The effect of zinc oxide on rooster semen cryopreservation

ABSTRACT

1. Deleterious effects from the freeze-thawing process on post-thawed sperm quality attributes are main limiting factors in cryopreservation. The current study was conducted to determine the effect of semen extender containing zinc oxide (ZnO) on post-thaw rooster sperm quality indices.

2. Semen samples from six, 60-week-old broiler breeder roosters were collected weekly during five successive weeks. The samples were mixed and divided into three equal parts and diluted with semen extender containing different levels of ZnO; 0 (ZnO-0), 1 (ZnO-1) or 2 (ZnO-2) µg/ml. After thawing, motility and velocity parameters, plasma membrane functionality, apoptotic like changes, mitochondrial membrane potential (MMP), and DNA fragmentation index (DFI) were evaluated.

3. Results showed that the addition of ZnO in the extender quadratically affected (P < 0.01) total motility (TM), progressive motility (PM), and average path velocity (VAP) with the highest values were noted in the ZnO-1 group. Levels of ZnO quadratically affected percentages of live (P < 0.01), apoptotic (P < 0.03) and dead (P < 0.10) spermatozoa, where the highest percentage of live, and the lowest percentage of apoptotic or dead spermatozoa was for the ZnO-1 group. Although adding ZnO quadratically affected plasma membrane functionality and MMP (P < 0.01), it did not affect (P > 0.05) DFI.

4. In conclusion, there were some beneficial effects of ZnO supplementation in semen extender on post-thawed rooster sperm quality which may result in a better freezability.     

The effect of sodium heparin on equine articular cartilage.

This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no significant increase was found among the treatment groups (P =0.782). For gentamicin, no significant difference in percentage GAG loss was found between the control and three of the five treatment groups (P =0.667). The percentage GAG loss in the sodium heparin treated AC explants was greater than for any of the gentamicin-treated AC explants. It can be concluded that sodium heparin sulphate stimulates an increase in GAG release from equine articular cartilage explants, though no firm conclusions can be drawn on its use in treating equine infectious arthritis. Copyright Harcourt Publishers Ltd.     

超低温冷冻对牛卵母细胞组蛋白乙酰化和膜蛋白CD9表达的影响

本试验旨在探讨玻璃化冷冻对牛体外成熟卵母细胞(MⅡ期)组蛋白乙酰化和膜蛋白CD9表达的影响。牛MⅡ期卵母细胞采用OPS法冷冻,即卵母细胞于10%EG+10%DMSO溶液中预处理30s,然后再移入玻璃化溶液EDFSF30中处理25s,以OPS为承载器投入液氮中。毒性组卵母细胞未投入液氮,其他过程与冷冻组相同,新鲜牛MⅡ期卵母细胞为对照组。卵母细胞解冻后,利用免疫荧光染色法检测存活细胞DNA组蛋白乙酰化;qRT-PCR和Western blot检测CD9mRNA蛋白的表达。结果表明:冷冻组卵母细胞形态正常率(93.8%)和存活率(92.7%)显著低于毒性组(100.0%,97.2%)和对照组(100.0%,98.5%)(P<0.05),而毒性组与对照组之间无显著差异。超低温冷冻后,卵母细胞DNA组蛋白乙酰化水平显著上升(P<0.05),CD9mRNA与蛋白表达显著降低(P<0.05)。以上显示,玻璃化冷冻不但降低了牛体外成熟卵母细胞的存活率,而且改变了DNA组蛋白乙酰化和膜蛋白CD9表达水平。     

离心去脂处理对不同发育阶段猪孤雌激活胚胎冷冻保存效果的影响

通过对不同发育阶段猪孤雌激活胚胎进行离心去脂处理,比较胚胎冷冻效果,优化冷冻体系。以猪孤雌激活胚胎为材料,比较实施电激活后第1天（2～3细胞）、第2天（4～6细胞）、第3天（6～8细胞）进行离心处理,取培养到第6天所得到扩张囊胚进行玻璃化冷冻保存,未离心处理直接进行冷冻的胚胎作为对照组。结果显示第3天离心组和第2天离心组所得的囊胚形成率（30．23％,28．22％）虽好于第1天离心组和不处理组（21．73％,22．43％）,但4个试验组差异不显著（P〉0．05）;第3天离心组和第2天离心组的冷冻后胚胎复苏率（28．57％,20．56％）显著高于不处理组（7．73％）、第1天离心组（12．24％）,差异显著（P〈0．05）;第3天离心组的解冻后囊胚内细胞计数（30．40）显著高于对照组（18．25）、第1天离心组（16．60）,差异显著（P〈0．05）,高于第2天处理组（23．71）,但差异不显著（P〉0．05）。结果表明,在孤雌激活后第3天进行离心处理,对胚胎发育到囊胚没有影响,且进行冷冻后所得复苏率及胚胎内细胞团数较好。     

德国牧羊犬精子超微结构及超低温冷冻对精子超微结构的影响

为了研究超低温冷冻前后德国牧羊犬精子超微结构的变化,采用扫描电镜和透射电镜对冷冻前后德国牧羊犬精子超微结构变化进行研究,结果显示在扫描电镜下,鲜精整体呈“蝌蚪状”,头部呈扁卵圆形.在透射电镜下精子纵切面头部呈“楔形”,头部细胞膜由外向内分别由质膜、顶体外膜、项体内膜和核膜组成.精子尾部中段横切面由外向内可见线粒体鞘膜、9束外周致密纤维,9对轴丝和2根中央微管.精子头长约为5.40 μm,头宽3.26 μm,颈长1.25 μm,颈宽0.55 μm,尾部中段长11.34 μm,中段直径0.87 μm,尾部长55.70 μm,线粒体螺旋数为44旋.鲜精和冷冻前精子头部、颈部和尾部形态变化差异均不显著(P＞0.05).超低温冷冻后精子头部、颈部和尾部发生形态变化的精子数极显著高于鲜精和冻前精子数(P＜0.01).冻后顶体发生明显形态变化的精子占54.21％,高于发生颈部形态变化(10.61％)和尾部形态变化(28.83％)的精子数.结果表明鲜精和冻前精子形态变化不显著,而超低温冷冻后精子头部、颈部和尾部发生形态变化的精子数显著高于鲜精和冻前精子数.     

犬精液冷冻解冻技术研究

试验以Tris-果糖稀释液为基础稀释液,研究了甘油浓度(4%、6%和8%)、冷冻起始温度(-80℃和-113℃)、冷冻时精液在液氮面上的高度(2 cm4、cm和6 cm)和解冻方法(30℃、12s和65℃、5 s)对犬精液冷冻保存效果的影响。结果表明,犬的精子冷冻保存以6%的甘油浓度、-80℃的始冻温度、在液氮面上2 cm的高度进行冷冻和65℃5 s的解冻方法为好,冷冻-解冻后精子的活率可达0.450 0±0.044 7。     

Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis (OA). Apoptotic pathways in chondrocytes are multi-faceted, although some cascades appear to play a greater in vivo role than others. Various catabolic processes are linked to apoptosis in OA cartilage, contributing to the reduction in cartilage integrity. Recent studies suggest that beta1-integrin mediated cell-matrix interactions provide survival signals for chondrocytes. The loss of such interactions and the inability to respond to IGF-1 stimulation may be partly responsible for the hypocellularity and matrix degradation that characterises OA. Here we have reviewed the literature in this area of cartilage cell biology in an effort to consolidate the existing information into a plausible hypothesis regarding the involvement of apoptosis in the pathogenesis of OA. Understanding of the interactions that promote chondrocyte apoptosis and cartilage hypocellularity is essential for developing appropriately targeted therapies for inhibition of chondrocyte apoptosis and the treatment of OA.     

The effect of cryopreservation on the capacitation status and epithelial cell attachment capability of dog spermatozoa

Freezing and cooling of spermatozoa during cryopreservation for artificial insemination causes ultrastructural changes in the acrosome and plasma membrane which reduces longevity and fertility. Cryopreservation-induced capacitation-like changes and reduced ability of spermatozoa to bind to the cells of the reproductive tract of the bitch may contribute to the reduced fertility of cryopreserved spermatozoa. Previous studies in the dog have investigated the effects of extending and cooling spermatozoa on the plasma membrane but often only after freeze-thawing and not in conjunction with an assessment of their ability to bind to uterine tube epithelial explants. This study investigated the effect of each stage of the cryopreservation process on capacitation and attachment to the reproductive tract of the bitch. The capacitation status of spermatozoa was studied over time after cryopreservation using a chlortetracycline and Hoechst 33258 stain. The ability of spermatozoa to bind to uterine tube epithelial explants was studied using Hoechst 33342 stain. Extending, cooling and freeze-thawing promoted capacitation and decreased the spermatozoal binding ability. The effect of each stage appeared to be cumulative with the freeze-thawing stage being the most dramatic. The results suggested that the cumulative effect of each stage of the cryopreservation process results in the promotion of capacitation before spermatozoa have reached the site of fertilisation, and therefore spermatozoa have reduced ability to attach to epithelial cells. These effects are contributory factors to the reduced fertility of cryopreserved spermatozoa.     

狐精液冷冻保存研究进展

就国内外的狐精液冷冻技术研究进行综合阐述,以期为我国未来养狐业精液冷冻技术的发展提供借鉴。     

Suppressive effect of hyaluronan on chondrocyte apoptosis in experimentally induced acute osteoarthritis in dogs

Dogs receiving anterior cruciate ligament transection (ACLT) were treated with either intravenous (IV) or intraarticular (IA) administration of hyaluronan (HA), and differences in appearance of chondrocyte apoptosis of the stifle joint were investigated. Chondrocyte apoptosis was detected using flow cytometry as well as by staining with TdT-mediated dUTP nick end labeling (TUNEL). The percentage of apoptotic chondrocytes in dogs with ACLT was significantly higher than that in intact (non-ACLT) dogs. Dogs treated with IA or IV injection of HA after ACLT had fewer apoptotic chondrocytes than non-treated dogs after ACLT. It was suggested that ACLT-induced apoptosis of chondrocytes was suppressed by HA administration of either IA or IV.     

猪精液冷冻保存研究进展

根据国内外的研究进展,本文简要阐述了猪精液冷冻保存的机理和操作方法,对影响猪冷冻精液质量的因素进行了分析,同时指出了目前在猪精液冷冻保存技术的理论和实践中存在的问题,并提出了该技术中需要解决的问题和展望。     

褪黑素、谷胱甘肽对猪精液冷冻保存效果的影响

本试验旨在研究褪黑素、谷胱甘肽两种抗氧化剂对精子冷冻的保护效应。以成年杜洛克公猪为研究对象,在猪精液冷冻稀释液中先后单独、联合添加褪黑素、谷胱甘肽,解冻后精子质量通过检测活率、活力、顶体完整性、线粒体活性以及活性氧（ROS）含量来判定。结果表明：分别单独添加0.25mg/mL褪黑素、5mmol/L谷胱甘肽,或联合添加0.125mg/mL褪黑素和1mmol/L谷胱甘肽,均能减少精液冷冻过程中ROS的生成,显著提高冷冻精子解冻后的质量（P〈0.05）,其中联合添加效果显著优于单独添加效果。     

Effects of thermal energy on chondrocyte viability

OBJECTIVE: To determine the critical temperature that reduces chondrocyte viability and evaluate the ability of chondrocytes to recover after exposure to the critical temperature. SAMPLE POPULATION: Cartilage explants obtained from the humeral heads of 30 sheep. PROCEDURES: In a randomized block design, 318 full-thickness cartilage explants were collected from 30 humeral heads of sheep and cultured for up to 14 days. On the first day of culture (day 0), explants were subjected to temperatures of 37 degrees , 45 degrees , 50 degrees , 55 degrees , 60 degrees , or 65 degrees C for 5 minutes by heating culture tubes in a warming block. The ability for chondrocytes to recover after exposure to the critical temperature was determined by evaluating viability at days 0, 1, 3, 7, and 14 days after heating. Images were analyzed by use of confocal laser microscopy. RESULTS: Analysis of images revealed a significant decrease in live cells and a significant increase in dead cells as temperature increased. Additionally, the deepest layer of cartilage had a significantly lower percentage of live cells, compared with values for the 3 most superficial layers. Chondrocytes did have some ability to recover temporarily after the initial thermal insult. CONCLUSIONS AND CLINICAL RELEVANCE: A strong relationship exists between increasing temperature and cell death, with a sharp increase in chondrocyte death between 50 degrees and 55 degrees C. Chondrocytes in the deepest cartilage layer are most susceptible to thermal injury. The threshold of chondrocyte recovery from thermal injury is much lower than temperatures reached during chondroplasty by use of most radiofrequency energy devices.     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。     

The effect of supplementing layer diets with shark cartilage or chitosan on egg components and yolk lipids

1. An experiment was designed to evaluate the effects of the addition of shark cartilage (SC) or chitosan (CH) to layer diets on egg component weights, yolk lipids and hen plasma lipids. 2. Hy-Line laying hens (80) were used during a 56 d feeding trial. Treatments were: basal diet (BD), BD + 20 g/kg SC, BD + 30 g/kg SC, BD + 20 g/kg CH and BD + 30 g/kg CH. Eggs were analysed on d 14, 28, 42 and 56. 3. Egg weight and egg component weights were not affected by these treatments throughout the experimental period. 4. After 14d of experimental feeding, cholesterol levels were higher in eggs from birds given BD + 20 g/kg CH and BD + 30 g/kg CH than in those from birds given BD. 5. Furthermore, eggs from hens given BD + 20 g/kg SC or BD + 20 g/kg CH were higher in palmitic and stearic acids and lower in oleic acid than those from birds fed on BD. After 56 d feeding, however, palmitic and stearic acid contents in eggs from hens given any of the supplemented diets were lower than in those from hens given BD, and oleic acid in eggs from hens given BD + 20 g/kg SC, BD + 30 g/kg SC and BD + 30 g/kg CH was higher than in those from birds fed on BD. 6. Plasma cholesterol and triacylglycerol levels were not significantly affected by dietary treatment. 7. Shark cartilage or chitosan at up to 30 g/kg in layer diets did not affect egg component weights (yolk, white and shell) and total lipid contents. During the period from 42 to 56d of experimental feeding, diets containing up to 30 g/kg chitosan reduced egg yolk contents of cholesterol, palmitic and stearic acids and increased the content of oleic acid.     

鸭精液冷冻保存试验研究

以笼养金定鸭为材料,对鸭精液冷冻保存稀释液、防冻剂进行筛选,并研究了最佳平衡时间和解冻温度,同时还对公鸭精子在抗冻性方面的差异进行了初步探讨.结果发现:以10%二甲基亚砜做防冻剂,精子冻后活力最高,达0.7以上,经小群输精、孵化测定,受精率达65%.平衡时间10 min、解冻温度40C时,鸭精子冻后活力最好.公鸭在精子抗冻性方面存在极显著差异(P＜0.01),因此要在抗冻性方面对公鸭进行选择.     

In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage

Objective: To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone. Study Design: Experimental study. Animals: Equine cadaver larynges (n=13). Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F_max). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F_max. The force applied was released, left VCE performed, and photographs repeated. Arytenoid left:right angle quotients (LRQ) and glottic cross‐sectional area ratios (CSAR) were calculated at each force level in each condition (PL and VCE‐PL). Results: Mean LRQ and CSAR for both PL and VCE‐PL increased with increasing force, initially rapidly before plateauing at ～50% of F_max. LRQ and CSAR were significantly greater for VCE‐PL than for PL (P<.001). When VCE was performed before PL, 12% less force was required to achieve an LRQ of 0.8, and 45% less for a CSAR of 0.8. Conclusions: In vitro, VCE performed before PL enables the arytenoid cartilage to be abducted to a greater degree for a given PL suture force.     

Ex vivo investigation of the use of hydrothermal energy to induce chondrocyte necrosis in articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of horses

OBJECTIVE: To evaluate the use of hydrothermal ablation of articular cartilage for arthrodesis in horses through investigation of the effects of joint lavage with physiologic saline (0.9% NaCI) solution (80 degrees C) for various treatment times on chondrocyte viability in the articular cartilage of the metacarpophalangeal and metatarsophalangeal joints of cadaveric horse limbs. Sample Population-7 pairs of metacarpophalangeal and 8 pairs of metatarsophalangeal joints from 8 Thoroughbreds. PROCEDURE: The horses were euthanatized for reasons unrelated to musculoskeletal disease. On a random basis, 1 joint of each pair underwent intra-articular lavage for 5, 10, or 15 minutes with heated saline solution (80 degrees C); the other joint underwent sham treatment of similar duration with saline solution at 22 degrees C (control). Cartilage samples from the distal articular surface of metacarpus III (or metatarsus III), the proximal surface of the proximal phalanx, and the lateral and medial proximal sesamoid bones were assessed for chondrocyte viability via confocal microscopy and viability staining following enzymatic digestion. RESULTS: Compared with the control joints, findings of both viability assays indicated that the percentage of sites containing viable chondrocytes in heat-treated joints was decreased.Treatment hazard ratios of 0.048 (confocal microscopy) and 0.2 (digestion assay) were estimated. Histologically, periarticular soft tissues had minimal detrimental effects after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Ex vivo intra-articular lavage with saline solution at 80 degrees C resulted in the death of almost all articular chondrocytes in the joint. This technique may be a satisfactory method for extensive cartilage ablation when performing arthrodesis by minimally invasive techniques.