Yellowish myotis presented four reproductive stages and long-term sperm storage in cauda epididymis. Aiming to investigate the morphofunctional aspects of the epididymis and spermatozoa of yellowish myotis, 48 adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil, for histomorphometric evaluations of the epididymis and sperm analysis. Yellowish myotis spermatozoa were observed in the caput and corpus epididymis only during the Mature and Regressed stages, coinciding with increased epithelial height in these stages, indicating their possible role in sperm maturation. In contrast, spermatozoa were found in the cauda epididymis during all reproductive stages, with the epithelium maintaining a consistent height, which suggests that this region is adapted for long-term sperm storage. The spermatozoa remained alive and motile for up to 25 days during the Regressed and Early Rest stages. Mating period in yellowish myotis occurs during the Rest stage, when spermatozoa in the cauda epididymis exhibit reduced DNA fragmentation and minimal chromatin abnormalities. During this period, sperm with wider heads and longer midpieces dominate, reflecting selection for traits that would enhance fertilization success. These results reinforced the importance of cauda epididymis physiology for sperm survival and future events of capacitation and fertilization.

FOXR1 belongs to the large conserved F-box family of DNA binding transcription factors that are involved in various developmental processes and diseases. The gene is important for embryogenesis in a fish model, brain development in mammals, and human FOXR1 was shown to possess oncogenic properties when it is abnormally expressed as a fusion gene. Earlier work on Foxr1 expression in mouse and human suggested roles for the protein in embryogenesis and sexual reproduction. We generated a mouse gene deletion model that revealed an embryonic lethal phenotype with partial penetrance and normal fertility in persistent homozygous male mutants. The results suggest that Foxr1 is functionally redundant in adult male gonads but needed for normal early embryo development and post-natal viability. We discuss our results in the context of publicly available human and rodent genomic and genetic data.

To identify the optimal in vitro maturation (IVM) duration for bovine oocytes with different nuclear maturation speeds (NMS), this study assessed how varying IVM durations (24, 28, and 32 h) affect developmental competence and embryo quality in oocytes with fast- or slow-predicted NMS classified via machine learning. Developmental competence was evaluated through cleavage rates, first cleavage timing and patterns, and blastocyst formation under individual culture. Embryo quality was assessed via differential staining of inner cell mass and trophectoderm and expression analysis of quality-related genes in formed blastocysts. For oocytes with slow-predicted NMS, extending IVM to 28 h increased cleavage rates and accelerated first cleavage timing (p < 0.01). The lower blastocyst formation rates of oocytes with slow-predicted NMS matured for 24 h improved when IVM reached 28 h, becoming comparable to fast-predicted NMS oocytes. However, extended IVM decreased expression of pluripotency-related genes (e.g., NANOG and OCT4; p < 0.01) regardless of predicted NMS. In conclusion, extending IVM duration to 28 h improved developmental competence of slow-predicted NMS oocytes, highlighting the importance of fertilization timing relative to nuclear maturation completion, though it reduced expression of key pluripotency genes. Individualized IVM protocols based on predicted NMS can enhance bovine embryo production efficiency.

Embryo genome activation (EGA) is a crucial event implicated in proper embryonic development. Similarly, the DNA Damage Response (DDR) is essential for correcting or preventing occasional errors during embryonic cell division that could result in embryo development arrest or the propagation of genetic abnormalities. Although both EGA and DDR are regulated by epigenetic mechanisms, the role of microRNAs (miRNAs) in these processes has not been explored in pig embryos. Herein, we assessed the abundance of miRNAs linked to embryo development and DDR across four developmental stages: Day 2 (D2), Day 3 (D3), and Day 4 (D4), and at the blastocyst stage, as well as after DNA damage induction. mRNA levels of EGA-related genes confirmed our timepoints as representing EGA timeframe, while immunofluorescence for γH2AX validated DNA damage induction. Significant decrease in blastocyst rate and total cell number per blastocyst was detected in UV-exposed embryos. Analysis of miRNA abundance revealed increased levels of miR-200a-5p on D3, which were partially maintained on D4. Both miR-15a and miR-24-3p increased on D4, but their levels were downregulated in UV-exposed embryos at the same stage of development. In blastocysts, UV exposure upregulated miR-29a-3p and miR-344b-3p. Together, these findings provide the first characterization of miRNAs expression in porcine embryos during EGA and following DNA damage induction.

This study aimed to assess differences in the immunostaining intensity of follicle-stimulating hormone receptor (FSHr) and leutenizing hormone receptor (LHr) receptors in the ovarian follicles of Bos indicus cows with high or low antral follicle counts (AFCs). Ovaries from cyclic Nelore cows (N = 20) were obtained from a local slaughterhouse and classified based on AFC (≥ 3 mm) into high- (≥ 30 follicles, N = 10) and low- (≤ 15 follicles, N = 10) AFC groups. Immunohistochemical studies were performed for FSHr and LHr. Immunostaining intensity was measured using ImageJ software with the IHC Profiler plugin, and pixel intensity was measured on a scale of 0 (darkest) to 255 (lightest). An interaction was observed between the AFC group and follicular developmental stage for FSHr immunostaining intensity, with preantral follicles from the low-AFC group showing highest immunostaining intensity (p < 0.0001). The FSHr immunostaining intensity of antral follicles from the low-AFC group was higher than that of the high-AFC group (p = 0.03). LHr immunostaining intensity also was higher in the low-AFC group than in the high-AFC group (p = 0.002). These findings suggest that ovarian follicle characteristics of low-AFC cows have distinct characteristics that could affect their response to reproductive treatments.

Cumulus-oocyte complexes (COCs) used for in vitro production (IVP) of bovine embryos originate from antral follicles of different sizes, leading to variations in developmental competence. To address this, pre-in vitro maturation (pre-IVM) allows oocytes with additional time to acquire developmental competence. Given the role of follicular fluid-derived extracellular vesicles (EVs) in ovarian follicle communication, which has been shown to vary in content and function across folliculogenesis, we investigated whether EVs from early versus late antral follicles influence COCs during pre-IVM. EV supplementation significantly altered gene expression in cumulus cells and oocytes. In cumulus cells, affected pathways included MAPK signaling, Gap junctions, Cytokine-cytokine receptor interaction, Axon guidance, cAMP, and Cushing syndrome. In oocytes, fewer genes were altered, with effects on Inositol phosphate metabolism, p53 signaling and Cholesterol metabolism. Despite these changes, no significant effects of the EV treatment were noted on oocyte chromatin configuration and developmental competence, except for a significant increase of mitochondrial membrane potential (Δψm) in blastocysts. In conclusion, EV supplementation during pre-IVM significantly altered the transcriptional profile of COCs, with EVs from early follicles modulating the expression of genes regulating cumulus cell proliferation and gap junctions, while EVs from late follicles impacted pathways associated with meiotic resumption, cumulus cell expansion, and apoptosis. Along with improved Δψm in blastocysts, these results support a positive effect of EVs on bovine COCs, but further research is needed to better characterize the functional consequences, mainly in terms of the effects of early versus late follicle-derived EVs on oocyte developmental potential.

Among the eight types of development in sponges, the least common and least studied is direct, non-larval development during viviparity. To supplement our knowledge of this rare type of demosponge development, we present here a description of the embryonic development of four species of the genus Craniella (Demospongiae, order Tetractinellida) from the deep-sea in the Northeast Atlantic. Craniella development is asynchronous within one sponge. Mature oocytes are polylecithal and isolecithal. Embryonic development occurs in a dense double-layer follicle: layers of flattened cells and a thick layer of collagen. The cleavage is total, unequal, and asynchronous. It is characterized by collagen layers penetrating inside the embryo and surrounding blastomeres. As a result of cleavage, an oval-shaped apolar stereoblastula is formed. At the stereoblastula stage, embryonic sclerocytes secrete the first megascleres, long thin oxeas, radially positioned. Later, the embryo is divided into the peripheral, intermediate, and central zones. In the intermediate zone, choanocyte chambers, lacunes, and canals of the aquiferous system are formed. The fully formed juveniles have a subspherical to flattened shape with cone-shaped outgrowths on the surface. Unlike the adults, juveniles lack cortical microxeas and have characteristic anamonaenes spicules. The juveniles exit the mother's body through the exhalant canals of the aquiferous system. Trial Registration: Not applicable.

Small extracellular vesicles (sEVs) function as critical regulators of ovarian follicular development. Although several pathways, including one involving neutral sphingomyelinase (nSMase), contribute to sEV production, the specific pathway active in ovarian follicles has not been clearly identified. In this study, we investigated GW4869, a specific inhibitor of nSMase activity, to determine its impact on sEV production by mouse mural granulosa cells (MGCs), the primary source of follicular sEVs. We also examined how nSMase inhibition affects the in vitro growth of oocyte‒granulosa cell complexes (OGCs) derived from secondary follicles. Transcripts encoding nSMases (Smpd2 and Smpd4) were detected in MGCs, and GW4869 treatment significantly reduced sEV production in MGC monolayer cultures. Control OGCs developed into antral follicle-like structures, with the antrum-like structure separating granulosa cells into cumulus-like and MGC-like cells. However, GW4869 treatment impaired OGC development. MGC-like cells from GW4869-treated OGCs exhibited significantly lower Cyp19a1 levels, whereas adding MGC-derived sEVs promoted Cyp19a1 expression. These results suggest that nSMase activity, likely involving Smpd2 and Smpd4, is required for sEV production by MGCs and that follicular sEVs may regulate Cyp19a1 expression in MGCs.

Spexin (SPX) is adipokine linked with the regulation of metabolism and reproduction. However, its role in the corpus luteum (CL) is unknown. Our aim was to determine the expression of SPX and galanin receptor 2 and 3 (GALR2/3), in the porcine CL during the luteal phase, its regulation and the effect of SPX on the luteal steroidogenesis. We demonstrated that SPX was higher at the mRNA level in the middle and late luteal phases, opposite to GALR2, while GALR3 protein was decreased with luteal phase progression. We observed SPX and its receptors in the cytoplasm of small and large luteal cells. Insulin, luteinizing hormone, and progesterone (P4) stimulated SPX and GALR2 mRNA levels, while prostaglandin F2 decreased the GALR2 transcript. Moreover, SPX decreased P4 secretion by reducing HSD3B protein levels via GALR2 and protein kinase A and directly stimulated STAR, CYP11A1, and aromatase protein expression with no effect on oestradiol secretion. SPX increased GALR2 protein levels, mitogen-activated kinase phosphorylation, and inhibited protein kinase B while modulating protein kinase A phosphorylation. To sum up, SPX is a new, important factor in luteal cell function by regulating steroid synthesis and may be an important player in metabolically related fertility control.

Dairy cows often experience a period of negative energy balance (NEB) during the post-calving period, which can significantly impact economic outcomes due to extended calving-to-conception intervals and overall reduced fertility. This reduction is due, in part, to the impact on uterine biology by high nonesterified fatty acids (NEFA) and beta-hydroxybutyrate concentration. The uterine fluid (UF) contains small extracellular vesicles (UF-EVs) that, through their cargo, including microRNAs (miRNAs), respond to metabolic stress, affecting the uterine environment. This study aimed to assess the long-term impact of NEB intensity on the uterine environment of dairy cows. Post-partum dairy cows were classified based on NEFA concentrations in their blood during the 3 weeks post-calving as having either Low or High NEB. At 30 and 60 DPC, the synchronization protocol was started, and UF samples were collected (corresponding to ~15 days after initiation of the synchronization protocol) to isolate UF-EVs and uterine epithelial cells for miRNA and transcriptome profiling. We also investigated whether UF-EVs could modulate epithelial uterine naïve cells. Our results indicate that the uterine environment of dairy cows experiencing a High NEB post-calving is unfavorable for embryo development at 60-day post-calving. Importantly, we show that UF-EVs can reproduce this phenotype in epithelial uterine naïve cells, suggesting that UF-EVs may act as modulators of the uterine response to metabolic challenges.

We characterized the morphogenetic processes of larval metamorphosis and the development of the olynthus and heterocoelic aquiferous system (AS) in Sycettusa hastifera (syconoid) and Paraleucilla magna (leuconoid) (Porifera, Calcarea). Metamorphosis and development up to the olynthus were similar in both species. During metamorphosis, apparently, the juvenile's cell lineages were established by invagination of the ciliated pole into the larval cavity, while posterior pole cells covered the juvenile. Ciliated cells seemed to differentiate into an inner cell mass (ICM) (forming choanoblasts and scleroblasts), while granular cells seemed to form mainly the pinacoderm. Then, cavitation of the ICM formed the asconoid AS of the olynthus. Choanocyte chamber morphogenesis for the syconoid and leuconoid AS seemed to be epithelial, but followed different paths. In S. hastifera, the chambers were formed by folding the primordial choanoderm towards the external portion of the sponge, whereas in P. magna, invagination of the choanoderm into the spongocoel segregated groups of choanocytes into spherical chambers. The morphogenesis of the heterocoelic AS in these calcareans is likely different from that in Demospongiae (leuconoid AS is established via mesenchymal-to-epithelial morphogenesis) but similar to Homoscleromorpha (AS arises through epithelial morphogenesis). Characterizing the post-embryonic development in these species is the first step toward understanding the mechanisms that regulate the ontogeny and evolution of Porifera's primary synapomorphy: the aquiferous system.

Aging refers to the irreversible changes in the structure and function of organisms over time. Under the influence of social and economic factors, the apparent effect of aging on female fertility has been widely recognized; however, its effect on male fertility has not received sufficient attention. Spermatogenic stem cells can differentiate and produce gametes continuously for a long time in a man's life, but more significant cumulative effects often occur over time. These age-related effects mainly manifest as increased chromosomal abnormalities and DNA damage, lower levels of sex hormones, impaired testicular function, and reduced spermatozoa count and quality. In the past, more attention has been paid to some of the macroscopic changes associated with aging. In this review, we will focus on the cellular level, discussing the effects of aging on male germ cells and the changes in the spermatogenic microenvironment, which consists mainly of Sertoli and Leydig cells.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.

The increasing focus on understanding spermatozoa mechanisms in rodents aims to enhance reproductive stability and support conservation efforts, particularly for ecologically significant and declining species like the red-rumped agouti. We aimed to evaluate the interaction between capacitation media and time, testing BSA concentrations-low (4 mg/mL) and high (15 mg/mL)-with or without 2 mM calcium chloride (CaCl) across three time points. Epididymal sperm were distributed into five groups: without capacitation agent (WCA), high BSA (HBSA), high BSA with CaCl (HBCa), low BSA (LBSA), and low BSA with CaCl (LBCa), each incubated for 1, 3, or 6 h. Total sperm motility was significantly higher only in media with CaCl. However, only LBCa maintained high progressive motility. LBCa and HBCa maintained membrane integrity, mitochondrial functionality, and reduced reactive oxygen species levels, but only LBCa increased glutathione levels. HBSA, HBCa, LBSA, and LBCa improved the sperm capacitation rate, but LBCa yielded the highest proportion of capacitated sperm and acrosome-reacted cells after 6 h. Hyperactivation rates were higher in both LBCa and HBCa after 6 h. Therefore, the optimal condition for red-rumped agouti sperm capacitation is a low BSA concentration (4 mg/mL) supplemented with 2 mM calcium chloride following a 6 h incubation.

Circular RNAs are associated with important pathophysiological characteristics of gestational diabetes mellitus (GDM). This study preliminarily explored circular polyribonucleotide nucleotidyltransferase 1 (circ-PNPT1) expression in placental tissues and serum of GDM patients and its role in regulating the miR-144-3p/UBE2G1 axis to affect endothelial dysfunction in GDM. Twenty GDM patients and 20 pregnant women with normal glucose tolerance were enrolled. Human umbilical vein endothelial cells (HUVECs) were cultured with high glucose (HG) to establish an in vitro GDM model, then treated with short hairpin-circ-PNPT1 and a lentiviral empty plasmid. Circ-PNPT1 levels, HUVEC viability, endothelial function, and oxidative damage were assessed using RT-qPCR tube formation, CCK-8, flow cytometry, and Transwell assays, respectively. Normally cultured and HG-cultured HUVEC samples underwent small RNA sequencing to analyze differentially expressed miRNAs. The possible miRNAs and mRNAs downstream of circ-PNPT1 were screened using bioinformatics and verified using RT-qPCR, dual luciferase reporter assay, and Ago2-RIP. GDM patients exhibited highly expressed circ-PNPT1 and UBE2G1, and weakly expressed miR-144-3p in placental tissues and serum. In vitro, HG-treated HUVECs displayed highly expressed circ-PNPT1 and cellular dysfunction, as evidenced by reduced cell survival, enhanced apoptosis, decreased cell migration and angiogenesis, an elevated MDA level, and downregulated SOD and GSH-Px levels. Circ-PNPT1 knockdown alleviated HG-induced HUVEC dysfunction. circ-PNPT1 might target and bind miR-144-3p; miR-144-3p might target and bind UBE2G1. Additionally, circ-PNPT1 might act as a competing endogenous RNA for miR-144-3p to regulate UBE2G1 expression in HG-induced HUVECs. Taken together, circ-PNPT1 modulates HG-induced HUVEC dysfunction via the miR-144-3p/UBE2G1 axis.

The circadian clock is not functional during preimplantation development because lack of transcription prevents the feedback loop required for circadian cyclicity. Moreover, transcript abundance for the core clock genes (CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2) declines in the embryo after embryonic genome activation. Nonetheless, transcripts for each of the clock genes are present in the embryo. The potential for translation of these transcripts in the bovine embryo was evaluated by assessing whether circadian clock genes are associated with polysomes and whether these transcripts are preferentially retained as development proceeds to the blastocyst stage. Transcript abundance declined at the eight-cell stage for BMAL1 and CRY1 and at the morula stage for PER1, PER2, and CRY2. Before embryonic genome activation at the eight-cell stage, a large fraction of transcripts for each of the genes was associated with polysomes. At specific later stages of development, there was less transcript associated with polysomes than with other fractions. This was true for PER1 at the morula and blastocyst stage, PER2 at the morula stage, and CRY1 at the eight-cell stage. The percent of transcripts associated with polysomes was also calculated. This value was lower after the two-cell stage for CLOCK and PER1. Based on the decrease in transcript abundance and proportional association with polysomes after the two-cell stage, it was concluded that capacity for translation of circadian clock genes declines in the preimplantation embryo as development proceeds. Thus, de novo synthesized proteins involved in the circadian clock mechanism are unlikely to play an important function in the preimplantation embryo following embryonic genome activation.

Visfatin, a multifunctional adipokine, plays a crucial role in ovarian physiology. This study examines its effects on the level of selected angiogenesis and apoptosis factors in the porcine corpus luteum (CL). Using in vitro cultures of luteal cells, we analyzed the effects of visfatin on key angiogenic factors (VEGF family, PDGF, bFGF2, ANG, and iNOS) and their receptors. Additionally, we assessed luteal cell viability and apoptosis by measuring caspase activity, DNA fragmentation, and transcript levels of apoptotic factors. We noted that visfatin mostly decreases the expression of angiogenic factors and their receptors during the luteal phase except for VEGFC, VEGFR3 on Days 2-3 of the estrous cycle, and VEGFB, VEGFD, VEGFR1, VEGFR2, FGFR2 on Days 14-16 of the estrous cycle, which were upregulated. Moreover, visfatin inhibits apoptosis by downregulating caspase-3, -8, -9, BAX, and caspase 3/7 activity while increasing BCL-2 and luteal cells viability. Treatment with FK866, nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, largely disrupted observed effects, highlighting the NAMPT-dependent function of visfatin. Moreover, visfatin suppressed VEGF-A levels via insulin receptor and MAPK, while insulin receptor, MAPK, and AKT pathways mediated the inhibition of caspase 3/7 activity. These findings suggest that visfatin regulates apoptosis and angiogenesis in the porcine CL.

Phospholipase D2 (PLD2) modulates cytoskeletal dynamics and membrane trafficking processes by converting phosphatidylcholine (PC) into phosphatidic acid (PA) and choline within somatic cells. Nonetheless, the role in oocyte meiosis remains largely unknown. Here, we demonstrate that PLD2 is selectively targeted to the meiotic spindle in mouse oocytes. The knockdown of PLD2 via the specific morpholino oligonucleotides (MOs) or its inhibition with VU 0364739 led to a marked increase in α-tubulin acetylation and induced a meiotic arrest at metaphase I (MI), accompanied by misaligned chromosomes. These defects were effectively rescued by the ectopic expression of Pld2 complementary RNA (cRNA). Furthermore, our findings implicate the Pld2 MO-induced alterations in the AKT-GSK3-Tau signaling cascade in oocytes. The overexpression of a gain-of-function GSK3β mutant (GSK3β) and a Tau-phosphorylation-enhancing mutant (Tau) substantially reversed the increased microtubule acetylation and the reduced rate of the first polar body extrusion (PBE) in oocytes lacking PLD2 activity. Additionally, the co-immunoprecipitation revealed a direct physical interaction between PLD2, GSK3β, and Tau in mouse oocytes. Together, PLD2 finely regulates α-tubulin acetylation through the modulation of the AKT-GSK3β-Tau signaling axis, thereby preserving an optimal microtubule dynamic equilibrium and ensuring the fidelity of the spindle apparatus function during oocyte meiosis.

In vitro models to study the oviduct are challenged by cellular dedifferentiation, a complex coculture system for embryo production, limited cell lifespan, and/or very complex methodologies. Hence, we aimed to develop an in vitro oviductal model using the magnetic bioprinting system, a three-dimensional (3D) culture system. Using the bovine epithelial and stromal oviductal cells (BOEC and BOSC, respectively), we produced the Oviductal Magnetic Spheroid (OMS), a duo somatic cell spheroid aggregate with self-organization capacity. The OMS showed to be viable for 21 days and recapitulated the oviductal tissue features after 7 days in culture, such as a simple epithelial cell layer facing outwards, expressing ciliation (acetylated tubulin positive) and secretory marker (oviduct-specific glycoprotein 1). Although the responsiveness for hormonal treatment with estradiol and progesterone in an estrous cycle-dependent way might require further improvements, the OMS offers an ethical and practical alternative as a three-dimensional oviductal in vitro model to study oviductal physiology, and maybe, a future platform to test therapies and a technology aiming to improve fertility and assisted reproduction success.

Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic secretome, and EVs were subsequently harvested. In vitro assays demonstrated that PGE2-preconditioned ET-eMSCs and their EVs inhibited α-smooth muscle actin expression, reduced collagen I deposition, and restored key endometrial markers of receptivity and proliferation. These findings establish the first evidence that PGE2-enhanced ET-eMSC-derived EVs can exert anti-fibrotic effects in an in vitro model of equine endometrial fibrosis. This study provides a robust translational framework for developing targeted regenerative therapies for fibrotic diseases across species, with potential applications in human reproductive medicine.

Forskolin (FSK), l-carnitine (LC), and Conjugated Linoleic Acid (CLA) are lipid modulators that reduce cellular lipid content. This study characterizes the lipid profile and mRNA content of immature and in vitro matured cat oocytes with or without a MIX of these modulators. Ovaries were collected, COC retrieved, and allocated into three groups: immature (IM), matured without (CONT), or with FSK, LC, and CLA (MIX). Lipid analysis was performed using High-performance thin-layer chromatography, gene expression by RT-qPCR, and oocytes stained with Nile Red, Oil Red O, and Sudan Black B. Eight lipid subclasses were detected in all groups. Esterified sterol was more abundant in CONT than IM, with intermediate levels in MIX. Triacylglycerol was higher in MIX than IM, while monoacylglycerol increased in both CONT and MIX. In MIX, DGAT1, FABP3, and PLIN2 were upregulated compared to IM. Moreover, in IM, DGAT1 was downregulated and FABP3 upregulated relative to CONT. FABP3 levels were higher in MIX than CONT. Oil Red O and Sudan Black B staining showed reduced lipid content in MIX compared to CONT, while Nile Red detected no difference. In conclusion, feline oocytes subjected to MIX during in vitro maturation exhibited increased triacylglycerol levels and enhanced FABP3 expression.