Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology

SIMPLE SUMMARY: Notwithstanding extensive research into fertility problems in mares, pregnancy rates have remained low mainly because of endometrial inflammation (endometritis). In the field of equine research, endometrial explants have been used to carry out in vitro studies of the mare’s endometrium. However, there has been no wide-ranging assessment of relative stability of this model over time. The aim of this study was to perform an in-depth transcriptomic assessment of endometrial explants over a culture period of 72 h and assess if they are representative of the whole mare. Explants at 24 h demonstrated significant changes when compared to biopsies at 0 h as expected. Even though gene expression changes were seen between 24 and 48 h of culture, prior to this window changes were dominated by the effects of explanting and culture and subsequently, transcription was generally compromised. Our results, therefore have defined the optimal period when explants can be used to study equine endometritis and how the endometrium is modulated during inflammation. It highlights the use of abattoir derived samples to understand the physiology and pathophysiology of the equine endometrium, negating the need to collect repeated uterine biopsies from living mares. ABSTRACT: Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F(2α) as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h

New insights into the phylogeny of Entamoeba species provided by analysis of four new small-subunit rRNA genes.

Sequences of small-subunit rRNA genes have been obtained for four new isolates of Entamoeba. Phylogenetic analyses give new insights into the evolution of these organisms. A novel Entamoeba from pigs in Vietnam that produces uninucleate cysts proved to be unrelated to other uninucleated cyst-producing species. Revival of the name Entamoeba suis for this organism is proposed. Instead of being related to Entamoeba polecki, it shares a recent common ancestor with the non-encysting Entamoeba gingivalis in a lineage that is basal to the tetranucleate cyst-producing clade. This suggests that species producing cysts with four nuclei are descended from an ancestor that produced cysts with a single nucleus. An Entamoeba from a horse was isolated in culture. No cysts were observed in the original stool sample but the sequence is placed unequivocally within the clade of tetranucleate cyst-producing species with no other sequences being specifically related. Revival of the name Entamoeba equi for this organism is proposed. The Entamoeba ecuadoriensis sequence was found to be the most closely related to Entamoeba histolytica and Entamoeba dispar, as predicted, despite the organism having been an environmental isolate originally assigned to Entamoeba moshkovskii. Finally, a partial E. polecki gene sequence from a pig proved to be virtually identical to that of Entamoeba struthionis from an ostrich, suggesting that the latter name is a synonym

FREEZING OF DONKEY SEMEN (EQUUS ASINUS)

Por muchas décadas, el desarrollo y utilización de la inseminación artificial en loséquidos, especialmente con semen congelado, estuvo restringido, principalmente, porimposiciones de las asociaciones de criadores. Recientemente, las legislaciones de criadoresde équidos en varios países se han tornado más flexibles, permitiendo el registro deproductos oriundos de semen congelado. En el Brasil, frente a ese nuevo cambio, laprincipal asociación de criadores de burros (Associação Brasileira de Criadores de JumentoPêga) revisó sus conceptos y comenzó a permitir la utilización de esta biotecnología.Asimismo, en muchos países, el mayor interés en el asno o burro como semental estárelacionado a la producción de mulares, pues estos animales son deseables en el mediorural, debido a que reúnen las mejores características del burro y del caballo. Los primerostrabajos en congelamiento de semen de asnos utilizaron dilutores a base de yema dehuevo y glicerol, y ampolletas de vidrio como sistema de envase, basados en la metodologíade congelamiento de toros. Sin embargo, pese al tiempo transcurrido, pocas investigacioneshan sido dirigidas a esta especie, en especial a biotecnologías del semen. Enesta revisión de literatura se discuten las principales técnicas de congelamiento de semende équidos y se describen estudios referentes al congelamiento de semen de laespecie asnal.For decades, the development and use of the artificial insemination in the equine, especially with frozen semen, was restricted due to impositions of equine breeders associations that opposed the use of the technique. Recently, these legislations have become more flexible in several countries, allowing the registration of products originating from frozen semen. In Brazil, based on these changes, the main donkey breed association (Brazilian Breeders Association of the Pêga Donkeys) revised their concepts and started to allow the use of this biotechnology. The current interest in many countries for the donkey sire is the production of mules, because their acceptability as these animals inherits suitable characteristics of both donkeys and horses. The first reports on donkey frozen semen used extenders based on egg yolk and glycerol, packed in glass ampoules, and followed the existing methodology for freezing bull semen. However, despite of the elapsed time, few research works have been carried out on this species, especially on semen. This literature review discussed the main techniques of freezing equine semen and describes studies on freezing of sperm of asinine species

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Freezing of donkey semen (Equus asinus)

For decades, the development and use of the artificial insemination in the equine, especially with frozen semen, was restricted due to impositions of equine breeders associations that opposed the use of the technique. Recently, these legislations have become more flexible in several countries, allowing the registration of products originating from frozen semen. In Brazil, based on these changes, the main donkey breed association (Brazilian Breeders Association of the Pêga Donkeys) revised their concepts and started to allow the use of this biotechnology. The current interest in many countries for the donkey sire is the production of mules, because their acceptability as these animals inherits suitable characteristics of both donkeys and horses. The first reports on donkey frozen semen used extenders based on egg yolk and glycerol, packed in glass ampoules, and followed the existing methodology for freezing bull semen. However, despite of the elapsed time, few research works have been carried out on this species, especially on semen. This literature review discussed the main techniques of freezing equine semen and describes studies on freezing of sperm of asinine species.Por muchas décadas, el desarrollo y utilización de la inseminación artificial en loséquidos, especialmente con semen congelado, estuvo restringido, principalmente, porimposiciones de las asociaciones de criadores. Recientemente, las legislaciones de criadoresde équidos en varios países se han tornado más flexibles, permitiendo el registro deproductos oriundos de semen congelado. En el Brasil, frente a ese nuevo cambio, laprincipal asociación de criadores de burros (Associação Brasileira de Criadores de JumentoPêga) revisó sus conceptos y comenzó a permitir la utilización de esta biotecnología.Asimismo, en muchos países, el mayor interés en el asno o burro como semental estárelacionado a la producción de mulares, pues estos animales son deseables en el mediorural, debido a que reúnen las mejores características del burro y del caballo. Los primerostrabajos en congelamiento de semen de asnos utilizaron dilutores a base de yema dehuevo y glicerol, y ampolletas de vidrio como sistema de envase, basados en la metodologíade congelamiento de toros. Sin embargo, pese al tiempo transcurrido, pocas investigacioneshan sido dirigidas a esta especie, en especial a biotecnologías del semen. Enesta revisión de literatura se discuten las principales técnicas de congelamiento de semende équidos y se describen estudios referentes al congelamiento de semen de laespecie asnal