Comparison of the SMLM technique and the MSSR algorithm in confocal microscopy for super-resolved imaging of cellulose fibres.

Nowadays, the use of super-resolution microscopy (SRM) is increasing globally due to its potential application in several fields of life sciences. However, a detailed and comprehensive guide is necessary for understanding a single-frame image's resolution limit. This study was performed to provide information about the structural organisation of isolated cellulose fibres from garlic and agave wastes through fluorophore-based techniques and image analysis algorithms. Confocal microscopy provided overall information on the cellulose fibres' microstructure, while techniques such as total internal reflection fluorescence microscopy facilitated the study of the plant fibres' surface structures at a sub-micrometric scale. Furthermore, SIM and single-molecule localisation microscopy (SMLM) using the PALM reconstruction wizard can resolve the network of cellulose fibres at the nanometric level. In contrast, the mean shift super-resolution (MSSR) algorithm successfully determined nanometric structures from confocal microscopy images. Atomic force microscopy was used as a microscopy technique for measuring the size of the fibres. Similar fibre sizes to those evaluated with SIM and SMLM were found using the MSSR algorithm and AFM. However, the MSSR algorithm must be cautiously applied because the selection of thresholding parameters still depends on human visual perception. Therefore, this contribution provides a comparative study of SRM techniques and MSSR algorithm using cellulose fibres as reference material to evaluate the performance of a mathematical algorithm for image processing of bioimages at a nanometric scale. In addition, this work could act as a simple guide for improving the lateral resolution of single-frame fluorescence bioimages when SRM facilities are unavailable.

Contributions of dysfunctional plasticity mechanisms to the development of atypical perceptual processing.

Experimental studies of sensory plasticity during development in birds and mammals have highlighted the importance of sensory experiences for the construction and refinement of functional neural circuits. We discuss how dysregulation of experience-dependent brain plasticity can lead to abnormal perceptual representations that may contribute to heterogeneous deficits symptomatic of several neurodevelopmental disorders. We focus on alterations of somatosensory processing and the dynamic reorganization of cortical synaptic networks that occurs during early perceptual development. We also discuss the idea that the heterogeneity of strengths and weaknesses observed in children with neurodevelopmental disorders may be a direct consequence of altered plasticity mechanisms during early development. Treating the heterogeneity of perceptual developmental trajectories as a phenomenon worthy of study rather than as an experimental confound that should be overcome may be key to developing interventions that better account for the complex developmental trajectories experienced by modern humans.

Bicarbonate and BSA increase the capacitation pattern and acrosomal exocytosis in boar sperm after 120 min of incubation.

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly influence in vitro sperm capacitation in this species.

Second verse, same as the first: learning generalizable relational concepts through functional repetition.

The ability to learn and flexibly apply sophisticated concepts is thought by many to be what differentiates humans from all other animals. A basic assumption underlying this belief is that some "lower-order" associative learning mechanisms link perceptual events to specific reactions, whereas the kinds of verbalizable concepts that humans form depend on "higher-order" cognitive processes that rely less on perception and more on rational thought. Evidence in support of this interpretation comes largely from experiments in which animals either fail to learn or generalize concepts that humans readily learn, or learn them with great difficulty. Here, we argue that the formation of generalizable relational concepts may depend more on an individual's capacity to shift attention than on the possession of representational processes that are unique to humans. Studies of relational concept learning in non-human animals show that they can learn generalizable concepts when conditions are favorable. In particular, repetition of similar training experiences appears to facilitate attentional redirection, thereby enabling animals to flexibly reenact past events and to judge the similarity of items within stimulus sets. The conditions that promote concept learning in humans may differ substantially from those experienced by most other animals. This does not imply, however, that either (1) conceptual learning mechanisms differ qualitatively from other learning mechanisms, or (2) that the processes that lead to concept formation in humans differ significantly from those present in other species.

Comment on: "Variability in humpback whale songs reveals how individuals can be distinctive when sharing a complex vocal display" [J. Acoust. Soc. Am. 153, 2238-2250 (2023)].

Lamoni, Garland, Allen, Coxon, Noad, and Rendell [(2023). J. Acoust. Soc. Am. 153, 2238-2250] analyzed variations in humpback whale song structure to assess how individual singers can produce distinctive patterns that communicate desirable individual qualities to potential mates. Their analyses revealed that singers rarely produced individually specific sound patterns and that singers varied subjectively distinctive structural features of songs differently across years. These findings provide the strongest evidence to date that singing humpback whales are not varying song structure in ways that reliably reveal individual singers' physical or cognitive characteristics. Surprisingly, the authors appear to reach the opposite conclusion. Objective strategies for quantitatively comparing song properties are crucial for evaluating competing hypotheses regarding the nature and function of humpback whale songs, but the value of such strategies is reduced when the objectivity of the analyses is suspect and when negative evidence is framed as supporting prior beliefs.

All units are equal in humpback whale songs, but some are more equal than others.

Flexible production and perception of vocalizations is linked to an impressive array of cognitive capacities including language acquisition by humans, song learning by birds, biosonar in bats, and vocal imitation by cetaceans. Here, we characterize a portion of the repertoire of one of the most impressive vocalizers in nature: the humpback whale. Qualitative and quantitative analyses of sounds (units) produced by humpback whales revealed that singers gradually morphed streams of units along multiple acoustic dimensions within songs, maintaining the continuity of spectral content across subjectively dissimilar unit "types." Singers consistently produced some unit forms more frequently and intensely than others, suggesting that units are functionally heterogeneous. The precision with which singing humpback whales continuously adjusted the acoustic characteristics of units shows that they possess exquisite vocal control mechanisms and vocal flexibility beyond what is seen in most animals other than humans. The gradual morphing of units within songs that we observed is inconsistent with past claims that humpback whales construct songs from a fixed repertoire of discrete unit types. These findings challenge the results of past studies based on fixed-unit classification methods and argue for the development of new metrics for characterizing the graded structure of units. The specific vocal variations that singers produced suggest that humpback whale songs are unlikely to provide detailed information about a singer's reproductive fitness, but can reveal the precise locations and movements of singers from long distances and may enhance the effectiveness of units as sonar signals.

Cognitive control of song production by humpback whales.

Singing humpback whales are highly versatile vocalizers, producing complex sequences of sounds that they vary throughout adulthood. Past analyses of humpback whale song have emphasized yearly variations in structural features of songs made collectively by singers within a population with comparatively little attention given to the ways that individual singers vary consecutive songs. As a result, many researchers describe singing by humpback whales as a process in which singers produce sequences of repeating sound patterns. Here, we show that such characterizations misrepresent the degree to which humpback whales flexibly and dynamically control the production of sounds and sound patterns within song sessions. Singers recorded off the coast of Hawaii continuously morphed units along multiple acoustic dimensions, with the degree and direction of morphing varying across parallel streams of successive units. Individual singers also produced multiple phrase variants (structurally similar, but acoustically distinctive sequences) within song sessions. The precision with which individual singers maintained some acoustic properties of phrases and morphing trajectories while flexibly changing others suggests that singing humpback whales actively select and adjust acoustic elements of their songs in real time rather than simply repeating stereotyped sound patterns within song sessions.

Intra-individual variation in the songs of humpback whales suggests they are sonically searching for conspecifics.

Observations of animals' vocal actions can provide important clues about how they communicate and about how they perceive and react to changing situations. Here, analyses of consecutive songs produced by singing humpback whales recorded off the coast of Hawaii revealed that singers constantly vary the acoustic qualities of their songs within prolonged song sessions. Unlike the progressive changes in song structure that singing humpback whales make across months and years, intra-individual acoustic variations within song sessions appear to be largely stochastic. Additionally, four sequentially produced song components (or "themes") were each found to vary in unique ways. The most extensively used theme was highly variable in overall duration within and across song sessions, but varied relatively little in frequency content. In contrast, the remaining themes varied greatly in frequency content, but showed less variation in duration. Analyses of variations in the amount of time singers spent producing the four themes suggest that the mechanisms that determine when singers transition between themes may be comparable to those that control when terrestrial animals move their eyes to fixate on different positions as they examine visual scenes. The dynamic changes that individual whales make to songs within song sessions are counterproductive if songs serve mainly to provide conspecifics with indications of a singer's fitness. Instead, within-session changes to the acoustic features of songs may serve to enhance a singer's capacity to echoically detect, localize, and track conspecifics from long distances.

Spectral interleaving by singing humpback whales: Signs of sonar.

The duplex sonar model of humpback whale song proposes that broadband units within songs function differently from narrowband units. Specifically, this model suggests that singing humpback whales interleave constant frequency (CF) units, which can generate prolonged reverberation focused at specific frequencies, with less reverberant broadband units that minimally overlap with the focal frequencies of preceding and following CF units (referred to as spectral interleaving) to increase the efficacy of song as a sonar source. Here, it is shown that singers recorded off the coast of Hawaii in 2015 devoted most of their time singing to spectrally interleaving broadband elements of units around quasi-CF components that consistently generated persistent reverberant tails. Singers maintained reverberant CF streams in specific frequency bands when units contained broadband elements and when singers switched from producing pairs of alternating reverberant units to producing a single reverberant unit. Additionally, singers showed the ability to flexibly control where acoustic energy was concentrated within broadband components in ways that minimized spectral overlap with the focal frequencies of reverberant tails. The consistency and precision with which singing humpback whales interleaved broadband and reverberant CF elements of units confirm two novel predictions of the duplex sonar model.

Comment on "A standardized method of classifying pulsed sounds and its application to pulse rate measurement of blue whale southeast Pacific song units" [J. Acoust. Soc. Am. 146, 2145-2154 (2019)].

Patris, Malige, Glotin, Asch, and Buchan [(2019). Acoust. Soc. Am. 146, 2145-2154] proposed a technique for classifying and describing pulsed sounds produced by whales that can improve the precision and objectivity of acoustic measurements from song units. Their analyses revealed that blue whales produce units at precise pulse rates. The structure and precision of the song phrase they describe is remarkably similar to what is seen in song production by chickadees. In both species, precise control of shifts in pulse rate may produce reverberation-related cues that enable listeners to localize singers. The techniques developed by Patris and colleagues thus can provide more accurate measures as well as insights into how animals produce and use songs.

Performance anxiety and the plasticity of emotional responses.

Current psychological theories of performance anxiety focus heavily on relating performers' physiological and mental states to their abilities to maintain focus and execute learned skills. How task-specific expertise and past experiences moderate the degree to which individuals become anxious in a given performance context are not well accounted for within these theories. This review considers how individual differences arising from learning may shape the psychobiological, emotional, and cognitive processes that modulate anxious states associated with the performance of highly trained skills. Current approaches to understanding performance anxiety are presented, followed by a critique of these approaches. A connectionist model is proposed as an alternative approach to characterising performance anxiety by viewing performers' anxious states at a specific time point as jointly determined by experience-dependent plasticity, competition between motivational systems, and ongoing cognitive and somatic states. Clarifying how experience-dependent plasticity contributes to the emergence of socio-evaluative anxiety in challenging situations can not only help performers avoid developing maladaptive emotional responses, but may also provide new clues about how memories of past events and imagined future states interact with motivational processes to drive changes in emotional states and cognitive processing.

Modeling and Control of a Micro AUV: Objects Follower Approach.

This work describes the modeling, control and development of a low cost Micro Autonomous Underwater Vehicle (µ-AUV), named AR2D2. The main objective of this work is to make the vehicle to detect and follow an object with defined color by means of the readings of a depth sensor and the information provided by an artificial vision system. A nonlinear PD (Proportional-Derivative) controller is implemented on the vehicle in order to stabilize the heave and surge movements. A formal stability proof of the closed-loop system using Lyapunov's theory is given. Furthermore, the performance of the µ-AUV is validated through numerical simulations in MatLab and real-time experiments.

Modulatory Effects of Breed, Feeding Status, and Diet on Adipogenic, Lipogenic, and Lipolytic Gene Expression in Growing Iberian and Duroc Pigs.

Meat quality depends on tissue composition which is in turn influenced by different factors, such as diet, genotype, age, or sex. We evaluated the effects of breed, 24 h fasting, and dietary energy source (HO: oleic acid versus CH: carbohydrates) on the expression of candidate genes involved in adipogenesis, lipogenesis, and lipolysis in the adipose tissue from Iberian and Duroc growing pigs. The Iberian pigs showed greater feed intake, backfat thickness, and saturated fatty acids (SFA) content in the subcutaneous fat, whereas the Duroc pigs had greater ham weight and polyunsaturated fatty acids (PUFA) content. In both breeds, the diet induced changes in the fatty acid (FA) composition of subcutaneous fat samples. The HO group had higher monounsaturated fatty acids (MUFA) and oleic acid, and lower SFA than the CH group. Regarding gene expression, breed and feeding status (fasting versus postprandial) had significant effects on gene expression, with quantitative interactions between them, while diet showed negligible effects. In general, adipogenic and lipogenic genes were upregulated in the Iberian pigs and in postprandial samples. In contrast, the expression of lipolytic genes showed complex interaction effects. Our results agree with the phenotypic differences between the Iberian and Duroc breeds and with the inhibition of lipogenesis by fasting. Quantitative interactions between breed and feeding status effects were observed, which indicates a different response to fasting of the two breeds, with the obese Iberian breed showing a more stable expression of lipogenic genes. These results highlight the complexity of lipid metabolism regulation, especially in relation to lipolysis processes.

Regularities in responding during performance of a complex choice task.

Systematic variations in the rate and temporal patterns of responding under a multiple concurrent-chains schedule were quantified using recurrence metrics and self-organizing maps to assess whether individual rats showed consistent or idiosyncratic patterns. The results indicated that (1) the temporal regularity of response patterns varied as a function of number of training sessions, time on task, magnitude of reinforcement, and reinforcement contingencies; (2) individuals showed heterogeneous, stereotyped patterns of responding, despite similarities in matching behavior; (3) the specific trajectories of behavioral variation shown by individuals were less evident in group-level analyses; and (4) reinforcement contingencies within terminal links strongly modulated response patterns within initial links. Temporal regularity in responding was most evident for responses that led to minimally delayed reinforcers of larger magnitude. Models of response production and selection that take into account the time between individual responses, probabilities of transitions between response options, periodicity within response sequences, and individual differences in response dynamics can clarify the mechanisms that drive behavioral adjustments during operant conditioning.

Modeling possible effects of atypical cerebellar processing on eyeblink conditioning in autism.

Autism is unique among other disorders in that acquisition of conditioned eyeblink responses is enhanced in children, occurring in a fraction of the trials required for control participants. The timing of learned responses is, however, atypical. Two animal models of autism display a similar phenotype. Researchers have hypothesized that these differences in conditioning reflect cerebellar abnormalities. The present study used computer simulations of the cerebellar cortex, including inhibition by the molecular layer interneurons, to more closely examine whether atypical cerebellar processing can account for faster conditioning in individuals with autism. In particular, the effects of inhibitory levels on delay eyeblink conditioning were simulated, as were the effects of learning-related synaptic changes at either parallel fibers or ascending branch synapses from granule cells to Purkinje cells. Results from these simulations predict that whether molecular layer inhibition results in an enhancement or an impairment of acquisition, or changes in timing, may depend on (1) the sources of inhibition, (2) the levels of inhibition, and (3) the locations of learning-related changes (parallel vs. ascending branch synapses). Overall, the simulations predict that a disruption in the balance or an overall increase of inhibition within the cerebellar cortex may contribute to atypical eyeblink conditioning in children with autism and in animal models of autism.

The ability to recognize objects from bottlenose dolphin (Tursiops truncatus) echoes generalizes across multiple orientations in humans and neural networks.

Object constancy, the ability to recognize objects despite changes in orientation, has not been well studied in the auditory modality. Dolphins use echolocation for object recognition, and objects ensonified by dolphins produce echoes that can vary significantly as a function of orientation. In this experiment, human listeners had to classify echoes from objects varying in material, shape, and size that were ensonified with dolphin signals. Participants were trained to discriminate among the objects using an 18-echo stimulus from a 10° range of aspect angles, then tested with novel aspect angles across a 60° range. Participants were typically successful recognizing the objects at all angles (M = 78 %). Artificial neural networks were trained and tested with the same stimuli with the purpose of identifying acoustic cues that enable object recognition. A multilayer perceptron performed similarly to the humans and revealed that recognition was enabled by both the amplitude and frequency of echoes, as well as the temporal dynamics of these features over the course of echo trains. These results provide insight into representational processes underlying echoic recognition in dolphins and suggest that object constancy perceived through the auditory modality is likely to parallel what has been found in the visual domain in studies with both humans and animals.

Learning to discriminate frequency modulation rate can benefit and worsen pitch acuity.

Participants were trained to discriminate frequency modulation rates (FM-rate training) or Gabor patch orientations (visual training) in a same-different task for two different training lengths. Test discriminations involved trains of FM sweeps with identical modulation rates, but different frequencies. FM-rate training enhanced test accuracy (relative to visual) when sweep trains contained frequencies similar to training. For extended FM-rate training, the opposite was true for trains shifted one octave higher. In contrast to previous work, generalization of learning to the untrained dimension (pitch) was not well accounted for by conceptual learning. Mechanisms of stimulus learning may better explain the current cross-dimensional generalization.

Evaluation of Different Thawing Protocols on Iberian Boar Sperm Preserved for 10 Years at Different Liquid Nitrogen Levels.

The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN2) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN2 levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN2 levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN2, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN2. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN2.

MicroRNA expression in specific segments of the pig periovulatory internal genital tract is differentially regulated by semen or by seminal plasma.

microRNAs play pivotal roles during mammalian reproduction, including the cross-talk between gametes, embryos and the maternal genital tract. Mating induces changes in the expression of mRNA transcripts in the female, but whether miRNAs are involved remains to be elucidated. In the current study, we mapped 181 miRNAs in the porcine peri-ovulatory female reproductive tract: Cervix (Cvx), distal and proximal uterus (Dist-Ut, Prox-Ut), Utero-tubal-junction (UTJ), isthmus (Isth), ampulla (Amp), and infundibulum (Inf) when exposed to semen (natural mating (NM) or artificial insemination (AI-P1)) or to infusions of sperm-free seminal plasma (SP): the first 10 mL of the sperm rich fraction (SP-P1) or the entire ejaculate (SP-E). Among the most interesting findings, NM decreased mir-671, implicated in uterine development and pregnancy loss prior to embryo implantation, in Cvx, Dist-UT, Prox-UT, Isth, and Inf, while it increased in Amp. NM and SP-E induced the downregulation of miR-let7A-1 (Dist-UT, Prox-UT), a regulator of immunity during pregnancy. miR-34C-1, a regulator of endometrial receptivity gene expression, was increased in Dist-UT, UTJ and Amp (NM), in Prox-UT (AI-P1), and in Amp (SP-P1). miR-296, a modulator of the inflammatory response and apoptosis, was upregulated in the UTJ (all treatments). NM elicited the highest miRNA activity in the sperm reservoir (UTJ), suggesting that key-regulators such as miR-34c or miR-296 may modulate the metabolic processes linked to the adequate preparation for gamete encounter in the oviduct. Our results suggest that SP should be maintained in AI to warrant miRNA regulation within the female genital tract for reproductive success.

Effect of Milk-Feeding Frequency and Calcium Gluconate Supplementation on Growth, Health, and Reproductive and Metabolic Features of Holstein Heifers at a Rearing Farm.

We compared the effects of milk-feeding in 288 Holstein calves (72 per group) which were fed twice (2F) or thrice (3F) daily, with or without the addition of hydrogenated fat-embedded calcium gluconate (G) supplemented in the starter food and in the daily diet up to the age of 9 months, on the calves' metabolism, growth, health, and reproductive efficiency up to first pregnancy. The calves received 6 L of milk replacer (130 g/L) and had ad libitum access to water and textured calf starter with or without gluconate. Gluconate supplementation promoted a "catch-up" in growth in supplemented calves compared to their counterparts that did not receive gluconate. Gluconate appeared to reduce animal metabolic stress during key events, such as weaning and transfer into open-door pens, reducing fructosamine (352.61 vs. 303.06 in 3FG and 3F, respectively; p = 0.028) and urea (3F revealed the highest values compared with the other three groups: 19.06 for 3F vs. 13.9 (2F), 13.7 (2FG), and 14.3 (3FG), respectively, p = 0.002) from weaning onwards. The feeding of dairy calves with milk replacer three rather than two times per day tended to be associated with better health from weaning to 4 months old; parameters such as ultrasound lung score and calf health score improved over time (p < 0.001). Thrice-daily feeding with milk replacer tended to reduce the number of artificial inseminations per pregnancy in heifers by 0.2 points (p = 0.092). We confirmed significant correlations between early health and growth parameters and reproductive efficiency and a positive correlation between body weight and average daily weight gain and the thickness of the back fat layer in young heifers (r = 0.245; p < 0.0001; r = 0.214; p < 0.0001 respectively). Our study was conducted on a commercial farm with reasonably effective animal management, so baseline welfare was likely satisfactory.