scholarly journals Effects of Arsenite and Antioxidants on Sperm Motility, Plasma Membrane Integrity, Mitochondrial Activity, and Lipid Peroxidation in Pigs

2017 ◽  
Vol 27 (5) ◽  
pp. 517-523 ◽  
Author(s):  
Han-Su Kim ◽  
Yu-Sub Lee ◽  
Sang-Hee Lee ◽  
Hee-Tae Cheong ◽  
Choon-Keun Park ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Plasma Membrane Integrity

scholarly journals Effects of the addition of docosahexaenoic acid and ?-tocopherol on quality of equine spermatozoa stored at 5°C

2020 ◽  
Vol 41 (1) ◽  
pp. 167 ◽  
Author(s):  
Breno Fernandes Barreto Sampaio ◽  
Bruno Gomes Nogueira ◽  
Maria Inês Lenz Souza ◽  
Eliane Vianna da Costa-e-Silva ◽  
Carmem Estefânia Serra Neto Zúccari
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Docosahexaenoic Acid ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Membrane Composition ◽  
Artificial Vagina

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and ?-tocopherol (?-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; ?-Toh; DHA/?-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of ?-Toh; : 10 ng mL-1 of DHA + 2 mM of ?-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/?-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), ?-Toh (1,890.8 ± 749.5) and DHA/?-Toh (1,821.1 ± 627.2). In conclusion, ?-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

20 LIPID PEROXIDATION AND GENERATION OF HYDROGEN PEROXIDE FROM SUBFERTILE STALLION SPERMATOZOA DURING STORAGE AT REFRIGERATION TEMPERATURE

2013 ◽  
Vol 25 (1) ◽  
pp. 157 ◽  
Author(s):  
P. N. Guasti ◽  
C. P. Freitas-Dell'aqua ◽  
R. R. D. Maziero ◽  
G. A. Monteiro ◽  
F. P. Hartwig ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Sperm Cells ◽  
Band Pass Filter ◽  
Membrane Lipid Peroxidation ◽  
Pass Filter ◽  
Plasma Membrane Integrity

The aim of this study was to evaluate the generation of hydrogen peroxide (H2O2) and membrane lipid peroxidation of subfertile spermatozoa stored at 5°C for 24 h. Semen samples, collected from 5 subfertile stallions (≤40% of conception rate), were diluted in a skim-based extender (BotuSemen; Botupharma) and stored in a passive transport container at 5°C over a period of 24 h. Sperm motility was determined by computer-assisted semen analysis (CASA; IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA) for total motility (TM), progressive motility (PM), and rapid sperm (RAP). Plasma membrane integrity, generation of H2O2, and membrane lipid peroxidation were determined by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Franklin Lakes, NJ, USA). For evaluation of acrosome and plasma membrane integrity, samples were stained with Hoechst 33342 dye (H33342; Molecular Probes, Eugene, OR, USA), iodide propidium (IP; Sigma, St. Louis, MO, USA), and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). For the assessment of generation of H2O2, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma), H33342, and IP were added to the sperm suspension. For the assessment of sperm lipid peroxidation, samples were stained with C11-BODIPY581/591 (Molecular Probes), H33342, and IP. Samples were evaluated after semen collection (0 h) and after 24 h of cooled storage. A total of 10 000 gated events were analyzed per sample by flow cytometry. The green fluorescence (FL1) was collected through a 580-nm band-pass filter and the red fluorescence (FL3) through a 635-nm band-pass filter. Statistical analysis was performed using GraphPad Prism version 4.03 (2005; GraphPad Software Inc., La Jolla, CA, USA), through paired t-test to identify the significant differences (P ≤ 0.05). In general, sperm motility parameters of TM and RAP, and acrosome and plasma membrane integrity significantly decreased after 24 h of refrigeration at 5°C (P ≤ 0.05). Interestingly, no differences were found in PM at 0 and 24 h (P ≥ 0.05). The percentage of sperm cells with high generation of H2O2 did not differ at 0 and 24 h (P ≥ 0.05), whereas the percentage of sperm cells with membrane peroxidation increased (0.56 ± 0.3 v. 2.24 ± 1.3; P ≤ 0.05) at these periods. The percentage of viable sperm cells with low generation of H2O2 had a significant decrease from 22.6 ± 11.2 at 0 h to 0.08 ± 0.1 after 24 h of storage (P ≤ 0.05), although no differences were found in the percentage of viable sperm cells without membrane peroxidation (P ≥ 0.05). In conclusion, the cooled storage of subfertile spermatozoa for 24 h drastically decreased the number of viable spermatozoa with low generation of H2O2 and increased the percentage of membrane lipid peroxidation, which is related to the decrease in sperm motility and increase in dead sperm. These results make it difficult to use refrigerated semen of subfertile stallions with poor semen quality in commercial breeding programs. São Paulo Research Foundation (FAPESP) is acknowledged for supporting this research.

scholarly journals Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Bruna Farias Brito ◽  
Bárbara Mara Bandeira Santos ◽  
Leonardo Alves Rodrigues Cabral ◽  
David Baruc Cruvinel Lima ◽  
Cristiane Clemente De Melo Salgueiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Plasma Membrane Integrity ◽  
Significant Difference ◽  
Motility Parameters

Background:  Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P > 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes.

87 OSMOTIC SENSITIVITY OF OKAPI SPERMATOZOA AND DEVELOPMENT OF CRYOPRESERVATION PROTOCOLS USING CRYOMICROSCOPY

2008 ◽  
Vol 20 (1) ◽  
pp. 124 ◽  
Author(s):  
L. M. Penfold
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Repeated Measures ◽  
Membrane Integrity ◽  
Latin Square ◽  
Crystal Formation ◽  
Plasma Membrane Integrity ◽  
Power Of The Test ◽  
Before And After ◽  
Cooling Effects

Okapi would benefit from artificial insemination with frozen-thawed sperm in cases where aggression prevents mating or where individuals are geographically disparate. Effective sperm cryopreservation is a prerequisite to this goal. Ejaculates (n = 20) were collected from 7 anesthetized adult male okapi housed individually, or with a female for breeding, throughout the year by electroejaculation, and semen and sperm parameters were assessed. Semen aliquots were centrifuged; resuspended in 500 µL of PBS with the osmolarity adjusted to 35, 75, 150, 600, 1200, and 2400 mOsm; and incubated for 30 min before returning to isosmotic conditions. Semen was extended in TEST containing 1%, 2%, or 4% glycerol with or without 0.5% Equex (Minitube, Verona, WI, USA); 5-µL aliquots were cooled in a Latin square design on a Linkam BCS 196 cryomicrostage (Linkam Scientific, Tadworth, Surrey, UK) at 20�C min–1 to –6� –12�C at which point ice crystal formation was induced (seeded), and cooled further to –70�C before warming at 50�C min–1 to 35�C (okapi body temperature). To investigate cooling effects only, raw ejaculate was cooled to –6�C without seeding and warmed to 35�C. Percent sperm motility and plasma membrane integrity (PMI) were recorded before and after treatments. Differences were examined using one-way repeated measures analysis of variance. No differences in motility, total sperm numbers, or percent normal morphology were observed throughout the year (P > 0.05), although the power of the test was low so that negative findings should be interpreted cautiously. Mean semen volume was 1.3 � 0.19 mL, sperm motility was 29 � 3.2%, with a PMI of 39 � 6.8%; 48 � 2.8% were morphologically normal. High proportions of non-motile, plasma membrane-damaged cells were noted in every ejaculate, and whiplash motility, possibly indicating spontaneous capacitation, was observed in several ejaculates 1 h after collection. Motility was dramatically reduced on either side of isosmotic conditions and was more sensitive to osmotic pressure than was plasma membrane integrity. Cooling of raw ejaculate to sub-zero temperatures without freezing did not result in any loss of motility or PMI, indicating cold tolerence. Superior results were obtained when sperm were frozen-thawed in TEST containing 4% glycerol with 0.5% Equex. Findings indicate that okapi semen collected by electroejaculation routinely contain high numbers of non-motile and plasma membrane-damaged spermatozoa, apparently unrelated to season or the length of time since the male was housed with a breeding female. Okapi spermatozoa are remarkably intolerant of departures from isosmotic conditions, indicating a lack of ability to regulate or withstand volume excursions during osmotic stress events; however, cooling to sub-zero temperatures in the absence of cryoprotectant did not reduce percent sperm motility or PMI, indicating resistance to cold shock. Increasing and maintaining proportions of motile, membrane-intact spermatozoa prior to and during cryopreservation will be critical for development of freezing protocols for this species.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function

2019 ◽  
Vol 31 (9) ◽  
pp. 1434
Author(s):  
Andressa Dalmazzo ◽  
João D. A. Losano ◽  
Daniel S. R. Angrimani ◽  
Isabel V. A. Pereira ◽  
Marcelo D. Goissis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Expression ◽  
Membrane Integrity ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Mitochondrial Activity ◽  
Hormone Binding ◽  
Plasma Membrane Integrity ◽  
Gene And Protein Expression ◽  
Acrosome Integrity

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

The effects of chronic administration of ghrelin on rat sperm quality and membrane integrity

2009 ◽  
Vol 59 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Arash Kheradmand ◽  
Majid Taati ◽  
Homayoon Babaei
Keyword(s):  
Plasma Membrane ◽  
Treatment Group ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Chronic Administration ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Significant Difference

AbstractAlthough ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the effects of chronic administration of ghrelin on the motility, plasma membrane integrity and concentration of rat spermatozoa. 45-d male Wistar rats were scheduled for the study and were divided into control and treatment groups. In the treatment group, 1 nmol of ghrelin was administered as sc injection for 10 consecutive days or vehicle (physiological saline) to the control rats. Sperm collection was achieved by killing of the rats on days 15, 25 and 50 after first injection. Total sperm motility and forward progressive movement did not exhibit significant difference during the experiment, although, there was a tendency for greater motion rate on d 15 and 25 in the treated rats compared to the control group. Plasma membrane integrity (HOS-reacted spermatozoa) was significantly higher in the treated animals, especially on day 15 as well as day 25, because of possible antioxidant properties of ghrelin. This value was statistically higher on day 15 than that of day 25 (P <0.05). Likewise, there was a significant correlation between the FPM (P <0.0001, r = 0.79) and TSM (P <0.01, r = 0.52) with the HOS test percentage in the treatment group. It was not observed statistically difference in the sperm concentration between groups during all of the experimental days. In conclusion, chronic administration of ghrelin (similar to induced by energy deficiency such as fasting) increased the integrity of sperm membrane, however, the sperm motility and concentration did not display any alterations.

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

2010 ◽  
Vol 13 (4) ◽  
pp. 571-579 ◽  
Author(s):  
W. Kordan ◽  
M. Lecewicz ◽  
R. Strzeżek ◽  
A. Dziekońska ◽  
L. Fraser
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Mitochondrial Function ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Platelet Activating Factor ◽  
Computer Assisted ◽  
Atp Content ◽  
Plasma Membrane Integrity ◽  
Semen Extender

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa 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 × 10-3M, 1 × 10-4M, 1 × 10-5M and 1 × 10-6M) 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 × 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 × 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 × 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.

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