Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen-thawed feline spermatozoa.

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit - Cat 4MID® Kit (SPQI - 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at -80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen-thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long-lasting motility.

The morphology, morphometry and functionality of fresh and cryopreserved wisent (Bison bonasus) epididymal spermatozoa.

Epididymal spermatozoa obtained post mortem are considered a valuable source of genetic material which is often irrevocably lost. This makes these gametes constitute a key element in protection and restitution programs. The wisent (Bison bonasus, Linnaeus 1758) is a species that survived in zoos after extinction from its natural habitat. This resulted in a narrowing of the genetic pool of the whole population, which is at present derived from only 12 ancestors. Currently, wisent protection programs are aimed at preserving the genetic diversity by establishing a germplasm bank. The objective of this study was to comprehensively characterize the morphology, morphometry and functionality of wisent epididymal spermatozoa and evaluate the effectiveness of their cryopreservation in extender based on Tris buffer and chicken egg yolk. The median total number of spermatozoa obtained from one individual was 1985.0 × 106 (62.5 × 106-7452.0 × 106). These gametes were characterized by median: 40.0% (0.5-70.0%) subjective motility, 69.8% (32.5-90.0%) viability and 54.3% (10.5-83.3%) normal morphology. The sperm head had a median size of 5.0 µm (3.5-6.7 µm) width, 8.5 µm (6.4-11.3 µm) length and 36.9 µm2 (23.7-48.6 µm2) surface area. The viable population of the obtained gametes was characterized by median values 53.2% (4.5-80.3%) of intact sperm membrane, 50.8 (26.0-76.6%) of intact acrosome, 0.4% (0-98.7%) of fragmented chromatin, 5.9% (0.0-88.8%) of cells with high mitochondrial potential and 42.1% (8.3-63.7%) without lipid peroxidation. The viable population of the frozen/thawed gametes was characterized by median values: 18.4% (2.4-57.9%) of intact sperm membrane, 35.1 (11.9-56.7%) of intact acrosome, 0.07% (0-89.2%) of fragmented chromatin, 12.8% (0.0-49.7%) of cells with high mitochondrial potential and 16.3% (2.2-53.6%) without lipid peroxidation. Due to the material originating from a relatively large number of wild individuals, the research presented here contributed to the description of certain species standards for the assessment of wisent epididymal spermatozoa. The presented effect of cryopreservation on these gametes justifies the use of an extender based on Tris buffer with the addition of chicken egg yolk. The obtained effects are satisfactory from the point of view of preserving valuable genetic material and their use in ART.

The Lambda Variant in Argentina: Analyzing the Evolution and Spread of SARS-CoV-2 Lineage C.37.

The second wave of COVID-19 occurred in South America in early 2021 and was mainly driven by Gamma and Lambda variants. In this study, we aimed to describe the emergence and local genomic diversity of the SARS-CoV-2 Lambda variant in Argentina, from its initial entry into the country until its detection ceased. Molecular surveillance was conducted on 9356 samples from Argentina between October 2020 and April 2022, and sequencing, phylogenetic, and phylogeographic analyses were performed. Our findings revealed that the Lambda variant was first detected in Argentina in January 2021 and steadily increased in frequency until it peaked in April 2021, with continued detection throughout the year. Phylodynamic analyses showed that at least 18 introductions of the Lambda variant into the country occurred, with nine of them having evidence of onward local transmission. The spatial--temporal reconstruction showed that Argentine clades were associated with Lambda sequences from Latin America and suggested an initial diversification in the Metropolitan Area of Buenos Aires before spreading to other regions in Argentina. Genetic analyses of genome sequences allowed us to describe the mutational patterns of the Argentine Lambda sequences and detect the emergence of rare mutations in an immunocompromised patient. Our study highlights the importance of genomic surveillance in identifying the introduction and geographical distribution of the SARS-CoV-2 Lambda variant, as well as in monitoring the emergence of mutations that could be involved in the evolutionary leaps that characterize variants of concern.

Characterization of novel proteases identified by metagenomic analysis from dairy stabilization ponds.

Cheese whey is the main by-product of dairy industries. It is used as a raw material for other value-added products, like whey protein concentrate. By using enzymes, this product can be further treated to obtain new higher value products, like whey protein hydrolysates. Proteases (EC: 3.4) represent a large segment of industrial enzymes, since they are used in several industries, including food. In this work, we describe three novel enzymes identified using a metagenomic approach. Metagenomic DNA from dairy industry stabilization ponds were sequenced, and the predicted genes were compared against the MEROPS database, focusing on families commercially used to produce whey protein hydrolysates. From a total of 849 candidates, 10 were selected for cloning and expression and three showed activities with both the chromogenic substrate, azocasein, and whey proteins. Particularly, Pr05, an enzyme from the yet uncultured phylum Patescibacteria, showed activity that is comparable to a commercial protease. All these novel enzymes could represent an alternative for dairy industries to produce value-added products from industrial by-products. KEY POINTS: • Over 19,000 proteases were predicted in a sequence-based metagenomic analysis. • Three proteases were successfully expressed and showed activity with whey proteins. • The enzyme Pr05 showed hydrolysis profiles of interest for food industry.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

A Comparative Study of the Effects of Osaterone Acetate and Deslorelin Acetate on Sperm Kinematics and Morpho-Functional Parameters in Dogs.

Benign prostatic hyperplasia (BPH) is one of the most common problems in older male dogs that often has a huge impact on their health and welfare. This article presents a comparison between osaterone acetate (Ypozane®; Virbac®)(OA) and deslorelin acetate (Suprelorin®; Virbac®)(DA), medications that are the main therapeutic alternative to castration in dogs with BPH. Forty dogs were divided into four groups: I-negative control (five dogs without BPH); II-positive control (10 individuals diagnosed with BPH); III-15 dogs treated with DA, and IV-10 individuals treated with OA. Semen fractions were collected on days 0 (day of treatment), 7, 14, and 21, and weeks 8, 12, 16, and 20. Macroscopic, microscopic and CASA analyses were performed. Both DA and OA significantly affected the properties of the canine ejaculate. The DA lead to the lack of libido and had lesser effects to the sperm function before it caused azoospermia, whereas OA had no effect on libido and only temporary reduction in seminal plasma volume was observed, which resulted in temporary deterioration in the percentage of motile and progressive spermatozoa.

Dead or Alive? A Review of Perinatal Factors That Determine Canine Neonatal Viability.

The perinatal period has a critical impact on viability of the newborns. The variety of factors that can potentially affect the health of a litter during pregnancy, birth, and the first weeks of life requires proper attention from both the breeder and the veterinarian. The health status of puppies can be influenced by various maternal factors, including breed characteristics, anatomy, quality of nutrition, delivery assistance, neonatal care, and environmental or infectious agents encountered during pregnancy. Regular examinations and pregnancy monitoring are key tools for early detection of signals that can indicate disorders even before clinical signs occur. Early detection significantly increases the chances of puppies' survival and proper development. The purpose of the review was to summarize and discuss the complex interactions between all elements that, throughout pregnancy and the first days of life, have a tangible impact on the subsequent fate of the offspring. Many of these components continue to pose challenges in veterinary neonatology; thus, publications presenting the current state of knowledge in this field are in demand.

Identification of key microorganisms in facultative stabilization ponds from dairy industries, using metagenomics.

Wastewater stabilization ponds are a natural form of wastewater treatment. Their low operation and maintenance costs have made them popular, especially in developing countries. In these systems, effluents are retained for long periods of time, allowing the microbial communities present in the ponds to degrade the organic matter present, using both aerobic and anaerobic processes. Even though these systems are widespread in low income countries, there are no studies about the microorganisms present in them and how they operate. In this study, we analised the microbial communities of two serial full-scale stabilization ponds systems using whole genome shotgun sequencing. First, a taxonomic profiling of the reads was performed, to estimate the microbial diversity. Then, the reads of each system were assembled and binned, allowing the reconstruction of 110 microbial genomes. A functional analysis of the genomes allowed us to find how the main metabolic pathways are carried out, and we propose several organisms that would be key to this kind of environment, since they play an important role in these metabolic pathways. This study represents the first genome-centred approach to understand the metabolic processes in facultative ponds. A better understanding of these microbial communities and how they stabilize the effluents of dairy industries is necessary to improve them and to minimize the environmental impact of dairy industries wastewater.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

Changes in Body Surface Temperature Associated with High-Speed Treadmill Exercise in Beagle Dogs Measured by Infrared Thermography.

Evaluation of body surface temperature change in response to exercise is important for monitoring physiological status. The aim of the study was to assess the influence of high-speed treadmill exercise on body surface temperature using infrared thermography (IRT) in selected body regions of healthy Beagle dogs, taking into account gait and recovery time. Thermographic images of the dogs were taken before exercise (BE), after walk (AW), after trot (AT), after canter (AC), just after second walk (JAE), 5 min after exercise (5 AE), 15 min after exercise (15 AE), 30 min after exercise (30 AE), 45 min after exercise (45 AE), and 120 min after exercise (120 AE). Body surface temperature was measured at the neck, shoulder, upper forearm, back, chest, croup, and thigh. Statistical analysis indicated the highest temperature at the upper forearm, shoulder, and thigh, and the lowest on the croup, back, and neck. The peak values of surface temperature in all ROIs were at AC and JAE and the lowest at 120 AE. The study demonstrated that body surface temperature was influenced by high-speed physical exercise on a treadmill and IRT was a viable imaging modality that provided temperature data from specific body regions. The proximal forelimb and hindlimb were the most influenced by exercise.

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle.

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Long-Term Administration of Abacavir and Etravirine Impairs Semen Quality and Alters Redox System and Bone Metabolism in Growing Male Wistar Rats.

Highly active antiretroviral therapy (HAART) is used in HIV-infected patients. Alongside the prolongation of patients' life, adverse side effects associated with long-term therapy are becoming an increasing problem. Therefore, optimizing of HAART is extremely important. The study is aimed at evaluating the toxicity of abacavir and etravirine in monotherapy on the reproductive system, liver, kidneys, and bones in young, sexually mature, male rats. Thirty-six 8-week-old male Wistar rats randomized into three 12-animal groups received either normal saline (control), abacavir 60 mg/kg (AB group), or etravirine 40 mg/kg (ET group) once daily for 16 weeks. Semen morphology, oxide-redox state parameters (MDA, SOD, catalase, GPx, glutathione, GSH/GSSG ratio) in tissue homogenates (testes, liver, kidneys), and serum samples were studied. In bones, microcomputed tomography and a four-point bending test were performed. Total sperm count, sperm concentration, motility, and sperm morphology did not differ significantly in AB or ET groups compared to the control. In the flow cytometry of semen, an increased percentage of cells with denatured DNA was noticed for both tested drugs. However, no significant changes of oxide-redox state in testicular homogenates were found, except of increased SOD activity in the AB-receiving group. Additionally, ET significantly altered catalase and GPx in the liver and SOD activity in kidneys. Abacavir decreased catalase in the liver and GSH levels in kidneys. AB caused significant changes to bone microarchitecture (bone volume fraction, trabecular number, connectivity density, total porosity) and increased Young's modulus. Etravirine had a greater impact on macrometric parameters of bones (tibial index, mid-tibial diameter, femur length). After 4 weeks in the ET group, a lower 1,25-dihydroxyvitamin D3 serum concentration was found. The results showed that abacavir and etravirine disturb oxidative stress. An increase in the percentage of sperms with chromatin damage suggests decreased fertility in rats receiving the studied drugs. Both drugs affected bone formation in growing rats. Additionally, etravirine disturbed vitamin D metabolism.

ß-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. ß-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding ß-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with ß-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-ß-galactoside and lactose. The activity levels of one of these novel ß-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic ß-galactosidases from diary stabilization ponds' metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries' by-products.

Redescription of Echinocoleus hydrochoeri (Travassos, 1916) (Nematoda: Trichuridae) from Hydrochoeris hydrochaeris Linnaeus, 1766 (Rodentia: Caviidae) from Argentina.

Twenty-eight Capillariinae species have been recorded in rodents; 1 of these species was reported from a caviomorph rodent, Hydrochoeris hydrochaeris (capybara), and placed in the genus Echinocoleus by Moravec (1982). However, both original description and subsequent contributions of Echinocoleus hydrochoeri are poor and incomplete. In this paper, this species is redescribed, and a new geographical distribution is reported. The redescription is based on morphologic and morphometrical features; intestine ends in a cloaca beside ejaculatory duct, caudal bursa composed of 2 large ventrolateral lobes with a fleshy internal part and a membranous external part (they are not united dorsally with a membrane), 1 pair of caudal papillae, terminal part of cylindrical cirrus ornamented with thin and thick spines (and particular pattern distribution), sclerotized spicule in male, and vulvar appendage in female, and 3 bacillary bands (1 ventral and 2 lateral). Generic and specific analyses were performed to establish new standards for future studies on the systematic position of Capillariinae species. This study presents new morphological information and a new record of a capillariid species from Argentina.