Early postnatal nutrition is crucial for the growth and development of lambs, and artificial milk formulas are widely used as alternatives to breast milk in intensive sheep production. However, the molecular and microbial mechanisms underlying the differences between breast milk and formula feeding remain unclear. This study aimed to compare the fecal metagenomic and whole blood transcriptomic profiles of lambs fed breast milk (BF group) and commercial formula (FF group) from 4 to 45 days of age, to provide a theoretical basis for optimizing formula compositions. A total of 6 lambs were randomly divided into two groups (n = 3 per group), with body weight and body dimensions measured at 45 days of age, followed by fecal metagenomic sequencing and whole blood transcriptomic sequencing. The results showed that BF lambs had significantly higher body weight, body length, heart girth, and chest width than FF lambs. Metagenomic analysis revealed that at the phylum level, Bacteroidetes was enriched in FF lambs, whereas Firmicutes predominated in BF lambs. Differential abundance was also observed at the genus level (higher Desulfovibrio in FF lambs) and the pathway level, with BF lambs enriched in quorum sensing and FF lambs showing higher abundances of pathways related to ubiquinone and other terpenoid-quinone biosynthesis. Moreover, transcriptomic analysis identified 3290 differentially expressed genes (DEGs) between the two groups, with DEGs mainly enriched in metabolic pathways, mTOR signaling pathway, osteoclast differentiation, B cell receptor signaling pathway and MAPK signaling pathway. Collectively, compared with FF, BF enhanced lamb growth, optimized gut microbiome structure and modulated blood transcriptomic profiles related to metabolism, signaling and immunity. These findings highlight the key microbial taxa and functional pathways modulated by breastfeeding, providing valuable insights for the development of more effective milk formula alternatives.

This study investigated Proanthocyanidin B2 (PCB2), a potent antioxidant flavonoid, using sheep oocytes as a model to assess its effects on oocyte developmental competence, oxidative stress levels, mitochondrial function, and early apoptosis. During in vitro maturation (IVM), supplementation with 5 μg/mL PCB2 significantly increased the first polar body extrusion rate compared to concentrations of 0, 2.5, and 7.5 μg/mL (P < 0.05). Further analysis revealed that 5 μg/mL PCB2 significantly reduced reactive oxygen species (ROS) levels while increasing glutathione (GSH) levels (P < 0.05). Additionally, this treatment enhanced mitochondrial activity, endoplasmic reticulum function, mitochondrial membrane potential, and cortical granule dynamics (P < 0.05). To elucidate the underlying mechanisms, Smart-seq RNA sequencing was employed to identify differentially expressed mRNAs (DEMs) in oocytes treated with 5 μg/mL PCB2. Compared to the control, 585 DEMs (293 upregulated and 292 downregulated) were detected. Transcriptomic analysis suggested that PCB2 promotes cytoplasmic maturation by enhancing EGFR-mediated activation of the PI3K/AKT pathway, thereby mitigating oxidative stress and preserving mitochondrial function.

为了丰富我国绵羊遗传资源,开展绵羊品种繁育和新品系培育工作,从澳大利亚引进纯种萨福克胚胎331枚.并对进口冷冻胚胎的质量、受体处理、技术员操作等因素对胚胎移植妊娠率的影响进行了应用研究.随机将受体羊分为4组,在相同的环境、饲养和管理条件下,采用舍饲加放牧的饲养模式开展相应的应用研究.结果表明,4组胚胎中A级胚胎占45%～47%,B级胚胎占35%～36%,C级胚胎占17%～19%;4组受体羊同期发情移植率分别为88.10%,82.83%,88.42%和83.49%;移植受胎率分别为58.10%,73.17%,48.81%和41.76%,平均受胎率为55.46%.从以上实验数据可以看出,在相同的饲养环境、胚胎质量、受体状态的条件下,技术人员的操作水平对受胎率产生极大的影响,差异极显著.本文对羊胚胎移植中的技术环节和水平进行试验和针对性的讨论,以期对该技术的生产应用提供参考.

B型超声诊断作为羊妊娠诊断技术之一,广泛应用在羊输精后的30～45d,妊娠诊断准确率能够达到生产应用的要求.本试验采用兽用直肠超声B超仪,对100只经子宫角内窥镜输精的肉羊进行妊娠检查,同时随机抽取40只经B超妊娠检查怀孕羊血液采用早孕诊断试剂进行妊娠检查,比较两种不同妊娠检查方法的效果.结果表明:子宫角内窥镜输精的肉羊30d兽用直肠超声B超仪的准确率为100%,早孕诊断试剂妊娠检查准确率为93.05%.由以上数据可知,配种后30～45d,使用B超进行早期妊娠诊断能够取得较高的准确率.早孕检测试剂盒作为新的妊娠诊断方法,虽然在准确性方面略低于B超妊娠诊断法,但该法以简便、节约劳力、速度快等优势也不失为一种羊的早期妊娠诊断的补充方法之一.

旨在异源表达羊妊娠相关糖蛋白5(ovPAG5),并对其蛋白结构及功能进行预测分析,为ovPAG5抗体制备和羊早孕诊断技术的研发提供技术支撑.利用生物信息学方法对ovPAG5基因的编码序列进行密码子偏好性分析,根据宿主细胞HEK293的密码子偏好性对ovPAG5基因进行优化,全基因合成后构建重组表达载体pcDNA3.1(-)-ovPAG5,转染至HEK293细胞中诱导表达重组蛋白并纯化,进一步利用生物信息学软件对ovPAG5蛋白的结构及功能进行预测.结果显示,ovPAG5基因有效密码子数(NEC)和密码子适应指数(CAI)分别为51.91和0.78,表明该基因密码子选择偏性较弱.优化后ovPAG5基因密码子适用指数(CAI)由0.78提高到0.96,GC总含量由48％提高到57％,并成功合成优化的ovPAG5基因,大小与预期结果相符.重组质粒测序结果显示,其核苷酸序列与优化后基因完全一致,氨基酸未发生突变.表达的重组蛋白ovPAG5经SDS-PAGE和Western Blot鉴定分析,其相对分子质量约52 kDa.生物信息学预测分析结果显示,ovPAG5蛋白具有2个糖基化修饰位点、12个B细胞抗原表位以及31个氨基酸残基组成的信号肽.其二级结构由无规则卷曲(40.81％)、延伸链(31.74％)、α-螺旋(22.17％)和β转角(5.29％)组成.本研究为今后.vPAG5抗体的制备和羊早孕检测技术的研发奠定了基础.

为建立一种简便、经济、无创的应用于绵羊早期妊娠诊断的快速检测技术,以绵羊妊娠相关糖蛋白7(O-vine pregnancy-associated glycoproteins-7,ovPAG7)核酸适配体为分子识别探针,建立一种间接竞争酶联适配体(Indi-rect competitive enzyme-linked aptamer assay,ic-ELAA)检测ovPAG7的新方法,并对ovPAG7包被质量浓度、包被缓冲液、封闭条件、适配体浓度、链霉亲和素-辣根过氧化物酶(SA-HRP)稀释比例等检测条件进行优化,将建立的方法应用于人工授精后28 d的中国美利奴羊血清检测,检测结果与妊娠相关糖蛋白-酶联免疫吸附测定(PAG-ELISA)商业化试剂盒和B超检测法进行比较.结果显示,在ovPAG7包被质量浓度为2.0μg/ml、包被缓冲液为50 mmol/L碳酸盐缓冲液(pH 9.6)、1.0％牛血清蛋白质封闭2 h、适配体浓度为40 nmol/L、SA-HRP稀释比例为1:40000(体积比)、室温下竞争反应时间为40 min等优化条件下,ovPAG7在缓冲液体系和血清体系中质量浓度的线性范围均为0.1～50.0 ng/ml(R2>0.994),半数抑制质量浓度(IC50)分别为1.40 ng/ml和2.11 ng/ml,检测限分别为0.11 ng/ml和0.19 ng/ml.ovPAG7在空白样品中加标回收率为96.4％～106.8％,相对标准偏差小于6.0％.妊娠诊断结果显示,ic-ELAA法的诊断敏感性、特异性和准确率分别为96.0％、83.3％和91.2％,与B超检测结果一致性较高(Kappa值=0.810).

本文概述了新疆生产建设兵团第六师(简称第六师)肉羊产业的基本情况,分析了制约肉羊产业发展的因素,针对存在的问题提出对策和建议,以期为促进第六师肉羊产业发展提供参考.

BACKGROUND: Developmental stage and cryopreservation method have significant impact on the pregnancy rate after transfer of embryos produced in vivo. OBJECTIVE: To determine the pregnancy outcomes from ovine embryos cryopreserved at different developmental stages. MATERIALS AND METHODS: Embryos at different developmental stages were obtained from donor ewes through simultaneous estrus treatment and laparoscopic artificial insemination. Embryos, either cryopreserved via vitrification or slow freezing method, were implanted into recipient ewes. The pregnancy rate was determined 35 days after transfer. RESULTS: The pregnancy rate of developing embryos increases after transfer from the morula stage, early blastocyst to expanded blastocyst stages (64.9%, 73.9% and 81.3%, respectively). However, cryopreservation significantly decreases the pregnancy rate of embryos at all three developmental stages, and there is no significant difference among developmental stages (43.9%, 43.7%, 52.9%, respectively). There is also no significant difference in the pregnancy rate between slowly-frozen embryos and vitrified embryos. CONCLUSION: The pregnancy outcomes of embryo transfer is better at the expanded blastocyst stage than at earlier stages. However, no difference is observed in the pregnancy rate of embryos at different developmental stage after cryopreservation, either by slow freezing and vitrification. Cryopreservation methods for ovine embryos, both slow freezing and vitrification, need further improvement.

Pregnancy-associated glycoproteins (PAGs) are widely used as powerful markers for early pregnancy diagnosis in livestock. To improve expression efficiency of recombinant ovine pregnancy-associated glycoprotein-7 (ovPAG-7) in HEK293 cells, the codon usage bias of the ovPAG-7 gene was analyzed using bioinformatic approaches, after which the DNA sequence encoding ovPAG-7 was designed, synthesized, and expressed in HEK293. The structure and function of ovPAG-7 were predicted using bioinformatics software and online databases. The results showed that the effective number of codons (NEC) of the ovPAG-7 gene was 56.82, indicating that the ovPAG-7 gene was weakly biased. ovPAG-7 gene had 26 biased codons (relative synonymous codon usage (RSCU) > 1), 15 of which were biased towards G/C at the third position. After codon optimization, the codon adaptation index of the ovPAG-7 gene increased from 0.74 to 0.96, and its GC content changed from 46.6 to 58.6%. The amino acid sequence encoded by the optimized gene was entirely consistent with those published in Gen Bank. Western blot analysis indicated that the recombinant ovPAG-7 protein with a relative molecular mass of 48 kDa was successfully expressed in HEK293 cells. The bioinformatics prediction results showed that ovPAG-7 protein contained 3 N-glycosylation sites, 13 B-cell epitopes, and a signal peptide consisting of 15 amino acids at the N terminus. The secondary structure of the ovPAG-7 protein was predicted to consist of random coils (46.85%), extended strands (32.05%), α-helices (16.16%), and β-turns (4.93%). This study provided a tool for the screening of monoclonal antibodies and functional research on ovPAG-7.

通过三个品种绵羊:11只萨福克、16只白杜泊和7只黑杜泊的超数排卵(简称超排)试验,探究同一超排方案对不同品种绵羊的影响.试验结果表明:萨福克、白杜泊和黑杜泊平均回收胚胎数分别为7.45、11.16和7.29枚;平均黄体数最多的是白杜泊为13.13个,黑杜泊和萨福克分别为11.14和10.27个;萨福克、白杜泊和黑杜泊的平均可用胚数和可用胚率分别为6.55、9.13和6.68枚,84.55％、83.88％和92.87％,且上述各类指标数据间差异均不显著(P>0.05).对萨福克羊再进行重复超排,4次超排平均回收胚胎数依次为7.45、9.75、5.12和1.10枚,第一、二次超排回收胚胎数显著高于第四次(P<0.05),第一、二、三次相互间差异不显著(P>0.05);第二次超排A级胚数显著高于第四次(P<0.05),其余各次间差异不显著(P>0.05);各次回收B级胚数间差异不显著(P>0.05).说明三个品种用同一超排方案,其效果有一定差别,但达不到显著水平;萨福克羊经过四次连续超排后,其回收胚胎数和胚胎质量均明显降低.

[目的]对bPAG16基因进行优化和合成,构建proEM-bPAG16重组表达载体,将重组质粒转染至HEK293细胞中,获得bPAG16重组蛋白,为家畜早期妊娠诊断技术的研发提供技术支撑.[方法]通过生物学技术对bPAG16基因密码子进行优化和人工合成,经T4?DNA连接酶将bPAG16基因插入到proEM载体中,构建重组载体proEM-bPAG16,将其转染至HEK293细胞中进行瞬时表达,重组蛋白经Ni-IDA亲和柱纯化后,采用SDS-PAGE、Western?Blot检测其表达效果.[结果]优化后的bPAG16基因密码子适用指数(CAI)由原来的0.77提高到0.96,GC含量由48％提高到58％.重组载体proEM-bPAG16双酶切后分别获得1179?bp和4369?bp的2条片段,与预期值一致;重组质粒测序结果显示,其核苷酸序列与优化后基因完全一致,氨基酸未发生突变;SDS-PAGE和Western?Blot鉴定结果显示,获得重组蛋白bPAG16(rbPAG16)相对分子质量约48?kDa,经Ni-IDA亲和层析纯化后,其纯度达到90％以上.[结论]通过密码子优化策略及重组蛋白技术高效表达了rbPAG16,为奶牛早孕快速检测技术的研发奠定了基础.

萨福克肉羊是世界上公认的、优良的杂交改良父本,国内各地陆续引进了种羊或胚胎.亟待MOET(Multiply Ovulation and Embryo Transfer,MOET)技术开展纯种扩繁或种羊培育,而影响MOET技术环节的因素较多,包括超排药物筛选、剂量、超排程序、超排次数、季节、供体输精方式、受体同期发情和受胎率等.本试验研究结果表明:FSH药物对获得胚胎数方面无明显差异(P<0.05),且国产FSH价格更便宜,每枚胚胎的成本较进口FSH有较大优势;在供体超排效率和获得可用胚胎数、受体移植成功率等方面差异不明显(P<0.05);在供体输精方式比较上,采用腹腔镜输精技术的供体获得可用胚胎数、卵子受精率和胚胎质量方面与传统方法比较差异极显著(P<0.01).说明FSH药物筛选、超排程序、输精方式和受体移植等几个方面影响MOET效率,为MOET技术在生产中的应用提供技术依据.

随着规模化养羊产业的不断推进,羊繁殖率低、群体遗传改良进程缓慢、发情不集中的问题已成为影响养羊业发展的重要制约因素,绵羊的同期发情及定时输精技术日益受到规模化养殖场的重视.自20世纪50年代中期绵羊同期发情技术建立以来,同期发情技术在提高绵羊产羔数、缩短繁殖间隔、最大幅度地提高羊只年产羔数等方面发挥了重要作用.文章主要对绵羊同期发情及定时输精技术研究进展进行了综述.

研究同期发情处理中国美利奴羊(新疆军垦型)的外周血生殖激素含量变化与发情时间的关系,探讨中国美利奴羊(新疆军垦型)最佳人工输精时间.分别采集经孕酮海绵栓+ PMSG肌注法处理母羊在埋栓前1d、埋栓后7d、埋栓后13 d(撤栓0h)和撤栓/PMSG肌注48 h的外周血,应用ELISA方法检测外周血中雌二醇(E2)、促卵泡素(FSH)、促黄体素(LH)、孕酮(P)的质量浓度,分析试验母羊在同期发情处理前后外周血中生殖激素含量变化,统计分析生殖激素质量浓度的相关性和4个时间点的差异显著性.研究结果,对孕酮海绵栓+PMSG肌注法处理前后试验母羊外周血生殖激素的ELISA检测,与埋栓前1d、埋栓后7d、埋栓后13 d(撤栓0h)相比,撤栓/PMSG肌注48 h试验羊外周血中E2 、FSH、LH含量显著升高(P＜0.05),P含量显著下降(P＜0.05),且不同个体间不同激素含量的波动范围明显减小,达到相对一致水平,与该时间点的中国美利奴羊(新疆军垦型)集中发情现象相吻合.结果 表明,孕酮阴道海绵栓+PMSG处理后的48 h是中国美利奴羊(新疆军垦型)集中发情的高峰时间,也是最佳输精时间.

[目的]解决天然牛妊娠相关糖蛋白(bPAG)不易分离纯化的问题,获得高效异源表达且具有良好免疫反应性的bPAG6,为国产bPAG检测产品的研发提供技术支持.[方法]根据宿主细胞对密码子的偏好性,利用MaxCodonTM在不改变氨基酸序列的前提下,对bPAG6基因密码子进行优化,采用全基因合成技术扩增优化后的bPAG6基因,构建proEM-bPAG6重组载体,并转染至HEK293细胞中进行高效表达;然后通过SDS-PAGE电泳、Western blotting和ELISA检测重组蛋白的表达效果及免疫反应性,并利用在线生物信息学分析软件对重组蛋白的结构及功能进行预测.[结果]优化后bPAG6基因的密码子适用指数(CAI)由0.80提高到0.96,G+C含量由49.4％提高到58.8％,其蛋白编码区(CDS)长度为1137 bp,共编码379个氨基酸残基.将全基因合成技术扩增获得的bPAG6基因插入proEM载体构建proEM-bPAG6重组载体,经EcoR I和Hind III双酶切鉴定可获得2条与预期结果相符的片段[proEM载体(4369 bp)和bPAG6基因(1176 bp)],测序结果也显示其碱基序列与优化后的bPAG6基因完全一致.proEM-bPAG6重组载体转染HEK293细胞表达获得的重组蛋白bPAG6分子量约46 kD.重组蛋白bPAG6可被抗bPAG6抗体识别,即具有良好的免疫反应性.重组蛋白bPAG6信号肽由N端的前15个氨基酸残基组成;存在5个N-糖基化位点,其N-糖基化修饰主要位于57Asn、73Asn、102Asn、124Asn和181Asn位点处;重组蛋白bPAG6为分泌性蛋白,无跨膜螺旋区,其二级结构由无规则卷曲(42.74％)、延伸链(31.93％)、α-螺旋(19.26％)和β-转角(6.07％)组成.[结论]通过基因优化及真核表达获得的重组蛋白bPAG6具有良好免疫反应性,可作为抗原应用于牛早期妊娠快速诊断产品的研发.

The aim of this research was to investigate the changes in reproductive hormone receptor expressions of the ovary and hormone concentrations between oestrous cycle pattern of two different sheep breeds in China. Ovarian tissues were collected from Chinese Merino (Junken type) and Hu sheep with different reproductive states in spring and autumn. Serum samples were assayed for oestrogen (E2), progesterone (P), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations by radioimmunoassay during spring. The ovarian expression of hormone receptors (ER alpha, ER beta, PR, LHR and FSHR) was analysed using real-time reverse transcription polymerase chain reaction (RT-PCR). In Chinese Merino, there was no corpora lutea and ovulation point on the surfaces of ovaries in spring and low basal levels of both LH and P in serum. ER alpha, ER beta and FSHR were expressed significantly higher in Merino ovaries during anoestrus compared with oestrous or luteal phases of Hu sheep (p p < 0.01). However, both varieties of sheep exhibited a similar tendency to secrete E2 and FSH. Compared with Hu sheep, FSH levels were slightly higher in Merino serum. In Hu sheep, ER alpha, ER beta, FSHR, LHR and PR expressed in luteal phase ovaries during spring were significantly lower (p < 0.05, p p < 0.001) than autumn. Interestingly, LHR and PR expressed in anoestrous ovaries were similar to that in oestrous phase of both sheep breeds. The above results suggest that seasonal reproductive sheep increased the expression of E2 and FSH receptors in ovary during spring may enhance the effects of E2 and FSH on follicular development. It is likely that this enhancement prevents the ovary from progressing to the luteal phase.

The aim of the study is to explore LEPR expression characteristics in seasonal estrus of sheep. Chinese merino(seasonal estrus sheep)and Duolang sheep(perennial estrus sheep)were selected to determine the expression changes of LEPRb mRNA in hypothalamus,ovaries and uterus in different stage of estrus cycle by quantitative real-time PCR. The results showed that during the whole estrus cycle,except the metaestrus,the LEPRb levels in the ovarian and uterus were found to be significantly lower than the levels in hypothalamus of Chinese Merino,but the results in metaestrus showed the reverse. Although the expression level of LEPRb varied in the same tissue of 2 cultivars during estrus stage,the trend was similar. Moreover,the LEPRb expression levels in different tissues of Chinese Merino were significantly higher than those in Duolang sheep. The results indicate that the expression variation of LEPRb is correlated with sheep estrus.

根据leptin基因在GenBank中的已知序列设计两对引物,采用PCR-SSCP技术在常年发情的湖羊和季节性发情的阿勒泰羊群体中进行单核苷酸多态性(SNPs)检测,对筛查到的SNP位点进行基因型与绵羊季节性发情的关联分析.结果表明,与湖羊相比,阿勒泰羊leptin基因第1内含子上有3个连续碱基TTG的插入和C/T碱基突变;第3外显子3上发生G/T碱基突变,编码氨基酸由缬氨酸变成亮氨酸.Leptin外显子2扩增片段上检测到AA、AB、BB三种基因型,BB基因型在阿勒泰羊群体中属于优势基因型;对两个群体进行基因型频率独立性x2检验,差异极显著(P＜0.001),说明BB基因型是影响季节性发情的有利基因型.研究结果提示,绵羊品种中Leptin基因序列的差异性可能是造成绵羊季节性发情的原因之一,可作为常年发情绵羊品种选育的辅助标记.

本试验旨在对绵羊多胎湖羊与单胎细毛羊孕角子叶面积、子叶内DNA含量及子宫、胎儿重量与早期妊娠之关系的分析,揭示绵羊多胎妊娠的生物学基础,以便在生产上采取相应措施,从而达到提高绵羊繁殖力的目的.

[Objective] The aim of this study was to analyze the different factors influencing the results of In Vitro Fertilization(IVF)in dairy cattle in order to establish an efficient IVF system.[Method] Influences of oocytes from different sources,different treatment ways of semen,different culture systems and different fertilization time and different fertilization density on IVF were summarized.[Result] Differences of cleavage rates and blastocyst rates of oocytes collected from slaughterhouse and live body were not significant.The sperms were treated by percoll and upriver.There were no significant differences at cleavage rates and blastocyst rates between the two groups.The cleavage rates and blastocyst rates in SOF and BO were not significantly different between the two groups.The blastocyst rate was not significantly different between 8 h and 10 h and between 10 h and 12 h.The former cleavage rates and blastocyst rates was higher than the latter.Putting 20 matured oocytes cells into 100 μl fertilization droppings lowered cleavage rates and blastocyst rates and the differences were significant.[Conclusion] Along with the prolonging of fertilization time,cleavage rates and blastocyst rates lowered gradually and differences between them were not significant.8 h fertilized blastocyst rate was higher than 12 h fertilization;putting 20 matured oocytes cells into 100 μl fertilization droppings could have better IVF effects.