Investigations into the role of platelet-activating factor (PAF) in the peri-conception period of the mare.

Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility, but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separation (LC-MS/MS) was performed on equine follicular fluid (FF), and PAF was quantitatively detected. Subsequently, untargeted high-resolution mass spectrometry-based lipidomic analysis was carried out to quantify PAF in different sized pre-ovulatory follicles, whereby different molecular species of PAF, PAF(14:0) and PAF(16:1), were both seen to be increasing with follicle diameter. These findings suggest that PAF within FF is increasing as preovulatory follicles approach ovulation. Additionally, immunofluorescence staining identified the PAF Receptor (PAFR) in the luminal pericellular, apical and basal aspect of equine oviductal epithelial cells. Lastly, an equine oviductal epithelial organoid model was generated, and showed that the addition of PAF significantly increased the ciliary beat frequency (CBF) (Hz), an action consistent with a role for PAF in embryo migration. It is proposed that the local action of PAF on the ciliated cells of the oviduct propels both the oocyte and the conceptus towards the uterus. In the mare, it appears that PAF is a contributor during the periconception period, potentially being a mediator in the mechanisms of ovulation and in the dialogue of very early pregnancy.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa.

The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

What is driving the global decline of human fertility? Need for a multidisciplinary approach to the underlying mechanisms.

An intense period of human population expansion over the past 250 years is about to cease. Total fertility rates are falling dramatically all over the world such that highly industrialized nations, including China and the tiger economies of SE Asia, will see their populations decline significantly in the coming decades. The socioeconomic, geopolitical and environmental ramifications of this change are considerable and invite a multidisciplinary consideration of the underlying mechanisms. In the short-term, socioeconomic factors, particularly urbanization and delayed childbearing are powerful drivers of reduced fertility. In parallel, lifestyle factors such as obesity and the presence of numerous reproductive toxicants in the environment, including air-borne pollutants, nanoplastics and electromagnetic radiation, are seriously compromising reproductive health. In the longer term, it is hypothesized that the reduction in family size that accompanies the demographic transition will decrease selection pressure on high fertility genes leading to a progressive loss of human fecundity. Paradoxically, the uptake of assisted reproductive technologies at scale, may also contribute to such fecundity loss by encouraging the retention of poor fertility genotypes within the population. Since the decline in fertility rate that accompanies the demographic transition appears to be ubiquitous, the public health implications for our species are potentially devastating.

Governance of advanced materials: Shaping a safe and sustainable future.

The past few decades of managing the uncertain risks associated with nanomaterials have provided valuable insights (knowledge gaps, tools, methods, etc.) that are equally important to promote safe and sustainable development and use of advanced materials. Based on these insights, the current paper proposes several actions to optimize the risk and sustainability governance of advanced materials. We emphasise the importance of establishing a European approach for risk and sustainability governance of advanced materials as soon as possible to keep up with the pace of innovation and to manage uncertainty among regulators, industry, SMEs and the public, regarding potential risks and impacts of advanced materials. Coordination of safe and sustainable advanced material research efforts, and data management according to the Findable, Accessible, Interoperable and Reusable (FAIR) principles will enhance the generation of regulatory-relevant knowledge. This knowledge is crucial to identify whether current regulatory standardised and harmonised test methods are adequate to assess advanced materials. At the same time, there is urgent need for responsible innovation beyond regulatory compliance which can be promoted through the Safe and Sustainable Innovation Approach. that combines the Safe and Sustainable by Design concept with Regulatory Preparedness, supported by a trusted environment. We further recommend consolidating all efforts and networks related to the risk and sustainability governance of advanced materials in a single, easy-to-use digital portal. Given the anticipated complexity and tremendous efforts required, we identified the need of establishing an organisational structure dedicated to aligning the fast technological developments in advanced materials with proper risk and sustainability governance. Involvement of multiple stakeholders in a trusted environment ensures a coordinated effort towards the safe and sustainable development, production, and use of advanced materials. The existing infrastructures and network of experts involved in the governance of nanomaterials would form a solid foundation for such an organisational structure.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100%), whereas polymer E100 demonstrated dose-dependent behaviour (20-90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42% at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Population decline: where demography, social science, and biology intersect.

In brief: Over the past half century, the world has witnessed an unprecedented decline in human fertility rates. This analysis reviews the various socioeconomic, cultural, and biological factors involved in driving this change and considers whether low fertility rates are a temporary or permanent feature of our future demographic profile. Abstract: Since the early 1960s, the world has witnessed the spectacular collapse of human fertility. As a result of this phenomenon, several countries are already seeing their population numbers fall and more will follow in the coming decades. The causes of this fertility decline involve a complex interplay of socio-economic, environmental, and biological factors that have converged to constrain fertility in posterity's wake. Since large numbers of offspring are no longer needed to compensate for high infant mortality in contemporary society, couples have opted to have small families in a quality-over-quantity investment in their progeny's future. Simultaneously, increases in female education, the enhanced participation of women in the paid workforce, and a resultant delay in childbearing has placed limits on achievable family size. Progressive urbanization, the improved availability of contraceptives, and the socio-economic pressures experienced by young adults in ageing societies are also contributing to fertility's demise. These factors, together with the individualism that pervades modern society and the increasing social acceptability of voluntary childlessness, have firmly established a low fertility ethos in most post-transition countries. Since none of these forces are about to relent, it looks as if extremely low fertility might be with us for some time to come. This may have long-term consequences. The lack of selection pressure on high fertility genotypes, the ability of ART to retain poor fertility genotypes within the population, and sustained exposure to reproductive toxicants in modern industrialized environments may all contrive to leave a permanent mark on the fecundity of our species.

The Global Decline in Human Fertility: The Post-Transition Trap Hypothesis.

Over the past half a century many countries have witnessed a rapid fall in total fertility rates, particularly in the world's most advanced economies including the industrial powerhouses of Eastern Asia and Europe. Such nations have now passed through the first and second demographic transitions and are currently exhibiting fertility rates well below the replacement threshold of 2.1, with no sign of recovery. This paper examines the factors responsible for driving these demographic transitions and considers their impact on both fertility and fecundity (our fundamental capacity to reproduce). I argue that because the first demographic transition was extremely rapid and largely driven by socioeconomic factors, it has had no lasting impact on the genetic/epigenetic underpinnings of human fecundity. However, the second demographic transition will be different. A series of conditions associated with low fertility societies, including relaxed selection pressure for high-fertility genotypes, the indiscriminate use of assisted reproductive technologies to treat human infertility, and environmental contamination with reproductive toxicants, may impact our genetic constitution in ways that compromise the future fecundity of our species. Since any fundamental change in the genetic foundations of human reproduction will be difficult to reverse, we should actively pursue methods to monitor human fecundity, as sub-replacement fertility levels become established across the globe.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Sperm chromatin structure assay (SCSA®) and flow cytometry-assisted TUNEL assay provide a concordant assessment of sperm DNA fragmentation as a function of age in a large cohort of approximately 10,000 patients.

BACKGROUND: Sperm DNA integrity is increasingly seen as a critical characteristic determining reproductive success, both in natural reproduction and in assisted reproductive technologies (ART). Despite this awareness, sperm DNA and nuclear integrity tests are still not part of routine examinations for either infertile men or fertile men wishing to assess their reproductive capacity. This is not due to the unavailability of DNA and sperm nuclear integrity tests. On the contrary, several relevant but distinct tests are available and have been used in many clinical trials, which has led to conflicting results and confusion. The reasons for this are mainly the lack of standardization between different clinics and between the tests themselves. In addition, the small number of samples analyzed in these trials has often weakened the value of the analyses performed. In the present work, we used a large cohort of semen samples, covering a wide age range, which were simultaneously evaluated for sperm DNA fragmentation (SDF) using two of the most frequently used SDF assays, namely the TUNEL assay and the sperm chromatin structure assay (SCSA®). At the same time, as standard seminal parameters (sperm motility, sperm morphology, sperm count) were available for these samples, correlations between age, SDF and conventional seminal parameters were analyzed. RESULTS: We show that the SCSA® and TUNEL assessments of SDF produce concordant data. However, the SDF assessed by TUNEL is systematically lower than that assessed by SCSA®. Regardless of the test used, the SDF increases steadily during aging, while the HDS parameter (High DNA stainability assessed via SCSA®) remains unchanged. In the cohort analyzed, conventional sperm parameters do not seem to discriminate with aging. Only sperm volume and motility were significantly lower in the oldest age group analyzed [50-59 years of age]. CONCLUSIONS: In the large cohort analyzed, SDF is an age-dependent parameter, increasing linearly with aging. The SCSA® assessment of SDF and the flow cytometry-assisted TUNEL assessment are well correlated, although TUNEL is less sensitive than SCSA®. This difference in sensitivity should be taken into account in the final assessment of the true level of fragmentation of the sperm nucleus of a given sample. The classical sperm parameters (motility, morphology, sperm count) do not change dramatically with age, making them inadequate to assess the fertility potential of an individual.

RéSUMé: CONTEXTE: l'intégrité de l'ADN des spermatozoïdes est de plus en plus considérée comme une caractéristique essentielle déterminant le succès de la reproduction, tant dans la reproduction naturelle que dans les techniques de reproduction assistée (AMP). Malgré cette prise de conscience, les tests d'intégrité nucléaire des spermatozoïdes ne font toujours pas partie des examens de routine pour les hommes infertiles ou fertiles souhaitant évaluer leur capacité de reproduction. Cette situation n'est pas due à l'indisponibilité des tests. Au contraire, plusieurs tests pertinents mais distincts sont disponibles et ont été utilisés dans de nombreux essais cliniques, ce qui a donné lieu à des résultats contradictoires et à une certaine confusion. Les raisons en sont principalement le manque de normalisation entre les différentes cliniques et entre les tests eux-mêmes. En outre, le petit nombre d'échantillons analysés dans ces essais a souvent affaibli la valeur des analyses effectuées. Dans le présent travail, nous avons utilisé une vaste cohorte d'échantillons, couvrant une large tranche d'âge, évalués simultanément pour la fragmentation de l'ADN des spermatozoïdes à l'aide de deux des tests les plus fréquemment utilisés, à savoir le test TUNEL et le test de la structure de la chromatine des spermatozoïdes (SCSA®). Parallèlement, comme les paramètres séminaux standard (motilité, morphologie, numération) étaient disponibles pour ces échantillons, les corrélations entre l'âge, le niveau de fragmentation et les paramètres séminaux conventionnels ont été analysées. RéSULTATS: Nous montrons que les évaluations SCSA® et TUNEL produisent des données concordantes. Cependant, le SDF évalué par TUNEL est systématiquement plus faible que celui évalué par SCSA®. Quel que soit le test utilisé, la fragmentation augmente régulièrement au cours du vieillissement, alors que le paramètre HDS (« High DNA stainability¼ évalué par le test SCSA®) reste inchangé. Dans la cohorte analysée, les paramètres spermatiques conventionnels ne semblent pas varier avec le vieillissement. Seuls le volume et la mobilité des spermatozoïdes étaient significativement plus faibles dans le groupe d'âge le plus élevé analysé [50­59 ans]. CONCLUSIONS: Dans la grande cohorte analysée, la fragmentation de l'ADN spermatique est un paramètre dépendant de l'âge, augmentant linéairement avec le vieillissement. L'évaluation du SDF par SCSA® et l'évaluation via le test TUNEL assistée par cytométrie de flux sont bien corrélées, bien que le TUNEL soit moins sensible que le SCSA®. Cette différence de sensibilité doit être prise en compte dans l'évaluation finale du niveau réel de fragmentation du noyau des spermatozoïdes d'un échantillon donné. Les paramètres classiques du sperme (motilité, morphologie, nombre de spermatozoïdes) ne changent pas de façon spectaculaire avec l'âge, ce qui les rend inadéquats pour évaluer le potentiel de fertilité d'un individu.

Multigenerational effects of TiO2 rutile nanoparticles on earthworms.

Nanoparticles have gained considerable attention as one of the pollutants released into the environment through consumer products. This study describes the sub-chronic and generational effects of TiO2 (rutile) nanoparticles on earthworms over a 252-day duration, with exposure ranging from 0.1 to 1000 mg kg-1. Results indicate that sub-chronic exposure (28 days) of TiO2 nanoparticles did not cause notable adverse effects on the weight, reproduction, and tissue accumulation in parent earthworms. However, the F1 generation displayed remarkable growth and maturity retardation during their early developmental stages, even at lower nano-TiO2 (rutile). Significant impacts on the reproduction of the F1 generation were observed solely at the highest concentration (1000 mg kg-1), which is predicted to be below the highest exposure scenario. Moreover, long-term (252 days) exposure resulted in considerable bioaccumulation of Ti metal in the F1 generation of E. fetida. This study uncovers the negative effects of TiO2 rutile nanoparticles on earthworms across two generations, with pronounced effects on the growth, maturity, and bioaccumulation in the F1 generation compared to the parent generation. These findings suggest the potential induction of toxic effects by TiO2 rutile nanoparticles, emphasizing the sensitivity of juvenile parameters over adult parameters in toxicity assessments. Furthermore, the study highlights the urgent need for comprehensive evaluations of the longer-term toxicity of nanoparticles on terrestrial organisms. Implementing multigenerational studies will contribute significantly to a better understanding of nanoparticle ecotoxicity on environmental organisms.

Development of a model for studying the developmental consequences of oxidative sperm DNA damage by targeting redox-cycling naphthoquinones to the Sertoli cell population.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations.

Enhanced Recovery After Surgery (ERAS®) Society Consensus Guidelines for Emergency Laparotomy Part 3: Organizational Aspects and General Considerations for Management of the Emergency Laparotomy Patient.

BACKGROUND: This is Part 3 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy using an enhanced recovery after surgery (ERAS) approach. This paper addresses organizational aspects of care. METHODS: Experts in management of the high-risk and emergency general surgical patient were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and MEDLINE database searches were performed for ERAS elements and relevant specific topics. Studies were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses and large cohort studies, and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. RESULTS: Components of organizational aspects of care were considered. Consensus was reached after three rounds of a modified Delphi process. CONCLUSIONS: These guidelines are based on best current available evidence for organizational aspects of an ERAS® approach to patients undergoing emergency laparotomy and include discussion of less common aspects of care for the surgical patient, including end-of-life issues. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Consensus Guidelines for Perioperative Care for Emergency Laparotomy Enhanced Recovery After Surgery (ERAS®) Society Recommendations Part 2-Emergency Laparotomy: Intra- and Postoperative Care.

BACKGROUND: This is Part 2 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy (EL) using an Enhanced Recovery After Surgery (ERAS) approach. This paper addresses intra- and postoperative aspects of care. METHODS: Experts in aspects of management of high-risk and emergency general surgical patients were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and Medline database searches were performed for ERAS elements and relevant specific topics. Studies on each item were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses, and large cohort studies and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. Some ERAS® components covered in other guideline papers are outlined only briefly, with the bulk of the text focusing on key areas pertaining specifically to EL. RESULTS: Twenty-three components of intraoperative and postoperative care were defined. Consensus was reached after three rounds of a modified Delphi Process. CONCLUSIONS: These guidelines are based on best available evidence for an ERAS® approach to patients undergoing EL. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen.

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare's ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa.

Abstract: Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation. Lay Summary: Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.