During cryopreservation, boar sperm are readily subjected to cold shock, ice crystal formation, osmotic changes, and oxidative stress, leading to a significant decline in the post-thaw sperm quality. The addition of cryoprotective agents to the freezing extender can effectively alleviate cryodamage and improve sperm motile performance. In this study, we first employed a metabolomic analysis to identify metabolites potentially associated with sperm cryoinjury, and then explored their roles and underlying mechanisms in amelioration of sperm cryodamage. Briefly, by way of a LC-MS-based comparative analysis among fresh semen, once frozen-thawed samples, and repeatedly frozen-thawed samples, we identified ergothioneine (EGT) as a metabolite strongly correlated with sperm cryoinjury. Supplementation of the freezing extender with different concentrations of EGT (0, 100, 200, 500, and 1000 μM) significantly improved post-thaw sperm motility, motion parameters, and the acrosome and plasma membrane integrity, with 200, 500, and 1000 μM EGT notably enhancing sperm antioxidant capacity and energy metabolism, thereby effectively alleviating sperm cryoinjury. The subsequent mechanistic investigation revealed that EGT exerted its protective effects by activating the AMPK signaling pathway, while the addition of an AMPK inhibitor markedly attenuated the protective effects. Collectively, this study demonstrates that the endogenous metabolite EGT alleviates the cryodamage of boar sperm via the AMPK signaling pathway, providing robust scientific evidence for introduction of endogenous antioxidant metabolites into the freezing extender and shedding light on development of safer and more effective strategies for the sake of semen cryopreservation.
Ovum pick-up (OPU) technical variability limits the efficiency and consistency of bovine in vitro embryo production (IVP). Here, we established a sequential refinement framework for OPU by systematically evaluating key technical parameters across seven cumulative experiments in Holstein cows. An 18 G aspiration needle achieved significantly higher cumulus-oocyte complex (COCs) recovery than 20 G and 22 G needles (53.05% vs. 40.03-45.79%; p < 0.05). 90° improved recovery compared with non-vertical insertion (55.64% vs. 48.86%), while insertion to one-third to one-half follicle depth and bevel-down orientation further enhanced recovery efficiency (56.52% and 57.97%, respectively; p < 0.05). Polyvinyl chloride (PVC) tubing provided superior COCs recovery (63.29%) and morphological quality, with a tubing length of 1.2-1.5 m. Vacuum pressure of 50 mmHg resulted in higher COCs recovery (70.27%) and blastocyst rate (40.65%), significantly outperforming the previously used 70 mmHg setting. In contrast, excessive aspiration pressure (80 mmHg) was associated with mitochondrial dysfunction, disrupted cortical granule distribution, and increased apoptosis (p < 0.05), indicating impaired oocyte nuclear and cytoplasmic maturation under excessive mechanical stress. Notably, recovery efficiency progressively increased from 53.05% to 70.27% throughout the sequential optimization process throughout the sequential refinement process. Collectively, this study provides a field-based refinement approach for OPU procedures and demonstrates the association between aspiration parameters and bovine oocyte developmental competence under the tested conditions.
This study aimed to determine whether increased water temperature during the reproductive period impairs male reproductive function in Onychostoma macrolepis and to evaluate whether dietary selenium nanoparticles (SeNPs) alleviate heat stress-induced reproductive damage. As water temperature increased during the reproductive period, sperm total motility, progressive motility, beat cross frequency (BCF), and plasma membrane integrity decreased by 42.3%, 66.5%, 41.7%, and approximately 55%, respectively, from May 9 to July 2. Increased water temperature also impaired testicular antioxidant status, as shown by an approximately 60% decrease in T-AOC and an approximately 110% increase in MDA content on July 2, together with structural damage, mitochondrial abnormalities, and increased protein levels of HSP70 and NF-κB p65. Dietary supplementation with SeNPs at 0.1, 0.3, and 0.9 mg/kg alleviated heat stress induced reproductive damage, with 0.3 mg/kg showing the most pronounced effects. At this level, SeNPs improved sperm quality, antioxidant status, and fertilization capacity, increasing fertilization rate by approximately 20% on June 15 and by more than 100% on July 2. Further analysis showed that SeNPs significantly increased the levels of selenate, SeCys2, MeSeCys, and SeMet in the testis. In vitro experiments further revealed that MeSeCys was the most effective selenium species in maintaining sperm motility, stabilizing ROS levels, and improving fertilization capacity. Overall, these findings indicate that heat stress is a key factor leading to reduced reproductive performance in O. macrolepis, whereas SeNPs mitigate reproductive damage by reducing oxidative stress, with MeSeCys likely serving as a key bioactive selenium species.
The objective was to determine whether combining equine chorionic gonadotropin (eCG) and 48-h temporary calf removal (CR) at the end of an estradiol and progesterone (P4)-based estrous synchronization protocol for fixed-time AI (TAI) improves pregnancy per AI (P/AI) in suckled multiparous and primiparous Nellore cows. Suckled cows (n = 2643) with adequate body condition score (>95% with BCS ≥ 2.5 on a 5-point scale) were assigned to receive CR, 300 IU of eCG, or eCG + CR on day -2 (D-2) followed by measurement of follicular diameter and estrus expression by TAI (D0), serum P4 concentrations on D7, pregnancy per AI (P/AI) on D30, and calf weaning weights. At TAI, eCG-treated cows had larger follicle diameter (13.5 mm) than CR-treated cows (13.2 mm vs. eCG + CR: 13.1 mm, SEM: 0.2), P < 0.05. Estrus expression was greater in CR and eCG + CR treated cows than in eCG-treated cows (eCG: 59.5b% vs. CR: 77.8a% vs. eCG + CR: 75.0a%). Among primiparous cows, eCG-based treatment (eCG and eCG + CR) resulted in greater serum P4 concentrations on D7 than CR alone. Despite these physiological differences, P/AI was similar among treatments (eCG: 49.8% vs. CR: 53.6% vs. eCG + CR: 49.4%). Calves from cows treated with CR and eCG + CR were lighter (unadjusted weaning weights: 184.1 and 186.2 kg, respectively) than calves from eCG-treated cows (189.2 kg). In conclusion, combining eCG and CR did not improve P/AI compared with either treatment alone. Temporary 48-h calf removal increased estrus expression but did not improve fertility and reduced calf weaning weight. In contrast, eCG alone resulted in heavier calves at weaning, supporting its use over temporary 48-h calf removal in estrous synchronization programs when calf weaning weight is also considered.
This study evaluated the reproductive performance and clinical safety of a novel recombinant eCG (r-eCG) in ewes. Two weeks prior to study onset (Day 0: intravaginal progesterone device insertion), 45 ewes were randomly assigned to negative control (NC; no eCG; n=10), positive control (PC; 400IU commercial non-recombinant eCG; n=20), or treatment group (T; 400IU r-eCG; n=15). Ewes were inseminated with fresh-diluted semen (200×106 sperm). Ultrasonography assessed follicle count/diameter (days 11, 13), ovulation (day 13), corpus luteum (CL) number/characteristics (day 20), and pregnancy (day 44). Serum progesterone was measured on day 20; Group T underwent clinical/hematological evaluations (days 0, 20, and 44). Estrus signs were more frequent (P<0.01) in T (100%) than NC (50%), while PC (85%) did not differ. Ovulation rates were similar between T (93%) and PC (95%), but higher than NC (50%; P<0.05). On day 13, largest follicle diameter did not differ (P>0.05) among groups; however, fewer follicles > 2 mm occurred in PC than NC and T (P<0.05). CL number was higher in T than PC (2.0±0.3 vs. 1.0±0.0, P<0.01), while NC did not differ (1.0±0.25). Conversely, pregnancy rate was higher (P<0.05) in PC (70%) than T (28.6%) and NC (20%). Among ovulated ewes, progesterone concentration and progesterone/CL ratio did not differ among groups (P=0.92 and P=0.08, respectively). No relevant r-eCG-related clinical or hematological alterations occurred. In conclusion, 400IU r-eCG effectively induces estrus and ovulation without adverse effects; however, the discrepancy between ovulatory response and pregnancy rate underscores the need for further study refinement to improve fertility.
The plastics pollution becomes a global environmental issue with both microplastics (MPs) and nanoplastics (NPs) pose a great risk to animal health. Therefore, we tried to determine the impacts of MPs and NPs on reproductive health of a farm poultry species-Japanese quail (Coturnix japonica) from the perspective of the regulation of hypothalamic-pituitary-testicular (HPT) axis. One-week chicks were exposed to environmental relevance concentration of 0.4 mg/kg and 8 mg/kg polystyrene MPs and polystyrene NPs respectively for five weeks. The results showed MPs and NPs accumulated in the brain and testis in a dose-dependent manner, associated with disruption of both the blood-brain barrier and the blood-testis barrier. Moreover, MPs and NPs exposure caused histopathological injury to the hypothalamus, pituitary and testis resulting in oxidative stress leading to testicular necroptosis and increasing sperm deformity, which induced the decrease of Gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and testosterone (T) implying HPT axis disruption and reproductive dysfunction. Furthermore, transcriptome analysis showed that GnRH signaling pathway and steroid hormone biosynthesis pathway were disturbed in the testis. These findings confirmed the adverse influence of MPs and NPs on avian reproductive health and underscored the urgency and necessity of mitigating MPs and NPs impacts.
This study evaluated whether the administration of human chorionic gonadotropin (hCG) during early pregnancy accelerates ovulatory dynamics and alters serum progesterone concentrations in mares with dominant follicles. Thirty pregnant embryo-recipient mares with follicles ≥35 mm during early gestation were randomly assigned to receive either 1500 IU hCG IV (hCG group; n = 17) or saline solution IV (control group; n = 13). Mares were monitored by serial ultrasonography to determine the ovulatory response and the interval to secondary ovulation. Serum progesterone concentrations were analyzed using two complementary approaches: a chronological post-treatment analysis and an analysis synchronized with ovulation timing. The proportion of mares that ovulated within 48 h after treatment was significantly higher in the hCG group than in the control group (82.4% vs. 0%; P < 0.0001). Likewise, the interval between treatment and secondary ovulation was shorter in hCG-treated mares (median: 1.7 days; IQR: 1.6-1.8) than in control mares (median: 22 days; IQR: 20-23; P < 0.0001). The interval between primary and secondary ovulation was also shorter in hCG-treated mares (median: 38 days; IQR: 34-43) than in control mares (median: 54 days; IQR: 51-60; P = 0.0008). The chronological post-treatment analysis revealed a significant group × time interaction for serum progesterone concentrations (P < 0.0001). When the analysis was synchronized with ovulation timing, progesterone concentrations increased similarly over time in both groups (time effect: P < 0.0001), with no significant group × time interaction (P = 0.28). These findings demonstrate that dominant follicles present during early pregnancy remain responsive to exogenous gonadotropic stimulation, allowing secondary ovulation and, consequently, the formation of supplementary corpora lutea. Although hCG administration altered the temporal pattern of serum progesterone concentrations by accelerating ovulatory events, post-ovulation progesterone dynamics remained equivalent between groups when the analysis was synchronized with ovulation timing.
Boar sperm are prone to functional deterioration during incubation, which is commonly associated with reduced motility, impaired mitochondrial function, and insufficient adenosine triphosphate (ATP) production. This study investigated whether glycine supplementation improves boar sperm progressive and linear motility by enhancing mitochondrial energy metabolism through the 5-aminolevulinic acid (5-ALA)/heme-associated mitochondrial pathway. Boar sperm samples were incubated with different concentrations of glycine (0, 0.1, 1, and 10 mM) at 37 °C for 6 h, and sperm motility was assessed at 0, 1, 3, and 6 h. Mitochondrial membrane potential (MMP), ATP levels, 5-ALA levels, heme levels, and mitochondrial complex IV activity were also evaluated. Compared with the control group, addition of 1 mM glycine significantly increased sperm total motility, progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and linearity (LIN) after 3 h of incubation (P < 0.05), whereas 10 mM glycine treatment reduced sperm motility. Glycine supplementation also significantly enhanced MMP, ATP levels, 5-ALA levels, heme levels, and complex IV activity (P < 0.05). To further evaluate the involvement of the 5-ALA/heme-associated mitochondrial pathway, exogenous 5-ALA supplementation and pharmacological inhibition of 5-aminolevulinate synthase (ALAS) and 5-aminolevulinate dehydratase (ALAD) were performed. Exogenous 5-ALA reproduced several glycine-induced improvements in sperm motility and mitochondrial energy metabolism, whereas addition of ALAS or ALAD inhibitors attenuated the beneficial effects of glycine on sperm motility, ATP levels, and heme levels (P < 0.05). In addition, artificial insemination with glycine-supplemented semen significantly increased total born litter size without significantly affecting sow pregnancy rate. These findings demonstrate that glycine improves boar sperm progressive and linear motility by enhancing mitochondrial energy metabolism. Therefore, glycine may serve as a promising metabolic additive for improving boar sperm quality and reproductive performance.
This study evaluated the effects of dietary genistein supplementation on laying performance, eggshell quality, antioxidant capacity, and ovarian steroidogenesis in 66-week-old Hy-Line Brown laying hens. A total of 384 hens were randomly allocated to four treatments: a basal diet (control) or diets supplemented with 80, 120, or 400 mg/kg genistein, with 6 replicates of 16 birds each. Genistein supplementation at all levels significantly increased laying rate and reduced feed conversion ratio and broken egg rate (P < 0.05). Regarding egg quality, 80 and 120 mg/kg genistein decreased egg shape index, while all treatments increased shell ratio, and 80 mg/kg enhanced blunt-end shell thickness (P < 0.05). Plasma superoxide dismutase (SOD) activity was elevated by 80 and 400 mg/kg genistein (P < 0.05). In the liver, all genistein treatments increased SOD, glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC), and reduced malondialdehyde (MDA) levels (P < 0.05). In the ovary, 80 mg/kg increased GSH-Px activity, while 80 and 400 mg/kg enhanced catalase (CAT) activity (P < 0.05). Moreover, 80 and 120 mg/kg genistein increased plasma progesterone (P4) concentrations (P < 0.05). Expression of steroidogenic genes was also upregulated, with increased mRNA levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD). Corresponding increases in protein abundance were observed for StAR, CYP11A1, and 3β-HSD at specific supplementation levels (P < 0.05). In conclusion, dietary genistein improved laying performance and selected eggshell quality traits, enhanced antioxidant capacity, and was associated with increased plasma progesterone concentrations and altered expression of ovarian steroidogenesis-related factors in late-laying hens.
Ruminants are among the livestock most at risk of microplastics (MPs) exposure due to the ingestion of contaminated forage, particularly with polyethylene terephthalate (PET). Yet, despite this high risk, the potential effects of microplastics on reproductive function in these species remain largely unknown. Here, we studied how environmentally relevant PET-MPs impact placental functionality and cellular responses in an in vitro sheep model. First, virgin PET was mechanically processed through progressive milling and sieving to obtain two fractions of 20-50 μm or <20 μm, which were characterised in size, distribution, and crystal structure; then ovine term placental cells were exposed to both sizes of PET-MPs for 24-48 h. MTT revealed that the smaller PET-MPs reduced cell viability even at low concentrations (1 μg/mL), whereas the larger fraction induced cytotoxicity only at 50 μg/mL. Placental cells diminished migratory capacity in the presence of smaller PET-MPs. Oxidative stress assessment further demonstrated a reduced ability to metabolize H2O2, along with a decrease in mitochondrial membrane potential, indicating compromised mitochondrial function; additionally, apoptosis was also slightly increased following exposure to the <20 μm PETs. Overall, our findings show that PET-MPs, particularly those below 20 μm, impair multiple functional and metabolic endpoints in ovine term placental cells, highlighting the risk to reproductive health in ruminants chronically exposed to microplastic-contaminated feed.
The aim of this study was to compare luteal blood perfusion (BP) estimated by subjective human evaluation, ImageJ pixel analysis, and a deep learning (DL)-based system. Color Doppler ultrasound (CDU) examinations utilized herein were from three independent studies where CDU videos were collected at embryo transfer (Day 7; early luteal phase; ELP; n = 129), or on Days 15 (mid luteal phase; MLP; n = 48) and 20 (late luteal phase; LLP; n = 47) of pregnancy. Each corpus luteum (CL) was classified as cavitated (CV; cavity ≥10% of luteal area) or non-cavitated (NC; <10%). Luteal BP was estimated using three methods: Human evaluation, ImageJ pixel counting, and a DL pipeline for frame selection, CL segmentation, and BP quantification. Positive linear relationships were observed among BP estimation methods in all datasets (r ≥ 0.52; P < 0.001) and within CL cavity classifications (r ≥ 0.62; P < 0.001). Strong relationships were consistently observed in the LLP dataset (r ≥ 0.79; P < 0.001). No relationship was detected between circulating progesterone (P4) and BP at ELP or MLP (P ≥ 0.14), regardless of the estimation method. Positive linear relationships (P ≤ 0.001) with P4 were observed for Human, ImageJ, and DL estimates at LLP. In conclusion, DL-derived BP estimates showed strong linear relationships, satisfactory agreement, and biological validity comparable to Human and ImageJ methods, supporting its use for automated assessment of luteal BP in cattle.
Litter size is an economically important trait in sheep and is closely associated with ovarian follicular development and granulosa cell (GC) function. This study investigated the association between GDF9 polymorphisms and litter size, and examined the post-transcriptional regulation of GDF9 by miR-6388 in ovine GCs. Variants were initially identified by Sanger sequencing in 20 ewes, and subsequently genotyped in 377 three-year-old ewes, including 231 Sonid (SN) sheep and 146 Ujimqin (UM) sheep for association analysis. Candidate miRNAs targeting litter size-associated variants in the GDF9 3'UTR were predicted, and the miR-6388-GDF9 interaction was evaluated using dual-luciferase reporter assays. RT-qPCR, Western blotting, EdU incorporation, and flow cytometry were used to assess endogenous GDF9 expression and GC function. Twelve single-nucleotide polymorphisms were identified, including the putatively novel variant g.42114076C > G. The linkage disequilibrium block comprising g.42116936C > T, g.42113821T > A, and g.42113962G > A polymorphisms of GDF9 was significantly associated with litter size in both SN and UM sheep, whereas the c.477G > A was associated with litter size only in UM sheep. Reporter assays showed that the GDF9 3'UTR region carrying the G allele of g.42113962G > A was more responsive to miR-6388-mediated repression than the region carrying the A allele. miR-6388 overexpression reduced GDF9 mRNA and GDF9 protein levels, inhibited GC proliferation, altered cell-cycle distribution, and promoted apoptosis, whereas miR-6388 knockdown increased GDF9 expression and GC proliferation and reduced apoptosis. These cellular changes were accompanied by altered expression of cell-cycle and apoptosis-related genes and TGF-β signaling-related components.
Assisted reproductive technologies (ARTs) have the potential to accelerate genetic improvement in selective equine breeding programmes while addressing specific reproductive challenges. One recent assisted reproductive technique in domestic horse breeding is Ovum Pick-Up combined with Intracytoplasmic Sperm Injection (OPU-ICSI). Despite their advantages, the implementation of these new procedures, like more established breeding methods, raises ethical concerns relating to aspects like animal welfare, staff safety, professional and veterinary practice integrity, which must be monitored and assessed. In this paper, we propose and discuss an ethical checklist for self-assessment to ensure that OPU procedures are conscientiously performed and monitored from an ethical standpoint. Based on three key ethical dimensions - Animal welfare ethics, Professional ethics, and Procedural ethics - the checklist has been developed and tested through an iterative process involving a panel of 14 experts, both in OPU procedures and applied ethics. The checklist is designed to achieve several objectives, including reducing procedural risks and protecting animal welfare, while the self-assessment approach enables the professionals involved to highlight potential issues that might otherwise be overlooked in daily practice and ensure that OPU procedures adhere to the highest ethical standards.
Transzonal projections (TZPs)-mediated communication between oocytes and cumulus cells plays a critical role in oocyte maturation and developmental competence, and disruption of this communication during in vitro maturation (IVM) is considered a major factor limiting oocyte quality. This study investigated the effects of Procyanidin B2 (PCB2) on porcine oocyte maturation and the underlying mechanisms. PCB2 supplementation significantly increased polar body extrusion (71.49 ± 5.75% vs. 87.23 ± 2.67%, P < 0.05, in the Control and PCB2 groups, respectively) and blastocyst cell numbers (28.00 ± 2.60 vs. 35.58 ± 2.17, P < 0.05). In addition, PCB2 reduced intracellular reactive oxygen species levels (58.60 ± 2.75 vs. 46.41 ± 2.06, P < 0.001) and increased glutathione content in porcine oocytes (73.75 ± 1.19 vs. 76.93 ± 0.78, P < 0.05). PCB2 also promoted cumulus expansion (P < 0.05) and increased TZP abundance during IVM (P < 0.05). Furthermore, we discovered that gap junctional uncoupler carbenoxolone (CBX) treatment impaired oocyte maturation (P < 0.05), cumulus expansion (P < 0.05), oocyte viability (P < 0.001), and lipid content (P < 0.001), whereas PCB2 partially rescued these defects (P < 0.05). Collectively, these findings demonstrate that PCB2 improves porcine oocyte maturation through the combined interaction between improved redox homeostasis, enhanced cumulus cell function, cellular metabolism, and cumulus-oocyte communication.
