Dietary profile of n‐3 series LC‐PUFAs in rainbow trout under regular stripping condition: Semen production and quality,hepato‐somatic index,haemato‐immunologic values,oxidative stress and fatty acid composition of liver,muscle and semen

In this study, the semen production and quality, hepato‐somatic index, haemato‐immunologic values, oxidative stress and the fatty acid contents in liver, muscle and semen of rainbow trout fed diets supplemented n‐3 series long‐chain polyunsaturated fatty acids (LC‐PUFAs) under regular stripping condition were investigated. For this aim, three diets (Control, D1 and D2) were prepared. These diets were contained n‐3 LC‐PUFAs (as a percentage of dietary total fatty acid) at 3.14%, 7.84% and 13.63% respectively. Experimental fish were fed with the control and test diets. The highest hepato‐somatic index, spermatologic (semen pH and volume, sperm motility and density), haematologic (haematocrit value, haemoglobin, erythrocyte count, corpuscular volume, haemoglobin and its concentration in corpuscular), immunologic (nitroblue tetrazolium activity, leucocyte count, phagocytic index, protein and immunoglobulin [IgM] in total plasma) and antioxidants (reduced glutathione, catalase, glutathione peroxidase and superoxide dismutase) values were found in fish fed the D2, D1 and control diets respectively (p < 0.01). Increase in the dietary n‐3 LC‐PUFAs was not significantly (p > 0.01) increased the oxidative stress (malondialdehyde) in fish. The results indicated that the n‐3 LC‐PUFAs at 13.63% level of total fatty acid in the diet could increase the semen production and quality, hepato‐somatic index, haematologic and immunologic values, and the n‐3 LC‐PUFA contents in liver, muscle and semen of rainbow trout broodstock under regular stripping condition.     

AMPK激活剂在绵羊精液冷冻保存中的作用研究

旨在研究AMPK激活剂二甲双胍（metformin，Met）和阿卡地新（acadesine，AICAR）对绵羊精液冷冻保存效果的影响。本研究首先在冷冻基础稀释液中分别添加不同浓度（0、100、200、300、400、500 μmol·L^-1）的Met和AICAR，冷冻解冻后根据精子活力、运动性能和结构完整性指标筛选出最佳的添加浓度（400 μmol·L^-1 Met、200 μmol·L^-1 AICAR）；然后分别使用不同的冷冻稀释液（对照组：稀释液；Met组：含400 μmol·L^-1 Met的稀释液；AICAR组：含200 μmol·L^-1 AICAR的稀释液）冷冻精液，解冻后检测精子中AMPK蛋白表达、顶体酶活性、代谢指标、线粒体功能以及抗氧化酶活性。结果表明，稀释液中添加400 μmol·L^-1 Met和200 μmol·L^-1AICAR均可显著提高解冻后精子活力、运动性能及精子结构完整性（P<0.05），其中400 μmol·L^-1 Met组精子总活力达43.20%，顶体完整率为91%，质膜完整率为46%。与对照组相比，Met组和AICAR组解冻后精子中AMPK磷酸化水平显著升高（P<0.05）；顶体酶活性显著提高（P<0.05）；丙酮酸水平显著下降（P<0.05），乳酸脱氢酶活性、乳酸以及ATP含量均显著升高（P<0.05）；与对照组相比，Met和AICAR组稀释液更有利于维持线粒体膜电位（P<0.05），提高ATP酶（P<0.05）以及抗氧化酶的活性（P<0.05）。添加适当浓度的AMPK激活剂可以提高绵羊精液冷冻保存的效果。     

Effect of ascorbic acid supplementation on zootechnical performance,haematological parameters and sperm quality of lebranche mullet Mugil liza

The feed provided to breeding fish is one of the main factors influencing the quality of fish gametes. This study was carried out to evaluate the influence of ascorbic acid on growth, haematological parameters and sperm quality of Lebranche mullet males (Mugil liza). Six diets with different levels of ascorbic acid (0; 53; 107; 216; 482 and 708 mg/kg) were tested in triplicate for 75 days. During spermiation (first gonadal maturation), 144 individuals (205.7 ± 11.5 g and 25.7 ± 0.4 cm) were randomly distributed in 18 experimental tanks. Growth parameters were evaluated at the beginning and end of the experiment. Fish blood was collected to analyse glucose, total protein and erythrocyte count (EC) (n = 9). Fish (n = 12) from each treatment were euthanized to determine hepatosomatic index (HSI) and gonadosomatic index (GSI). Semen was collected to evaluate spermatic density, cell membrane integrity and sperm motility. No difference (p > .05) was found on growth parameters, GSI, HSI and total protein. However, EC was lower in fish fed without ascorbic acid (the control group). Ascorbic acid supplementation provided positive effects on sperm characteristics. Fish from treatments with 53 and 107 mg/kg presented the best results for motility time (133.30 ± 4.25 and 135.00 ± 2.77 respectively). Treatments with 107, 216 and 708 mg/kg provided the best results for motility rate (92.0 ± 2.9%, 93.0 ± 5.8% and 93.0 ± 5.8% respectively). Supplementation with 107 and 216 mg/kg provided the best results for plasma membrane integrity (70.12 ± 9.10% and 76.3 ± 3.1% respectively). Lower spermatic density was observed in fish without ascorbic acid supplementation, although no difference was found in sperm density among the treatments with ascorbic acid (p < .05). Considering these results, supplementation of dietary ascorbic acid between 107 and 216 mg/kg optimizes the spermatic quality in males of lebranche mullet.     

Sperm cryopreservation of the noble Scallop Chlamys nobilis by a programmable freezing method: Effect of cryoprotectant

A study on Chlamys nobilis sperm cryopreservation by a programmable freezing method was conducted under laboratory condition. Four cryoprotectant agents (dimethyl sulfoxide [DMSO], methanol [MET], propanediol[PG] and ethylene glycol [EG]) and four concentrations (5%, 10%, 20% and 30%) were evaluated for their ability to retain sperm motility, movement characteristics and fertility. Results showed that cryopreserved sperm total motility produced by DMSO and MET at 5%, 10% and 20% were higher than other cryoprotectant treatment groups (CPA groups), as well as rapid sperm percentage. The curvilinear (VCL) and straight line (VSL) velocity produced by DMSO at 5% significantly higher than other CPA groups (p < 0.05), while no significant differences were found for average path (VAP) velocity. The lateral head displacement (ALH) in all CPA groups was similar and without significant difference (p > 0.05), as well as the beat‐cross frequency (BCF). A significant higher fertilization rate was produced in DMSO than that in MET at same concentration (p < 0.05), and no significant differences were found for differing concentrations of the same cryoprotectant (p > 0.05). Overall, 5%‐20% DMSO was more suitable for Chlamys nobilis sperm programmable cryopreservation when the calcium‐free Hanks’ balanced salt solution was used as the extender, and 10°C/min from 0°C to ?80°C was used as freezing rate. The findings presented in this study will benefit conservation programs for Chlamys nobilis.     

不同品种公猪睾丸、精液品质发育规律及相关性研究

为明确公猪睾丸在选育中的意义，研究公猪睾丸参数、精液质量参数的发育性规律以及两者间的关联性。选择936头分布在280～1 409日龄的生产公猪，其中589头杜洛克、233头长白、114头大白。对睾丸和精液质量参数采用一般线性模型，以品种和日龄分组为固定因素进行多变量分析；使用二次项模型分品种分别对睾丸总体积和有效精子数进行发育曲线拟合；控制日龄变量，对睾丸与精液质量参数进行偏相关性分析。在睾丸大小方面：杜洛克>大白>长白，其中杜洛克与大白差异不显著（P>0.05），但两者均极显著（P<001）高于长白；在精液质量整体比较上：长白优于杜洛克优于大白，但差异不显著（P>0.05）；但在体积、密度、活力和畸形率上，三品种公猪均存在显著（P>0.05）或极显著(P<0.01)差异；睾丸随日龄增长呈先增大后萎缩趋势，精液整体质量随日龄增长呈下滑趋势；三品种公猪睾丸的大小均与精液量和有效精子数呈正相关，且大部分具有显著（P<0.05）或极显著性(P<0.01)。同日龄时期，三品种公猪睾丸的大小顺序为杜洛克>长白>大白，其中长白大于大白具有普遍性； 综合来看，三品种的精液质量比较结果为长白优于杜洛克优于大白，其中长白优于大白具有普遍性；公猪睾丸萎缩临界日龄可用于公猪淘汰依据，选育公猪时可重点选留睾丸较大者。     

虎雪兰玻璃化超低温法脱毒技术的研究

以兰属花卉虎雪兰组培原球茎为试材,采用玻璃化超低温法对兰花病毒脱除进行了研究,以期为虎雪兰玻璃化超低温法脱毒体系的建立提供参考依据。结果表明:在蔗糖浓度0.5 mol·L-1预培养4 d,然后在蔗糖0.6 mol·L-1加载液冰上处理50 min,之后转入PVS2溶液冰上玻璃化处理120 min,再液氮冷冻40 min,37℃水浴解冻3 min,最后卸载液(1/2MS+1.2 mol·L-1蔗糖)卸载20 min,待恢复培养后,原球茎成活率可达到65%以上,随机检测不同处理样品的脱毒率可达97%。     

野猪粪便中乳酸菌的分离鉴定及特性研究

【目的】筛选安全的、具有优良特性的乳酸菌菌株,进一步研发益生制剂,为饲料添加剂等动物相关产品提供资源。【方法】从我国黑龙江省大兴安岭地区采集野猪粪便样品13份,编号后置于4℃保温箱迅速运回实验室,利用MRS培养基分离纯化乳酸菌。使用细菌基因组DNA提取试剂盒提取分离菌株的基因组DNA,利用细菌16S rDNA通用引物进行PCR鉴定,将扩增得到的序列测序后在NCBI上使用BLAST与GenBank数据库中序列进行对比分析,确定各菌株的分类学地位。将鉴定后的乳酸菌菌株分别接种于酸性（pH 3.0）和含胆盐（0.3%）的MRS培养基,在不同条件下评价乳酸菌菌株的耐酸、耐胆盐特性。将过夜培养的乳酸菌于室温条件下静置,在不同时间测定其OD_600nm,进行自凝集能力评价;过夜培养的菌株分别与致病性埃希氏大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌3种致病菌混合后于室温静置,进行共凝集能力检测。在体外,分别进行乳酸菌菌株对Caco-2细胞和IPEC-J2细胞的黏附能力测定,评价不同菌株的黏附能力。通过测定乳酸菌菌株对致病性埃希氏大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌3种致病菌的抑菌环直径,评价分离菌株的抑菌活性。通过体内外试验评价乳酸菌菌株的安全性。在体外,分别以模式菌株嗜酸乳杆菌和金黄色葡萄球菌作为阴性对照和阳性对照,将3株乳酸菌菌株和对照菌株在血平板上划线,37℃厌氧孵育18—24 h,观察细菌菌落周围是否形成溶菌环,评价分离菌株的溶血特性。使用文献中已报道的毒力因子引物对分离的乳酸菌菌株进行PCR扩增,检测是否存在毒力因子的编码基因,评估分离菌株的安全性。在体内,将过夜培养的乳酸菌连续饲喂7周龄的BALB/c小鼠21 d,分别测定小鼠的初始体重和最终体重,观察计算体重变化情况;饲喂21 d后,解剖获取小鼠的脾脏、肝脏和肾脏计算器官指数,评价分离乳酸菌菌株的体内安全性。【结果】从野猪粪便中分离得到3株对酸和胆盐具备一定耐受力的乳酸菌,经鉴定分别为蒙氏肠球菌（Enterococcus mundtii）、耐久肠球菌（Enterococcus durans）和黏膜乳杆菌（Lactobacillus mucosae）。3株乳酸菌菌株均表现出较强的自凝集能力和对致病菌的共凝集能力,同时对Caco-2细胞和IPEC-J2细胞均表现出较强的黏附能力,抑菌试验结果显示黏膜乳杆菌对3种致病菌均具备较强的抑菌活性。经体内外安全性评价,3株乳酸菌菌株无溶血性,且均未检测到毒力基因,经其连续饲喂的小鼠行为表现正常、状态良好,其中,与对照组相比,黏膜乳杆菌饲喂后小鼠增重显著。【结论】从大兴安岭野猪粪便中分离的3株乳酸菌（特别是黏膜乳杆菌）具有良好的特性和安全性,具备进一步开发益生菌制剂的潜力。     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.