[Effects of Long-term Combined Application of Organic and Inorganic Fertilizers on Soil Carbon Pool and Greenhouse Gas Emissions in Orchards].

Applying organic fertilizer can increase the contents of soil organic carbon (SOC) and active organic carbon, which are crucial for strengthening soil quality and fertility. Four treatments were established:no fertilization (CK), single application of organic fertilizer (M), single application of chemical fertilizer (NPK), and combined application of organic and inorganic fertilizers (MNPK). The changes in SOC and active components under long-term combined application of organic and inorganic fertilizers were investigated, as were the effects of various fertilization measures on greenhouse gas emissions. Moreover, we evaluated the variation in the soil carbon pool management index (CPMI). Total organic carbon (TOC), microbial biomass carbon (MBC), dissolved organic carbon (DOC), easily oxidized organic carbon (EOC), and particulate organic carbon (POC) increased by 82.84%, 66.30%, 21.12%, 93.28%, and 145.80%, respectively, when compared to those in the CK treatment. The NPK treatment had no discernible effect on SOC and organic carbon components. The combined application of organic and inorganic materials could enhance LI, CPI, and the soil carbon pool management index, with the increase in LI and CPI being the primary reason for the increase in CPMI. Correlation analyses revealed that soil organic carbon components and CPMI were significantly positively correlated with greenhouse gas emissions. The combined application of organic and inorganic materials enhanced cumulative CO2 emissions and warming potential (GWP) but decreased GHGI and yielded a maximum of 56365 kg·hm-2. Compared with that in the CK treatment (29073 kg·hm-2), apple yield in MNPK increased by 93.87%. Therefore, applying organic and inorganic fertilizers in dryland apple orchards can improve the accumulation of organic carbon and stabilize the soil carbon pool, which is more beneficial to the sustainable development of orchards.

[Correlation analysis of Poor Prognosis and Immunotherapy of lncRNAs Related with m 6A Modification in Cervical Cancer].

Objective: To study the correlation between N 6-methyladenosine (m 6A)-modification-associated long non-coding RNAs (lncRNAs) and poor prognosis and immunotherapy in cervical cancer based on data mining of The Cancer Genome Atlas (TCGA) cervical cancer dataset, so as to assess effectively the prognosis of cervical cancer patients and the feasibility of immunotherapy. Methods: We identified m 6A-modification-associated lncRNAs correlated to the prognosis of cervical cancer by conducting bioinformatics analysis of cervical cancer samples from the TCGA datasets and constructed a prognostic risk model of cervical cancer accordingly. Results: A total of 343 m 6A-modification-associated lncRNAs were identified from the samples of 304 cervical cancer patients. Univariate Cox regression analysis showed that 26 out of the 343 m 6A-modification-associated lncRNAs were significantly associated with the prognosis of cervical cancer patients. We identified 7 m 6A-modification-associated lncRNAs, including DLEU1, AC099850.4, DDN-AS1, EP300-AS1, AC131159.1, AL441992.2, and AL021707.6 through Lasso regression analysis and then developed a prognostic risk model based on them. According to the Kaplan-Meier survival analysis, cervical cancer patients in the low-risk group exhibited significantly improved overall survival (OS) in comparison with those in the high-risk group ( P<0.001). The area under the curve ( AUC) of receiver operating characteristic (ROC) curve analysis demonstrated the high sensitivity and credibility of the risk model. Multivariate Cox analysis showed that the risk score was an independent prognostic factor of cervical cancer patients. Tumor immune dysfunction and exclusion (TIDE) analysis predicted that the high-risk group would benefit more from immunotherapy. In addition, we found that immune checkpoint PD1 was associated with the expression of m6A-modification-related lncRNAs such as DDN-AS1, and the expression was higher in the high-risk group ( P<0.05). Conclusion: The prognostic risk model constructed on the basis of the aforementioned 7 m 6A-modification-associated lncRNAs can be used to effectively predict the prognosis of cervical cancer patients and assess the efficacy of immunotherapy targeting PD1.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation.

Boar sperm are susceptible to oxidative damage caused by reactive oxygen species (ROS) during storage. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important therapeutic target, because it is a cellular metabolism energy sensor and key signalling kinase in spermatozoa. We evaluated the effects of rosmarinic acid (RA), an antioxidant, on boar sperm during liquid storage to determine whether it protects boar sperm via AMPK activation. Boar ejaculates were diluted with Modena extender with different concentrations of RA and stored at 17°C for 9 days. Sperm quality parameters, antioxidant capacity, energy metabolism, AMPK phosphorylation and fertility were analysed. Compared with the control, 40 µmol/L significantly improved sperm motility, plasma membrane integrity and acrosome integrity (p < .05). The effective storage time of boar sperm was up to 9 days. On the third and seventh days, the sperm with RA exhibited increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, mitochondrial membrane potential (ΔΨm) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, whereas malondialdehyde (MDA) content was significantly decreased (p < .05). Western blot showed that RA, as well as AICAR (AMPK activator), promoted AMPK phosphorylation, whereas Compound C (AMPK inhibitor) inhibited this effect. The sperm-zona pellucida binding experiment showed that 40 µmol/L RA increased the number of sperm attached to the zona pellucida (p < .05). These findings suggest meaningful methods for improved preservation of boar sperm in vitro and provide new insights into the mechanism by which RA protects sperm cells from oxidative damage via AMPK activation.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway.

Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10-6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.

Effects of sulfanilamide on boar sperm quality, bacterial composition, and fertility during liquid storage at 17°C.

Sulfanilamide (SA) is an effective broad-spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.

Effect of addition of melatonin on liquid storage of ram semen at 4°C.

Adding a certain amount of antioxidants to semen extender has been shown to improve semen quality. The aim of present study was to elucidate whether the supplementation of melatonin to the Tris-based extender (CTR) could enhance the quality of ram spermatozoa during storage at 4°C. Ram semen samples were collected and diluted with CTR extender containing different concentrations (0, 0.05 (M 0.05), 0.1 (M 0.1), 0.2 (M 0.2) or 0.4 (M 0.4) mM) of melatonin. Sperm routine indicators, mitochondrial activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were analysed in control and melatonin treatment groups. The higher per cent of motility, plasma membrane integrity, mitochondrial activity and T-AOC activity was observed in M 0.05, M 0.1 and M 0.2 groups compared to control group at 5 days of storage (p < 0.05), while lower percentage of MDA content was observed among these groups (p < 0.05). In addition, there were no significant differences in acrosome integrity among the control and M 0.05, M 0.1 and M 0.2 groups during the experiment. The above results show that the addition of 0.05, 0.1, 0.2 mM of melatonin is beneficial to the preservation of ram semen during liquid storage at 4°C mainly through antioxidative stress.