cell viability. Cell proliferation capacity was assessed using Ki-67 immunocytochemistry. The expression levels of cell cycle-related proteins, including CDK2, CDK4, cyclin D, and cyclin E, were analyzed by Western blotting. Additionally, apoptosis-related proteins, including phosphorylated p53 and BCL2, were evaluated to investigate the apoptotic activity of chrysin treatment. Results: Chrysin treatment significantly reduced the viability and migration of CMT-U27 cells and decreased the number of Ki-67-positive cells in a dose-dependent manner. Following chrysin treatment, the expression of CDK2, CDK4, cyclin D, and cyclin E was significantly reduced, indicating that cell cycle progression was inhibited. Additionally, chrysin increased the expression of phosphorylated p53 and decreased BCL2 expression. Conclusions: Chrysin exhibited significant anticancer effects in canine mammary tumor-derived CMT-U27 cells through the inhibition of cell proliferation, accompanied by G2/M phase accumulation and downregulation of cyclin-CDK complex components. In addition, chrysin altered apoptosis-related signaling molecules, including p53 and BCL2. These results suggest that chrysin may have potential as a therapeutic candidate for canine mammary tumors.
Background: Sperm cooling is a critical step in cryopreservation, as exposure to temperatures below 15℃ can induce cold shock-associated damage, including phospholipid scrambling and lipid peroxidation. Goat sperm are particularly vulnerable to cooling-induced injury because of their membrane characteristics. This study evaluated the effects of cooling rate and interval agitation during cooling in frozen-thawed goat sperm. Methods: The samples were cooled at 0.5℃/min (0.5 Cool) or 0.1℃/min (0.1 Cool) and agitated every 15 min during cooling until 5℃, then, the samples were cryopreserved in liquid nitrogen. After thawing, sperm motility was assessed using computer-assisted sperm analysis (CASA) and plasma membrane integrity, acrosomal integrity, intracellular H2O2 levels, mitochondrial activity, mitochondrial membrane integrity, and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Results: In results, there were no significant differences in sperm motility, plasma membrane integrity, acrosomal integrity, and mitochondrial function among the treatment groups. However, intracellular H2O2 levels were significantly lower in agitation-treated groups than non-agitated groups both 0.5 Cool and 0.1 Cool treatment groups (p < 0.05), whereas there were no significant differences between cooling rate groups. Conclusions: In conclusion, these results indicate that interval agitation during the cooling process could reduce intracellular reactive oxygen species in frozen-thawed goat sperm.
Background: This pilot study aims to develop an automated, multi-task deep learning system as a proof of concept for both the classification of pregnancy status and gestational day prediction in Korean crossbred goats. Methods: Two 1-year-old goats were followed longitudinally until mid to late gestation. A complete data set containing 11,092 high-resolution image frames (6,647 pregnant and 4,445 non-pregnant) was created from the video sequences. The dataset was segmented at the frame level with 80% allocated to the training partition (n = 8,875), 10% to the validation partition (n = 1,110), and 10% to the test partition (n = 1,107). Multi-task Four convolutional neural networks (ResNet18, EfficientNet-B0, MobileNetV3-Large, and ConvNeXt-Tiny) were trained following a multi-task learning strategy with the AdamW optimizer, OneCycle scheduling, and a hybrid Binary Cross-Entropy and Huber loss function. Results: Accuracy was achieved for the four architectures, with more than 99.5%. The MobileNetV3-Large model performed the best in terms of frame-level classification accuracy, with 100% sensitivity and specificity. For the regression-based task for predicting gestational days, EfficientNet-B0 got the best performance with a frame-level Mean Absolute Error (MAE) of 1.3 days. The video-level MAE of 0.882 days and the competitive video MAE of 1.204 days were the outcomes for the aggregated video sequences using ConvNeXt-Tiny and EfficientNet-B0, respectively. Conclusions: The present pilot study lays the foundation for the methodological feasibility of small optimized deep learning models for accurate monitoring of fetal development and for the automation of reproductive diagnostics.
Background: Miniature pigs are widely used as biomedical models because of their anatomical and physiological similarities to humans; however, hematologic and serum biochemical data for newly developed or regionally derived populations remain limited. The Jeju miniature pig, derived from Korean Jeju native pigs, is an emerging laboratory animal with potential value in veterinary and translational research. This study aimed to characterize hematologic and serum biochemical profiles in healthy Jeju miniature pigs and to evaluate age- and sex-associated physiological variation. Methods: Blood samples from clinically healthy animals were analyzed for complete blood count and serum biochemical parameters, and animals were stratified by age and sex to assess demographic influences. Results: Age was the primary determinant of variation across most parameters. Juvenile animals exhibited higher lipid-related markers and alkaline phosphatase activity, whereas adults showed increased creatinine and total protein concentrations, consistent with physiological maturation. Sex-related effects were modest and largely confined to erythrocyte-associated indices. Comparative analysis demonstrated that these age-related patterns were broadly consistent with those reported in established miniature pig breeds, including Göttingen and Yucatan pigs. Conclusions: These findings provide baseline physiologic information for Jeju miniature pigs and improve understanding of age- and sex-associated hematologic and biochemical variation in this emerging biomedical animal model, supporting its application in experimental and translational research settings.
Abstract Background: Africa is the fastest-growing continent, with more than half of the global population growth expected to occur in the region by 2050. The increasing population and lifestyle changes translate to high demand for food. However, Africa does not produce enough food to cater to its population. The current study sought to bridge knowledge gaps in the dairy and beef production systems by addressing factors affecting semen production in bulls used for AI at the Kenya Animal Genetic Resources Center. Methods: A total of 88 bulls met the inclusion criteria (> 16 months, healthy, and in active semen collection). The breed breakdown was as follows; Ayrshire (n = 39), Friesian (n = 34), Jersey (n = 7), Boran (n = 3), and Sahiwal (n = 5). Bull characteristics (age, breed, body weight, BCS), testicular attributes (SC and testicular echotexture), and semen parameters (semen volume, forward progressive motility, sperm concentration, and post-thaw motility) were determined, and results were analyzed using R statistical software. Results: Semen volume significantly increased with body weight, age and SC (p < 0.001-0.05). Sperm concentration significantly decreased with increased BCS (p < 0.05). Breed significantly affected semen volume and sperm concentration (p < 0.01-0.05). Hyperechoic testicular lesions noticed in older bulls did not significantly affect semen quality (p > 0.05). Conclusions: These findings will inform bull selection and culling criteria, highlighting that bulls presumed overdue for culling can still produce high-quality semen (the oldest bull at the time of data collection was aged 140 months). Further studies elucidate the relationship between increased testicular echotexture and semen parameters.
In this case report we describe the clinical condition of a 5-year-old Nelore bull with marked enlargement of the right side of the scrotum. After ultrasound evaluation, a hydrocele involving the right testicle was observed. The right testis and epididymis were surgically removed along with the tunica vaginalis and histological examination of each segment was performed. On macroscopic examination gray nodules were found in the tunica vaginalis and adhesions of the visceral layer with the testis. On histopathological evaluation the nodules consisted of papillary neoplastic cells that had invaded the surrounding tissue. Based on these findings, the tumor was diagnosed as sarcomatoid mesothelioma. This is the first case confirmed of mesothelioma in the tunica vaginalis in a semen donor bull by histopathology in Brazil.
Background: Heat stress (HS), driven by global climate change, adversely affects reproductive performance in livestock and mammals by impairing gamete development and reducing fertility. Exposure of oocytes to elevated temperatures during in vitro maturation (IVM) can induce persistent cellular stress that compromises subsequent embryonic development. However, the involvement of ATF5-mediated UPRmt signaling and GRP75-associated ER-mitochondria communication during early bovine embryogenesis under HS remains unclear. Methods: In this study, bovine cumulus–oocyte complexes (COCs) were matured for 22 h at either 38.5℃ (control; Con) or 41.5℃ (HS), followed by in vitro fertilization and embryo culture under standard conditions. Results: HS exposure during IVM significantly reduced cleavage and blastocyst formation rates (p < 0.01). Embryos derived from HS-exposed oocytes exhibited decreased mitochondrial membrane potential (ΔΨm), increased reactive oxygen species (ROS) production, and mitochondrial calcium (Ca2+) accumulation, suggesting disruption of GRP75 (known as HSPA9)-mediated ER-mitochondria communication. These embryos also showed increased expression of HSPA9 and HSPD1 at the cleavage stage, indicating persistence of stress signals beyond fertilization. Immunofluorescence analysis revealed enhanced nuclear localization of ATF5 in blastocysts, while qPCR analysis demonstrated increased expressions of ATF5-mediated UPRmt-related genes, including LONP1, HSPD1, and HSPA9. Conclusions: Collectively, these findings demonstrate that heat stress during oocyte maturation induces persistent mitochondrial dysfunction associated with GRP75-mediated mitochondrial Ca2+ dysregulation and ATF5-mediated UPRmt activation, which may represent an adaptive compensatory response in surviving bovine embryos.
Background: The morphological characteristics of sperm are essential for assessing sperm quality, which partially influences reproductive efficiency variables such as litter size in sows. Recently, deep-learning-based object detection algorithms have been explored to detect and classify sperm morphological features, with the training of these models requiring sperm microscopy image data. The performance of these models in detecting morphological features was significantly affected by the size of the dataset and the image quality of the images. Methods: This study proposed a deep-learning-based super-resolution (SR) algorithm to enhance the quality of sperm microscope images. The model was trained using a dataset consisting of high-resolution (HR) original and low-resolution (LR) images generated by downscaling the original images through bicubic interpolation. The SR results of the test dataset were compared with those of the original HR images to evaluate their performances using the peak signal-to-noise ratio (PSNR) and structural similarity index measure (SSIM). Statistical analyses were conducted to examine the performance differences based on the training models and dataset types. Results: This study indicated that the SR images showed no significant differences in PSNR (p = 0.9740) and SSIM (p = 0.9864) compared with the original HR images in same magnification. Moreover, increased processing speed was observed with reductions in model hyperparameters. While processing SR images improved spatial resolution across various microscopic magnifications, the overall image quality did not exceed that of the original HR images. Conclusions: SR models applied to sperm microscopy images outperformed conventional SR algorithms. These findings suggest that SR algorithms hold promise for improving the quality of LR microscopic images in future deep-learning-based object-detection algorithms.
Background: Myostatin (MSTN) gene is a major inhibitor of skeletal muscle growth, and variants within its promoter region may affect transcriptional activity and muscle-related traits. Mongolian native horses have adapted to endurance and harsh environmental conditions, whereas Halla horses exhibit enhanced musculature owing to crossbreeding with Thoroughbreds. MSTN promoter variations in these breeds have not been sufficiently investigated, highlighting a critical research gap that motivated the present study. Methods: Two single nucleotide polymorphisms (SNPs) in the MSTN promoter region were examined in 19 Mongolian and 4 Halla horses using PCR amplification and Sanger sequencing. Additionally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway annotations were used to characterize gene functions. Results: Two promoter region SNPs were identified in MSTN gene. These distribution patterns represent preliminary observations within the sampled populations, providing a valuable foundation for future investigations. Functional annotation further supported the regulatory relevance of MSTN through its involvement in muscle growth inhibition and the TGF-β/ACVR2B/SMAD pathway. Conclusions: This study provides the first baseline characterization of MSTN promoter polymorphisms in Mongolian and Halla horses. The g.26T>C variant was identified in Mongolian horses, whereas the shared g.156T>C SNP represents a regulatory variant observed in both breeds, pending confirmation in broader populations. However, due to the limited sample size, we consider these findings to be preliminary observations rather than definitive evidence of breed-specific adaptation. The results of this study are expected to serve as fundamental data for establishing future genetic information–based breeding and conservation strategies for Mongolian horses.
Background: When chemically defined media are used for in vitro maturation (IVM), fetal calf serum (FCS) and bovine serum albumin (BSA) are often replaced with synthetic macromolecules such as polyvinyl alcohol (PVA). However, the developmental competence of oocytes under these conditions is typically reduced, with lower blastocyst formation rates. This study aimed to quantify the transcripts of GDF9, BMP15, and OOSP1 in oocytes, and GREM1, PTGS2, PFKP, AREG, EREG, HAS2, VCAN, PTX3, ADAM10, and ADAM17 in cumulus cells. Methods: Oocytes were divided into three groups: immature oocytes at the germinal vesicle stage (GV), in vivo matured oocytes (IVMO), and oocytes matured in vitro in IVM medium supplemented with 10% FCS, 4 mg/mL BSA, or 1 mg/mL PVA. For the IVMO group, ten donor cows were superovulated with follicle-stimulating hormone (FSH), and cumulus–oocyte complexes (COCs) were recovered by ovum pick-up (OPU) 19-20 hours after gonadorelin administration. Gene expression was evaluated in oocytes and cumulus cells using quantitative real-time PCR. Results: The relative transcript levels of GREM1, PTGS2, PFKP, and AREG were significantly higher (p < 0.05) in cumulus cells from IVMO compared with those from GV or IVM oocytes. Additionally, oocytes matured in vitro in medium supplemented with FCS showed increased (p < 0.05) expression of GREM1 and AREG compared with those cultured with BSA or PVA. Conclusions: FCS supplementation during IVM positively influenced the transcription of GREM1 and AREG. However, the superior expression profile observed in cumulus cells from in vivo matured oocytes highlights the need for improved IVM media and culture conditions to enhance oocyte competence.
Background: Cryptorchidism is the most common congenital abnormality affecting male reproductive development in dogs. Castration is generally recommended for affected individuals, and many cases are identified incidentally during elective procedures. A better understanding of the epidemiology and clinical characteristics of cryptorchidism may enhance awareness among veterinarians and dog owners, thereby contributing to improved surgical management and postoperative care. Methods: Clinical data were retrospectively collected from dogs diagnosed with cryptorchidism between 2018 and 2025. Cryptorchidism was classified according to the anatomical location and laterality of the undescended testis. Breed-related associations were evaluated by comparing distribution patterns between purebred and mixed-breed dogs using Fisher’s exact test and odds ratios analyses. Results: Among 618 dogs undergoing castration, 80 were diagnosed with cryptorchidism (12.94%). Right-sided unilateral cryptorchidism was the most common presentation (n = 55, 8.90%), followed by left-sided unilateral cryptorchidism (n = 17, 2.75%), and bilateral cryptorchidism was the least frequent (n = 8, 1.29%). Undescended testes were more frequently located in the inguinal region (n = 53, 66.25%) than in the abdominal cavity (n = 27, 33.75%). No statistically significant breed-specific differences in anatomical location were detected when compared with mixed-breed dogs. Conclusions: This study characterizes dog cryptorchidism based on anatomical location and laterality, providing baseline clinical data on breed-related distribution patterns that may support future studies of clinical decision-making.
Background: The rise in annual mean temperatures caused by global warming has increased the risk of heat stress in livestock. Dairy cows are particularly susceptible, with both productivity and reproductive performance adversely affected. Previous studies have reported that THI ≥ 72 is generally considered the onset of heat stress. Against this background, the present study examines conception rate variations of dairy cows in relation to the THI in Jeolla-do region through 2024. Methods: The THI for the Jeolla-do region was collected from the Korea Meteorological Administration Data Portal, and artificial insemination conception records were compiled for 35,053 cows from 39 farms across Gwangju Metropolitan City and eight counties in Jeolla-do region (Gochang, Gwangju, Yeonggwang, Naju, Hampyeong, Muan, Jangseong, and Yeongam). Seasonal artificial insemination conception rates (spring: March-May, summer: June-August, autumn: September-November, and winter: December-February) were compared and analyzed in relation to the regional THI values (n = 35,053). Results: The THI threshold for heat stress in dairy cows (THI ≥ 72) persisted from May through September, and all locations, except Yeongam and Naju, reached their annual maximum THI in August. In Jangseong, THI remained ≥ 72 for the longest period, lasting 175 days. In most locations, the highest monthly conception rate occurred in July. By season, conception rates were highest in summer (June-August) at 58.5% and lowest in autumn (September-November) at 43.5%. Conclusions: The pronounced reduction in conception rates observed in autumn appears to be a consequence of the cumulative heat stress of the summer, which continues to impair reproductive performance even in autumn. Although farms commonly deploy cooling systems such as ventilation fans and sprinklers during the summer, management of heat stress may be neglected in autumn despite animals continuing to experience thermal stress, thus contributing to the observed decline in conception rates. These findings underscore the need for vigilant herd management and heat‑stress mitigation not only in summer but also in autumn. The results can serve as baseline data for future research on the effects of heat stress on dairy cattle and on strategies for its mitigation.
Background: Cigarette smoke is a well-known reproductive toxicant that causes structural and functional damage to the testis through oxidative stress and apoptotic pathways. This study aimed to evaluate whether apple seed extract (ASE) can mitigate testicular damage induced by tobacco smoke extract (TSE) in a mouse model. Methods: Adult male mice were orally administered TSE to induce testicular injury. ASE was delivered intraperitoneally, either as a post-treatment or concurrently with TSE. Histological evaluation (H&E staining), immunohistochemistry (IGF-1 and VEGF), and immunofluorescence (TNF-α, Caspase-3, and BCL-2) were conducted to assess tissue morphology, growth factor expression, and apoptotic signaling. Results: TSE exposure led to degeneration of the seminiferous tubules, suppressed IGF-1 and VEGF expression, and increased pro-apoptotic markers (TNF-α, Caspase-3), along with reduced BCL-2 expression. ASE treatment restored seminiferous tubule architecture, enhanced the expression of growth and anti-apoptotic factors, and attenuated apoptotic signals. These restorative effects were particularly significant in the post-treatment groups. Conclusions: ASE demonstrated protective and reparative effects against TSE-induced testicular toxicity by modulating oxidative and apoptotic pathways. These findings highlight ASE as a promising natural therapeutic candidate for ameliorating smoke-related male reproductive dysfunction.
Developing reliable models that capture the complexity of maternal–embryonic interactions and implantation is essential for deepening our understanding of early embryonic development as well as the underlying mechanisms of reproductive disorders. Conventional two-dimensional (2D) culture systems, however, fall short in accurately replicating the dynamic and multilayered in vivo microenvironment. In contrast, three-dimensional (3D) organoid technologies have recently emerged as a transformative approach, offering structurally and functionally relevant platforms that better reflect physiological conditions. This review highlights the latest strategies, innovations, and methodological advances in employing 3D organoids to model maternal–embryonic communication and implantation processes. It also outlines their growing potential in research applications and personalized medicine, particularly within the context of assisted reproductive technologies. Furthermore, the review discusses future directions for organoid-based studies from the endometrium, oviduct, ovary, testis, and epididymis have been established, their integration with gametes and embryos marks a new frontier. Cross-species work, especially your pioneering studies on endometrial organoids and lacunoids/cystoids, opens translational opportunities for understanding maternal–embryonic crosstalk, implantation biology, and fertility disorders. The review will discuss current advances, technical challenges, and future directions toward interconnected organoid systems (organoid-on-chip).
Background: Pyometra, involves pus accumulation that can lead to death if detected late, making early diagnosis and management crucial, and it is more common in older dogs. The Axiom™ Canine HD Array was used in the study to determine single nucleotide polymorphisms (SNPs) associated with pyometra, with the aim of providing foundational data for the future development of SNP markers. Methods: Samples were collected from 95 dogs of 26 breeds in South Korea, and SNP genotypes were analyzed for the final two SNPs (AX-168186923, AX-168208364). To verify the sequences and genotypes of the selected SNPs, we conducted a sequencing analysis of 96 dogs from 11 breeds previously reported to have been affected by the disease. To confirm the linkage disequilibrium (LD) between the selected SNPs and the influence of non-synonymous SNP (nsSNP), candidate gene function and, protein structure change prediction analyses were performed. Results: Sequence and genotype analyses of the selected SNPs revealed differences in genotype frequencies among the varieties. A strong LD block was formed between the SNPs, confirming that both SNPs were genetically associated. Candidate gene ABCC4 analysis revealed that ABCC4 transports prostaglandins. An analysis of the three-dimensional protein structure affected by the nsSNP (AX-168186923) showed that the amino acid changed from Methionine to Valine, but the overall protein structure remained unchanged. Conclusions: The SNPs selected in this study can be used to identify genetic factors, associated with pyometra and vulnerable breeds. This is expected to reduce companion animal related costs and the psychological burden on owners.
Background: Mitoquinone mesylate (MitoQ), a synthetic analog of coenzyme Q10 with antioxidant properties, has demonstrated significant improvements in mitochondrial penetration compared to coenzyme Q10, demonstrating potential medicinal benefits. We investigated the effects of MitoQ on neonatal testicular development using ex vivo mouse testicular fragments (MTFs). Methods: Testicular cell differentiation was assessed by culturing MTF from 5-day-old male mice in vitro with 0.01-1 μM MitoQ for 5 days. Cell differentiation, proliferation, and distribution within the testes were assessed using immunohistochemical staining and analysis of gene and protein expression. Results: Histological analysis revealed that 0.01-1 μM MitoQ induced germ cell miogenesis in the seminiferous tubules, increasing the number of Sycp3-positive cells and reducing the genetic expression of the undifferentiated marker Sall4. Sertoli and Leydig cell markers increased at the genetic and protein levels, as confirmed by immunohistochemical staining. In addition, MitoQ increased the expression of antioxidant regulatory and antioxidant enzyme-related factors. Conclusions: Overall, MitoQ can induce the expression of antioxidant factors in in vitro cultured neonatal testes while simultaneously enhancing germ cell differentiation, Sertoli cell proliferation, and Leydig cell maturation and proliferation.
Background: Cryopreserved semen and embryos are essential tools in livestock reproduction, enabling genetic improvement and herd management. Although these materials are theoretically stable in liquid nitrogen (LN2), viability often decreases over time, particularly in farm settings. Micro-ice crystals (MICs) are hypothesized to form under poor LN2 handling conditions, potentially compromising the survival of frozen genetic resources. However, the extent and impact of MIC accumulation have not been thoroughly quantified. Methods: This study evaluated MIC accumulation and its effects on the viability of cryopreserved bovine semen and embryos under different LN2 storage environments and conditions. MIC content was measured by filtering 10 L of LN2 through nonwoven fabric and weighing the retained crystals and debris. The viability of sperm and embryos were assessed using a computer-assisted semen analysis (CASA) and blastocoel re-expansion. Results: MIC content was 3.5 times higher in farm-stored LN2 than in laboratory LN2, with significantly more debris also detected. Progressive motility and velocity parameters (VCL, VAP, VSL) were similarly reduced. Blastocyst survival dropped significantly under farm conditions after six months (42.4%) compared to laboratory storage (84.4%, p < 0.05). These findings suggest a strong correlation between MIC accumulation and decreased post-thaw viability of cryopreserved materials. Conclusions: MICs formed in LN2 due to environmental exposure and poor handling can severely impair the viability of cryopreserved sperm and embryos. Regular filtration and improved LN2 management, especially in farm environments, are essential to reduce MIC-related damage. These practices may enhance the long-term usability and reliability of genetic resources in livestock breeding programs.
