Optimized protocol for high-vacuum scanning electron microscopy analysis of polyacrylamide hydrogel-attached sperm cells in a binary system.

A protocol for the analysis of a binary system comprising polyacrylamide hydrogel-attached sperm cells using high-vacuum scanning electron microscopy (SEM) is presented. This protocol focuses on optimizing the SEM procedure to obtain accurate and detailed imaging of the sperm cells and their interactions with the hydrogel scaffold. The methodology involves a stepwise sample preparation, including sample dehydration through a gradual exchange of ethanol/water ratios, followed by the application of a conductive metal coating. By employing this modified protocol, the traditional use of acetone dehydration, which may introduce chemical alterations to the materials, is avoided. The proposed approach enables a comprehensive evaluation of the morphology and interactions within the biological system in contact with the soft material scaffold. Furthermore, the potential application of this protocol extends to the study of other mammalian reproductive cells or cells of different origins adhered to hydrogel scaffolds. RESEARCH HIGHLIGHTS: Novel SEM protocol reveals precise imaging of sperm-hydrogel attachment in a binary system, enhancing our understanding of cell-material interactions. By optimizing SEM procedures, the protocol achieves precise imaging of sperm-hydrogel interactions using ethanol/water dehydration and a conductive metal coating. This modified approach enables a thorough assessment of morphology and interactions in the binary system,extending its potential applicability to other reproductive cells on hydrogelscaffolds.

Biointerfacial behavior of stallion spermatozoa adhered to hydrogel surfaces: Impact of the hydrogel chemical composition and the culture medium.

Novel soft materials based on hydrogel are proposed to enhance the selection of high-quality stallion sperm based on their adhesion capacity. The hydrogel surfaces are derived from polyacrylamide (PAAm), which is copolymerized with neutral and ionic co-monomers to modify the interfacial properties. The hydrogels undergo characterization through FTIR spectroscopy, assessment of swelling capacity, and wettability under various experimental conditions. Sperm adhesion capacity on the hydrogels is examined through several parameters including the percentage of bound sperm (%Sp) to hydrogels, tail oscillation intensity and flagellar movement. The biointerfacial properties of sperm-hydrogel systems vary based on the chemical composition of hydrogel as well as the components present in the culture medium. High %Sp and excellent metabolic activity of the spermatozoa are observed on hydrogel surfaces that possess moderate hydrophilicity. Specifically, a cationic hydrogel in BGM3 culture medium and a neutral surface in BGM3 medium supplemented with BSA exhibit favorable outcomes. Scanning Electron Microscopy (SEM) reveals the normal morphology of the head and tail in spermatozoa adhered to the hydrogel. Therefore, these hydrogel surfaces are potential materials for selecting stallion sperm with high quality, and their application could be extended to the study of other mammalian reproductive cells.

Semen Quality and Freezability Analyses in the Ejaculates of Two Poitou Donkeys in the Southern Hemisphere.

The Baudet du Poitou is a vanishing donkey breed recognized for engendering robust working mules. In Chile, only two pure breed Poitou males exist, which belong to the Chilean army and are used for mule production. We performed an extensive sperm and seminal analysis of these two jackasses aged 3 and 6 years and investigated the use of a simple hypometabolic extender for sperm cryopreservation. Computer-assisted sperm analysis showed high motility, velocity, and linearity in sperm movement. The seminal plasma analysis revealed that sodium and chloride were the main electrolytes, and globulins were the main metabolites. Active and variable enzymatic activity was observed. New information is reported about gamma-glutamyltransferase, aspartate aminotransferase, zinc, and magnesium concentrations in seminal plasma of Poitou donkeys. Ejaculates among jackasses showed some variability due to individual variability and different stages in sexual maturation according to age. The freezability index analysis based in viability, total motility and progressive motility with Botucrio extender (57.1 ± 11.0%; 56.6 ± 20.0%; and 22.6 ± 10.3%, respectively) were significantly higher (p < 0.05, p < 0.0001, and p < 0.0001, respectively) than with HM-0 extender (42,6 ± 11.4%; 14.9 ± 5.1%; and 1.0 ± 2.5%, respectively). We report new information on Poitou donkey semen and cryopreservation in the Southern Hemisphere that could be useful in donkey breeding and conservation programs to develop strategies that improve the effectiveness of population management of this breed.

Semen quality and freezability analysis during breeding and non-breeding seasons in heavy draft stallions in southern Chile.

The aim of this study was to evaluate seasonal changes in basic parameters of sperm quality and freezability behaviour of ejaculates from 10 fertile heavy draft stallions. A total of 140 ejaculates were collected, processed and evaluated during both the breeding (September-November) and non-breeding seasons (April-June). Fresh semen was evaluated for volume, concentration, total spermatozoa per ejaculate, plasma membrane integrity and total sperm motility. Cryopreserved samples were evaluated for plasma membrane integrity and sperm motility by the CASA system, and for the freezability index (FI), which was defined as the decreased ratio of viability after freezing-thawing. In fresh ejaculates, only viability showed significantly higher values in the breeding than in the non-breeding season (64.0% ± 15.0% vs. 58.6% ± 12.0%, respectively; p < .05). The sperm post-thawing analysis of viability and total motility parameters showed no significant changes linked to the season. However, the FI analysis showed that the ejaculates collected in the non-breeding season had higher cryoresistance characteristics than those collected in the breeding season. Results suggest that the presence of some cryoprotective factor/s in heavy draft horse ejaculates could be modulated by seasonality, with higher protective effects in the non-breeding season.