A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

Angrimani, Daniel de Souza Ramos; Bicudo, Luana de Cássia; Luceño, Núria Llamas; Leemans, Bart; Nichi, Marcílio; Vannucchi, Camila Infantosi; Soom, Ann Van

doi:10.1016/j.cryobiol.2021.12.001

Cited by 2 publications

(2 citation statements)

References 46 publications

(58 reference statements)

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“…These characteristics were analyzed simultaneously using a modification of the triple fluorescent procedure described by Angrimani et al [41], which includes the fluorochromes PI, fluorescein isothiocyanate peanut (Arachis hypogaea) agglutinin (FITC-PNA), and 5,5 ,6,6tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1). All reagents were purchased from Sigma-Aldrich (Bornem, Belgium) and the detailed composition of HEPES-TALP can be found in the study by Rijsselaere et al [34].…”

Section: Plasma Membrane Integrity Acrosome Integrity and Mitochondri...mentioning

confidence: 99%

“…After incubation, a 10 µL aliquot was mounted on a pre-warmed slide, covered with a coverslip, and evaluated using a Leica DMR epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a mercury lamp at a magnification of 1000×. One hundred spermatozoa per sample were counted and classified into eight different classes according to their staining patterns (Table 1) [41]. Intact plasma membrane was obtained by adding classes I to IV, acrosome integrity by adding classes I-II-V-VI, and high MMP by adding classes I-III-V-VII.…”

Section: Plasma Membrane Integrity Acrosome Integrity and Mitochondri...mentioning

confidence: 99%

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Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs

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Hassan

Wydooghe

et al. 2022

Animals

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This study evaluated how semen selection by single layer centrifugation (SLC) with Canicoll affects semen freezability in dogs. A total of eighteen ejaculates, collected from dogs with optimal and suboptimal semen quality (optimal: normal morphology (NM) ≥ 80%, n = 9; suboptimal: NM between 60 and 79%, n = 9), were divided into two aliquots and subjected to standard centrifugation or SLC before cryopreservation. Motility, NM, membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were improved in fresh samples after SLC, regardless of semen quality, but at the expense of some good quality spermatozoa. After thawing, NM and membrane integrity were improved in SLC-selected semen in both semen qualities. Interestingly, MMP was also higher but only in optimal quality semen. Still, spermatozoa from suboptimal quality semen did not survive freezing to the same extent as spermatozoa from optimal quality semen, even after selecting superior spermatozoa. Semen selection with Canicoll is, therefore, an effective technique to isolate a subpopulation of high-quality spermatozoa and obtain sperm samples of better quality after thawing, but is not sufficient to improve the intrinsic inferior freezability of suboptimal quality semen. So far, eighteen pups were born after insemination with SLC-selected frozen-thawed semen, proving that these selected spermatozoa remain fertile.

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Section: Plasma Membrane Integrity Acrosome Integrity and Mitochondri...mentioning

confidence: 99%

Section: Plasma Membrane Integrity Acrosome Integrity and Mitochondri...mentioning

confidence: 99%

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs

Domain

Hassan

Wydooghe

et al. 2022

Animals

Self Cite

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Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals

Egyptien

Dewals

Ectors

et al. 2023

Animals

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Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

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A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs

Cited by 2 publications

References 46 publications

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs

Influence of Single Layer Centrifugation with Canicoll on Semen Freezability in Dogs

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals

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