OBJECTIVE:This study aimed to elucidate the impact of obesity and weight loss on autophagic processes in ovarian granulosa cells (GCs), given the crucial roles that these cells and this process play in oogenesis. METHODS:Three experimental groups were used: a control group (ND group), a high-fat diet-induced obese mouse model group (HFD group), and a swimming exercise group (SE group). Immunohistochemistry (IHC) and Western Blotting (WB) were used to detect protein expression in GCs. RESULTS:LC3B expression was higher in the HFD and SE groups than in the ND group (p<0.01), but lower in the SE group than in the HFD group (p<0.05). p62 expression was lower in the HFD and SE groups than in the ND group (p<0.01 and p<0.05, respectively), but higher in the SE group than in the HFD group (p<0.05). The LC3B-II/I ratio was elevated in the HFD group versus the ND and SE groups (p<0.05). p62 expression was lower in the HFD group than in the SE and ND groups (p<0.05 and p<0.01, respectively), and lower in the SE group than in the ND group (p<0.01). In IHC, the p-mTOR/mTOR ratio was lower in the HFD group than in the ND group (p<0.05), but higher in the SE group than in the ND and HFD groups (p<0.01). The AMPK/AMPK ratio was higher in the HFD group than in the ND group (p<0.01), but lower in the SE group than in the HFD group (p<0.01). In WB, the p-mTOR/mTOR ratio was lower in the HFD and SE groups than in the ND group (p<0.01), but higher in the SE group than in the HFD group (p<0.05). p-RPS6/RPS6 ratio was lower in the HFD group than in the ND group (p<0.01), but higher in the SE group than in both groups (p<0.01). CONCLUSIONS:Our findings suggest that obesity activates granulosa cell autophagy via AMPK/mTOR signaling, while weight loss partially restores homeostasis.

To preliminarily elucidate the bidirectional metabolic effects caused by changes in PFKM expression, provide research clues for further investigation of the molecular mechanisms through which PFKM regulates porcine skeletal muscle metabolism and myogenic differentiation, and offer a reference for identifying candidate genes associated with meat production traits and studying gene regulation in pig breeding, this study compared skeletal muscle protein expression profiles between Large White and Bama pigs. Candidate regulatory factors related to muscle growth and energy metabolism were screened, and the effects of altered muscle-type phosphofructokinase (PFKM) expression on metabolic homeostasis and myogenic differentiation in porcine skeletal muscle satellite cells (SMSCs) were preliminarily evaluated. Longissimus dorsi muscle tissues from Large White and Bama pigs were analyzed using iTRAQ-based proteomics. A total of 2040 reliably quantified proteins were identified, of which 51 were relatively upregulated in Large White pigs and 73 were relatively upregulated in Bama pigs. Functional enrichment analysis showed that the differentially expressed proteins were mainly involved in glycolysis, mitochondrial energy metabolism, protein synthesis, and the regulation of muscle fiber structure and function. PFKM was therefore selected as a key candidate differentially expressed protein. Porcine SMSC models comprising a PFKM knockdown group (PFKM-KD), a PFKM overexpression group (PFKM-OE), and a normal control group (PFKM-CON) were subsequently established. Glucose consumption and lactate accumulation in the culture medium, ATP levels, reactive oxygen species (ROS), mitochondrial membrane potential, apoptosis, mitochondrial dynamics-related proteins, and myogenic differentiation markers were then examined. Compared with the PFKM-CON group, the PFKM-OE group showed significantly increased glucose consumption and lactate accumulation, together with significant increases in ROS levels, mitochondrial membrane potential, and apoptosis, whereas ATP levels were significantly reduced. In the PFKM-KD group, glucose consumption and lactate accumulation were significantly decreased, accompanied by reductions in mitochondrial membrane potential, ROS, ATP levels, and apoptosis. PFKM overexpression mainly induced oxidative stress, ATP depletion, and increased apoptosis, whereas PFKM knockdown primarily reduced mitochondrial membrane potential, ROS, and ATP levels, indicating a relatively low-metabolic state. Both treatments were accompanied by dysregulated expression of the mitochondrial dynamics-related proteins DRP1, MFN2, and OPA1, although their patterns of change were not identical. Western blotting and immunofluorescence consistently showed that the expression levels of the myogenic differentiation markers MyoD and MYH were significantly lower in both the PFKM-KD and PFKM-OE groups than in the control group, suggesting that either excessive or insufficient PFKM expression may impair the myogenic differentiation potential of SMSCs. In conclusion, changes in PFKM expression are closely associated with glycolysis-related metabolism, energy and redox homeostasis, mitochondrial function-related indicators, and myogenic differentiation capacity in porcine SMSCs. The normal biological function of PFKM may therefore depend on its expression being maintained within an appropriate range.

Cadmium (Cd), an environmental toxicant, is known to cause significant damage to reproductive systems in human and animals. However, the detrimental effects of maternal Cd exposure during gestation on male offspring and the underlying mechanisms remain unknown. In this study, pregnant mice were exposed to Cd (32 mg/l) in drinking water throughout gestation to investigate the mechanisms underlying testicular and sperm injury and repair in male offspring. Results showed that maternal Cd exposure caused growth inhibition in male offspring at birth, accompanied by testicular DNA damage and upregulation of Rad51, a key protein involved in homologous recombination repair. After delivery, when Cd was removed, the male offspring exhibited compensatory growth and development. At 7 weeks of age, despite abnormally enlarged seminiferous tubules, spermatogonial stem cell meiosis arrest, and reduced total sperm motility, DNA damage levels, DNA repair capacity, and the motility function of surviving sperm were all at normal levels. Compared with controls, the offspring at 7 weeks of age showed no alterations in global 5-methylcytosine (5-mC) of sperm, whereas DNA methylation of Line1 were decreased. Meanwhile, DNA methylation of imprinted genes H19 and Peg3, which regulate sperm quality and post-fertilization embryonic development, remained normal. Cd exposure during gestation led to transcriptomic abnormalities in the testes of newborn male offspring, but these abnormalities were gradually repaired during postnatal growth and development. These findings indicate that although gestational Cd exposure impairs reproductive function of male offspring, postnatal growth and development, coupled with DNA repair mechanisms, can partially restore their reproductive potential.

This study investigated the effects of short-term serum starvation on autophagy, functional protein expression, and cellular metabolism in porcine granulosa cells (GCs). Fluorescence microscopy revealed that 7.5% and 2.5% serum concentrations significantly increased autophagosome formation compared to the 10% serum group (P < 0.01), which was suppressed by the autophagy inhibitor 3-MA (P < 0.01). Western blot analysis demonstrated elevated LC3B and reduced p62 protein levels under serum starvation, indicating autophagy activation, while p-mTOR/mTOR decreased and p-AMPK/AMPK increased (P < 0.05 or P < 0.01), suggesting AMPK/mTOR pathway involvement. Additionally, serum starvation enhanced the expression of functional proteins (FSHR, HIF-1α, 3β-HSD) and steroidogenesis (E2, P4), which were attenuated by 3-MA (P < 0.05). Reactive oxygen species (ROS) and apoptosis increased significantly under severe starvation (2.5% serum, P < 0.01), while mild starvation (7.5% serum) elevated ATP levels (P < 0.01). Mitochondrial membrane potential declined in the 2.5% serum group (P < 0.01), with autophagy inhibition further reducing it. These findings indicate that short-term serum starvation induces protective autophagy via AMPK/mTOR signaling, enhancing GCs' functional protein expression and steroidogenesis, whereas severe starvation promotes oxidative stress and apoptosis.

Obesity, a globally prevalent chronic disease, disrupts systemic homeostasis and impairs female fertility, yet the mechanisms linking adipose dysfunction to ovarian reserve remain unclear. Using high-fat diet-induced obese C57BL/6 mouse models (HFD) and exercise-diet intervention models (SE group), we systematically evaluated obesity-associated reproductive deficits. Histomorphological analysis revealed that HFD mice exhibited ovarian atrophy, increased atretic follicles, and reduced primordial/antral follicle counts, which were partially restored by SE intervention. TEM demonstrated lipid droplet accumulation and mitochondrial heterogeneity in HFD ovaries, with residual vacuolization persisting despite SE-mediated improvement. Superovulation assays demonstrated reduced oocyte production in HFD mice, accompanied by impaired in vivo maturation and blastocyst formation. Immunofluorescence revealed abnormal spindle assembly and heterogeneous mitochondrial distribution in HFD oocytes, potentially associated with elevated ROS. Mechanistically, HFD downregulated folliculogenesis regulators (BMP-15, HIF-1α, PTEN/AKT/FoxO3) while upregulating metabolic stress markers (Chemerin, CMKLR1). Western blot confirmed reduced ovarian protein acetylation and BMP-15/HIF-1α expression in HFD mice, with partial recovery following exercise-diet intervention. These findings demonstrate obesity-induced dual impairments: mitochondrial-ROS dysfunction compromising oocyte competence and BMP-15/HIF-1α suppression disrupting follicular survival through PTEN-AKT-FoxO3 signaling. Although exercise-diet intervention improved metabolic parameters and oocyte quality, residual abnormalities highlighted irreversible impairments. Our study identifies obesity as a driver of ovarian aging and emphasizes the fertility-enhancing potential of combined exercise-diet intervention in obese female mice.

Intermittent fasting (IF) as a dietary intervention with potential health benefits has garnered significant attention in recent years. This study investigated the effects of varying fasting intensities on skeletal muscle growth using mouse models. Compared to the normal-diet (ND) control group, short-term fasting induced feeding amount-dependent alterations in skeletal muscle autophagy markers, characterized by elevated LC3B expression, reduced p62 levels, and decreased p-mTOR/mTOR ratio. Notably, short-term mild fasting (sMF) significantly upregulated myogenic (MYH, MyoD) and adipogenic (LPL, PPARγ) differentiation markers, whereas short-term severe fasting (sSF) suppressed myogenic markers without significantly affecting adipogenic factors. Pharmacological modulation using 3-methyladenine (3-MA) and rapamycin (RAPA) confirmed the critical role of autophagy in myogenic and adipogenic processes. Multi-cycle IF studies revealed that intermittent mild fasting (IMF) enhanced metabolic efficiency (evidenced by increased feed conversion ratio), elevated organ indices of gastrocnemius and quadriceps femoris muscles, and reduced groin fat. IMF also promoted intramuscular adipogenesis and myofiber remodeling. In contrast, intermittent severe fasting (ISF) impaired glucose tolerance, decreased triglyceride levels and aspartate aminotransferase (AST) activity, inhibited myofiber growth, and exhibited no significant effect on intramuscular adipogenesis. Our findings demonstrate that IMF enhances skeletal muscle mass and reduces visceral adiposity through mTOR-autophagy axis, providing an optimized fasting regimen for metabolic health and body composition regulation.

Maternal cadmium exposure during pregnancy has been demonstrated to have detrimental effects on offspring development. However, the impact of maternal cadmium exposure on offspring oocytes remains largely unknown, and the underlying mechanisms are not fully understood. In this study, we found that maternal cadmium exposure during pregnancy resulted in selective alteration in epigenetic modifications of mouse oocytes in offspring, including a decrease in H3K4me2 and H4K12ac, as well as an increase in DNA methylation of H19. Although ROS levels and mitochondrial activity remain at normal levels, the DNA damage marker γH2AX was significantly increased and the DNA repair marker DNA-PKcs was remarkably decreased in offspring oocytes from maternal cadmium exposure. These alterations are responsible for the decrease in the quality of mouse oocytes in offspring induced by maternal cadmium exposure. As a result, the meiotic maturation of oocytes and subsequent early embryonic development are influenced by maternal cadmium exposure. RNA-seq results showed that maternal cadmium exposure elicits modifications in the expression of genes associated with metabolism, signal transduction, and endocrine regulation in offspring ovaries, which also contribute to the disorders of oocyte maturation and failures in early embryonic development. Our research provides direct evidence of transgenerational epigenetic inheritance of cadmium reproductive toxicity in mouse germ cells.

This study investigated the effects of long-term serum starvation on autophagy, metabolism, and differentiation of porcine skeletal muscle satellite cells (SMSCs) and elucidated the role of autophagy in skeletal muscle development. Our findings provide a theoretical basis for improving meat production in domestic pigs. The SMSCs isolated and preserved in our laboratory were revived and divided into six groups based on the culture medium serum concentration to simulate varying levels of serum starvation: 20% serum (control group), 15% serum (mild serum starvation group), 5% serum (severe serum starvation group), and their autophagy inhibition groups supplemented with 3-methyladenine. After 96 h of culture, the apoptosis rate, mitochondrial membrane potential, reactive oxygen species, and ATP were measured to evaluate the effects of serum starvation on the SMSCs’ metabolism. Additionally, the levels of autophagy-related proteins, autophagosomes, and autolysosomes were measured to investigate the impact of long-term serum starvation on autophagy. The expression of proteins associated with myogenic and adipogenic differentiation (MHC, MyoD1, peroxisome proliferator-activated receptor γ, and lipoprotein lipase) as well as lipid content were also determined to investigate the effects of long-term serum starvation on SMSC differentiation. The results showed that long-term serum starvation induced autophagy through the AMPK/mTOR signaling pathway, accelerated cell metabolism and apoptosis, exacerbated reactive oxygen species accumulation, and inhibited myogenic and adipogenic differentiation of SMSCs. Moreover, these effects were positively correlated with the level of serum starvation. In addition, serum starvation-induced autophagy moderately promoted the myogenic and adipogenic differentiation of SMSCs; however, these effects were insufficient to counteract the inhibition of cell differentiation by long-term serum starvation. This study provides insight into leveraging serum starvation as a stressor to regulate muscle growth and metabolism in domestic pigs.

The acquisition of animal genome-editing individuals is a complex and time-consuming process. The use of genome-editing technology alone can only obtain edited cells. In order to efficiently obtain animals with edited genomes, it is necessary to combine it with other related technologies. This chapter systematically introduces some technologies related to animal genome editing, such as microinjection technology, somatic cell nuclear transfer technology, RNA interference, and other technologies, and systematically expounds on the operation process, precautions, and existing problems of related technologies in combination with examples in experimental work. We hope to provide constructive suggestions for the subsequent improvement of relevant technologies.

为研究精液生产中温度管理对猪精子质量的影响及机制,将采集后的猪精液样品等分成3份并分别于17℃(对照组)、22℃(公猪站实验室温度)和37℃(用于稀释液预温的水浴锅温度)下保存24 h.在0,6,18和24 h分别取样检测猪精子活率、活力、精子质膜和顶体膜完整性、细胞内ROS水平及精子凋亡率.在保存24 h后取样检测精子p-AMPKa、LKB-1、CaMKKβ、Bax、Bcl-2、Hsp70和Hsp90蛋白表达量.结果表明,与17℃相比,22℃下保存精子活率和活力在24 h后显著下降(P＜0.05).与17℃和22℃相比,37℃下保存精子各质量参数均显著下降(P＜0.05)且出现质量损伤的时间更早,细胞内ROS水平显著升高(P＜0.01),保存18 h后精子早期凋亡水平显著降低(P＜0.05)而晚期凋亡水平显著升高(P＜0.05).与17℃和37℃相比,22℃保存24 h后精子p-AMPKa水平显著升高(P＜0.05).与17℃和22℃相比,37℃保存24 h后AMPK上游激酶LKB-1和CaMKKβ的表达量显著降低(P＜0.05),凋亡蛋白Bcl-2、Bax和Caspase-3及热休克蛋白Hsp70表达量显著下降(P＜0.05).综合以上结果,精液生产过程中17～22℃下保存不会显著影响猪精子质量,精子通过LKB-1途径激活AMPK调节凋亡和热休克蛋白的表达来应答温度的升高.37℃下保存促进了精子早期凋亡向晚期凋亡的转化,破坏了精子的调节机制,不利于精子的存活.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

兽医学科人才的培养应服务于国家战略需求和社会经济发展需要,同时人才的职业发展规划也要与社会生产实践的要求相结合.新时代背景下,兽医人才在保障动物健康和生产、人类健康和食品安全等方面将承担更重的责任,这对兽医人才的培养也提出了新的要求.我国新冠疫情防控的成功实践启示我们:基础知识和能力扎实、实践经验丰富和科研攻关能力强的人才队伍是取胜的关键.为此,文章将教学与科研和生产实际结合起来,以教学为基础、以科研为延伸、以解决生产问题为导向,对"兽医产科学"课程的教学模式进行探索,以期为符合新时代要求的兽医人才培养提供理论和实践的参考.

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 μM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm’s total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

为了构建猪PPARD基因编辑载体并进行基因编辑效率检测,本研究利用改良的双位点编辑CRISPR/Cas9载体系统,根据PPARD基因结构和序列特点,设计2个能靶向切割PPARD基因的sgRNA序列,将2个PPARD sgRNA表达盒以串联的形式连接到一个CRISPR/Cas9载体中.将构建的质粒转染PK15细胞,提取各组细胞DNA,PCR扩增突变区域后,通过序列测定在DNA水平检测载体编辑效率.分别提取各组细胞总RNA及细胞总蛋白,利用qRT-PCR及Western blot在基因表达及蛋白表达水平上检测重组载体的基因编辑效率.结果发现,PPARD-2KO组DNA总突变率为45.83％(22/48);与正常对照组相比,PPARD基因编辑组中PPARD mRNA显著下降84％(P＜0.01),PPARD蛋白表达量显著下降61％(P＜0.01).本研究利用改良的CRISPR/Cas9载体系统成功构建了高效PPARD基因编辑载体,并且未经药物筛选即可在细胞上实现高效率基因编辑,将大幅提高后续筛选单细胞克隆效率,推进对PPARD基因的功能研究.

This study aimed to investigate the effects of different levels of autophagy induced by transient serum starvation on the metabolism, lipid metabolism, and differentiation of porcine skeletal muscle satellite cells (SMSCs) to preliminary elucidate the role and function of autophagy in the regulatory network of skeletal muscle development. Different levels of autophagy were induced by controlling the serum concentration in the culture system for 24 h. Apoptosis, membrane potential, reactive oxygen species (ROS), ATP, and myogenic and lipogenic differentiation markers were monitored to determine if autophagy affected the metabolism and differentiation of SMSCs. Autophagy was induced in SMSCs via serum starvation (5%, 15%), as evidenced by decreased p62 and mTOR phosphorylation levels and increased LC3B lipidation and AMPK phosphorylation levels. Transmission electron microscopy revealed the presence of autophagosomes, and the rates of morphologically abnormal nuclei and mitochondria gradually increased with the decrease in serum concentration, the number of autophagic lysosomes also increased, indicating that 5% serum starvation induced severe autophagy, while 15% serum starvation induced mild autophagy. Compared with the control group and 15% serum-starved SMSCs, SMSCs undergoing 5% serum starvation had the highest intracellular ATP and ROS levels, the highest percentage of apoptotic cells, and the lowest membrane potential. The 15% serum-starved SMSCs had the highest membrane potential, but the percentage of apoptotic cells did not change significantly compared with the control group. The levels of the myogenic markers MyoD1 and MHC were significantly higher in 15% serum-starved SMSCs than in serum-sufficient SMSCs and the lowest in the 5% serum-starved SMSCs. The lipid contents (measured by Oil Red O staining and quantification of triglycerides) and lipogenic markers Peroxisome Proliferators-activated Receptors γ and Lipoprotein Lipase were also significantly higher in SMSCs undergoing 15% serum starvation than in the control group, and the lowest in the 5% serum-starved SMSCs. Different levels of starvation stress induce different levels of autophagy. Mild autophagy induced by moderate serum starvation promotes the metabolism and differentiation of SMSCs, while severe autophagy renders SMSCs more apoptotic, abnormal metabolism and suppresses SMSC differentiation into adipocytes or myocytes, and reduces lipid metabolisms. Our study suggests that autophagy plays a role in skeletal muscle development and may help design strategies for improving meat production traits in domestic pigs.

XRCC1 is a molecular scaffold protein that plays a central role in the base excision repair pathway. Here， we examined the subcellular localization of XRCC1 and investigated its effects on meiotic maturation of oocytes， subsequent embryonic development， and global DNA methylation in mice. XRCC1 was found to be localized mainly in the nuclei during oocyte maturation and subsequent embryonic development. Blocking XRCC1 by microinjection with antibodies partly reduced oocyte germinal vesicle breakdown （GVBD） and embryo cleavage， and also disrupts DNA demethylation in the 2-cell embryos. The results indicates that XRCC1 is mainly located in the nuclei at the interphase of cell cycle， and XRCC1 with normal function contributes to the maintenance of the quality of mouse oocytes during meiosis and the normal development of early embryos.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Stanniocalcin-1 (STC-1) is a glycoprotein hormone involved in calcium/phosphorus metabolism and direct inhibition of bone and muscle growth. The aim of this study was to investigate the STC-1 gene with respect to the regulatory mechanisms of porcine growth metabolic pathways involving autophagy. Western blotting was used to detect the expression of autophagy and mitochondrial function-related proteins, and flow cytometry was used to detect mitochondrial function-related. Changes in the autophagosome and mitochondrial were observed by electron microscopy. The expression of the autophagy-related proteins was detected by confocal microscopy. The results showed that Pink1, Parkin and LC3B expression was increased; SQSTM1/P62 expression was reduced. Electron microscopy revealed that the cells in the serum starvation group all produced autophagosomes. The fluorescence intensity of GFP-LC3B and GFP-Parkin increased. The Bax/Bcl-2 ratio, Pink1 and Parkin protein levels were profoundly reduced in the STC-KO. In addition, the increase in Mfn2, OPA1, DRP1 and LC3B proteins was attenuated; the increase in the apoptosis rate and amount of active oxygen was attenuated; the decrease in membrane potential; the decrease in ATP was reversed; the fluorescence intensity of GFP-LC3B and GFP-Parkin was increased. These results indicate that autophagy can be caused by serum starvation. Knocking out the porcine STC-1 gene had an obvious antiapoptotic effect on cells, the inhibition of serum starvation-induced autophagy. This is the first study to show that the porcine STC-1 gene confers self-protection in the absence of nutrients. To provide a theoretical basis for studying the effect of STC-1 on pig growth and development.

CRISPR/Cas9系统是近年来快速发展的一种简单高效的基因编辑系统.在哺乳动物中,同时靶向敲除多基因的研究仍然较为匮乏.为优化哺乳动物中靶向多基因的敲除系统的构建,本研究在已有的CRISPR/Cas9载体的基础上进行升级改造,将4个U6启动子介导的小向导RNA(small guide RNA,sgRNA)表达框串联至一个载体中的形式制备了多基因位点编辑载体,分别选取家猪(Sus scrofa)的去乙酰化酶3基因(sirtuin 3,Sirt3)和围脂滴蛋白1基因(perilipin 1,Plin1)的2个sgRNA,构建了2个猪Sirt3 sgRNA的基因编辑载体(S2)、2个猪Plin1 sgRNA基因编辑载体(P2)和同时包括上述4个sgRNA的基因编辑载体(S2P2),上述载体分别转染猪肾细胞PK15,以转染未装载靶点质粒的细胞为对照组(CON组),在各实验组细胞目的基因的DNA水平、RNA水平和蛋白水平检测目的基因敲除效率.结果表明,各实验组细胞目的基因DNA中均检测出突变,S2P2组和S2组中Sirt3基因突变率分别为33％和26％,S2P2组和P2组中Plin1基因突变率分别为33％和24％;对目的基因RNA水平与蛋白水平的检测结果表明,与CON组相比,所有实验组的目标基因mRNA及蛋白表达量均有所下降,差异极显著(P<0.01),其中S2组与S2P2组间Sirt3基因表达量差异不显著;P2组与S2P2组间Plin1基因表达量差异不显著.本研究通过改良的CRISPR/Cas9载体系统构建了可以同时对猪Sirt3基因和Plin1基因高效编辑的载体,有助于后续开展多基因功能的研究.

为揭示17℃液态保存过程中猪精子质量和功能持续下降的机制,本试验采集9头在役的不同品种公猪(长白、大白和杜洛克)的精液48份,使用BTS溶液等体积稀释后分为3份,其中的两份分别使用反复冻融(方法A)和低渗处理(方法B)方法杀死精子并离心获得稀释液A和B,将另一份精液样品离心分离精浆(Seminal Plasma,SP)和精子.分别使用不同体积比例稀释液A:精子(1:1、4:1和19:1),稀释液B:精子(1:1、4:1和19:1),SP:精子(1:1和4:1)和BTS:精子(4:1)重悬离心后的猪精子,以SP和BTS重悬的精液及原精液样品作为对照,所有精液样品于17℃下保存3 d.结果表明:随着保存时间的延长,所有稀释液和稀释倍数处理均损伤了精子活率和活力(P<0.01)及质膜完整性(P<0.05).稀释液A比B引起的精子脂质过氧化水平更低(P<0.05).5倍稀释精子,稀释液B的精子活力较BTS组降低(P<0.05),稀释液B引起的精子细胞内活性氧族(ROS)水平比稀释液A更高(P<0.05),而精液总抗氧化物(TAC)水平更低(P<0.05).与其他稀释倍数处理比,5倍稀释引起的精子脂质过氧化水平更低(P<0.05).稀释液A组,20倍稀释比其他稀释倍数引起的精子细胞内ROS水平更高(P<0.05),精液TAC水平更低(P<0.05).稀释液B组,5倍稀释比其他稀释倍数降低精子活力(P<0.05),5倍和2倍稀释精液TAC水平均低于20倍稀释组(P<0.05).相关性分析表明,精液TAC水平与精子质膜完整性正相关(R=0.147,P<0.05),与精子凋亡水平负相关(R=-0.183,P<0.05).综上,在17℃液态保存过程中,死精子可能通过引起活精子细胞内ROS水平升高或细胞膜脂质过氧化造成活精子质量和功能的损伤,而精液中的TAC可能在对抗以上损伤中发挥积极作用.