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The worldwide climate is thought to be drastically changing as a result of the global temperatures, a phenomenon known as "global warming". Thermal stress is a crucial obstacle facing buffalo cyclicity. Investigation of the molecular regulation concerning proliferation and apoptosis of corpus luteum (CL) is not fully comprehended in buffaloes. We aimed to (1) study mRNA expression of candidate genes related to proliferation (PGR, AGTR1, and LHCGR) and apoptosis (TNFα, BAX, FASLG, CASP3, AGTR2 and PTGS2), (2) explore effect of thermal stress on the expression of HSP70, NOS1, NOS2 mRNAs, NO and SOD concentrations in CL homogenate during different stages of CL. For this, ovaries (n = 70) were collected in pairs from buffaloes during cold and hot seasons. According to morphology of CL, samples were divided into: early, mid, and late. For RNA isolation, NO and SOD concentrations, small sections from CL stages were frozen in - 80 °C. The results showed that PGR, AGTR2, TNFα, BAX, cALP2beta and PTGS2 mRNAs decreased (P < 0.001) at different stages of CL at hot season. The decline of AGTR2 associated with decreased NOS2 mRNA, which consequently affected TNFα, BAX, and CASP3 mRNAs. Apoptosis might be affected by direct effect of AGTR2 on CASP3 during thermal stress. We supposed that NO had a regulatory role during early and late stages of CL. It could be concluded that thermal stress (THI > 68) changed the expression of proliferation and apoptosis genes of CL in Egyptian buffaloes. Finally, the thermal stress in cold or hot seasons has marked impact on CL dynamics.
Sex sorting of sperm is a valuable reproductive biotechnology for improving herd productivity and genetic management in livestock. Recent advances have explored Toll-like receptor 7/8 (TLR7/8) activation using resiquimod (R848) as a biological approach to selectively modulate sperm function. However, its application in swamp buffalo remains limited. This study aimed to evaluate the effectiveness of R848 combined with discontinuous Percoll density gradient centrifugation (PDGC) for enriching Y-bearing sperm in frozen-thawed swamp buffalo semen and to determine the optimal concentration to maximize enrichment without compromising sperm quality. Frozen-thawed semen from five swamp buffalo bulls (Bubalus bubalis carabanensis) was incubated in tris-based medium containing 0, 0.03, 0.3, and 1 μM R848 at 37°C for 45 min. Following incubation, samples were subjected to PDGC (45%/90%) to isolate motile sperm populations. The pellet fraction was reprocessed and evaluated for sperm kinematics using computer-assisted sperm analysis. The relative abundance of Y-bearing sperm was quantified by SYBR-based quantitative polymerase chain reaction targeting the BRY4a gene, normalized against Glyceraldehyde-3-phosphate dehydrogenase. Quantitative polymerase chain reaction analysis demonstrated that treatment with 0.03 μM R848 resulted in a significant increase in BRY4a expression (approximately 2.6-fold) compared to other groups (p < 0.05), indicating enhanced Y-chromosome enrichment. Sperm motility parameters showed no significant differences in total and progressive motility between treated groups (0, 0.03, and 0.3 μM) and post-thawed controls (p > 0.05). However, velocity-related parameters such as average path velocity and curvilinear velocity increased significantly, while linearity decreased, reflecting a hyperactivation-like motility pattern. In contrast, the highest concentration (1 μM) negatively affected overall sperm motility and kinematic performance. The combination of low-dose R848 (0.03 μM) and PDGC effectively enhances the relative proportion of Y-bearing sperm in frozen-thawed swamp buffalo semen without detrimental effects on motility. This approach represents a cost-effective and field-applicable alternative to conventional sperm sexing methods, with potential applications in buffalo breeding and conservation programs. Further validation using larger sample sizes and fertility trials is recommended.
The discovery of female germline stem cells in the mature murine ovary reignited discussion of postnatal oogenesis. Due to extremely low numbers, the use of an appropriate, highly reliable dissociation method is crucial in guaranteeing the recovery of these putative cells. This study aimed to determine which mechanical and enzymatic dissociation method would give an ovarian cell suspension with the highest number of viable cells in a prepubertal porcine model, followed by flow cytometric detection of a rare DDX4-positive cell population indicative of putative female germline stem cells. It was discovered that viability was increased using the GentleMACS homogeniser as compared to the Ultra-Turrax T18 homogeniser, and that viability increased further when using Liberase DH as compared to collagenase IV digestion. No further improvement in viability of recovered cells resulted from altering other experimental parameters such as enzyme incubation time (30 min or 90 s), blocking buffer (DPBS or HBSS), overnight incubation temperature (37 °C or 38.5 °C), cell detachment with or without trypsin, and centrifugation speed (1000 g, 300 g, or no centrifugation). The optimised retrieval protocol therefore used GentleMACS homogenisation with 30 min Liberase DH digestion, DPBS for blocking steps, incubation at 38.5 °C, trypsin recovery of cells, and centrifugation at 300 g. Flow cytometry identified a rare viable DDX4-positive cell population. This study provides an optimised approach for generating highly viable porcine ovarian single-cell suspensions, which may facilitate subsequent analysis of rare ovarian cell populations, including DDX4-positive cells consistent with putative female germline stem cells. Further studies, including additional marker analysis and functional assays, are required to determine the identity and functional properties of the DDX4-positive population.
While fast in vitro embryo development has been associated with improved outcomes, the relationships of slow in vitro embryo development and vitrified versus fresh in vitro produced (IVP) embryo transfer to foaling percentage and foal sex are less explored. To determine the relationships of (1) day of blastocyst formation (D7-11 after intracytoplasmic sperm injection (ICSI)) and (2) vitrification and warming method (one- and three-step) versus fresh transfer with pregnancy, early pregnancy loss (EPL), foaling percentage and foal sex. Retrospective clinical study. Blastocysts (n = 201) were collected on days 7-11 after ICSI of in vitro matured oocytes from Warmblood mares and transferred either fresh or vitrified-warmed into recipient mares on day 4 after ovulation. Pregnancy, EPL, foaling percentage, and foal sex were compared using multivariable generalised linear mixed-effects logistic regression models. Day of blastocyst formation was significantly associated with pregnancy outcome at D14 (p = 0.048), D42 (p = 0.046) and foaling percentage (p < 0.001). The odds of foaling decreased with developmental age, with significantly lower odds after D11 blastocyst transfer (OR = 0.0045, 95% CI 0.00034-0.061; p < 0.0001). Slow embryo development was associated with a significantly higher proportion of female offspring. The odds of obtaining a filly versus a colt were 5.6-fold higher for D10 blastocysts than D7 (95% CI 1.14-25.0; p = 0.034). Transfer of fresh versus vitrified-warmed blastocysts, using a one- or three-step protocol, did not significantly affect pregnancy or foaling outcome. Small sample size per day of blastocyst formation. Slow in vitro embryo development is associated with decreased pregnancy and foaling outcomes following transfer of D11 IVP blastocysts. Transfer of D10 IVP blastocysts yields acceptable foaling percentages and is associated with a higher proportion of female offspring. Vitrification is effective for preserving equine embryos and allows the use of a simplified one step warming protocol.
Mesenchymal stem cells (MSCs) derived from reproductive tissues are promising for regenerative and reproductive applications; however, ovarian MSCs from large animal models remain poorly characterized. The present study aimed to isolate and characterize the ovine ovarian MSCs and to evaluate passage-dependent changes in their morphology, clonogenicity, growth kinetics, immunophenotype, stemness-associated gene expression, and differentiation potential, while identifying an optimal passage for molecular analyses. This is the first report of isolation and characterization of MSCs from sheep ovary to the best of our knowledge. Plastic-adherent cells were isolated from sheep ovaries, cultured under standard conditions, and established as ovine ovarian mesenchymal stem cells. Based on morphological uniformity and proliferative stability, passage 3 (P3) cells were selected for molecular characterization using immunocytochemistry (ICC), flow cytometry, and quantitative RT-PCR (qRT-PCR). Clonogenicity was assessed by CFU-F assays, growth kinetics by population doubling time (PDT), and differentiation potential under adipogenic, osteogenic, and chondrogenic induction. Early passages (P1-P3) exhibited typical fibroblast-like morphology, high proliferative capacity, strong CFU-F efficiency, and short PDTs. Beyond passage 4, PDT increased, and clonogenicity declined. Molecular analyses of P3 cells confirmed strong expression of MSC markers (CD73, CD90, CD105, and CD44), pluripotency/stemness-associated genes (OCT4, SOX2, and NANOG), and absence of hematopoietic markers (CD34 and CD45). A transient CD34 transcript expression detected at early passages was completely lost by P3, indicating stabilization of the mesenchymal phenotype. The isolated MSCs showed efficient differentiation into adipogenic, osteogenic, and chondrogenic lineages. Prolonged culture induced morphological alterations and reduced proliferative and clonogenic potential. These findings establish the sheep ovary as a reliable source of MSCs and identify P3 as the optimal passage for molecular and functional studies, supporting the translational use of MSCs in reproductive and regenerative medicine.
As a member of the Groucho/TLE family, Transducin-like enhancer of split 3 (TLE3) functions as a transcriptional co-repressor that is highly expressed in the testis. It recruits histone deacetylases (HDACs) and binds histones to mediate chromatin remodeling, thereby regulating gene expression. To investigate the physiological function of TLE3 in spermatogenesis, we generated germ cell-specific conditional knockout (cKO) mouse models using Stra8-Cre and Vasa-Cre drivers. However, Tle3 cKO male mice exhibited grossly normal development and fertility. Histological examination revealed intact testicular architecture and normal spermatogenic progression in the knockout mice. Moreover, immunofluorescence analyses of key germ cell marker proteins, including DDX4 (pan-germ cell), PLZF (undifferentiated spermatogonia), c-Kit (differentiating spermatogonia), γH2AX (meiosis recombination initiation) and PNA (acrosome in spermatids), showed normal germ cell distribution and differentiation. Furthermore, TUNEL assays for apoptosis detection revealed no significant difference between control and cKO mice. Collectively, our findings demonstrate that TLE3 is dispensable for male germ cell development and spermatogenesis.
Cattleyak, a hybrid between cattle and yak, can adapt to the harsh environment of the highlands and also exhibit high production performance. However, its male infertility problem has not yet been solved, which limits the application of its hybrid vigor. In this work, we cloned the OTUD6A gene of cattleyak and analyzed its expression patterns and proliferative effect on Sertoli cells and Leydig cells. The expression level of OTUD6A in testicular tissues, Sertoli cells and Leydig cells were significantly lower than those in yak. Over-expression of OTUD6A in Sertoli cells and Leydig cells of cattleyak significantly increased their proliferative activity and upregulated the proliferation-associated genes. Furthermore, OTUD6A overexpression resulted in significant upregulation of tight junction-related genes (TJP1,TJP2,CLDN11,CLDN6,OCNLD) in Sertoli cells of cattleyak, and the expression of testosterone synthesis-related genes (CYP11A1 and SF1) in Leydig cells were significantly downregulated, indicating that OTUD6A can influence cell function by upregulating the genes related to tight junctions in Sertoli cells and downregulating the genes related to testosterone synthesis in Leydig cells. These results indicate that the abnormal expression of OTUD6A in cattleyak may affect the functions of Sertoli cells and Leydig cells, thereby affecting spermatogenesis in cattleyak. This study provided a new theoretical basis for investigating the molecular mechanism by which OTUD6A regulates spermatogenesis in cattleyak and the molecular mechanism of spermatogenic arrest in cattleyak.
Bone mineral density (BMD) assessment in donkeys is poorly documented, and routine use of DEXA is limited. Aluminum-calibrated digital radiography may provide a practical quantitative alternative. The study aimed to estimate BMD in donkey metacarpal and metatarsal bones using aluminum-calibrated digital radiography and to evaluate the validity and agreement of millimeters of aluminum equivalent (mmAl) values against DEXA-derived BMD (DEXA-BMD) measurements. An ex vivo study was conducted on 20 distal limbs obtained from 5 skeletally mature donkeys. Metacarpal and metatarsal bones were imaged using both DEXA and digital radiography. Radiographic grey-scale values were converted to mmAl using a calibrated step-wedge reference. The relationship between mmAl values and DEXA-BMD was assessed using correlation and linear regression analyses. Agreement between modalities was evaluated using Bland-Altman analysis and intraclass correlation coefficients (ICC). The mean DEXA-BMD of the donkey's bone was 1.23 g/cm² for the metacarpal bones and 1.58 g/cm² for the metatarsal bones. Both imaging modalities demonstrated significantly higher BMD values in metatarsal bones compared with metacarpal bones (p< 0.05). No significant differences were observed between right and left sides in both modalities. The mmAl measurements demonstrated a strong positive correlation with DEXA-BMD across both metacarpal and metatarsal bones. Linear regression confirmed that mmAl values significantly predicted DEXA-BMD, with a high proportion of explained variance. ICC values indicated good agreement between methods. Following appropriate calibration, digital radiography can provide reliable estimates of BMD comparable to DEXA, supporting its potential utility as an accessible alternative for BMD assessment.
Foot-and-mouth disease (FMD) is a highly contagious transboundary viral disease that affects cloven-hoofed livestock, causing substantial economic losses. It is caused by an Aphthovirus under the family Picornaviridae. This study aimed to analyze the spatiotemporal distribution of FMD in Bangladesh, evaluate the influence of meteorological factors, and forecast future disease trends. FMD cases and meteorological variables were obtained from DLS and BMD, respectively. Spatial autocorrelation metrics, including Getis-Ord Gi*, Moran's I, LISA, and inverse distance weighting, were applied to identify disease clusters and risk zones. Correlation and regression analyses identified significant associations between FMD incidence and climatic factors. Relative humidity and temperature exhibited positive correlations with disease occurrence. Additionally, regression modeling revealed that both relative humidity and wind speed had a significant impact on FMD incidence. Predictive models, including ARIMA, Random Forest, and XGBoost, were applied, with XGBoost providing the most accurate forecasts (RMSE = 153.64), followed by Random Forest and ARIMA. FMD incidence peaked in March, with persistent southeastern hotspots and emerging northern clusters. IDW mapping showed elevated risk in southern regions, particularly during the pre-monsoon period, influenced by climatic factors. These insights can inform targeted surveillance and control strategies to mitigate the burden of FMD in Bangladesh.
Heat stress is a major factor impairing cattle fertility by reducing oocyte competence and embryo development leading to significant economic losses. Follicular fluid-derived extracellular vesicles (FF-EVs) are key mediators of intercellular communication within the ovarian follicle and may improve oocyte competence. However, their potential to mitigate naturally occurring seasonal heat stress remains unclear. This study evaluated the impact of supplementing the in vitro maturation medium with FF-EVs from small (3-5 mm) or large (>9 mm) follicles from normothermic conditions on bovine oocytes collected during summer. EVs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, cryo-electron microscopy, and flow cytometry. Oocyte competence was evaluated through oxidative status and DNA fragmentation, while embryo development and quality were assessed by developmental rates, redox status, differential cell count, apoptosis, and lipid content. Supplementation with FF-EVs derived from small follicles (Small FF-EVs) reduced intracellular reactive oxygen species (ROS) levels and DNA fragmentation in oocytes, while significantly increasing blastocyst rates. In addition, Small FF-EVs enhanced embryonic redox balance and reduced lipid content. In contrast, EVs from large follicles increased oxidative stress and DNA damage in oocytes, although they reduced ROS levels and lipid droplet volume in blastocysts without affecting embryo development rates. No significant differences were observed in total cell number or apoptosis rates. Overall, FF-EVs exert differential effects on oocyte competence and embryo development according to their follicular origin. In particular, Small FF-EVs enhance oocyte capacity to withstand naturally occurring heat stress and represent a promising strategy for improving reproductive outcomes under challenging environmental conditions.
Objectives were to evaluate the effects of replacing sulfate (STM) with hydroxychloride (HTM) sources of Cu, Mn, and Zn on health, reproduction, and survival in dairy cows. One-hundred and 41 Holstein cows were stratified by parity group prepartum as nulliparous or parous cows and, within parity, nulliparous cows were blocked by genomic breeding value for ECM yield and parous cows by recently completed lactation 305-d ECM. Within block, cows were assigned to STM or HTM, and prepartum diets contained (mean ± SD) 15 ± 1, 58 ± 2, and 66 ± 3 mg/kg of diet DM as Cu, Mn, and Zn, respectively, whereas postpartum diets contained 19 ± 3, 65 ± 15, and 77 ± 11 mg/kg diet DM as Cu, Mn, and Zn, respectively. Treatments were fed from 246 d of gestation to 105 DIM. Concentrations of nonesterified fatty acids (NEFA), BHB, haptoglobin, and ceruloplasmin were measured in plasma and those of total Ca (tCa) were measured in serum. Diseases were diagnosed in the first 105 d postpartum and survival was evaluated until 305 DIM. The estrous cycle of cows was synchronized and artificial insemination (AI) was performed on d 87 postpartum. On d 19 after AI, serum was analyzed for progesterone and blood mononuclear cells and cervical cells were analyzed for mRNA for interferon-stimulated genes (ISG). Treatment did not affect the concentrations of NEFA, BHB, tCa, and progesterone in blood. Feeding HTM reduced the concentrations of haptoglobin in the first 19 d postpartum (40.0 ± 9.1 vs. 25.7 ± 5.0 µg/mL) and that of ceruloplasmin only on d 6 postpartum (0.64 ± 0.03 vs. 0.58 ± 0.03 mg/mL). Feeding HTM reduced the risk of retained placenta (11.5 ± 6.3 vs. 3.8 ± 2.3%) and tended to reduce the risks of clinical (16.4 ± 9.7 vs. 4.0 ± 2.9%) and subclinical endometritis (29.8 ± 9.2 vs. 16.4 ± 5.7%). Cows fed HTM tended to have reduced rate (adjusted hazard ratio = 0.58; 95% CI = 0.33-1.04) and reduced risk of morbidity (51.7 ± 9.1 vs. 32.7 ± 7.1%) during first 105 d postpartum compared with feeding STM. Feeding HTM increased the relative expression of ISG by 1.7 to 2.0-fold in blood mononuclear cells on d 19 after AI in pregnant cows compared with STM; however, treatment did not affect the expression of ISG on cervical cells. Rate of pregnancy did not differ between treatments, but HTM tended to increase the proportion of pregnant cows by 305-d postpartum (68.8 ± 5.7 vs. 82.8 ± 4.7%) partially attributed to the reduced proportion of cows designated as do not inseminate (25.6 ± 5.3 vs. 9.8 ± 3.6%). Also, HTM reduced the rate (adjusted hazard ratio = 0.44; 95% CI = 0.20-0.96) and risk of leaving the herd by 305-d postpartum from 26.5 ± 9.6 in STM to 11.9 ± 5.0%. Replacing sulfate sources of Cu, Mn, and Zn with hydroxychloride sources of same trace minerals benefited early lactation health of cows in early postpartum which carried out to benefit reproduction and survival.
Zinc homeostasis is essential for oocyte maturation, activation, and genomic stability. Oocyte activation is physiologically triggered by calcium oscillations and a rapid exocytotic zinc release that coordinates meiotic exit and early embryogenesis. Although zinc chelation has been shown to induce oocyte activation in other mammalian species, its efficacy, optimal conditions, and developmental consequences in bovine oocytes remain poorly defined. Moreover, the interaction between zinc chelation-based activation and zinc availability during in vitro maturation (IVM) has not been systematically evaluated in cattle. This study aimed to optimize artificial oocyte activation (AOA) via zinc chelation using 1,10-phenanthroline (PHEN) and to evaluate the impact of zinc sulfate (ZnSO4) supplementation during IVM on parthenogenetic embryo development. Bovine cumulus-oocyte complexes were matured with different concentrations of ZnSO4 and activated using ionomycin or PHEN. Intracellular zinc levels in oocytes were measured, and blastocyst diameter and DNA fragmentation levels were assessed. Zinc supplementation significantly increased intracellular Zn2+ levels at 1.5 μg/mL ZnSO4 but did not improve cleavage or blastocyst rates. PHEN at 250 μM for 30 min induced zinc depletion and meiotic resumption yet produced lower developmental rates than ionomycin, whereas 250 μM for 60 min achieved comparable cleavage and blastocyst rates. Ionomycin combined with 1.0 or 1.5 μg/mL ZnSO4 significantly reduced blastocyst DNA fragmentation, while PHEN activated embryos maintained consistently low fragmentation levels. Collectively, these findings demonstrate that optimized zinc chelation can support bovine embryo development comparable to calcium-based activation, while zinc supplementation enhances intracellular zinc levels and genomic integrity rather than developmental yield.
As the threat of antimicrobial resistance (AMR) escalates globally, the influence of indiscriminate antimicrobial use (AMU) in livestock cannot be overlooked. Antimicrobial use practices are continually explored in larger food-producing animals; however, small ruminants (sheep and goats) receive comparatively less research attention. Our study addresses this gap by investigating small ruminant production practices in Nigeria and exploring how they affect the use of antimicrobials and alternatives. We adopted a mixed-methods study design. A semi-structured questionnaire was administered to 785 farmers. Following this, a focus group discussion (FGD) was conducted with 23 small ruminant industry stakeholders, which included farmers, para-veterinarians, and butchers. Participants were split into three round tables, with 7-8 participants per table. Of questionnaire respondents, 68% of farmers never vaccinated their flock against peste de petits ruminants (PPR) nor contagious caprine pleuropneumonia virus. Several health problems were regularly experienced by animals, including PPR, mastitis, and dermatophilosis. Diseases were mostly self-managed with antibiotics and herbs (> 70%) rather than through reliance on veterinary care (< 15%). More farmers (48%) used antibiotics than herbal remedies (14%) over the previous three months. Farmers' use of herbs was affected by their having low awareness of available options and how to use them appropriately. Perceived effectiveness also influenced farmers' choice between antibiotics and herbs, while economic considerations also led them to sell off sick animals before or during treatment. Among farmers who used animal health services, more farmers (59%) consulted unlicensed para-veterinarians and drugstore vendors rather than licensed government and private veterinarians (36%), a disparity attributed to the unavailability of qualified veterinary doctors. Most farmers had poor knowledge (62%), attitudes (47%), and practices (43%) towards AMU and AMR. We recommend conducting further studies to identify and investigate the efficacy of currently used herbs in treating common diseases. There is a crucial need to improve farmers' access to vaccines, veterinary care, and laboratory diagnostics. Barriers that hinder better compliance with regulations that govern the use of non-prescribed antimicrobials should be explored. Awareness programmes could be conducted to improve farmers' awareness of AMR and appropriate disease preventive practices.
Ovum pick-up (OPU) is a procedure that is now performed in mares worldwide to harvest oocytes for in vitro embryo production. There is a lack of consensus surrounding best practices for various procedural elements of the technique and recently there has been an increasing focus on the welfare aspects of the procedure. Our objective was to summarize current practice and to develop guidelines for OPU in the mare, using a modified GRADE approach to optimize efficiency and mare welfare. Published evidence on various aspects of the procedure was compiled, and to supplement the relative lack of publications, a survey was distributed to OPU practitioners on their clinical practice and experience with OPU. This data was reviewed alongside the authors' clinical experience and knowledge. A review of available literature and survey results from 71, predominantly European, respondents representing approximately 9531 OPU procedures over 12 months are described. Best practice guidelines are presented on aspects of the OPU procedure under the headings of mare selection, technical aspects, sedation and analgesia, complications, aftercare and laboratory aspects. Knowledge gaps are highlighted where the evidence was weak and/or insufficient. We conclude that ovum pick-up in mares appears efficient and generally safe when performed in practice, however scientific evidence underpinning several procedural elements is weak particularly related to the optimal analgesic and sedative regimen. This work provides the first international evidence-based recommendations for OPU in the mare and establishes a framework to guide future research priorities, with mare welfare as a central focus.
Improving fecundity and growth is a major goal in sheep breeding. Here, we established a zygote electroporation platform to deliver base editor ribonucleoproteins (BE RNPs) targeting the fecundity-associated BMPR1B(FecB) and growth-associated SOCS2 loci. Single-target validation achieved high editing efficiencies (95.0% for BMPR1B and 65.0% for SOCS2) without compromising embryonic development in vitro, although bystander edits were prevalent. However, dual-target editing introduced guide RNA (gRNA) crosstalk between co-delivered adenine base editor (ABE) and cytosine base editor (CBE) RNPs in preimplantation embryos. We obtained three live lambs after transfer of dual-editing blastocysts. Although all lambs carried the intended BMPR1Bp.Q249R mutation, these mutations were consistently accompanied by bystander edits. One lamb exhibited additional gRNA crosstalk at the BMPR1B locus. This lamb also harbored an unintended 13-bp frameshift deletion at the SOCS2 locus, replacing the desired point mutation with a predicted null allele. These results demonstrate that multiplex BE RNP electroporation in sheep zygotes generates unpredictable genomic outcomes, including bystander edits, gRNA crosstalk, and unintended indels. Consequently, current multiplex base editing systems, as implemented via co-electroporation of ABE and CBE RNPs, face significant challenges for routine application in sheep breeding.
This study describes embryo developmental progression and morphokinetic patterns in embryos derived from oocytes of young and aged domestic cats, a model for endangered felids, using a time-lapse monitoring system. Because maternal aging is associated with reduced oocyte competence, possibly linked to mitochondrial dysfunction that may impair fertilization and embryo development, the potential influence of the SIRT1 activator SRT1720 during in vitro maturation on embryo developmental progression was also evaluated under these conditions. Oocytes from young (1.13 ± 0.79 years) and old (7.0 ± 1.10) cats were matured in vitro with SRT1720 (1-μM, 0.1-μM, and 0.05-μM) and without, followed by in vitro fertilization. 8-Day embryo in vitro culture was noninvasively monitored using the MIRI® time-lapse system. Embryos from aged cats exhibited accelerated progression up to the morula stage; in contrast, embryos from young cats showed faster progression at later preimplantation stages and reached the hatched blastocyst stage. In young queens, embryos exposed to 0.1-μM showed numerically higher first cleavage and morula formation rates than controls, while 1-μM was associated with altered first cleavage timing and short cell cycles. In older queens, 0.1-μM maintained cleavage timing patterns comparable to untreated embryos but fail to produce hatched blastocysts, whereas 1-μM yielded a single hatched blastocyst despite signs of altered cell cycle dynamics. Overall, SRT1720 supplementation did not significantly influence embryo development under the tested conditions. Morphokinetic monitoring provided descriptive observations suggesting distinct age-related developmental profiles. These findings provide baseline information on feline developmental kinetics and may support further investigation of metabolic modulators in feline assisted reproduction.
Accurate diagnosis of prostatic disorders is essential for reproductive health and fertility management in male dogs. Ultrasonography (US) is widely used to evaluate the canine prostate; however, B-mode US alone lacks sufficient specificity to reliably distinguish benign from malignant conditions. Elastography, which quantifies tissue stiffness, may improve characterization of prostatic pathology and clinical decision-making. This study compared the diagnostic performance of B-mode US, strain elastography (SE), and 2D-shear wave elastography (2D-SWE) for differentiating normal, non-malignant, and malignant prostatic disorders. Thirty-four male dogs were assigned to three groups: normal prostate gland (NG; n = 9), non-malignant prostatic disorders (NMD; n = 20), and malignant prostatic disorders (MD; n = 5), based on clinical findings, cytology, bacterial culture, and histopathology when available. All dogs underwent B-mode US, SE using the Tsukuba elasticity scoring system and strain ratio (SR), and 2D-SWE. Diagnostic accuracy was assessed using receiver operating characteristic analysis. B-mode US showed limited specificity for distinguishing MD from NMD. Tsukuba elasticity scores tended to be higher in MD; however, intergroup overlap and non-significant SR differences limited the utility of SE. 2D-SWE revealed significantly greater stiffness in MD than in NG and NMD (P < 0.05), with area under the curve values of 0.83 (kPa) and 0.82 (m/s). Cut-off values of 18.76 kPa and 2.45 m/s yielded sensitivities of 80% and specificities of 86% and 79%, respectively. 2D-SWE may improve differentiation of malignant from non-malignant prostatic disorders and may serve as a valuable non-invasive adjunct for clinical and reproductive evaluation in male dogs.
This study investigated the effect of semen collection timing (06:00-08:00 h [morning] vs. 12:00-14:00 h [afternoon]) on semen quality in Thai native bulls under tropical heat stress conditions. Physiological responses, endocrine profiles, and sperm lipid peroxidation were also evaluated. Nine bulls were subjected to a multiple crossover design, with semen collection alternated between morning and afternoon periods across 12 sessions per animal. Environmental parameters, including temperature-humidity index (THI), were continuously monitored. Afternoon collections occurred under higher ambient temperature and THI (>81) than morning collections (77-80), accompanied by increased heart rate (93.88 vs. 79.75 beats/min; P < 0.001) and elevated average scrotal surface temperature (35.08 vs. 33.90 °C; P < 0.001). Serum cortisol concentrations did not differ between collection periods (2.36 vs. 2.18 ng/mL; P > 0.05), whereas testosterone levels were significantly higher in the afternoon (2.23 vs. 1.99 ng/mL; P < 0.05). Afternoon-collected semen exhibited higher total motility (82.80% vs. 76.02%; P < 0.05) and viability (83.68% vs. 77.24%; P < 0.05). However, acrosome integrity was significantly lower in afternoon collections than in morning collections (83.90% vs. 87.40%; P < 0.05), accompanied by higher malondialdehyde concentrations (1.15 vs. 0.84 µM/mL; P < 0.05). Midpiece abnormalities and bent tails with retained droplets were more prevalent in afternoon samples. These findings suggest a physiological trade-off between enhanced sperm motility and increased structural damage to sperm during afternoon semen collection under tropical heat stress. Morning collection may better preserve acrosome integrity and sperm morphology despite lower motility.