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1.
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.  相似文献   

2.
Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.  相似文献   

3.
The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.  相似文献   

4.
Contents Eight bull ejaculates were split to evaluate the effects of glycerol on sperm organelle function. Although glycerol protects sperm membranes during cryopreservation, preliminary data suggested that glycerol was detrimental to sperm organelles to varying degrees. To assess the compartmental effects, three organelle-specific fluorophores were used to analyze with (G+) and without glycerol (G–) in spermatozoa stored for 24 h at 5°C in an egg-yolk-based extender. The mitochondrial probe, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) was used to examine the level of mitochondrial metabolic function by it’s discrimination between high (J-aggregate staining, red-orange) and low membrane potentials (JC-1 monomeric staining, green); while acosomal reacted spermatozoa were identified using fluorescein-labelled lectin from arachis hypogaea, PNA-FITC. The proportions of living and dead spermatozoa were determined by staining with the combination of SYBR-14 and propidium iodide (PI). Split-plot analysis of variance revealed that within bulls, glycerol altered the proportions of sperm staining with each organelle-specific fluorophore to varying degrees. The proportion of spermatozoa labelled with SYBR-14, indicating intact plasmalemmae, were not affected by the addition of glycerol (p = 0.11). Although the total proportion of JC-1-labeled spermatozoa were similar in G– and G+ samples (p = 0.90), the presence of glycerol decreased the proportion of spermatozoa that exhibited J-aggregate staining (p < 0.01) while producing an increase in monomeric staining (p = 0.02). The proportions of acrosome-reacted spermatozoa, however, were greater in the G– samples than in the G+ samples as indicated by PNA-FITC (p = 0.03). These findings suggest that mitochondria, acrosomes and the plasmalemmae of unfrozen spermatozoa vary in their response to the addition of the cryoprotectant glycerol.  相似文献   

5.
This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.  相似文献   

6.
This study investigated the apoptosis-like events associated with cryopreservation process and their relationship with cryocapacitation in buffalo (Bubalus bubalis) sperm. A total of 49 semen ejaculates from seven bulls were studied for structural changes in sperm following cryopreservation. Apoptotic changes were detected by assays specific for translocation of phosphatidylserine (PS) to the cell surface, alterations in membrane permeability and mitochondrial membrane potential (MMP), and DNA integrity. A significant (p < 0.01) percentage of cryopreserved sperm showed externalization of PS and early apoptotic changes and lowered MMP when compared with the fresh sperm. Freezing and thawing of sperm increased permeability to YOPRO-1, an impermeant fluorescent dye. However, on TUNEL staining, cryopreserved sperm showed no breach in DNA integrity. The sperm capacitation status was evaluated by chlortetracycline (CTC) fluorescence pattern, in which a significant (p < 0.01) percentage of cryopreserved sperm were found to be capacitated. The capacitated sperm (Pattern B) was positively correlated with the aforementioned apoptotic events. In conclusion, cryopreservation process induced early apoptosis-like changes in buffalo sperm, and a close link exists between cryocapacitation and apoptosis during cryopreservation of sperm.  相似文献   

7.
A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150–1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa, as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350–1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.  相似文献   

8.
奶牛精子膜的分离及纯化   总被引：2，自引：0，他引：2  
柳荣  张鳞 《畜牧兽医学报》1997,28(5):409-415
本实验采用高压爆破匀浆法将精子膜与精子分离。分离后的精子膜粗品采用蔗糖密度梯离心法进行纯化。经电镜观察，膜制品均一性很好。经酶学分析，膜特异性酶－碱性磷酸酯酶在膜制品中含量很高。  相似文献   

9.
Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.  相似文献   

10.
The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 x 10(-3) M, 1 x 10(-4) M, 1 x 10(-5) M and 1 x 10(-6) M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 x 10(-3) M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 x 10(-3) M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 x 10(-3) M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.  相似文献   

11.
Tetraspanin CD9 is one of the egg membrane proteins known to be essential in fertilization process. The presence and localization of CD9 molecule in spermatozoa and its possible function in reproduction are still unclear. In our study, we describe the localization of CD9 on bull spermatozoa. In the immunofluorescence assay, the positive signal has been observed in the high proportion of sperm cells as a fine grains either on the apical part or through the entire anterior region of sperm head. CD9 recognized by monoclonal antibody IVA‐50 was detected on freshly ejaculated (83.4 ± 3.7%) and frozen‐thawed (84.3 ± 2.3%) sperm. The same reaction pattern was observed on sperm capacitated for 1 h, 2 h, 3 h and 4 h (83.6 ± 2.0%; 84.0 ± 1.5%; 85.7 ± 0.8%; 77.5 ± 10.8%). The presence of CD9 exclusively on plasma membrane of the bovine sperm has been detected by Western blot analysis of the protein fractions after the discontinuous sucrose gradient fractionation of the bull sperm. Moreover, probable role of the sperm CD9 molecule in fertilization process of cattle has been suggested as sperm treatment with anti‐CD9 antibody significantly reduced (by 25%, p ≤ 0.001) the number of fertilized oocytes compared to control group in fertilization assay in vitro.  相似文献   

12.
Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.  相似文献   

13.
Bull Spermatozoa under Mercury Stress   总被引：4，自引：0，他引：4  
Defective sperm function is the most prevalent cause of male infertility and is difficult to treat. This study was designed to evaluate the effect of mercuric chloride (HgCl2) at 50-300 micromol/l concentration range, in vitro, on the sperm membrane and DNA integrity, viability, reduced glutathione (GSH) content and acrosomal status of the bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid [phosphate-buffered saline (PBS), pH 7.2]. I recorded a meaningful increase in the lipid peroxidation (LPO) rate and a drastic fall in the spermatocrit values under mercury additions, dominantly at 300 microM mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage of viable spermatozoa (r = -0.9, p < 0.001). GSH content was significantly impaired. Data obtained from Comet assay [single-cell gel electrophoresis (SCGE)] technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 90% of DNA breaks were double-stranded. The correlation between LPO rate and percentage of DNA breaks was found to be 0.9 (p < 0.001). Results of the gelatin test indicate that mercury is capable of altering the integrity of acrosomal membranes, showing an abnormal acrosome reaction. In this regard, a strong correlation was found between LPO rate and percentage of halos (r = -0.9, p < 0.001). In conclusion, mercury proved to be a potential oxidant in the category of 'environmental factors' to bull spermatozoa. Hence, considering the widespread use of mercury and its compounds, these metals should be regarded with more concern.  相似文献   

14.
The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed and Biociphos Plus as compared with the Tris-egg yolk based diluent Biladyl, for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mul aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl results in higher sperm survival and longevity than the use of Andromed or Biociphos Plus.  相似文献   

15.
Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein-Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 12.2c computer-assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (p < 0.0001) and progressively (p < 0.0001) motile spermatozoa were encountered in the HF breed compared with the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross-frequency (p = 0.0007) combined with a higher lateral head displacement (p = 0.0015), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (p < 0.0001). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non-significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences.  相似文献   

16.
The development and use of modern techniques, such as intracytoplasmic sperm injection (ICSI), gene knockout and sperm fluorescence in situ hybridization with chromosome- specific probes, have significantly increased our knowledge about sperm defects. We describe a new oligoasthenoteratozoospermic defect in a bull. Because of its morphological characteristics the defect was named the multinuclear-multiflagellar sperm defect. All spermatozoa in the ejaculate were abnormal. Many of the spermatozoa had multiple nuclei and multiple sperm tails. All spermatozoa lacked an acrosome, and only seldom did spermatozoa have a mitochondrial helix in the midpiece area. Meiosis and spermiogenesis were severely affected in this otherwise phenotypically normal bull. The sperm defects resembled the phenotype of a targeted gene knockout Hrb(-/-) (HIV-1 Rev-binding/interacting protein) mutant mouse strain, which is expressed as sterility in males, while females remain fertile. Since the father of this bull has been extensively used in at least three countries the defective gene has possibly become widespread in the red and white breeds (Ayrshire, Swedish Red and White, Norwegian Red) in the Nordic countries. However, it is not proved that the father of this bull is a carrier of this defect.  相似文献   

17.
The effect of Mycoplasma bovis (Donetta strain) on the ability of bull spermatozoa to interact with zona pellucida-free hamster oocytes was studied in an in vitro assay. Ejaculates of semen from a fertile Holstein bull were used fresh on the day of collection (unextended semen) as well as diluted with egg yolk-citrate and used the following day (extended semen). The addition of M. bovis to both unextended and extended semen at a mycoplasma to sperm cell ratio of 10:1 significantly reduced sperm penetration rates and the mean number of sperm per penetrated egg. Similarly, the ability of spermatozoa to form pronuclei and the activation of penetrated oocytes were adversely affected by M. bovis. No apparent effect on sperm motility was detected. When M. bovis was added to the oocytes, there was a marked reduction in the sperm penetration rates and fertilization processes suggesting that the organism affects certain oocyte function(s). The results indicate that the presence of M. bovis in semen or in the female reproductive tract may affect fertilization. Moreover, the in vitro assay with hamster oocytes was found to be useful for demonstrating the effects of contaminating microbial agents on bovine fertilization processes.  相似文献   

18.
Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein–Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high‐density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD‐adjusted Bonferroni <0.05) with the non‐compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non‐compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.  相似文献   

19.
Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR‐14?PI staining; acrosomal membrane integrity using FITC‐conjugated Pisum Sativum Agglutinin?PI labelling; mitochondrial membrane potential (Δψm) by staining with JC‐1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = ?0.41) and with plasma membrane integrity (p = 0.01; r = ?0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.  相似文献   

20.
In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.  相似文献   

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