Reproduction in Domestic Animals最新文献

As the hybrid between cattle and yak, cattleyak is a typical male sterile mammal, and the underlying mechanism for its spermatogenic arrest is still unclear. In this study, the coding region of cattleyak TAF4B gene was cloned by RT-PCR and analysed by bioinformatics. To investigate the effects of TAF4B on cellular proliferation and differentiation, an expression vector was generated and introduced into undifferentiated spermatogonia (UDSPG) of cattleyak. The results showed that the protein encoded by TAF4B did not contain the signal peptide sequence. The expression level of TAF4B in UDSPG of cattleyak was lower than that in yak, while the overexpression of TAF4B in cattleyak promoted the proliferation activity of cattleyak UDSPG. Meanwhile, the expression of proliferation and meiosis-related genes was increased but the differentiation-related genes were decreased. Therefore, the aberrant expression of TAF4B in cattleyak UDSPG possibly impaired its proliferation and differentiation equilibrium and decreased its growth potentiality, thereby reducing the quantity of UDSPG and affecting spermatogenesis. This study provided a potential approach for further elucidation of the mechanism of spermatogenesis arrest and provided a new idea for solving the problem of male infertility in cattleyak.

Supplementing freeze diluents with certain antioxidants can maintain the quality of chilled sperm. The present study was an attempt to investigate the effect of Beltsville extender supplementation with the mitochondrial-targeted antioxidant 'Mitoquinol' on the quality parameters and fertility potential of rooster sperm during the cooling process. Semen samples were diluted in Beltsville extender, divided into five groups, and supplemented with 0, 1, 10, 100 and 1000 nM Mitoquinol. Samples were stored at 5°C for up to 50 h and then assayed for sperm motility, viability, mitochondrial function, membrane integrity and malondialdehyde concentration after 0, 25 and 50 h of cooling. To assess reproductive performance, artificial insemination was performed using sperm cooled for 25 h. The results showed no differences between groups at the beginning time. Extender supplementation with 10 and 100 nM Mitoquinol resulted in an improvement in total motility, progressive motility, membrane integrity, mitochondrial function and viability (p ≤ 0.05), as well as a lower malondialdehyde concentration (p ≤ 0.05) in comparison to the other groups during 25 and 50 h storage. Fertility rates were higher when roosters were inseminated with semen samples supplemented with 10 and 100 nM Mitoquinol, compared to the control group. Therefore, supplementing Beltsville extender with Mitoquinol (10 and 100 nM) effective in improving the quality and fertility potential of cooled rooster sperm.

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes.

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen-thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p < .05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p < .05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane.

In this study, we evaluated sheep sperm quality after using Tris-citrate-fructose-based extender with and without egg yolk, a Tris-citrate without fructose and with egg yolk and the commercial extender Biladyl®, preserving diluted semen at 15 and 23°C for different times (4, 24, 48 and 72 h). The results showed that the diluents with fructose and egg yolk gave the best results of seminal quality. Moreover, the production of ROS was higher for the temperature of 23°C compared to the temperature of 15°C (control). In addition, VCL and the percentage of spermatozoa with intact acrosome decreased with temperatures of 23°C. Finally, a drastic decrease in sperm quality was observed after 24 hours of preservation for most of the parameters evaluated.

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co-incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization-IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p > .05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p > .05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p > .05). In conclusion, in vitro exposure of boar sperm to OVGP1-functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

Premature acrosomal exocytosis in cryopreserved semen is one of the reasons attributed to low fertility among livestock. In the present study, we attempted to enhance the cryopreserved semen quality by selective removal of acrosome-reacted spermatozoa using FITC-PNA conjugated iron magnetic nanoparticles (MNPs). Further, the effect of nano purification on other sperm functional attributes was also assessed. Iron MNPs were prepared using co-precipitation method and dextran-coated MNPs were conjugated with FITC-PNA (0.04 mg/mL). A preliminary experiment was conducted to standardise the dose of FITC-PNA conjugated iron MNPs (0.02, 0.05, 0.1, 0.15, 0.2, 0.4 and 0.6 mg). Among the different doses used, 0.6 mg FITC-PNA conjugated iron MNPs significantly (p < 0.05) removed higher acrosomal reacted spermatozoa from the semen, and therefore, this dose was used in further experiments. Cryopreserved semen from Holstein Friesian breeding bulls (n = 6) were thawed and washed using Sperm-TALP to remove residual extender. Washed spermatozoa (2 × 10⁶) were exposed to 0.6 mg of FITC-PNA conjugated iron MNPs for 10 min at 37°C. The nano purified semen was assessed for various vital sperm parameters viz., viability, intracellular calcium, apoptosis, mitochondrial ROS and mitochondrial membrane potential using flow cytometry. We found that nanopurification using FITC-PNA conjugated iron MNPs significantly (p < 0.05) improved the sperm quality. The proportion of viable non-apoptotic spermatozoa with low intracellular calcium levels was significantly (p < 0.05) enriched in nano purified semen. Nano purification did not affect sperm mitochondrial membrane potential and ROS production. In conclusion, these preliminary findings indicate that FITC-PNA coated iron MNPs effectively removed acrosome reacted spermatozoa and significantly improved sperm functional attributes in the purified fraction.

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.