scholarly journals Freeze-thawing impairs the motility, plasma membrane integrity and mitochondria function of boar spermatozoa through generating excessive ROS

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Bin Zhang ◽  
Yan Wang ◽  
Caihong Wu ◽  
Shulei Qiu ◽  
Xiaolan Chen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromatin Structure ◽  
Membrane Integrity ◽  
Sperm Chromatin ◽  
Mitochondrial Activity ◽  
Atp Content ◽  
Plasma Membrane Integrity ◽  
Freeze Thaw ◽  
Boar Spermatozoa

Abstract Background Cryopreservation is an efficient way to store spermatozoa and is closely associated with the quality of sperm after the freeze-thaw process. During freeze-thaw cycling, excessive reactive oxygen species (ROS) are produced, and the effects of ROS on boar sperm during cryopreservation have not been identified. Results In this study, we evaluated the quality of boar spermatozoa in different steps of cryopreservation (extension, cooling, and thawing for 30 min and 240 min) with or without boar-sperm antioxidant (N-acetylcysteine (NAC)). The ROS levels, sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, ATP content, and sperm apoptosis were assayed. After thawing, the ROS level and sperm apoptosis were significantly increased, and the sperm motility, plasma membrane integrity, mitochondrial activity, sperm chromatin structure, and ATP content were significantly impaired compared with those at the extension period and cooling period. Moreover, the addition of N-acetyl L-cysteine (NAC) reversed these changes. Conclusion The freeze-thawing of boar spermatozoa impaired their motility, plasma membrane, mitochondrial activity, sperm chromatin structure and apoptosis by producing excessive ROS. Thus, the downregulation of ROS level by antioxidants, especially the NAC, is important for manufacturing frozen pig sperm to increase reproductive cells and livestock propagation, as well as to improve the application of frozen semen in pigs worldwide.

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.

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.

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.

scholarly journals The Effect of Different Concentrations of Caffeine, Pentoxifylline and 2’-Deoxyadenosine on the Biological Properties of Frozen-Thawed Canine Semen

2019 ◽  
Vol 19 (3) ◽  
pp. 733-746
Author(s):  
Marek Lecewicz ◽  
Rafał Strzeżek ◽  
Anna M. Majewska ◽  
Piotr S. Purpurowicz ◽  
Władysław Kordan
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Phosphodiesterase Inhibitors ◽  
Biological Properties ◽  
Kinematic Parameters ◽  
Plasma Membrane Integrity ◽  
Beneficial Effects

AbstractArtificial insemination (AI) and semen cryopreservation are the most accessible and commonly used techniques for breeding domestic animals. Among many parameters, such as plasma membrane integrity and acrosome structure, one of the key factors that determine the quality of frozen-thawed samples for artificial insemination is sperm motility. Sperm motility is one of the key parameters that determine the quality of frozen-thawed samples for AI. The total number of progressively motile spermatozoa in thawed canine semen is correlated with fertility. A variety of substances were used to compare sperm motility with the control. The aim of this study was to determine the effect of semen extender supplementation with motility stimulants, pentoxifylline (PTX), caffeine (CAF) and 2’-deoxyadenosine (DX), after different post-thaw incubation times (30, 60, 120 min) on the motility, selected kinematic parameters, plasma membrane integrity and mitochondrial membrane potential of cryopreserved canine spermatozoa. During attempts to improve the quality of cryopreserved semen, the applied substances exerted beneficial effects at a concentration of 10 mM. We demonstrated that both phosphodiesterase inhibitors, caffeine and pentoxifylline, as well as 2’-deoxyadenosine increased the motility and selected kinematic parameters of thawed canine spermatozoa.

51 STRESS PRECONDITIONING OF SPERMATOZOA WITH SUBLETHAL CONCENTRATIONS OF ETHANOL IMPROVE POST-THAW SPERM QUALITY IN BULL

2015 ◽  
Vol 27 (1) ◽  
pp. 118
Author(s):  
H. Vaseghi-Dodaran ◽  
M. Zhandi ◽  
M. Sharafi ◽  
E. Nejati-Amiri ◽  
A. Nejati-Javaremi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Thiobarbituric Acid ◽  
Sublethal Concentration ◽  
Mitochondrial Activity ◽  
Mild Stress ◽  
Dependent Manner ◽  
Plasma Membrane Integrity ◽  
The Difference

A variety of controlled mild stressors have been applied for activation of temporary response in oocytes, embryos, and somatic cells. So far, several stressors have been used to induce mild stress, including that of hydrostatic pressure, osmotic stress, mechanical stress, and oxidative challenges. Based on these evidences, we hypothesised that the ethanol in sublethal concentration would be capable of generating mild stress that may ultimately leads to an adaptive response in spermatozoa. To evaluate this hypothesis, semen samples (n = 24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled for each replicate. Pooled samples were divided into 5 equal parts and each part diluted with tris-glycerol-based (Optidyl®) extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9), and 0.15 (O-E15) % (vol/vol) ethanol and frozen. After thawing, sperm motility and velocity parameters (sperm class analysis), apoptosis status (Phospatidylserin Translocation Detection commercial kit), plasma membrane integrity (eosin-Nigrosin staining), malondialdehyde concentration (thiobarbituric acid reaction), and mitochondrial activity (rhodamine-123 and propidium iodide) were evaluated. The data were analysed using Proc Mixed of SAS 9.1 (version 9.1; SAS Institute Inc., 2002, Cary, NC, USA). Tukey's test was used to compare least squares means. As a result, the O-E9 group showed higher (85.2%) percentage of total motility compared with O-E0 (73.6%), O-E3 (51.9%), and O-E15 (67.5%) groups (P < 0.05). A highest (P < 0.05) percentage of live spermatozoa were observed in the O-E9 (62.9%) group as compared with O-E0 (49.4%), O-E3 (50.3%), and O-E15 (49.6%) groups, and also the proportion of apoptotic spermatozoa in the O-E9 (10.6%) group tended to be lowest as compared with those of O-E0 (15.6%), O-E3 (17.2%), O-E15 (14.1%) groups (P > 0.05). The plasma membrane integrity was higher (P < 0.05) in O-E9 (90.8%) compared with O-E3 (75%) and O-E15 (77.2%) groups; however, the difference was not significant when the O-E9 group was compared with the O-E0 group (83.2%; P > 0.05). Obtained results revealed that malondialdehyde level was lower in O-E3 (1.03%), O-E9 (0.63%), and O-E15 (0.89%) groups compared with the O-E0 (1.94%) group (P < 0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in O-E9 (57.7%) and O-E15 (57.5%) groups compared with O-E0 (49.1%) and O-E3 (38.2%) groups (P < 0.05). These results strongly suggest that supplementation of Optidyl® extender with sublethal concentration of ethanol influences post-thawed bull sperm quality in a dose-dependent manner. However, further studies are needed to empirically determine the effect of supplementation on fertilization and pregnancy outcome.

34 Cellular effects of antifreeze proteins type I and III in extender for sheep semen cryopreservation

2021 ◽  
Vol 33 (2) ◽  
pp. 124
Author(s):  
L. F. L. Correia ◽  
C. G. Espírito-Santo ◽  
R. F. Braga ◽  
V. L. Brair ◽  
C. J. C. de Paula ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromatin Condensation ◽  
Antifreeze Proteins ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Type I ◽  
Plasma Membrane Integrity ◽  
Ram Sperm ◽  
Perivitelline Membrane ◽  
Normally Distributed

Antifreeze proteins (AFP) have been included in extenders for sperm cryopreservation to prevent ice crystal formation. Thus, this study assessed the effects of supplementing semen extender with two concentrations of AFP types I and III on the quality of frozen–thawed ram sperm. The hypothesis is that various types and concentrations of AFP enhance cryopreservation of ram sperm. Semen was collected from 4 rams, pooled in 6 replicates, and allocated into 1 of 5 treatments: Control (CONT, without AFP); AFP type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg±mL−1]; or AFP type III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg±mL−1]. Straws were placed on a metal wire net frame at 37°C and placed in a refrigerator for 2h to cool them to 5°C (−0.25°C/min). After 2h for stabilisation, straws were cooled in liquid nitrogen vapor (−15.3°C/min) and subsequently immersed (−196°C). After thawing, samples from each treatment were evaluated microscopically (sperm kinetics, plasma membrane integrity, capacitation, hypoosmotic test, acrosome status and mitochondrial activity, chromatin condensation, morphology, binding to egg perivitelline membrane, and lipoperoxidation quantification). The normal distribution of residuals was determined by Shapiro-Wilk test and homoscedasticity by Levene’s test. Normally distributed variables were analysed with one-way analysis of variance (ANOVA), followed by Tukey’s test. The non-normally distributed were analysed by Kruskal–Wallis and Dunn’s test. The repeated-measures ANOVA in general linear model (GLM) was used to effects of concentration for each AFP type in paired samples. The Greenhouse-Geisser test was applied when sphericity was not considered, followed by the Sidak test. Values of P&lt;0.05 were considered significant. Treatments affected (P&lt;0.05) kinetic parameters, plasma membrane integrity, and morphology. Linearity was greater in AFPI-0.1 (56.6±3.1%, mean±s.e.m.), AFPI-0.5 (56.9±2.2%), and AFPIII-0.5 (64.7±6.2%) than in CONT (36.8±3.0%). Straightness was greater in all AFP-supplemented extenders (overall mean, 78.6±2.8%) than in CONT (63.2±0.8%). Plasma membrane integrity was greater in AFPI-0.1 (49.1±4.6%) and AFPI-0.5 (36.6±7.3%) compared with CONT (13.0±4.4%). All AFP groups had a greater percentage of normal sperm (overall mean: 74.3±1.3%) than CONT (65.3±1.9%). There were no significant differences in percentage of sperm with functional membrane (overall mean: 16.1±3.3%), normal acrosome (11.5±4.5%), mitochondrial activity (24.5±6.5%), chromatin condensation (98.8±0.4%), perivitelline membrane binding rate (194.0±44.5 sperm/mm2), and lipoperoxidation (556.7±20.5 TBARS ng±mL−1). In conclusion, the use of AFP, predominantly type I, had potential as a cryoprotectant for ram sperm, increasing sperm cell protection, with no adverse effects on potential fertilization capacity and did not increase reactive oxygen species. This research was funded by FAPERJ, CNPq, and CAPES (Finance Code 001).

scholarly journals Semen Quality of Garut Rams feed by Different Protein Sources and Their Implementation Potential in Small Farms of West Java

2020 ◽  
Vol 20 (1) ◽  
pp. 47-55
Author(s):  
Kurnia Bagus Ariyanto ◽  
Lilis Khotijah ◽  
Dewi Apri Astuti ◽  
Raden Iis Arifiantini ◽  
Jean-Baptise Menassol
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Black Soldier Fly ◽  
Hermetia Illucens ◽  
Brachiaria Humidicola ◽  
Plasma Membrane Integrity ◽  
Protein Sources ◽  
Sperm Plasma Membrane

ABSTRACT. Maggot Hermetia illucens (Maggot Black Soldier Fly, MBSF) is an alternative protein source besides soybean meal (SBM) which may be used as a feed for improving the quality of semen particularly in Garut rams to support prolific nature. The aims of this study were to analyzed and compare the impact of different protein sources in feed on semen quality of Garut rams, and to assess the prediction ability of Garut rams to serve ewe in small-scale breeders in West Java, Indonesia. This study was conducted using a completely randomized design with 3 treatments and 4 replications, consisted of Brachiaria humidicola (BH) grass and T1 (concentrate contains 20% of SBM), T2 (concentrate contains 10% of SBM and 10% of MBSF), and T3 (concentrate contains 20% of MBSF). The parameters measured were feed consumption, semen quality (macroscopic and microscopic characteristics), also a potential ability of rams to serve ewe. The results showed there were no significant effect on protein consumption, semen volume, semen pH, semen color and consistency, sperm mass movement, sperm motility, sperm concentration, sperm morphology, and prediction potential ability to serve ewe. However, the result showed a significant effect (P0.05) on sperm viability and sperm plasma membrane integrity. Sperm plasma membrane integrity of ram feed with T3 was better than T1 and T2 (P0.05). The prediction potential ability rams to serve ewes on MBSF treatment was 38 heads, while in T1 and T2 were 43 and 57 heads, respectively. In conclusion, MBSF can be an alternative source of protein besides SBM to improve the semen quality of Garut rams.  ABSTRAK. Maggot Hermetia illucens (Maggot Black Soldier Fly; MBSF) adalah sumber protein alternatif selain bungkil kedelai (SBM) yang dapat dipergunakan sebagai pakan untuk memperbaiki kualitas semen terutama pada domba Garut untuk mendukung sifat prolifik. Tujuan penelitian ini adalah menganalisis dan membandingkan dampak pemberian sumber protein berbeda terhadap kualitas semen domba Garut dan untuk menilai kemampuan domba Garut pejantan dalam melayani betina pada peternakan rakyat di Jawa Barat, Indonesia. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) dengan 3 perlakuan dan 4 ulangan yang terdiri dari rumput Brachiaria humidicola (BH) dan T1 (konsentrat mengandung 20% SBM), T2 (konsentrat mengandung 10% SBM dan 10% MBSF), dan T3 (konsentrat mengandung 20% MBSF). Parameter yang diukur adalah konsumsi pakan, karakteristik semen (makroskopis dan mikroskopis) serta potensi domba jantan melayani betina. Hasil Penelitian menunjukkan tidak ada perbedaan signifikan pada konsumsi protein pakan, volume semen, pH semen, warna dan konsistensi semen, gerakan massa sperma, motilitas sperma, konsentrasi sperma, morfologi sperma, dan prediksi potensi pejantan dalam melayani betina. Namun, hasil penelitian menunjukkan terdapat perbedaan (P0.05) pada viabilitas sperma dan membran plasma utuh sperma. Membran plasma utuh pada perlakuan T3 lebih baik dibandingkan perlakuan T1 dan T2 (P0.05). Prediksi potensi betina terlayani dari pejantan yang diberi pakan MBSF adalah 38 ekor, sedangkan yang diberi SBM dan kombinasinya adalah 43 dan 57 ekor. Kesimpulan penelitian ini adalah MBSF dapat menjadi alternatif sumber protein selain bungkil kedelai dalam memperbaiki kualitas sperma domba Garut.

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