Sexing sperm of domestic animals

The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.     

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.     

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas ® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.     

Diagnosis of mediastinal masses in dogs by flow cytometry

BACKGROUND: Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms. HYPOTHESIS: Flow cytometry of mediastinal mass aspirates may provide a definitive diagnosis of thymoma or lymphoma, reducing the need for biopsy. ANIMALS: Dogs with mediastinal masses presenting to the Veterinary Teaching Hospital/Animal Cancer Center were included in the study. METHODS: Aspirates obtained over 2 years that met the inclusion criteria (i.e. sufficient viable cells and a definitive diagnosis by means other than flow cytometry) were analyzed by flow cytometry to determine the percentage of cells expressing B- and T-cell markers, and co-expressing CD4 and CD8. RESULTS: All cases of thymoma (n = 6) consisted of > or = 10% lymphocytes coexpressing CD4 and CD8, a phenotype that is characteristic of thymocytes, whereas 6 of 7 lymphomas contained <2% CD4+CD8+ lymphocytes. The CD4+CD8+ lymphoma could be readily distinguished flow cytometrically from thymoma by light scatter properties. The phenotypes of the remaining lymphomas were CD4+ T cell (4), CD34+ (1) and B cell (1). CONCLUSIONS: Our studies demonstrate that flow cytometry is a useful tool for discriminating mediastinal masses. Lymphocyte-rich mediastinal masses could be unambiguously identified by flow cytometry in 13/13 cases.     

使用荧光染色和流式细胞术检测山羊精子

分别使用PI(碘化丙碇),FITC-PSA(异硫氰酸荧光素络合豌豆凝集素)和RH123(罗丹明)对山羊冷冻精子进行染色,再使用流式细胞仪进行检测、分析。试验发现,对于山羊的冷冻精子,PI染色阴性的为质膜完整的精子;FITC-PSA染色阴性的可能为顶体完整的精子,染色FITC-PSA+和FITC-PSA++,可能分别为顶体反应中的精子和顶体破损精子;RH123+应为线粒体无活性精子,有绿色荧光可能是因为染料残留在细胞膜上导致,而RH123++可能应为线粒体有活性的活精子。不同个体羊的精子对于低温敏感度不一样。不同个体羊之间的冻精顶体完整率、质膜完整率、线粒体有活性的精子比率之间差异显著,据此应选择合适的山羊个体进行精液冷冻。     

A study by flow cytometry of lymphocytes in sow colostrum

Mammary secretions contain leucocytes which may be of value to the neonate. The cells obtained from sow colostrum (1 to 2·5 × 10⁶ ml⁻¹) are mainly lymphocytes (10 to 25 per cent) and epithelial cells (more than 20 per cent). In milk, there are few lymphocytes (0·5 to 2 per cent) and mostly alveolar epithelial cells. The study of lymphocytes in the mammary secretions of sows has been made difficult by the high proportion of epithelial cells, which could not be separated from lymphocytes, and by a high background in membrane immunofluorescence labelling. This paper describes a method for the study of the cells in the mammary secretions of sows by flow cytometry. It showed that 70 to 90 per cent of colostral lymphocytes were T lymphocytes, with T8 lymphocytes predominating over T4, and that the ratio T4/T8 was significantly lower in colostrum (0·57) than in blood (0·80). There were no lymphocytes expressing interleukin-2-receptors in the colostrum of sows.     

Detection of canine interleukin-2 receptors by flow cytometry.

This study describes a method for detecting canine interleukin-2 receptors (IL-2R) by flow cytometry, using human recombinant IL-2 labeled with phycoerythrin (IL-2-PE). Peripheral blood mononuclear cells from four normal dogs were washed, incubated with IL-2-PE, and then washed to remove any unbound IL-2-PE. Flow cytometric analysis of the cells was performed with a 488 nm argon laser while gating on lymphocytes. Cells expressing the IL-2R were identified by their fluorescence as compared to cells stained with an anti-mouse immunoglobulin-G conjugated to phycoerythrin. The average percentage of resting cells expressing the IL-2R was found to be 21%. The addition of unlabeled human recombinant IL-2 to Day 3 phytohemagglutinin (PHA)-stimulated cells reduced the fluorescence intensity four-fold, thereby demonstrating the specificity of IL-2-PE for canine IL-2R. Following stimulation with optimal concentrations of PHA, the percentage of cells expressing the IL-2R increased daily and reached a maximum on Day 3 (76.4%). IL-2R density, as measured by mean fluorescence intensity, also increased and reached maximal levels on Days 2-3 (twenty-fold greater than resting cells). The binding, inhibition, and kinetic experiments provide evidence that human recombinant IL-2-PE is a useful tool for studying canine IL-2R expression. Thus, a one-step direct method for the flow cytometric detection and quantification of the canine IL-2R is now available.     

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.     

Sperm membrane functionality in the dog assessed by flow cytometry

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.     

Cell cycle analysis of bovine cultured somatic cells by flow cytometry

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions; 1) growth to 60-70% confluency (cycling), 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in G0 /G1, S and G2 /M. The majority was in G0/G1 regardless of cell type and treatment. Serum-starved or confluent cultures contained higher percentages of cells in G0/G1 (89.5-95.4%; P < 0.05). Percentages of cells in G0/G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in G0/G1 . Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in G0/G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in G0/G1, and indicate that a more efficient synchronization of the cells in G0/G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.     

Identification of Mycoplasma gallisepticum and M. synoviae by flow cytometry

Immunofluorescence and flow cytometric methods were examined to detect and distinguish Mycoplasma gallisepticum and M. synoviae. The procedure employed 24-hr broth cultures of each organism, direct immunofluorescence staining with either homologous or heterologous antiserum, and analyses by flow cytometry. The organisms were distinguishable on the basis of fluorescent profiles when stained with the appropriate antiserum.     

A new method for counting of quail leukocytes by flow cytometry

An automatic counting method was developed for fish blood cells using a fluorescent dye, 3, 3-dihexyloxacarbocyanine (DiOC (6)(3)), that selectively stain lipid bilayers in living cells. In the present study, the DiOC(6)(3) method was applied to quail (Cotumix cotumix japonica) blood cells. After quail blood cells were stained with DiOC(6)(3), absolute counts and relative proportions of erythrocytes, granulocytes, monocytes, and lymphocytes plus thrombocytes in whole blood were obtained by means of flow cytometry (FC). The number of each cell types by the FC was in good agreement with those counted microscopically. This method will offer new possibilities for routine blood cell counting for avian medicine.     

牦牛胚胎性别鉴定PCR反应体系的建立

研究旨在建立牦牛胚胎性别鉴定准确、可靠的PCR反应体系及研究切割取样对胚胎发育能力的影响.根据牦牛SRY和HSL基因序列分别设计合成2对巢式PCR引物作为性别鉴定引物和2对引物作为内标引物,通过优化PCR反应条件,对已知性别牦牛血液基因组DNA和牦牛杂种胚胎DNA采用巢式PCR进行性别鉴定.结果表明,血液样本性别鉴定与实际性别完全相符,准确率100%.从早期胚胎取8个细胞进行性别鉴定,结果雄性为45.8%(11/24),雌性为54.2%(13/24),取样后胚胎发育率为56.7%.结果说明已成功建立牦牛胚胎性别鉴定的PCR体系.     

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry

The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.     

Detection of activated platelets in canine blood by use of flow cytometry

OBJECTIVE: To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood. SAMPLE POPULATION: Blood samples from 10 dogs. PROCEDURE: Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine. RESULTS: A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.     

流式细胞术分析苜蓿相对DNA含量的样品处理方法

为制备用于流式细胞仪分析的高纯度苜蓿(Medicago sativa)细胞核悬液,筛选最适合流式细胞仪分析的苜蓿细胞核提取方法,选用江苏地方品种淮阴苜蓿为供试材料,分别采用酶解法和机械解离法提取苜蓿细胞核悬液;用5种细胞核分离缓冲液及两种混合酶解液制备样品,根据各自优缺点筛选最适宜的苜蓿细胞核提取法。在机械法所用的5种细胞核提取缓冲液中,LB01缓冲液提取效果最好,提取的细胞核形态及内部结构完整,获得的细胞核量多。利用两种混合酶液制备苜蓿细胞核悬液,均出现倍性明显的峰,混合酶液Ⅰ制备的单细胞核悬液优于混合酶液Ⅱ,且酶解法制备细胞核悬液,可获得数量更多更完整的原生质体。但利用酶解法制备细胞核原生质体,其悬浮液中杂质多,需时较长。结果表明,LB01缓冲液为最适合制备苜蓿细胞核悬液的提取缓冲液。     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry

The recognition of equine lymphocyte antigens by monoclonal antibodies (mAbs) directed against human CD11a, CD18, CD21, CD23, CD29 and DR, as well as mouse CD23 was studied by flow cytometry. Unlike anti-CD11a, -CD21, -CD23 and DR mAbs, anti-CD18 and CD29 mAbs labelled the same percentage of horse peripheral blood lymphocytes (PBL) as human PBL. Double-staining with anti-horse immunoglobulin antibodies showed that anti-CD21 and -CD23 mAbs are mainly bound to peripheral blood B lymphocytes. The seven mAbs were also tested on the lymph node and thymus cells. The molecular targets of anti-CD11a, CD18 and CD29 mAbs were confirmed by immunoprecipitation of the membrane proteins. Our results suggest that anti-CD18, -CD29 and -DR mAbs recognise similarly expressed molecular homologues on equine cells, but that anti-CD11a, -CD21 and -CD23 mAbs recognise either different molecules or homologues that are expressed at different levels on horse cells.