Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.     

Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Semen collected from Nili‐Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 μg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 μg/ml, tylosin 100 μg/ml, lincomycin 300 μg/ml and spectinomycin 600 μg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5°C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco‐spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5°C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5°C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5°C.     

牛、梅花鹿和山羊X、Y精子头部面积差异分析

用流式细胞仪分选有正常繁殖力的公牛(n=3)、公鹿(n=3)、公山羊(n=1)的精液,获得高纯度(≥91%)X和Y细管冷冻精液,对解冻后的X和Y精子进行常规染色制片,采用Motic Images Advanced 3.2软件自动测量X精子和Y精子头部面积。结果显示,公牛X精子的头部面积35.84μm2±4.12μm2,极显著高于Y精子(34.81μm2±3.72μm2)(P<0.01);梅花鹿X精子的头部面积33.29μm2±2.93μm2,极显著高于Y精子(32.90μm2±3.25μm2)(P<0.01);山羊X精子的头部面积28.53μm2±3.16μm2,也极显著高于Y精子(28.07μm2±3.19μm2)(P<0.01);不同分选纯度的公牛精液X精子之间、Y精子之间头部面积差异均不显著(P>0.05)。结论:牛、梅花鹿、山羊X精子头部面积均极显著高于Y精子。     

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

Evaluation of Breeding Value and Selection of Bull Dams in the Danish Dairy Breeds: I. Studies on Realized Efficiency of Bull Dam and Bull Sire Selection

Abstract

The relationship between twice the adjusted daughter yield deviation (DYD) of the son and the average of the estimated breeding values (EBV) of his parents was investigated for milk and fat yield. Deviations from expectations of DYD minus the pedigree index and the regression of DYD on the EBV for each of the parents were estimated. The EBV of each bull dam was calculated in several ways using different combinations of pedigree information and their own records. In all, 1693 bulls (880 Black and White (BW), 418 Red Danish (RD) and 395 Danish Jersey (DJ)) with Direct Updating indices for milk and fat yield were included in the investigation. Mean discrepancies between estimated and observed genetic merit for kg of milk yield were ?348 for BW, ?213 for RD and ?228 for DJ, when all of the bull dams' individual records were utilized in breeding value estimation. The corresponding mean discrepancies for kg of fat yield were ?14.7, ?11.5 and ?11.9, respectively. The discrepancies were positively correlated with the number of lactation records included in the dams' EBVs. Regression coefficients of bull DYDs on bull dam breeding values were significantly lower than expected for RD and BW regardless of the number of individual lactation records included in the dams' indexes for milk and fat yield. For all three breeds, the regression coefficients were lowest for fat yield. The most probable cause of overestimation in bull dam EBVs is preferential treatment, particularly in second and later lactations. To avoid this bias, estimation of a bull dam's breeding value should be based on pedigree information and on records from only her first lactation.     

高寒草甸区土壤水分动态的模拟研究

土壤水分在植物生长期间的动态变化,可分解为周期项和随机波动项两个部分。利用1992年土壤水分观测资料,以谐波分析法对周期项进行随时间序列的模拟处理,以剩余土壤水分(随机波动项)建立与自然降水量的线性回归关系后,依加法模式确定高寒草甸地区土壤水分动态变化的预报模型方程为: 对1992年土壤水分资料进行回代模拟,拟合率很高,平均相对误差为7.2％。对1993年土壤水分动态变化进行预测预报,准确率较高,其相对误差平均为8.9％。结果表明,该模拟方程,能准确预报土壤水分动态变化的过程,进而对研究草地物质能量循环等生态过程均具有一定的实用价值。     

桑叶各部位的水分和干物含量及主要元素的分布

为了更加准确地测算家蚕对桑叶的食下消化率和饲料转化效率以及桑叶作为食品与畜禽饲料其重金属含量的安全阈值,采用现代检测技术与相应的检测标准,对桑品种“桂桑优62”相同叶位成熟叶片的带皮叶肉、叶脉、叶柄等部位中水分和干物含量以及大量营养元素氮、磷、钾、钙、镁与常见重金属元素铬、铅、镉、汞、砷的分布规律进行了检测分析。桑叶各部位的含水率从高到低依次为叶柄、叶脉、带皮叶肉,而干物含量从高到低依次为带皮叶肉、叶脉、叶柄。桑叶的带皮叶肉部位是家蚕可食用部位,也是在叶片总质量中占比最高和干物质含量最高的部位,全叶干物质的70%～76%、大量营养元素的47%～87%和常见重金属元素的60%～91%均分布在这个部位。总体而言,桑叶中的氮、磷营养元素含量较为丰富,钙、镁、钾营养元素的含量较少;桑叶中的几种常见重金属元素含量由高到低依次是铬、铅、砷、镉和汞,但这些重金属元素的含量均在药品、食品和畜禽饲料的国家标准限量范围之内。     

Quantitative Histology Studies on the Test in Hybrid Bull of Wild and Domestic Yak

The testicular tissue of three types of yak bull (1/2 wild yak,cross 1/2 wild yak and domestic yak) were studied quantitatively at 6,12,18 and 24 months old. The results showed that the average values changed from breed to breed at the same age. But there were no significant difference. The volume density and the height of seminiferous tubule and epithelium increased with the age and testicular weight. The capacity rate of the testicular seminiferous tubule in three types were 78.71% ,75.78% and 78.58% respectively, which nearly reached the level of mature bull.     

The Effect of Parity of the Dam on Sexual Maturation,Serum Concentrations of Metabolic Hormones and the Response to Luteinizing Hormone Releasing Hormone in Bull Calves

The objectives of this study were to determine the effect of parity of the dam on age at which a scrotal circumference (SC) ≥ 28 cm was attained and the LH response to Luteinizing Hormone Releasing Hormone (LHRH) in bull calves. We also wanted to confirm, in a large group of bull calves, that the LH response to LHRH could be used to select early maturing bulls. Body weight and SC of the bull calves were measured every other week. At 15, 20 and 25 weeks of age, calves received 4.12 nm /kg body weight of LHRH ischio‐rectally and blood samples were taken every 15 min for 4 h. Calves from primiparous and multiparous dams were separated into two sub‐groups based on age at which an SC ≥ 28 cm was attained (early or late). An SC ≥ 28 cm was attained earlier in calves born to multiparous as compared with primiparous dams (p < 0.05). At 20 and 25 weeks of age, peak serum LH concentrations (LH‐peak) and area under the LH response curve (LH‐AUC) in response to LHRH were higher (p < 0.01) in calves born to multiparous as compared with primiparous dams. In calves born to multiparous dams the LH‐peak at 15 and 25 weeks of age and the LH‐AUC at 15 weeks of age were lower (p < 0.05) in calves that attained an SC ≥ 28 cm early as compared with late. The LHRH‐challenge test sensitivity and specificity ranged from 46% to 86%. We concluded that parity of the dam affected age at which SC ≥ 28 cm was attained and the LH response to LHRH in bull calves. Serum LH responses to LHRH at 15 and 25 weeks of age, in calves born to multiparous dams, show some promise for development into a test to select early maturing bull calves.     

新麦草种子成熟过程中活力变化的研究

１９９７年对新麦草种子成熟过程中活力变化进行了研究。研究结果表明：新麦草种子活力随着种子的成熟而逐渐增加并于种子获得最大干重时，活力达到最高。试验所选的指标均反映了新麦草种子活力变化状况，种子发芽率不能直接反映种子活力的变化，但它仍是检验种子活力不可缺少的指标。单项活力指标不能全面反映活力的变化，需采用多种指标，进行综合分析。     

Influence of Sample Preparation, Staining Procedure and Analysis Conditions on Bull Sperm Head Morphometry using the Morphology Analyser Integrated Visual Optical System

The importance of standardizing the procedures of sample and slide preparation for computer‐assisted morphologic analysis has been emphasized in human and veterinary andrology. The purpose of this study was to optimize slide preparation (dilution grade and sperm washing), staining procedures and analysis conditions (colour of light source and objective magnification) for the morphometric analysis of bull spermatozoa using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS). For experiment 1, one ejaculate was collected from one bull and diluted to 200 000–300 000 spermatozoa/μl. Slides were prepared and stained using seven different procedures: rapid Papanicolaou (PAP), rapid Papanicolaou with prolonged staining times (PAP+), Diff‐Quik (DIF), haematoxylin (HEM), Farelly (FAR), Spermac (SPER) and the modified GZIN (MGZIN) staining. All slides were analysed using a Hamilton Thorne Morphology Analyser IVOS equipped alternatively with a red, green or blue light source, and a 40× or 100× oil immersion objective. Recognition and digitization errors as well as morphometric parameters were determined. The IVOS was unable to detect DIF‐stained spermatozoa. The GZIN and the SPER staining as well as the blue light source led to unsatisfactory results. Among the staining methods examined, the FAR, HEM, PAP+, and PAP staining, preferably in combination with the green light source, and the 40× objective yielded optimal results concerning sperm recognition and digitization. The 100× objective did not allow reliable analysis of the sperm heads because of a frequently appearing digitization error. For experiment 2, three ejaculates were collected from each of three bulls and diluted to five dilution grades (100 000–500 000 spermatozoa/μl). An aliquot of each dilution grade was washed additionally. The percentage of correctly digitized sperm heads decreased with increasing spermatozoal concentration. However, the evaluation speed increased. The range of 200 000–300 000 spermatozoa/μl appeared to be a reasonable compromise for both criteria. Sperm washing failed to further improve the analysis results. Sperm head dimensions were influenced significantly by all variations of the methods in both experiments. In conclusion, using the proposed methods, the IVOS allows precise and reliable morphometric analyses of bull spermatozoa. The consistent application of these procedures may lead to an inter‐laboratory standardization and to further establishment of generally accepted morphometric criteria used in human andrology (e.g. World Health Organisation or strict criteria).     

粗毛褐孔菌化学成分研究

从粗毛褐孔菌乙醇提取物中分离鉴定了5个单体化合物,经过分析鉴定,分别为羊毛甾二烯二醇（I）、麦角甾醇（Ⅱ）、齿孔酸（Ⅲ）、D－阿拉伯糖醇（Ⅳ）、十八烷酸（V）。首次从该药材中获得各化合物,为药材的综合开发应用提供了科学依据。     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

广西右江河谷山羊圈养技术的研究

为充分利用广西农区丰富的秸秆、牧草、土地等资源,加快推进山羊业规模化、规范化和标准化生产,应用"一规范五推广"山羊圈养集成创新技术,引进隆林黑山羊和波尔山羊在右江河谷高热高湿地区圈养.建立了山羊圈养示范点43个,示范户203户,建设标准单舍10350m2,种植养羊牧草24.83 hm2.饲养基础母羊6012只,圈养经产母羊产羔率为199.05%.山羊人工授精冻精、鲜精配种受胎率为76.56和81.43%.波尔山羊与隆林山羊杂交一代羔羊初生体重2.56～2.98kg,比隆林山羊提高14.29～24.67%.制订了DB45/T244-2005山羊圈养技术规范.项目技术成果为实现广西山羊生产由传统山区向平原农区发展重心的战略性转移和山羊产业持续发展与资源永续利用提供了科学新方法和现实经验.     

广西右江河谷山羊圈养技术的研究

为充分利用广西农区丰富的秸秆、牧草、土地等资源,加快推进山羊业规模化、规范化和标准化生产,应用“一规范五推广”山羊圈养集成创新技术,引进隆林黑山羊和波尔山羊在右江河谷高热高湿地区圈养。建立了山羊圈养示范点43个,示范户203户,建设标准羊舍10350m^2,种植养羊牧草24.83 hm^2。饲养基础母羊6012只,圈养经产母羊产羔率为199.05%。山羊人工授精冻精、鲜精配种受胎率为76.56和81.43%。波尔山羊与隆林山羊杂交一代羔羊初生体重2.56-2.98kg,比隆林山羊提高14.29-24.67%。制订了DB45/T244-2005山羊圈养技术规范。项目技术成果为实现广西山羊生产由传统山区向平原农区发展重心的战略性转移和山羊产业持续发展与资源永续利用提供了科学新方法和现实经验。     

Consequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo Development

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N^w- l -nitro-arginine methyl-ester (10⁻⁷, 10⁻⁵ and 10⁻³  m l -NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of l -NAME (10⁻⁷  m ) during pre-maturation and/or maturation on embryo development and quality was also assessed. l -NAME decreased MII rates (78–82%, p < 0.05) when compared with controls without l -NAME (96%). Cleavage (77–88%, p > 0.05), Day 7 blastocyst rates (34–42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146–171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3–4 cells) increased with l -NAME treatment (p < 0.05). For oocytes cultured with l -NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26–34%) and Day 9 hatching rates (15–22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264–324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3–4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro . Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.     

Structural and Functional Events on the Porcine Zona Pellucida During Maturation, Fertilization and Embryonic Development: a Scanning Electron Microscopy Analysis

Porcine oocytes and pre-implantation embryos from the same, as well as from different animals, have an extremely heterogeneous morphology of the zona pellucida (ZP) surface, as shown by scanning electron microscopy. For years, it has been believed that this heterogeneous morphology plays an important role in the sperm-oocyte interaction. The aim of this study was to analyse the zona morphology and sperm-binding patterns on the porcine ZP. Oocytes were divided into four categories: immature, matured in vivo, or matured in vitro over a time period of 24 or 48 h. The zona morphology of early embryos grown in vivo or in vitro was also investigated. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between the different kinds of maturation in terms of these zona types. All oocytes exhibited extremely heterogeneous zona morphologies, with no clear trend. During subsequent in vivo embryo development, the zona surface changes from a porous structure to one with a compact surface, while the morphology of in vitro embryos remained compact at all stages of development. The analysis of the number and distribution patterns of spermatozoa trapped in the ZP revealed extremely variable patterns, regardless of the zona morphology. Differences were only present if sorted or unsorted spermatozoa were used for insemination. Regardless of the number of inseminated spermatozoa after sorting, only a few (1-2) could be detected on the ZP. Whether oocytes were matured in vivo or in vitro was not a relevant factor. Unsorted spermatozoa bound in higher numbers than sorted ones. The number was directly dependent on the number of spermatozoa used for insemination.     

牛体外成熟卵母细胞染色体形态学研究

[目的]为了给牛体细胞克隆和转基因克隆工作提供基础性材料.[方法]在显微镜下,从卵液中捡出卵丘-卵母细胞复合体,置入成熟培养液中进行成熟培养,处理培养不同时期的卵母细胞,所得的去掉卵丘的卵母细胞固定在载玻片上,染色、冲洗、晾干,在显微镜下观察.[结果]表明:成熟培养0 h到4 h大多数卵母细胞处于GV期(97.5%～87.8%),培养6 h以后GVBD发生了卵母细胞数量明显增多(51.6%),成熟培养8 h～12 h处于Pre-MI的卵母细胞逐渐减少(76.7%～43.2%),MI期卵母细胞逐渐增多(13.3%～50.0%).成熟培养16 h有17.8%的卵母细胞处于MII期,大部分细胞仍处于MI期(40.0%)和AI/TI期(42.2%).从16 h～24 h,MII期卵逐渐增多,培养24 h后大多数卵母细胞排出第一极体到达MII期(86.5%).[结论]在减数分裂过程中,染色体也发生了显著的形态变化.第一次减数分裂中期时的染色体清晰可数,进入后期时,分开的两团染色体各自凝集成染色质团,并且一直持续到末期,到达第二次减数分裂中期时又成为清晰可数的染色体状态.     

青贮,微贮,氨化与干玉米秸对育肥牛增重效果研究

为检验青贮、微贮、氨化与干玉米秸对育肥牛增重效果的差异,本试验选择改良去势公牛４８头,随机分为青贮组,微贮组、氨化组与对照组,经７０ｄ试验,结果表明,青贮组,微贮组与氨化组增重效果的均高于对照组,差异显著,但青贮组、微贮组与氨化组三者之间差异不显著,而以青贮组效果最好,微贮组次之,青贮组比对照组头日净增重多０．２４ｋｇ,增重效果提高２４．７８％,头日净增益多１．９８元,经济效益显著。