Effects of egg yolk and soybean lecithin on sperm quality determined by computer‐assisted sperm analysis and confocal laser scanning microscope in chilled canine sperm

In an attempt to increase the possibilities of a better sperm cryopreservation from these endangered small ruminant catalonian breeds, we are studying different strategies. One of them is to study the effect of the buffer system Tes-Tris (TEST) compared with the Tris and citric acid (TRIS) buffer system, testing simultaneously both systems in a 1% (w/v) soybean lecithin or in a 15% (v/v) powdered egg yolk–based media supplemented both with 5% glycerol and to assess also the inclusion of 5 mM of butylated hydroxytoluene (BHT) as an antioxidant on the cryopreservation media. However, the main objective of the present study was to test first the effect of the different media when the sperm were cultured 4 h at 5°C. Briefly, fresh ejaculates from 6 young bucks of Blanca de Rasquera breed (1 year old) were collected by an artificial vagina in favourable reproductive period and immediately mixed in equal quantities. Pooled ejaculates were split into 2 equal aliquots and washed by centrifugation (twice for 10 min at 600g) in TRIS- or TEST-based media without cryoprotectants and antioxidant. Afterward each pellet was split into 4 equal aliquots, re-suspended in TRIS or TEST, depending on the experimental group, soybean lecithin, or powdered egg yolk-based media, and supplemented or not with BHT and kept for 4 h at 5°C. Likewise, fresh ejaculates from 8 young rams (4 rams of Xisqueta and 4 rams of Aranesa breed, 1 year old) were collected and processed as buck semen samples. Sperm survival before cryopreservation was determined by eosine-nigrosine stain, and sperm motion parameters were analysed by a computer-assisted sperm analysis system (ISAS®). Six replications were performed in both species, and General Lineal Model (SAS®, Cary, NC, USA) was used for the statistical analysis. The highest sperm viability percentage (mean ± SE) on goat sperm cultured 4 h at 5°C was observed in the extender with TRIS buffer system in powdered egg yolk-based media supplemented with BHT (81.1 ± 2.8), not showing significant differences with the others extenders, except with the viability of the samples in the extenders with TEST buffer system in soybean lecithin-based media supplemented (56.5 ± 2.9; P < 0.001) or not with BHT (60.1 ± 5.1; P < 0.001). On the other hand, no significant differences on sperm viability were observed on ram sperm between treatments. Nevertheless, the sperm quality motion characteristics (data not shown) were quite different between all the treatments in both species. Considering that the present results are still preliminary, we suggest that more analysis should be made to explain how the different composition of the extenders affect sperm quality during the cryopreservation process. Supported by INIA (RZ2009-00008-00-00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundacion Carolina.

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Effect of Cholesterol and Equex-STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm

ISRN Veterinary Science ◽

10.1155/2013/280143 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 4

Author(s):

Lidia Gil ◽

Iván Galindo-Cardiel ◽

C. Malo ◽

N. González ◽

C. Álvarez

Keyword(s):

Sperm Quality ◽

Egg Yolk ◽

Computer Assisted ◽

Pig Liver ◽

Sperm Analysis ◽

Cryopreserved Sperm ◽

Fluorescence Test ◽

Acrosome Integrity ◽

Computer Assisted Sperm Analysis ◽

Economic Improvement

Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125–150 mM of cholesterol from pig liver and 0.5–0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6–8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100×106 spermatozoa) and thawed (21 s., 37∘C water bath). After thawing the following parameters were evaluated: viability (V), motility (computer assisted sperm analysis, CASA; % nonprogressive NP; % progressive MP), hipoosmotic swelling test (HOST), acrosome integrity (A), fluorescence test (FL), and resistance test (RT). Sperm quality was significantly affected by stallion (in the parameters V, VI, NP, MP, HOST, A, FL, and RT), extraction (VI, NP, MP, HOST, A, and FL), and the different combinations of Equex-STM-cholesterol (FL). We concluded that 0.5% of Equex-STM mixed with 125 mM of cholesterol has obtained better sperm quality results than those of original Martin's extender, showing a simple and economic improvement of this home-made practical seminal extender.

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3-D imaging of cells using a confocal laser scanning microscope and digital image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102560 ◽

1988 ◽

Vol 46 ◽

pp. 96-97

Author(s):

Thomas M. Jovin ◽

Michel Robert-Nicoud ◽

Donna J. Arndt-Jovin ◽

Thorsten Schormann

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Biochemical Processes ◽

Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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Minocycline-induced cutaneous hyperpigmentation: confocal laser scanning microscope analysis

Journal of the European Academy of Dermatology and Venereology ◽

10.1111/j.1468-3083.2006.01436.x ◽

2006 ◽

Vol 20 (4) ◽

pp. 435-439 ◽

Cited By ~ 7

Author(s):

RP Dodiuk-Gad ◽

HM Morentin ◽

J Schafer ◽

A Harel ◽

M Neudorfer ◽

...

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Microscope Analysis

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Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta

Animals ◽

10.3390/ani11071885 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1885

Author(s):

José Néstor Caamaño ◽

Carolina Tamargo ◽

Inmaculada Parrilla ◽

Felipe Martínez-Pastor ◽

Lorena Padilla ◽

...

Keyword(s):

Genetic Resource ◽

Sperm Quality ◽

Genetic Material ◽

Mitochondrial Activity ◽

Computer Assisted ◽

Kinematic Parameters ◽

Linear Mixed Effects ◽

Sperm Analysis ◽

Computer Assisted Sperm Analysis ◽

Analysis System

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

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