Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa – including sex sorting – as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry‐based assays. After sorting, oxidative stress increased from 26% to 33% in pre‐ and post‐incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post‐sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.     

流式细胞仪分离精子研究进展

本文概述了近年来用流式细胞仪分离动物X、Y精子的研究进展、存在的问题和应用前景。流式细胞仪分离精子的研究已经取得了很大进展 ,在某些动物 (如牛 )分离的精子已开始用于人工授精。尽管该技术还存在许多亟待解决的问题 ,但随着流式细胞仪的改进和与各项辅助生殖技术 (如手术授精、体外受精、胞质内精子注射、低剂量精子人工授精 )结合 ,性别分离精子必将在实践中得到广泛的应用 ,产生巨大的社会和经济效益     

Flow Cytometric Assessment of Acrosomal Status and Viability of Dog Spermatozoa

Contents
A procedure was attempted to simultaneously evaluate viability and acrosomal integrity of dog spermatozoa by flow cytometry and the dual staining technique using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide (PI). Three ejaculates were obtained from three dogs; each of which was divided into five aliquots and increasing concentrations (0–288 μmol/l) of lysophosphatidylcholine (LPC) were added to each one to artificially induce the acrosome reaction in different proportions of spermatozoa. Data obtained by flow cytometric analysis of each sample were compared with those obtained by microscopic evaluation under epifluorescence illumination and by light microscopy evaluation of smears stained with Spermac^® staining. Regression analysis was used to compare the flow cytometric assay with the epifluorescence and light microscopic techniques, and the results indicated that flow cytometry was highly correlated with the Spermac^® staining whereas the correlation with the epifluorescence microscopy was lower. In comparison with the Spermac^® staining, the results from this study validate flow cytometry as a precise method for evaluating the acrosomal integrity of canine spermatozoa.     

流式细胞仪测定奶牛全血多形核白细胞的吞噬功能和呼吸爆发作用

试验建立了用流式细胞仪同时测定奶牛全血中多形核白细胞（PMN）吞噬功能和呼吸爆发作用的微量检测方法。奶牛全血用二氢诺丹明处理后，与直径为1．75μm的蓝色荧光乳胶微球培养，PMN内的二氢诺丹明被呼吸爆发过程中产生的还原性物质转化成发绿色荧光的诺丹明（ROD）。用流式细胞仪计数PMN，仪器可在数秒钟内计数几十万个PMN，识别PMN是否吞噬蓝色荧光微球和／或含POD，并可检测每个PMN所吞噬的微球数量。当血液中含细胞松弛素B为1、2．5、5mg/L时，PMN的吞噬功能和呼吸爆发作用均下降。     

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 10⁶ sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 10⁶ spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.     

Simultaneous Flow Cytometric Analysis of Phagocytosis and Oxidative Burst Activity in Equine Leukocytes

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.     

Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine Spermatozoa Subjected to Ferrous Ascorbate

Quercetin (QUE) is a natural flavonol‐type flavonoid with antibacterial, anti‐inflammatory and anti‐aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS‐mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 μmol/l) in the presence or absence of a pro‐oxidant, that is ferrous ascorbate (FeAA; 150 μmol/l FeSO4 and 750 μmol/l ascorbic acid) during a 6‐h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer‐aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p < 0.001), viability (p < 0.001) and decreased the antioxidant parameters of the sperm samples (p < 0.001) but increased the ROS generation (p < 0.001), superoxide production (p < 0.001) and lipid peroxidation (p < 0.001). QUE administration resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (p < 0.01 with respect to the enzymatic antioxidants, p < 0.001 in relation to GSH) with a concentration range of 50–100 μmol/l QUE revealing to be the most effective. Our results suggest that QUE exhibits significant ROS‐scavenging and metal‐chelating properties which may prevent spermatozoa alterations caused by ROS, and preserve the functionality of male reproductive cells.     

Oxidative Stress During Pregnancy In The Sheep

During physiological pregnancy, all tissues and, mostly, placenta and foetus require high amounts of oxygen. Reactive oxygen species (ROS), generated both by mother and foetus, are implicated in foetal growth because they promote replication, differentiation and maturation of cells and organs. Nevertheless, ROS excess, if not properly counterbalanced, may lead to an alteration in cell constituents, with harmful effects both on mother and foetus.ROS exert a biphasic effect because adequate ROS concentration is essential for embryo development, implant, foetal defence against uterine infections, steroidogenesis, pregnancy maintainance and partum. On the other hand, an uncontrolled ROS generation, beyond physiological antioxidant defences, may lead to embryo resorption, placental degeneration with subsequent alteration in maternal‐foetal exchanges, delay in foetal growth, pregnancy interruption, stillbirths. This review investigates the mechanisms underlying ROS generation and effects, throughout physiological and pathological pregnancy in sheep, with a look to antioxidants and their importance in such a critical phase of the reproductive cycle of the sheep.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

Nitric Oxide and Superoxide Anion Production During Heparin‐Induced Capacitation in Cryopreserved Bovine Spermatozoa

The aim of this work was to quantify NO, O₂^? and ONOO^? production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O₂ uptake, and NO production. Conversely, O₂^? production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O₂ consumption (5.9 ± 0.6 nmol/min × 10⁷ cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10⁷ cells), and a five‐fold increase in O₂^? production (1.3 ± 0.07 nmol/min × 10⁷ cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O₂^? generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O₂ uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O₂^? production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.     

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.     

牛精子蛋白胶内酶切条件优化及问题分析

针对银染蛋白的胶内酶切质谱鉴定成功率较低的技术现状,对影响牛精子蛋白酶切鉴定的酶量、酶切时间、凝胶存储时间等影响因素进行了摸索,并优化了挖点、脱色、脱水、酶切消化和抽提肽段等操作细节,同时对质谱峰图中出现的污染问题进行了分析解决,提高了蛋白鉴定的成功率,为牛精子蛋白质的质谱鉴定奠定了技术基础。     

Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Gender Differences of Oxidative Stress Biomarkers and Erythrocyte Damage in Well-Trained Horses During Exercise

The aim of this study was to determine the effects of gender differences on the blood oxidative stress biomarkers, antioxidant defenses, and resistance of erythrocytes to hemolytic agents of trained horses before and after exercise. The study was carried out on nine mares and 14 stallions of Ukrainian Warmblood well-trained horses, involved in jumping, eventing, and dressage. Oxidative stress biomarkers, antioxidant defenses, and osmotic resistance of erythrocytes were assessed. Trained stallions showed a decrease in lipid peroxidation and higher glutathione reductase activity, whereas mares presented a higher superoxide dismutase activity after exercise. The resistance of erythrocytes was similar in female and male. No statistically significant differences were observed in the percentage of hemolyzed erythrocytes between after and before exercise. A correlation between the oxidative stress biomarkers and antioxidant defenses in the stallions after exercises were observed, which may indicate a protective response of superoxide dismutase and catalase against exercise-induced oxidative stress.     

The John Hughes Memorial Lecture: Aspects of Sperm Physiology—Oxidative Stress and the Functionality of Stallion Spermatozoa

Spermatozoa are vulnerable to oxidative attack because they contain an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation. In addition, functionally important proteins and DNA are also subject to oxidative modification and adduction by aldehydes, such as 4-hydroxynonenal (4HNE), generated as a consequence of the peroxidative process. The proteins adducted by 4HNE include elements of the mitochondrial electron transport chain, such as succinic acid dehydrogenase. The net result of such electrophilic attack is to stimulate generation of mitochondrial reactive oxygen species (ROS) in a self-perpetuating lipid peroxidation–ROS generation cycle that ultimately triggers the intrinsic apoptotic pathway, leading to a rapid loss of motility and cell death. A major point of difference between apoptosis in spermatozoa and somatic cells is that in the former, nuclear DNA is located in a compartment (the head) separate from the mitochondria and most of the cytoplasm (the midpiece). As a result, nucleases activated and released in the midpiece during apoptosis cannot gain access to the DNA in the sperm head in order to cleave the DNA. However, the ROS generated during apoptosis can readily gain access to the sperm nucleus and generate oxidative base adducts, typically 8-hydroxy, 2′-deoxyguanosine (8OHdG), which are converted into abasic sites by 8-oxoguanine glycosylase (OGG1), the only enzyme of the base excision repair pathway possessed by spermatozoa. These abasic sites subsequently become the foci of DNA fragmentation. Because defective sperm function and DNA damage are frequently associated with oxidative stress, there is a great deal of interest in the use of antioxidants in a therapeutic context. This presentation examines the fundamental relationships between oxidative stress and sperm function and considers the implications of recent findings for the management of sperm function and fertility in stallions.     

奶牛子宫内膜上皮细胞氧化应激模型的建立及奶牛脂肪间充质干细胞对其迁移的影响

【目的】探讨奶牛脂肪间充质干细胞(bovine adipose-derived mesenchymal stem cells, bAD-MSCs)对氧化应激条件下奶牛子宫内膜上皮细胞迁移能力的影响。【方法】使用0、5、25、50、100、200μmol/L H₂O₂处理奶牛子宫内膜上皮细胞2、4、6、8、12 h后,通过MTT试验检测细胞存活率、流式细胞术检测细胞内活性氧(ROS)水平来筛选H₂O₂诱导奶牛子宫内膜上皮细胞氧化应激模型的最佳条件。在细胞划痕试验中设立对照组、H₂O₂组、bAD-MSCs共培养组(1∶0.5)、bAD-MSCs共培养组(1∶1)、奶牛乳腺上皮细胞(MACT)共培养组(1∶0.5)和MACT共培养组(1∶1)共6组,分析奶牛子宫内膜上皮细胞迁移能力,并利用Western blotting检测细胞外蛋白调节激酶(Erk)和磷酸化细胞外蛋白调节激酶(pErk)蛋白的表达水平。【结果】MTT试验结果显示,4～12 h内50μmol/L...     

Preliminary Evaluation of a Unique Freezing Technology for Bovine Spermatozoa Cryopreservation

Artificial insemination (AI) and semen cryopreservation has significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality. Ten years ago, a unique freezing technology (UFT) was developed for the freezing of foodstuffs and other materials. Previous work from this laboratory has demonstrated the UFT to be a superior method of freezing for a number of cell types. In a preliminary study, the UFT was compared with the conventional freezing methodology of bovine semen. Semen samples were collected from an angus (Bull A) and a gelbivich bull (Bull B), prepared using a conventional bovine cryoprotectant, and frozen in the UFT or in liquid nitrogen (LN) mist. The samples were stored in LN before being thawed and assessed for the semen parameters of motility and forward progression. Preliminary results suggest the UFT is equivalent to current techniques in the cryopreservation and recovery of bovine semen, and with modification, possibly a superior technique for semen freezing. Further studies using larger sample populations, and using a CASA system to evaluate motility, forward progression and linearity are merited.