scholarly journals Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

2016 ◽  
Vol 68 (3) ◽  
pp. 620-628 ◽  
Author(s):  
L.S.L.S. Reis ◽  
A.A. Ramos ◽  
A.S. Camargos ◽  
E. Oba
Keyword(s):  
Plasma Membrane ◽  
Mitochondrial Membrane ◽  
Sexual Maturity ◽  
Wave Motion ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Circumference ◽  
Acrosomal Membrane ◽  
Sperm Membrane

ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion), scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot) and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342). The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA

2015 ◽  
Vol 27 (1) ◽  
pp. 222
Author(s):  
M. Bianchi Rodrigues Alves ◽  
A. Furugen Cesar de Andrade ◽  
R. Paes de Arruda ◽  
L. Batissaco ◽  
R. Lançoni ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Insulation ◽  
Testicular Degeneration

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P < 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P < 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P < 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P < 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.

319 VALIDATION OF THE CARBOXYFLUORESCEIN DIACETATE ASSOCIATED WITH PROPIDIUM IODIDE FOR MURINE SPERM ACROSOME AND PLASMA MEMBRANE INTEGRITY

2010 ◽  
Vol 22 (1) ◽  
pp. 315
Author(s):  
F. M. Sevciuc ◽  
C. M. Mendes ◽  
F. R. O. de Barros ◽  
W. B. Feitosa ◽  
R. Simões ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Propidium Iodide ◽  
Linear Regression Analysis ◽  
Membrane Integrity ◽  
Transgenic Animals ◽  
Exogenous Dna ◽  
Plasma Membrane Integrity ◽  
Standard Curve ◽  
Acrosomal Membrane ◽  
Simultaneous Evaluation

The spermatozoa is an ideal vehicle for genetic modification, production of transgenic animals, as well as a biotechnological tool for sperm-mediated gene transfer. However, in order to achieve successful sperm fertilization and exogenous DNA integration, it is necessary for viable cells to remain intact, allowing the sperm to penetrate the oocyte. Fluorescent probes allow evaluation of morphological and functional characteristics of cells, which can be evaluated separately or simultaneously. Therefore, the aim of this study was to validate the simultaneous evaluation of the integrity of plasma and acrosomal membranes of murine sperm using the probes carboxyfluorescein diacetate (CF) and propidium iodide (PI). In order to validate, a standard curve was performed. Sperm were obtained from epididymis and vas deferens from CD-1 mice (8 to 16 weeks of age). Recovered samples were diluted in PBS and then divided into 2 aliquots: one prepared with fresh semen (FS) and the other submitted to Percoll gradient (45%/90%) followed by flash-freezing in liquid nitrogen and thawing (FTP) to induce acrosome damage. Samples were prepared with the following average of FS:FTP: 100:0 (T100), 50 : 50 (T50), and 0 : 100 (T0). Samples were stained using 2 μL Hoescht 33342 (40 μLmL-1 in Dulbecco’s phosphate buffered saline), 3 μL of PI (0.5 mg mL-1 in PBS), 3 μL of CF (0.46 mg mL-1 in DMSO), and were incubated for 8 min at room temperature. After staining, the samples were placed on a slide, coverslipped, and evaluated immediately by epifluorescent microscopy. The Hoescht, PI, and CF fluorescence was detected using a filter with excitation at 352, 538, and 495 nm and emission at 455, 617, and 517 nm, respectively. Approximately 200 sperm cells per slide were examined and classified based on the fluorescence emitted from each probe. Spermatozoa CF+/IP- were considered as intact membranes, CF+/PI+ as acrosome membrane intact and plasma damaged, CF-/PI+ as damaged membranes, and CF-/PI- as acrosome membrane damaged and plasma intact. Hoeschst was used as positive dye. This experiment was replicated 6 times per group, and for statistical analyses, the data of plasma and acrosomal membrane integrity (dependent variables) in the treatments T0, T50, and T100 (independent variables) were submitted to simple linear regression analysis by STATVIEW software (SAS Institute Inc., Cary, NC, USA). The CFDA/PI probes were suitable for the analysis of acrosomal and membrane status of murine sperm and showed a high determination coefficient to plasma membrane integrity (R2 = 0.81; Y = 0.5412x + 6.375) and acrosome integrity (R2 = 0.85; Y = 0.5653x + 11.653). The described protocol was efficient for the simultaneous evaluation of plasma and acrosomal membrane integrity of murine spermatozoa, proving that CFDA can be employed to access acrosomal integrity as an alternative to FITC-PSA. Financial support: FAPESP.

108 EFFECT OF DIMETHYLFORMAMIDE AND METHYLFORMAMIDE ON OXIDATIVE STATUS OF MANGALARGA STALLIONS CRYOPRESERVED SEMEN

2010 ◽  
Vol 22 (1) ◽  
pp. 213
Author(s):  
F. A. Oliveira Neto ◽  
M. Nichi ◽  
E. G. A. Perez ◽  
J. R. C. Gurgel ◽  
G. H. Ferreira ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Plasma Membrane ◽  
Membrane Integrity ◽  
The Other ◽  
Lipid Peroxidation Product ◽  
Oxygen Species ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Peroxidation Product ◽  
Acrosome Integrity

Cryopreservation of equine semen has been widely studied by several research groups because of the large breed and individual variation in sperm freezability. A key factor in sperm cryopreservation is the high incidence of oxidative stress, an imbalance between reactive oxygen species (ROS) and antioxidant protection, which impairs sperm functionality by attacking plasma membrane, acrosome, mitochondria, and DNA. In order to study the resistance of equine spermatozoa to different reactive oxygen species (ROS), sperm samples from 4 Mangalarga stallions were collected using an artificial vagina. Samples were cryopreserved in extenders containing dimethylformamide (DMF) or methylformamide (MF). After thawing and washing, sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hidroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were evaluated using the 3-3′ diamino benzidine (DAB) stain, as an indicator of mitochondrial activity; the eosin nigrosin staining, to evaluate plasma membrane integrity; the simple stain (fast green/Bengal rose), to assess acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation product. Statistical analysis was performed using the Student t-test and LSD test. Results showed that sperm mitochondrial potential of frozen-thawed samples in MF was highly susceptible to the attack of hydroxyl radical and hydrogen peroxide. No effect of ROS was observed on membrane and acrosome integrity. On the other hand, samples cryopreserved in DMF showed no differences in susceptibility to ROS. When evaluating the main effects of different extenders, results showed a higher protective effect of the MF extender on acrosome integrity and mitochondrial potential (MF: 12.1 ± 2.2 and 7.8 ± 2.3% v. DMF: 3.4 ± 0.7 and 1.1 ± 0.7%, respectively, P < 0.05). However, a negative effect of MF extender was observed regarding the percentage of sperm showing intact membrane and TBARS content (MF: 2.0 ± 0.8% and 517 ± 115 ng/106 sperm v. DMF: 20.6 ± 1.7% and 118 ± 44 ng/106 sperm, respectively, P < 0.05). A strong negative correlation was found between TBARS and plasma membrane integrity (r = -0.88; P = 0.004) for samples cryopreserved in DMF, whereas a positive correlation was found between TBARS and sperm with full mitochondrial potential (r = 0.73; P = 0.04). Results of the present study indicate that DMF may play a role in the protection of sperm against the attack of ROS. However, such action is apparently limited to the plasma membrane. On the other hand, the MF-supplemented extender exerts an intracellular protection. Therefore, the antioxidant therapy, especially hydrogen peroxide and hydroxyl radical scavengers, may be an alternative to improve the post-thaw quality of MF-supplemented cryopreserved semen in stallions, by increasing extracellular antioxidant capacity. The authors thank Nutricell for financial support and the media used in the present experiment.

scholarly journals Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 529-536 ◽  
Author(s):  
Chihiro Kanno ◽  
Sung-Sik Kang ◽  
Yasuyuki Kitade ◽  
Yojiro Yanagawa ◽  
Yoshiyuki Takahashi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Fluorescence Microscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Bovine Spermatozoa ◽  
Acrosomal Integrity

SummaryThe present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen–thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE–PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE–PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE–PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE–PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

scholarly journals Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation

2020 ◽  
Vol 21 (17) ◽  
pp. 6415
Author(s):  
Filipa Bubenickova ◽  
Pavla Postlerova ◽  
Ondrej Simonik ◽  
Jitka Sirohi ◽  
Jiri Sichtar
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Plasma Protein ◽  
Seminal Plasma ◽  
Protein Fraction ◽  
Membrane Integrity ◽  
Protein Fractions ◽  
Heparin Binding ◽  
Plasma Membrane Integrity ◽  
Acrosomal Membrane

Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep−) fractions. The addition of three concentrations—125, 250, and 500 µg/mL—of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep− fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions.

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