Functional characterization of Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris haemocyte separated using density gradient centrifugation

Haemocytes from penaeid shrimp (Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris) were separated using a discontinuous Percoll gradient. Shrimp haemocytes were spontaneously adhered to glass, allowing slide preparations for staining and microscopic differential counting. Like other crustaceans, shrimp has three main populations differing in presence and size of cytoplasmic granules and each population seems to be biochemical or functionally compromised. Prophenoloxidase (proPO )activity was mainly located in large granules haemocytes (75%) while the small granules cells participate with 25%, but seem to be responsible for encapsulation. Haemocyte discrimination ability was tested using Sephadex? (Seph?), DEAE‐Seph? and CM‐Seph?. Only DEAE‐Seph? was encapsulated by shrimp haemocytes and provoked the release of proPO activating system, indicating the role of particle charge in the activation of shrimp immune response.     

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.     

两种不同方法处理牛分离精子对体外受精效果的影响

本试验旨在探讨采用2种不同方法处理牛分离精子对精子活力、浓度及体外受精（IVF）效果的影响。将体外培养成熟卵母细胞随机分为2组,与分别用洗涤法和Percoll法处理牛分离精子进行IVF,并观察处理后各组的精子活率、浓度和IVF后囊胚发育率。结果显示,洗涤法和Percoll法处理后精子活力和IVF后的囊胚发育率均无显著差异,但采用洗涤法处理后分离精子浓度极显著高于Percoll法,可显著提高分离精子利用效率,且方法简便、经济,应优先选择使用。     

Percoll法处理牛精子对体外受精胚胎发育的影响

牛冷冻精液解冻后，在Ｐｅｒｃｏｌｌ梯度液中４００ｇ离心２４ｍｉｎ有效地获得了５７．８７％±８．０％的精子回收率；对照组上游法精子回收率仅为１５．８７％±１．９％（Ｐ〈０．０５）。Ｐｅｒｃｏｌｌ和上游２种方法分离的精子在受精液中８，１８ｈ后的活率，分别为６０％，１０％和７０％，２０％，２种浓度Ｐｅｒｃｏｌｌ法分离的精子对不同成熟处理的卵母细胞体外受精子（ＩＶＦ）卵裂率的影响，精子最终浓度为２×１０＾     

萝卜游离小孢子培养中胚性小孢子的选择

研究了不连续perc0Ⅱ密度梯度离心方法在萝卜胚性小孢子选择上的应用。结果表明，经percoⅡ密度梯度离心后，萝卜小孢子被分为4层，每一层小孢子都位于两种密度的界面上，按小孢子的密度大小由上而下依次为四分体时期、单核-双核期、单核-双核-三核期及三核时期。同时研究了花蕾长度与平均核数的关系，选择出了适合萝卜游离小孢子培养的胚性小孢子。     

Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered.Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

洗涤及Percoll梯度离心后小鼠精子脂类含量的分析

为了分析ｐｅｒｃｏｌｌ梯度离心（ＰＧＣ）法分离小鼠附睾精子后，精子受精力提高的原因，本文测定了经洗涤和ＰＧＣ法分离后及进一步孵育后小鼠精子的胆固醇和磷脂含量，同时对附睾液和输精管液的胆固醇和磷脂含量、脂类的组成成份以及与精子共同孵育后对精子活力的影响也进行了测定。结果表明，附睾液和输精管液中存在着丰富的脂类小泡，含有高浓度的胆固醇和磷脂（０．２７３和０．７０４ｎｍｏｌｅ／μｇＤＮＡ），这些生理量的脂类与精子共同培养，能使精子失去活动力。用ＫＲＢ液洗涤后精子的胆固醇和磷脂含量分别为０．２３５和０．６９４ｎｍｏｌｅ／μｇＤＮＡ，用ＫＲＢ－ＢＳＡ洗涤和进一步孵育对精子胆固醇和磷脂含量无明显影响，二梯度ＰＧＣ法分离精子可显著降低精子的磷脂含量（０．３１５ｎｍｏｌｅ／μｇＤＮＡ）。     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

受精液和精子处理方法对水牛卵母细胞体外受精的影响

研究了不同受精液和精子处理方法对沼泽型水牛卵母细胞体外受精卵裂率的影响。结果表明,使用BO液和TALP液对卵裂率没有显著影响（P〉0.05）。Percoll离心法和离心洗涤法对卵母细胞受精卵裂率也没有显著影响（P〉0.05）。     

Isolation of granulocytes and mononuclear cells from the blood of dogs, cats, horses and cattle

A simple discontinuous Percoll density-gradient technique was adapted for isolation of granulocytes and mononuclear cells from cats, dogs, horses and cattle. Separation was accomplished at low speeds using a standard tabletop centrifuge. Cell purity was 100% for both granulocytes and mononuclear cells and cell viability exceeded 95%. Percent recovery of leukocytes ranged from 69 to 83%.