80 CHOLESTEROL-LOADED METHYL-BETA-CYCLODEXTRIN IMPROVES BOAR SPERMATOZOA CRYOPROTECTANT ADDITION AND REMOVAL TOLERANCE

2009 ◽  
Vol 21 (1) ◽  
pp. 140
Author(s):  
E. M. Walters ◽  
J. D. Benson ◽  
A. Rieke ◽  
J. K. Graham ◽  
J. K. Critser
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Tolerance Limits ◽  
Permeability Characteristics ◽  
Flow Cytometry Data ◽  
Osmotic Tolerance ◽  
Motile Cells ◽  
Boar Sperm ◽  
Chilling Sensitivity ◽  
Sperm Membrane

Difficulties associated with the cryopreservation of boar sperm include their sensitivities to osmotic stresses and chilling sensitivity. We investigated the effects of cholesterol-loaded cyclodextrin (CLC) on boar sperm motility and membrane integrity following exposure to various osmolalities. Samples were collected using the gloved hand method from crossbred boars, and ejaculates having greater than 75% motility were extended 1:3 with Androhep (Minitube Inc., Verona, WI) for this study. Samples were centrifuged at 700g for 5 min, and the resulting pellets were resuspended to 1.2 × 108 cells mL–1 in Androhep. Samples were then treated with 0, 1.5, or 3.0 mg of CLC/1.2 × 108 cells mL–1 for 10 min at room temperature. In experiment 1, samples were aliquoted into 1.5-mL centrifuge tubes, centrifuged at 700g for 5 min and the sperm exposed to Dulbecco’s PBS at different osmolalities for 5 min before being returned to 300 mOsm by adding Dulbecco’s PBS solutions at differing osmolalities. After returning the sperm to isosmotic conditions, sperm motility was analyzed. In experiment 2, samples were treated as in experiment 1 and following exposure to the various osmolalities, sperm were stained with Alexa 488-PNA and propidium iodide to determine sperm membrane integrity. Ten thousand sperm per treatment were analyzed by flow cytometry. Data were analyzed by standard ANOVA. The CLC-treated sperm (normalized means ± SEM; 33 ± 16, 80 ± 8, 86 ± 5, 100, 64 ± 4, 7 ± 3, 0 ± 0, respectively) exhibited greater percentages of motile cells following hypo-isosmotic exposure than control sperm (4 ± 1.6, 33 ± 9.6, 84 ± 7.1, 100, 37 ± 5.5, 3 ± 1.6, 0 ± 0, respectively), and there was a tendency for CLC-treated sperm (P = 0.0225) to maintain motility following hyper-isosmotic exposure. In addition, CLC-treated sperm (87 ± 4, 93 ± 1, 95 ± 1, 93 ± 2, 88 ± 4, 83 ± 3, 41 ± 9, respectively; P < 0.05) maintained greater percentages of membrane integrity following treatment with anisosmotic solutions compared with controls (29 ± 8, 63 ± 10, 81 ± 7, 92 ± 3, 73 ± 8, 44 ± 5, 21 ± 9, respectively). Using a combination of these osmotic tolerance data with previously published boar sperm membrane permeability characteristics, we mathematically modeled the number of steps needed for the addition or removal of cryoprotectants. Computer simulations indicate that an abrupt addition of 1 m glycerol will cause boar sperm to exceed their osmotic tolerance limits unless they are treated with 3 mg of CLC. Moreover, the addition of 1 m EG causes boar sperm to exceed all osmotic tolerance limits and therefore, the addition and removal of EG requires multiple-step protocols. However, the addition and removal of 1 m DMSO maintains volume excursions well within the osmotic tolerance limits with the addition of cholesterol (1.5 and 3 mg). Empirical data for addition of CPA have shown similar results as seen with the computer simulation. These data support the hypothesis that adding cholesterol to porcine sperm broadens their osmotic tolerance limits and potentially provide a mechanism to increase post-thaw survival of porcine sperm.

Kriopreservasi Semen Kambing Boer dengan Konsentrasi Pengencer Nira Aren dan Gliserol Berbeda

2019 ◽  
Vol 6 (1) ◽  
pp. 1
Author(s):  
Muhammad Riyadhi ◽  
Anis Wahdi ◽  
Muhammad Rizal
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Boer Goat ◽  
Palm Juice ◽  
Sperm Membrane ◽  
Live Sperm

ABSTRAK                                                                        Penelitian bertujuan untuk mengetahui efektivitas nira aren sebagai pengencer alternatif dalam proses pembekuan (kriopreservasi) semen kambing boer.Kriopreservasi semen kambing boer menggunakan pengencer tris-gliserol-kuning telur (P1 73-7-20%), nira aren-gliseol-kuning telur(masing-masing P2 74-6-20%, P3 73-7-20%, dan P4 72-8-20%) dan andromed (P5 tanpa mengandung kuning telur dan gliserol). Parameter evaluasi meliputi motilitas, viabilitas, dan membrane plasma utuh setelah pengenceran, ekuilibrasi dan thawing.  Evaluasi motilitas pasca thawing menunjukkan P5 52% berbeda nyata (P<0.05) dengan P1 42%, selanjutnya P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 8%, P3 6% dan P4 12%.  Viabilitas pasca thawing menunjukkan P5 65,4% tidak berbeda nyata (P>0,05) dengan P1 61,8%, akan tetapi P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 26,2%, P3 29,8%, dan P4 34%.  Membran plasma utuh (MPU) pasca thawing menunjukkan P5 66,2% tidak berbeda nyata (P>0,05) dengan P1 65,4%, akan tetapi keduanya berbeda sangat nyata (P<0.05) dengan P2 39%, P3 38%, dan P4 36,2%.  Disimpulkan kriopreservasi semen kambing boer dengan pengencer nira aren dan gliserol pada konsentrasi berbeda belum dapat dipergunakan sebagai sumber bibit berdasarkan standar nasional Indonesia.Kata Kunci : Kambing boer, semen, nira arenABSTRACTThe experiment was conducted to determine the effect of sugar palm juice as alternative extender for cryopreservation process of boer semen.Tris-glycerol-egg yolk (P1 73-7-20%), Sugar palm juice-glyserol-egg yolk (P2 74-6-20%, P373-7-20%, dan P4 72-8-20%), and andromed (P5) used as a extender  in the cryopreservation process of boer semen.  Sperm motility (%), live sperm (%) and sperm membrane integrity (%) were recorded after diluted, equilibration and freeze-thawing.  Result of post thawing motility showed that P5 52% was significantly different (P <0.05) with P1 42%, then P5 and P1 were significantly different (P <0.05) with P2 8%, P3 6% and P4 12%. Viability after thawing showed P5 65.4% was not significantly different (P> 0.05) with P1 61.8%, but P5 and P1 significantly different (P <0.05) with P2 26.2%, P3 29.8 %, and P4 34%. Spermmembrane integrity post-thawing showed P5 66.2% was not significantly different (P> 0.05) with P1 65.4%, but both were very significantly different (P <0.05) with P2 39%, P3 38% and P4 36.2%. Conclusions, sugar palm juice-glycerol-egg yolk with differentconcentrationsineffectively as an alternative extenderin cryopreservation of boer semen.Keywords: boer goat, semen, sugar palm juice

scholarly journals Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Heder Nunes Ferreira ◽  
José Carlos Ferreira-Silva ◽  
Jorge Motta Rocha ◽  
Pamela Ramos-Deus ◽  
Joane Isis Travassos Ribeiro ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Genetic Factors ◽  
Membrane Integrity ◽  
Sperm Dna Fragmentation ◽  
Fertility Rates ◽  
Livestock Species ◽  
Multiple Origins ◽  
Sperm Dna ◽  
Sperm Membrane

Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability.

scholarly journals Screening Mold Colonies by Using Two Toxicity Assays Revealed Indoor Strains of Aspergillus calidoustus Producing Ophiobolins G and K

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 683 ◽  
Author(s):  
Marja Johanna Salo ◽  
Tamás Marik ◽  
Ottó Bencsik ◽  
Raimo Mikkola ◽  
László Kredics ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Proliferation ◽  
Sperm Motility ◽  
High Performance ◽  
Biological Activities ◽  
Membrane Integrity ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Data ◽  
Toxic Substances ◽  
Boar Sperm

The occurrence and toxin production of the opportunistic pathogen Aspergillus calidoustus in Finnish buildings is not well documented in the literature. We tracked and identified four A. calidoustus colonies cultivated from indoor settled dusts and revealed the biological activities of crude biomass extracts. The toxic substances were identified as 6-epi-ophiobolin K, ophiobolin K, and ophiobolin G by high-performance liquid chromatography–mass spectrometry (HPLC-MS) based on chromatographic and mass spectrometry data (MS and MS/MS) on the crude extract of A. calidoustus strain MH34. A total of 29 fungal colonies collected from settled dust in an office room reported for indoor-air-related illnesses were screened for toxins that inhibited boar sperm motility in the BSMI (boar sperm motility inhibiting) assay and cell proliferation in the ICP (inhibition of cell proliferation) assays with PK-15 cells. Out of the 27 colonies tested as toxic, 12 colonies exhibiting conidiophores representative of the genera Chaetomium, Penicillium, and Paecilomyces were excluded from the study, while 13 colonies exhibited Aspergillus-like conidiophores. Biomass suspensions of these colonies were divided into two categories: Category 1 colonies (n = 4), toxic in the BSMI assay and the ICP assays, emitted blue fluorescence and grew at 37 °C; Category 2 colonies (n = 9), only toxic in the ICP assay, emitted orange fluorescence and exhibited limited or no growth at 37 °C. Colonies in Category 1 were pure-cultured, and the strains were named as MH4, MH21, MH34, MH36. Strain MH34 was identified as A. calidoustus by the internal transcribed spacer (ITS) sequences. Ethanol-soluble dry substances extracted from the biomass of the pure cultures exhibited a toxicological profile in the BSMI assay, SMID (sperm membrane integrity damage) assay, and ICP assay similar to that exhibited by pure ophiobolin A. Overall, the viable conidia of A. calidoustus in indoor settled dusts deserve attention when potentially hazardous mold species are monitored.

Tolerance of spermatozoa to hypotonic stress: role of membrane fluidity and correlation with cryosurvival

2015 ◽  
Vol 27 (2) ◽  
pp. 285 ◽  
Author(s):  
Harriëtte Oldenhof ◽  
Anna Heutelbeck ◽  
Anne-Kathrin Blässe ◽  
Heinrich Bollwein ◽  
Gunilla Martinsson ◽  
...  
Keyword(s):  
Membrane Fluidity ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Freeze Tolerance ◽  
Individual Variability ◽  
Temperature Range ◽  
Hypotonic Stress ◽  
Osmotic Tolerance ◽  
Bovine Spermatozoa

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15–30°C temperature range, whereas membrane stability was found to be decreased at 4 and 37°C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30°C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22°C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170 mOsm kg–1 among different stallions. Clear correlations were found for pre- versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions.

Treating boar sperm with cholesterol-loaded cyclodextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability

2011 ◽  
Vol 129 (3-4) ◽  
pp. 209-220 ◽  
Author(s):  
Cristina Tomás ◽  
Eva Blanch ◽  
Marta Hernández ◽  
María A. Gil ◽  
Jordi Roca ◽  
...  
Keyword(s):  
Tolerance Limits ◽  
Osmotic Tolerance ◽  
Boar Sperm

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

2005 ◽  
Vol 64 (1) ◽  
pp. 191-201 ◽  
Author(s):  
M. Spinaci ◽  
M. De Ambrogi ◽  
S. Volpe ◽  
G. Galeati ◽  
C. Tamanini ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Blastocyst Development ◽  
Boar Sperm ◽  
Sperm Membrane

Resveratrol protects boar sperm in vitro via its antioxidant capacity

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 417-424
Author(s):  
Linlin Sun ◽  
Xiaoteng Fan ◽  
Yao Zeng ◽  
Liqian Wang ◽  
Zhendong Zhu ◽  
...  
Keyword(s):  
Antioxidant Capacity ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
B Cell Lymphoma ◽  
Membrane Potentials ◽  
Cooling Process ◽  
Boar Sperm ◽  
Fast Cooling ◽  
Acrosome Integrity

SummaryThe objective of the present study was to elucidate whether resveratrol could facilitate the survival of boar sperm during liquid preservation and fast cooling processes. Boar semen were diluted with Modena extender containing different concentrations of resveratrol. Sperm motility was evaluated by visual estimation. Membrane integrity, acrosome integrity and mitochondrial membrane potentials were measured by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. Moreover, the levels of reactive oxygen species (ROS), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were measured using commercial assay kits. B-cell lymphoma protein-2 (BCL2) content was determined by western blotting. During liquid preservation at 17oC, the addition of 50 μM resveratrol to the Modena extender significantly improved sperm motility, membrane integrity, acrosome integrity, and sperm mitochondrial membrane potentials. Similar results were also observed in the 150 μM resveratrol group during the fast cooling process. Furthermore, addition of resveratrol led to a decrease of ROS and MDA, and an increase in the content of T-AOC and BCL2. These observations suggest that addition of resveratrol to Modena extender protects boar sperm against oxidative stress. The optimal concentrations of resveratrol are 50 μM and 150 μM during liquid preservation and fast cooling process, respectively.

scholarly journals Beneficial Role of Ginger Powder (Zingiber officinale) against Acephate-induced Reprotoxicity in Adult Male Rats

2021 ◽  
pp. 50-59
Author(s):  
V. Madhavi ◽  
U. Kanchana Ganga ◽  
S. B. Sainath ◽  
B. Kishori
Keyword(s):  
Body Weight ◽  
Sperm Motility ◽  
Sperm Count ◽  
Membrane Integrity ◽  
Male Rats ◽  
Sperm Viability ◽  
Marker Enzymes ◽  
Group I ◽  
Sperm Membrane

Aims: The present study was aimed to investigate the protective role of ginger against acephate-induced testicular toxicity in adult rats. Methodology: Rats were allocated into four groups where animals in group I served as controls, while animals in group II, III and group IV were treated as experimental rats. Rats in groups II, III and IV were treated with acephate (50mg/kg body weight), ginger (100mg/kg body weight) and combination of both acephate and ginger, respectively over a period of 60 days. After completion of experimental period sperm count, sperm viability, sperm motility, sperm membrane integrity, testicular steroidogenic marker enzymes (3β-HSD and 17β-HSD, serum testosterone and testicular architecture was performed in both control and experimental rats. Results: Relative weights of reproductive organs, sperm count, sperm viability, sperm motility and sperm membrane integrity were significantly decreased in acephate treated rats over controls. Acephate administration also reduced the circulatory levels of testosterone associated with a significant reduction in the testicular steroidogenic marker enzymes (3β-HSD and 17β-HSD) in rats. The testicular architecture was disrupted in acephate intoxicated rats. In contrast, ginger administration significantly recovered the acephate-induced suppressed selected reproductive parameters with increased circulatory levels of testosterone and restoration of sperm endpoints in as compared to acephate alone treated rats. No significant changes were observed in any of the selected reproductive endpoints in ginger treated rats as compared to controls. Conclusion: The results can be concluded that supplementation of ginger mitigates the negative effects of acephate on male reproductive health via amelioration of testicular setroidogenesis and spermatogenesis and epididymal sperm maturation events in rats.

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants

2016 ◽  
Vol 28 (12) ◽  
pp. 1990 ◽  
Author(s):  
D. Acha ◽  
M. Hidalgo ◽  
I. Ortiz ◽  
M. J. Gálvez ◽  
J. J. Carrasco ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Equus Asinus ◽  
Sperm Membrane ◽  
Freeze Test ◽  
Pooled Samples

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P < 0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P < 0.001), whereas DMSO affected sperm motility and membrane integrity (P < 0.001). DMFA 2.5% yielded higher (P < 0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

scholarly journals Exogenous Oleic Acid and Palmitic Acid Improve Boar Sperm Motility via Enhancing Mitochondrial Β-Oxidation for ATP Generation

Animals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 591 ◽  
Author(s):  
Zhendong Zhu ◽  
Rongnan Li ◽  
Chengwen Feng ◽  
Ruifang Liu ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Palmitic Acid ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Female Reproductive Tract ◽  
Positive Effects ◽  
Boar Sperm ◽  
Atp Generation ◽  
Acrosome Integrity

It takes several hours for mammalian sperm to migrate from the ejaculation or insemination site to the fertilization site in the female reproductive tract in which glucose, amino acids, and fatty acids are regarded as the primary substrates for ATP generation. The present study was designed to investigate whether oleic acid and palmitic acid were beneficial to boar sperm in vitro; and if yes, to elucidate the mechanism that regulates sperm motility. Therefore, the levels of oleic acid and palmitic acid, motility, membrane integrity, acrosome integrity, and apoptosis of sperm were evaluated. Moreover, the enzymes involved in mitochondrial β-oxidation (CPT1: carnitine palmitoyltransferase 1; ACADVL: long-chain acyl-coenzyme A dehydrogenase) were detected with immunofluorescence and Western blotting. Consequently, the ATP content and the activities of CPT1, ACADVL, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were also measured. We observed that CPT1 and ACADVL were expressed in boar sperm and localized in the midpiece. The levels of oleic acid and palmitic acid were decreased during storage at 17 °C. The addition of oleic acid and palmitic acid significantly increased sperm motility, progressive motility, straight-line velocity (VSL), membrane integrity, and acrosome integrity with a simultaneous decrease in sperm apoptosis after seven days during storage. When sperm were incubated with oleic acid and palmitic acid at 37 °C for 3 h, the activities of CPT1 and ACADVL, the ATP level, the mitochondrial membrane potential, the activities of MDH and SDH, as well as sperm motility patterns were significantly increased compared to the control (p < 0.05). Moreover, the addition of etomoxir to the diluted medium in the presence of either oleic acid or palmitic acid and the positive effects of oleic acid and palmitic acid were counteracted. Together, these data suggest that boar sperm might utilize oleic acid and palmitic acid as energy substrates for ATP production via β-oxidation. The addition of these acids could improve sperm quality.

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