Reproduction in Domestic Animals最新文献

In vitro maturation (IVM) of oocytes is crucial in livestock breeding. Oocytes obtained by IVM are more susceptible to oxidative stress than in vivo, leading to low maturation rates. Betaine from red beetroot acts as an antioxidant and methyl donor, regulating epigenetic modifications in cell physiology. This study investigates Betaine's effects on porcine oocyte IVM, embryo development and underlying molecular mechanisms. Results demonstrate that 16 mmol/L Betaine significantly enhances the first polar body extrusion, cleavage and blastocyst rates compared to the control and other concentrations. Betaine elevates normal cortical granule distribution, normal spindle assembly, normal chromosome arrangement and overall m6A levels during IVM. It increases the antioxidant gene expression and mitochondrial function and decreases reactive oxygen species levels. However, Betaine's beneficial effects were diminished by AMPK inhibitor compound C. In conclusion, Betaine enhances porcine oocyte IVM and early embryo development by enhancing the antioxidant capacity and mitochondrial function pathway.

This study aims to investigate the cryoprotective effect of Omega-3 nano-emulsion (Omega-3 NE) on stallion sperm quality, kinematic parameters, acrosome status, subcellular ultrastructure, oxidative/antioxidant markers, and semen microbiota. Forty ejaculates were collected, extended, and cryopreserved from 5 fertile Pure Egyptian stallions (Equus caballus). The ejaculates were divided into five groups: a control group (without additive) and four groups supplemented with 25, 50, 100, and 200 μg of Omega-3 NE/mL. The Omega-3 NE exhibited an average particle size of 51-146 nm, a PDI of 0.58, and a zeta potential of -31 mV. Omega-3 NE (200 μg/mL) significantly improved progressive motility, viability, and membrane integrity of stallion semen (p < 0.05). Additionally, supplementation with Omega-3 NE (200 μg/mL) led to a significant enhancement in post-thawed sperm kinematic parameters, including PM, DSL, VCL, and VSL, by 40%, 21.5%, 26.7%, and 20.7%, respectively, compared to the control group. The addition of 100 or 200 μg/mL Omega-3 NE to the media resulted in a higher percentage of live sperm with intact acrosomes. Additionally, all Omega-3 NE treatments significantly decreased the percentage of dead sperm with intact acrosomes as well as microbiota load (total bacterial count and coliform bacteria count) compared to the control (p < 0.01). Significant improvements in antioxidant status (TAC and CAT) and reduction of oxidative stress markers (MDA, NO, and H₂O₂) were observed in all Omega-3 NE groups compared to the control group (p < 0.05). Omega-3 NE (200 μg/mL) significantly reduced sperm apoptosis (p < 0.01) and preserved better subcellular integrity compared to the control and other treatment groups. The results suggest that Omega-3 NE at concentrations of 100-200 μg/mL can effectively enhance sperm cryo-resistance via enhancing sperm quality and kinematic variables, reducing oxidative stress and microbiota load, and maintaining sperm subcellular ultrastructure. The study highlights the potential of Omega-3 NE as a nanotechnology-based approach to boost assisted reproductive technologies in stallion breeding programmes.

Oxidative stress and redox homeostasis play crucial roles in ovarian function, influencing oocyte quality and developmental competence. This study evaluated the population-level associations between thiol-disulphide homeostasis, oxidative stress biomarkers, antioxidant enzyme activity, and oocyte quantity and quality in cattle. Blood and follicular fluid samples were analysed from cattle to assess total thiol (TTL), native thiol (NTL), disulphide (DS), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) levels. Oocytes were collected and classified based on morphological characteristics. Statistical analyses revealed a positive correlation between TTL and total oocyte count (TOC), suggesting that higher thiol availability enhances oocyte production. Conversely, elevated NTL levels were negatively associated with oocyte quality, indicating that an imbalance in thiol-disulphide homeostasis may impair follicular development. GPX activity exhibited a significant negative association with both TOC and high-quality oocyte count (HQOC), suggesting that excessive antioxidant activity might disrupt essential redox signalling pathways required for oocyte maturation. However, SOD, CAT and NO levels were not significantly correlated with oocyte count or quality, indicating a complex interplay between oxidative stress markers and reproductive efficiency. These findings demonstrate significant associations between thiol-disulphide balance, GPX activity, and both total and HQOCs, highlighting the relevance of redox status in follicular physiology. Further research is needed to explore targeted antioxidant interventions to optimise reproductive outcomes in livestock. Understanding the role of oxidative balance in oocyte maturation may contribute to improving assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and embryo transfer in cattle.

Niacin acts as an antioxidant that protects cells from oxidative damage. This study evaluated the effects of adding niacin to the equine semen freezing extender on sperm quality and gene expression after cryopreservation. Ejaculates from ten stallions were frozen using the INRA 96 extender (control) or extenders supplemented with 10- and 20-mM niacin. After thawing, sperm were analysed for motility, kinematics, viability, membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde and reactive oxygen species (ROS) concentrations, DNA integrity, sperm binding to bovine oviduct explants, and expression of apoptosis related (BCL2, BAX), mitochondrial (ROMO1), sperm binding (SPACA3) and DNA repair (OGG1) genes. Data were tested for normality (Shapiro-Wilk) and analysed by randomised block ANOVA followed by Tukey's test (p < 0.05). Machine learning algorithms (Logistic Regression, MLP, XGBoost, KNN and SVM) with SHAP analysis ranked the most influential parameters associated with sperm quality. The addition of 10 mM niacin improved sperm motility, mitochondrial activity, and the number of sperm bound to bovine oviduct explants, while reducing ROS levels and expression of BAX and ROMO1, and increasing BCL2 and SPACA3 genes. The 20 mM treatment also enhanced sperm binding and upregulated SPACA3 expression compared to the control. However, 20 mM niacin showed lower binding activity than 10 mM. Machine learning identified sperm binding to oviduct explants and SPACA3 expression as the most influential variables for classifying samples. In conclusion, both niacin concentrations improved equine cryopreserved sperm quality, although 10 mM showed superior antioxidant, anti-apoptotic, and functional effects, representing optimal supplementation level.

Artificial insemination in the pig industry requires frozen semen of better freezability and post-thaw quality. Several recent metabolomics studies were carried out to discover sperm freezability-related biomarkers. However, only a limited number of pig breeds were examined and results remain obscure. Here, boar semen samples were collected and quality evaluated for 3 Western breeds (Duroc, Landrace and Yorkshire), originating from a nucleus farm and a boar station. Post-thaw sperm from the nucleus farm exhibited significantly higher motility and vitality rates (p < 0.01), and also better acrosome integrity and plasma membrane integrity (p < 0.05). For the high and low freezability groups (36 samples in total), ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) on spermatozoa identified significantly differential metabolites (p < 0.05, including betaine), with 258 metabolites in Duroc, 126 in Landrace, and 215 in Yorkshire, which were significantly enriched in 11, 8 and 13 metabolic pathways (KEGG) (p < 0.05), respectively. Besides breed-specific pathways (Duroc: cysteine and methionine metabolism; Landrace: arginine and proline metabolism, tryptophan metabolism, lysine degradation, galactose metabolism and pyruvate metabolism; Yorkshire: steroid hormone biosynthesis, riboflavin metabolism and linoleic acid metabolism), two pathways common to 3 breeds (alanine, aspartate and glutamate metabolism, and pentose phosphate pathway) were found. Betaine was confirmed to be at a significantly higher level in the semen of high freezability for all three pig breeds (p < 0.05). Taken together, metabolites and metabolic pathways common and specific to commercial Western breeds were identified. Betaine was related to better spermatozoa freezability. Our findings provide the basis and insights into better understanding the role of metabolic molecules and pathways important to boar spermatozoa freezability.

Sex-sorted sperm and preimplantation sex determination are indispensable tools for improving reproductive efficiency and herd management in livestock production. The present study aimed to identify the most effective 'X' and 'Y' specific genes for sexing sorted bovine sperm and embryos. We selected five Y-specific (SRY, HSFY, TSPY, ZFY and OFD1Y) and four X-specific (PLCXD1, SHROOM 2, PLP and MAOA) genes and evaluated their specificity and sensitivity by PCR and qPCR analyses in X-sorted sperm, Y-sorted sperm, mixed sperm and female blood. HSFY and TSPY showed more pronounced and specific amplification in Y-sorted sperm DNA, making them robust Y-specific markers, while MAOA and SHROOM 2 were more specific to X-sorted sperm DNA and identified as effective X-specific markers. These results were further validated in buffalo IVF embryos, confirming their effectiveness in embryo sexing. The findings of this study can be applied in duplex or multiplex PCR assays, which minimise the risk of amplification failure and false negatives, providing a rapid and reliable toolkit for bovine sperm and embryo sexing. Implementing such a molecular approach can significantly benefit livestock management by improving breeding outcomes, reducing the costs and inefficiencies associated with undesired sex ratios.

In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and immotile spermatozoa after their injection into ovulated mouse oocytes. In both groups, injected horse spermatozoa activated the mouse oocytes. This was followed by the extrusion of the second polar body (2 PB) and the formation of maternal pronuclei (Mo-fPN-mouse female pronucleus); in parallel, the horse sperm heads rapidly decondensed in the murine cytoplasm and formed paternal pronuclei (Ho-mPN-horse male pronucleus), which were larger than the female ones. With the exception of one stallion tested, DNA damage has been detected in almost all Ho-mPNs originating from immotile spermatozoa. DNA in motile (even sporadically) spermatozoa was mostly undamaged. Moreover, when the xenogeneic zygotes cleave to the two-cell stage, the incidence of micronuclei in blastomeres mirrors the extent of DNA damage in paternal pronuclei. In conclusion, and contrary to the mouse, where sperm DNA is very resistant to damage, we do not recommend the use of immotile horse spermatozoa for ICSI. On the other hand, even the sporadically motile mouse spermatozoa have no damaged DNA and can thus be used for intragenic ICSI.

The objective of this study was to evaluate the effects of exogenous GnRH administration at the beginning of estrus synchronisation in mares during the spring transitional period. Estrus was synchronised using a progesterone releasing intravaginal device (PRID). The PRID was left in the vagina for 10 days, followed by an injection of 0.4 mg of cloprostenol at PRID removal. The GnRH group (n = 32) was subjected to intramuscular administration of 100 μg of the GnRH agonist triptorelin at PRID insertion, while the control group (n = 32) received 1 mL of sterile physiological saline solution. Ovulation was induced by an intramuscular injection of 3000 IU of human chorionic gonadotropin until the dominant follicle reached a diameter of 35 mm. The mares were examined and insemination was performed. Subsequently, insemination was carried out every 12 h until ovulation. Transrectal palpation and ultrasound were carried out 15 days after ovulation to confirm the presence or absence of an embryonic vesicle. The days of ovulation induction and insemination in the control group were more dispersed than in the GnRH group. Compared with the control group, the time of ovulation induction and insemination in the GnRH group were accelerated and concentrated. In summary, GnRH given at the beginning of the estrus synchronisation program significantly increased synchronisation of ovulation in mares; however, it did not increase pregnancy rates.

In vitro reproductive biotechnologies show promise for fertility preservation but still face challenges, including oxidative stress from high oxygen tension, which impairs cell viability and development. Antioxidants have been widely explored to attenuate oxidative stress during culture. Among them, anethole, a plant-derived phenylpropanoid, stands out for its promising properties. This review explores the mechanisms and applications of anethole in reproductive physiology and its potential to enhance in vitro reproductive systems. Findings indicate that anethole modulates key pathways and may improve outcomes in in vitro follicle culture, oocyte in vitro maturation and in vitro embryo culture. These insights support future research and the strategic inclusion of anethole in reproductive biotechnology protocols.

Follicle-stimulating hormone (FSH) plays an important role in regulating reproductive events, particularly follicular development and oocyte competence acquisition. Some studies using FSH protocols in zebu cattle were performed, but data regarding its application in the Red Sindhi breed are scarce and therefore warranted. In this context, this study aimed to assess the FSH administration regimen-multiple doses (T1-m) and single dose (T2-s) and coasting period (54 h vs. 102 h) in oocyte developmental competence in Sindhi females. A total of 80 mg of FSH was administered either as a single application (40 mg IM + 40 mg, SC) or compared to multiple applications (30 mg + 30 mg + 20 mg, IM). Animals that did not receive FSH treatment serve as controls (CT). The present data showed that both T1 and T2 applications resulted in a greater number of medium-sized follicles (7.80 vs. 8.57, p < 0.05), oocyte recovery (9.76 vs. 9.81), when compared to control (5.20; 6.30, respectively). Animals from T2 also had a greater number of aspirated follicles (12.52 vs. 8.70, p < 0.05), viable oocytes (7.33 vs. 4.45, p < 0.05) and blastocyst rates (43.22% vs. 29.11%, p < 0.05) than control animals. Our results showed that a reduced dose of FSH both single and multiple applications enhance oocyte developmental competence. Moreover, a single application of FSH combined with a longer coasting period offers practical advantages, making this approach more attractive for Sindhi breeding programmes.