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.

Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes.

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5-98.8%) and a mean Sp of 100% (95% CI = 92.0-100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3-99.5%) and a mean Sp of 90% (95% CI = 78.3-97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4-100%) and a mean specificity of 100% (95% CI = 100-100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies.

Development and evaluation of polyclonal antibody-based antigen detection ELISA and dot blot assays as a less costly diagnostic alternative for pathogenic Leptospira infection.

Leptospirosis is an infectious, zoonotic disease of worldwide distribution, the cause of which is infection by pathogenic Leptospira. In Chile, dairy cattle are recognized a significant source in the maintenance and transmission of this infection, which causes economic losses and represents an infection threat to workers in the dairy industry. The infection is underestimated in cattle, due to the lack of clinical, pathognomonic signs, as well as the low efficiency of current diagnostic techniques. In this study, we developed antigen ELISA and dot blot assays, based on polyclonal antibodies, to detect pathogenic Leptospira in the urine samples of dairy cattle. The proposed tests showed an acceptable diagnostic accuracy, based on an analytical sensitivity of 1·104 Leptospira per mL for ELISA, and 3.2·103 for dot blot. These results corresponded with those obtained by qPCR, and the use of urine samples allowed us to propose new diagnostic alternatives for pathogenic Leptospira infection at a low cost, which can provide information on active infection status, which is a key element in control programs both at individual and herd level.

Understanding the antibacterial mechanisms of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper and its alloys are natural and very well-proven antimicrobial materials. The mechanisms of action through which copper is highly effective have been described at the molecular and cellular level. However, both the design of the studies carried out and the nature of the microorganisms studied have meant that this research has been of limited scope. In the present study, we examined the action mechanisms of a copper ion treatment on the integrity of Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant animal pathogen. The copper ion treatment applied to MAP cells, resulted in nucleic acid degradation and disintegration, increased ROS production and protein alteration. However, the observed susceptibility of MAP to copper-based treatment was dose-dependent. Finally, it had no effect on the integrity of the MAP cell wall. This new evidence about the observed tolerance in the MAP cell wall against the copper ions, may help us to understand how we can improve the proposed copper-based treatment, and finally achieve a totally effective alternative to control MAP in calf´s milk.

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.

Temperature, but not excess of glycogen, regulates "in vitro" AMPK activity in muscle samples of steer carcasses.

Postmortem muscle temperature affects the rate of pH decline in a linear manner from 37.5°C to 0-2°C. The pH decline is correlated with the enzymatic degradation of glycogen to lactate and this process includes the metabolic coupling between glycogenolysis and glycolysis, and that are strongly upregulated by the AMPK. In this study, we used 12 samples previously characterized by have different muscle glycogen concentration, lactate and AMPK activity, selected from 38 steers that produced high final pH (>5.9) and normal final pH (<5.8) carcasses at 24 h postmortem. Moreover, we evaluated changes in the AMPK activity in samples from both categories incubated at 37, 25, 17 and 5°C and supplemented with exogenous glycogen. Finally, we analysed if there were structural differences between polymers from both categories. Our results showed that "in vitro" enzymatic AMPK activity evaluated at both 0.5 or 24 h was greater in samples from normal then high pH categories (p <0.01), and in all temperature of incubation analysed (17, 25 and 37°C). For other hand, a greater AMPK activity were obtained in samples incubated at 17 that 25 or 37°C, in normal carcasses at both 0.5 or 24 h (p < 0.01), as also in samples from carcasses categorized as high pH, but at 24 h (p < 0.05). Interestingly, AMPK activity was totally abolished at 5°C, independent of final pH category of carcasses, and was confirmed that the incubation temperature at which the maximum activity was obtained (p < 0.01), at least in carcasses with a normal pH is at 17°C. The enzymatic AMPK activity did not change in relation to excess glycogen (p > 0.05) and we did not detect structural differences in the polymers present in samples from both categories (p > 0.05), suggesting that postmortem AMPK activity may be highly sensitive to temperature and not to in vitro changes in glycogen concentration (p > 0.05). Our results allow concluding that normal concentrations of muscle glycogen immediately at the time of slaughter (0.5 h) and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, through the postmortem AMPK signalling pathway.

Analytical evaluation of an immunomagnetic separation PCR assay to detect pathogenic Leptospira in cattle urine samples obtained under field conditions.

Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

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.

Chronic Inflammatory Lameness Increases Cytokine Concentration in the Spinal Cord of Dairy Cows.

Lameness in dairy cows is an extremely painful multifactorial condition that affects the welfare of animals and economically impacts the dairy industry worldwide. The aim of this study was to determine the profile of cytokines in the spinal cord dorsal horn of dairy cows with painful chronic inflammatory lameness. Concentrations of 10 cytokines were measured in the spinal cord of seven adult dairy cows with chronic lameness and seven adult dairy cows with no lameness. In all cows lameness was evaluated using a mobility scoring system and registered accordingly. Immediately after euthanasia the spinal cord was removed and 20 cm of lumbar segments (L2-L5) were obtained. After dorsal horn removal and processing, cytokine quantification of tumor necrosis factor-alpha (TNF-α), interleukin-1alpha (IL-1α), interleukin 13 (IL-13), chemokine-10 (CXCL10/IP-10), chemokine-9 (CXCL9/MIG), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-21 (IL-21), interleukin-36ra (IL-36ra), and macrophage inflammatory protein-1 beta (MIP-1ß) was performed using a multiplex array. Lame cows had higher concentrations of TNF-α, IL-1-α, IL-13, CXCL10, CXCL9, IFN-α, and IFN-γ in their dorsal horn compared to non-lame cows, while IL-21 concentration was decreased. No differences in IL-36ra and MIP-1ß concentrations between lame and non-lame cows were observed. Painful chronic inflammation of the hoof in dairy cows leads to a marked increase in cytokine concentration in the dorsal horn of the spinal cord, which could represent a state of neuroinflammation of the Central Nervous System (CNS).

Spinal Reactive Oxygen Species and Oxidative Damage Mediate Chronic Pain in Lame Dairy Cows.

Lameness in dairy cows is a worldwide prevalent disease with a negative impact on animal welfare and herd economy. Oxidative damage and antioxidant system dysfunction are common features of many CNS diseases, including chronic pain. The aim of this study was to evaluate the levels of reactive oxygen species (ROS) and oxidative damage markers in the spinal cord of dairy cows with chronic inflammatory lameness. Locomotion score was performed in order to select cows with chronic lameness. Dorsal horn spinal cord samples were obtained post mortem from lumbar segments (L2-L5), and ROS, malondialdehyde (MDA), and carbonyl groups were measured along with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant response (TAR). Lame cows had increased levels of ROS, MDA, and carbonyl groups, while no differences were observed between lame and non-lame cows in SOD, GPx, CAT, and TAR activity. We conclude that painful chronic inflammatory lameness in dairy cows is associated with an increase in ROS, MDA, and carbonyl groups. Nonetheless, an association between ROS generation and dysfunction of the antioxidant system, as previously proposed, could not be established.

Early differential gene expression in beef Longissimus thoracis muscles from carcasses with normal (<5.8) and high (>5.9) ultimate pH.

The objective of this study was to investigate early postmortem (0.5 h) gene expression in beef Longissimus thoracis (LT) muscles from carcasses with NORMAL (<5.8) and HIGH (>5.9) ultimate pH (pHu). A total of 53 transcripts were differentially expressed (P-value <.05): 40 showed up-regulation and 13 showed down-regulation in HIGH pHu carcasses. Four up-regulated (PDK4, GADD45B, MAOA, METTL21C) genes were confirmed (P < .05) by q-PCR. HIGH pHu samples resulted with lower values in glycolytic potential and AMP-activated protein kinase activity compared to NORMAL at 0.5 and 24 h postmortem (P < .05). Functional pathway analysis showed that calcium transport and GADD45 signaling pathways are associated with the development of HIGH pH meat. Genes involved in stress-related signaling, such as GADD45B, METTL21C and MAOA were overexpressed. These genes are involved in stress signaling that might be affecting Ca2+ transport and oxidative metabolism pathways in HIGH pH muscles.

LED-based red light photostimulation improves short-term response of cooled boar semen exposed to thermal stress at 37°C.

Pre-treatment of boar semen with a red light photostimulation procedure increases its "in vivo" fertilising ability. However, "in vitro" conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short-term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620-630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca2+ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca2+ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short-term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.

Neuronal signaling repertoire in the mammalian sperm functionality.

The common embryonic origin has been a recurrent explanation to understand the presence of "neural receptors" in sperm. However, this designation has conditioned a bias marked by the classical neurotransmission model, dismissing the possibility that neurotransmitters can play specific roles in the sperm function by themselves. For instance, the launching of acrosome reaction, a fundamental sperm function, includes several steps that recall the process of presynaptic secretion. Unlike of postsynaptic neuron, whose activation is mediated by molecular interaction between neurotransmitter and postsynaptic receptors, the oocyte activation is not mediated by receptors, but by cytosolic translocation of sperm phospholipase (PLCζ). Thus, the sperm has a cellular design to access and activate the oocyte and restore the ploidy of the species by an "allogenic pronuclear fusion." At subcellular level, the events controlling sperm function, particularly the capacitation process, are activated by chemical signals that trigger ion fluxes, sterol oxidation, synthesis of cyclic adenosine monophosphate, protein kinase A activation, tyrosine phosphorylations and calcium signaling, which correspond to second messengers similar to those associated with exocytosis and growth cone guidance in neurons. Classically, the sperm function associated with neural signals has been analyzed as a unidimensional approach (single ligand-receptor effect). However, the in vivo sperm are exposed to multidimensional signaling context, for example, the GABAergic, monoaminergic, purinergic, cholinergic, and melatoninergic, to name a few. The aim of this review is to present an overview of sperm functionality associated with "neuronal signaling" and possible cellular and molecular mechanisms involved in their regulation.

Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5' AMP-activated protein kinase (AMPK).

This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 µM compound C AMPK inhibitor or 2 mM AMP+40 µM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP(+)), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.