Updates and Current Challenges in Reproductive Microbiome: A Comparative Analysis between Cows and Women.

The microbiota plays an important role in numerous physiological processes, pathogenesis, development, and metabolism in different animal species. In humans, several studies have demonstrated an association between the vaginal microbiota and fertility rates, and even success in assisted reproduction techniques. In the context of cattle reproduction, although few studies have addressed the microbiota in a healthy state (which is not associated with diseases that affect the reproductive tract of cows), changes in its composition also seem to influence fertility. This review aims to explain the importance of the reproductive microbiota in female bovines and what is available in the literature regarding its possible role in increasing fertility. What are the challenges involved in this process? Future perspectives on its use and manipulation as a selection or intervention tool. Will it be possible to one day extrapolate the findings to reality and apply them in the field? In short, understanding the role of the reproductive microbiota of female bovines can signal the prospect of increasing production, whether of milk or meat, from the same number of animals, as it can optimize reproductive efficiency and perhaps become an allied tool for the economic profitability and sustainability of livestock farming.

Bacterial microbiota and proinflammatory cytokines in the anal sacs of treated and untreated atopic dogs: Comparison with a healthy control group.

The pathogenesis of anal sacculitis has not been extensively investigated, although atopic dogs seem to be predisposed to the disease. The aim of this study was therefore to characterize and compare the bacterial microbiota and pro-inflammatory cytokines in the anal sacs of dogs from three groups (healthy dogs, untreated atopic dogs and atopic dogs receiving antipruritic treatment or allergen-specific immunotherapy) in order to determine whether changes could be at the origin of anal sacculitis in atopic dogs. Bacterial populations of anal sac secretions from fifteen healthy dogs, fourteen untreated and six treated atopic dogs were characterized by sequencing the V4 region of the 16S rRNA gene using Illumina technology. Proinflammatory cytokines were analyzed with the Luminex multiplex test. Community membership and structure were significantly different between the anal sacs of healthy and untreated atopic dogs (P = 0.002 and P = 0.003, respectively) and between those of untreated and treated atopic dogs (P = 0.012 and P = 0.017, respectively). However, the community structure was similar in healthy and treated atopic dogs (P = 0.332). Among the proinflammatory cytokines assessed, there was no significant difference between groups, except for interleukin 8 which was higher in the anal sacs of untreated atopic dogs compared to treated atopic dogs (P = 0.02), and tumor necrosis factor-alpha which was lower in the anal sacs of healthy dogs compared to treated atopic dogs (P = 0.04). These results reveal a dysbiosis in the anal sacs of atopic dogs, which may partially explain the predisposition of atopic dogs to develop bacterial anal sacculitis. Treatments received by atopic dogs (oclacitinib, desloratadine and allergen-specific immunotherapy) shift the microbiota of the anal sacs towards that of healthy dogs. Further studies are required to identify significant cytokines contributing to anal sacculitis in atopic dogs.

Fecal Microbial Communities of Nellore and Crossbred Beef Calves Raised at Pasture.

This study aimed to investigate the effect of age and genetics on the fecal microbiota of beef calves. Ten purebred Nellore (Bos taurus indicus) and ten crossbreed 50% Nellore-50% European breed (Bos taurus taurus) calves co-habiting on the same pasture paddock had fecal samples collected on days five (5 d), 14 d, 28 d, 60 d, 90 d, 180 d, 245 d (weaning) and 260 d after birth. All calves were kept with their mothers, and six Nellore dams were also sampled at weaning. Microbiota analysis was carried out by amplification of the V4 region of the 16S rRNA gene following high-throughput sequencing with a MiSeq Illumina platform. Results revealed that bacterial richness increased with age and became more similar to adults near weaning. Differences in microbiota membership between breeds were found at 60 d and 90 d and for structure at 60 d, 90 d, 245 d, and 260 d (p < 0.05). In addition, crossbreed calves presented less variability in their microbiota. In conclusion, the genetic composition significantly impacted the distal gut microbiota of calves co-habiting in the same environment, and further studies investigating food intake can reveal possible associations between microbiota composition and performance.

Current Understanding of Equine Gut Dysbiosis and Microbiota Manipulation Techniques: Comparison with Current Knowledge in Other Species.

Understanding the importance of intestinal microbiota in horses and the factors influencing its composition have been the focus of many studies over the past few years. Factors such as age, diet, antibiotic administration, and geographic location can affect the gut microbiota. The intra- and inter-individual variability of fecal microbiota in horses complicates its interpretation and has hindered the establishment of a clear definition for dysbiosis. Although a definitive causal relationship between gut dysbiosis in horses and diseases has not been clearly identified, recent research suggests that dysbiosis may play a role in the pathogenesis of various conditions, such as colitis and asthma. Prebiotics, probiotics, and fecal microbiota transplantation to modulate the horse's gastrointestinal tract may eventually be considered a valuable tool for preventing or treating diseases, such as antibiotic-induced colitis. This article aims to summarize the current knowledge on the importance of intestinal microbiota in horses and factors influencing its composition, and also to review the published literature on methods for detecting dysbiosis while discussing the efficacy of gut microbiota manipulation in horses.

Effect of dietary iron supplementation on the equine fecal microbiome.

Iron is an essential element for all living organisms, including bacteria, as several virulence factors and replication components are influenced by iron concentration. The objective of this study was to determine whether the composition and diversity of the fecal microbiota of adult horses are affected by supplemental dietary iron. Ten clinically healthy horses were randomly divided into a control and an iron-supplemented group (n = 5). The treated group was supplemented with oral ferrous sulphate monohydrate (720 ppm of iron), whereas the control group received 320 ppm of iron daily for 15 d. Fecal samples were collected before and 5, 10, 15, and 30 d after supplementation and frozen at -80°C. DNA was sequenced using an Illumina MiSeq platform and data were analyzed using the software Mothur and linear discriminant analysis (LDA) effect size (LEfSe). Iron supplementation caused no change in the overall composition of the fecal microbiota, but some minor changes were observed in the low-abundant bacteria, as well as an increased alpha diversity after 15 d of supplementation. Significant differences in community composition of the fecal microbiota over time were observed in both groups, highlighting the importance of a control group, as there are variables that cannot be controlled in microbiome studies.

Le fer est un élément essentiel pour tous les organismes vivants, y compris les bactéries, car plusieurs facteurs de virulence et composants de réplication sont influencés par la concentration en fer. L'objectif de cette étude était de déterminer si la composition et la diversité du microbiote fécal des chevaux adultes sont affectées par la supplémentation en fer alimentaire. Dix chevaux cliniquement sains ont été divisés au hasard en un groupe témoin et un groupe supplémenté en fer, n = 5 par groupe. Le groupe traité a reçu un supplément oral de sulfate ferreux monohydraté (720 ppm de fer) et le groupe témoin a reçu 320 ppm de fer par jour pendant 15 jours. Des échantillons fécaux ont été prélevés avant la supplémentation et 5, 10, 15 et 30 jours après la supplémentation puis congelés à −80 °C. L'ADN a été séquencé à l'aide de la plateforme Illumina MiSeq et les données ont été analysées à l'aide des logiciels Mothur et analyse de la fonction discriminante linéaire taille de l'effet LefSe. La supplémentation en fer n'a provoqué aucun changement dans la composition du microbiote fécal, mais certains changements ont été observés chez les bactéries peu abondantes, ainsi qu'une augmentation de la diversité alpha après 15 jours de supplémentation. Au fil du temps, des différences significatives dans la composition de la communauté bactérienne ont été observées dans les deux groupes, soulignant l'importance d'un groupe témoin, car il existe des variables qui ne peuvent être contrôlées dans les études sur le microbiome.(Traduit par les auteurs).

Impact of zinc oxide, benzoic acid and probiotics on the performance and cecal microbiota of piglets.

BACKGROUND: Intestinal health remains a key factor in animal production because it is essential for digestion, absorption and bacterial fermentation. Feed additives have been used to attenuate the weaning stress such as Zinc Oxide (ZnO) and benzoic acid (C7H6O2). The objective of this study was to evaluate the impact of of benzoic acid and probiotics (BA + P) on performance, diarrhea and cecal microbiota of piglets in the nursery phase (23 to 65 days). RESULTS: One hundred and sixty weaned piglets with an initial weight of 6.335 ± 0.698 kg and 23 days of age were submitted to four treatments: supplementation with 2500 ppm of Zinc oxide (ZnO), supplementation with a commercial blend of benzoic acid and probiotics (Bacillus licheniformis, Bacillus subtilis and Enterococcus faecium NCIMB 10415; Vevogut P®) (BA + P), supplementation with Zinc oxide plus benzoic acid and probiotics (ZnO + BA + P), and controls receiving only the basal diet without any supplementation. At 65 days of age, 32 piglets (n = 8 per treatment) were slaughtered for the evaluation of the cecal microbiota. Supplementation with ZnO and BA + P were associated with better feed conversion (P < 0.05) in the early stage (23 to 49 days) and with an improvement in all performance parameters over the entire experimental period. The occurrence of diarrhea was lower (P < 0.05) in the BA + P group. The 4 most abundant phyla along with unclassified bacteria represented 93% of all sequences. Firmicutes dominated the cecal microbiota of all groups, followed by Bacteroidetes. Richness represented by the observed number of genera and by the Chao index were statistically lower in ZnO and ZnO + BA + P supplemented animals compared to controls. The beta diversity analysis that compares similarities between bacterial communities demonstrated formation of two distinct clusters containing samples with and without supplementation with ZnO, confirming a strong influence of ZnO on the intestinal microbiota. CONCLUSION: The use of Benzoic acid with probiotics yields similar performance results with lower impact on the gut microbiota compared to ZnO, and it should be considered as a potential alternative in swine production.

Species-Level Gut Microbiota Analysis after Antibiotic-Induced Dysbiosis in Horses.

All current studies have used Illumina short-read sequencing to characterize the equine intestinal microbiota. Long-read sequencing can classify bacteria at the species level. The objectives of this study were to characterize the gut microbiota of horses at the species level before and after trimethoprim sulfadiazine (TMS) administration and to compare results with Illumina sequencing. Nine horses received TMS (30 mg/kg) orally for 5 days twice a day to induce dysbiosis. Illumina sequencing of the V4 region or full-length PacBio sequencing of the 16S rRNA gene was performed in fecal samples collected before and after antibiotic administration. The relative abundance and alpha diversity were compared between the two technologies. PacBio failed to classify the equine intestinal microbiota at the species level but confirmed Bacteroidetes as the most abundant bacteria in the feces of the studied horses, followed by Firmicutes and Fibrobacteres. An unknown species of the Bacteroidales order was highly abundant (13%) and deserves further investigation. In conclusion, PacBio was not suitable to classify the equine microbiota species but detected greater richness and less unclassified bacteria. Further efforts in improving current databanks to be used in equine studies are necessary.

Understanding the Intestinal Microbiome in Health and Disease.

This article provides readers with the basic concepts necessary to understand studies using recent molecular methods performed in intestinal microbiome assessment, with special emphasis on the high throughput sequencing. This review also summarizes the current knowledge on this topic and discusses future insights on the interaction between the intestinal microbiome and equine health.

Impact of a Single Oral Acute Dose of Aflatoxin B1 on Liver Function/Cytokines and the Lymphoproliferative Response in C57Bl/6 Mice.

Aflatoxin B1 (AFB1), a mycotoxin found in food and feed, exerts harmful effects on humans and animals. The liver is the earliest target of AFB1, and its effects have been evaluated in animal models exposed to acute or chronic doses. Considering the possibility of sporadic ingestion of AFB1-contaminated food, this study investigated the impact of a single oral dose of AFB1 on liver function/cytokines and the lymphoproliferative response in mice. C57BL/6 mice were treated with a single oral AFB1 dose (44, 442 or 663 µg AFB1/kg of body weight) on the first day. Liver function (ALT, γ-GT, and total protein), cytokines (IL-4, IFN-γ, and IL-17), histopathology, and the spleen lymphoproliferative response to mitogens were evaluated on the 5th day. Although AFB1 did not produce any significant changes in the biochemical parameters, 663 µg AFB1/kg-induced hepatic upregulation of IL-4 and IFN-γ, along with liver tissue injury and suppression of the lymphoproliferative response to ConA (p < 0.05). In conclusion, a single oral dose of AFB1 exposure can induce liver tissue lesions, liver cytokine modulation, and immune suppression in C57BL/6 mice.

Changes in the fecal microbiota of beef cattle caused by change in management and the use of virginiamycin as a growth promoter.

Several factors are known to affect the intestinal microbiota of cattle. However, how these changes occur over time is poorly understood. This study aimed to investigate the consequences of entrance into a feedlot and the effects of virginiamycin used as a growth promoter on the bovine fecal microbiota. Two batches of beef cattle (B1, n=50 and B2, n=36) entering a feedlot operation were randomly divided into two pens: one receiving virginiamycin and one group not receiving antibiotic (control group). Fecal samples were collected at arrival, mid feedlot and at exit to slaughter. The V4 region of 16S rRNA gene was amplified and sequenced. Escherichia coli strains isolated in samples from arrival and exit of B2 were also isolated and used as indicators of antimicrobial susceptibility. Marked changes in membership and structure of fecal microbiota occurred following entrance into the feedlot. At mid feedlot, virginiamycin affected bacterial community membership, but not structure, suggesting that the antibiotic had a stronger effect on the rare, but not on the most abundant species. The use of virginiamycin had no demonstrable effect on antibiotic resistance in E. coli. The differences found between batches are suggestive that variations in study conditions are important and can strongly affect the intestinal microbiota.

Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies.

Recent studies performed in humans and rats have reported that exercise can alter the intestinal microbiota. Athletic horses perform intense exercise regularly, but studies characterizing horse microbiome during aerobic conditioning programs are still limited. Evidence has indicated that this microbial community is involved in the metabolic homeostasis of the host. Research on ergogenic substances using new sequencing technologies have been limited to the intestinal microbiota and there is a considerable demand for scientific studies that verify the effectiveness of these supplements in horses. L-carnitine and chromium are potentially ergogenic substances for athletic humans and horses since they are possibly able to modify the metabolism of carbohydrates and lipids. This study aimed to assess the impact of acute exercise and aerobic conditioning, associated either with L-carnitine or chromium supplementation, on the intestinal microbiota of fillies. Twelve "Mangalarga Marchador" fillies in the incipient fitness stage were distributed into four groups: control (no exercise), exercise, L-carnitine (10g/day) and chelated chromium (10mg/day). In order to investigate the impact of acute exercise or aerobic conditioning on fecal microbiota all fillies undergoing the conditioning program were analyzed as a separate treatment. The fillies underwent two incremental exercise tests before and after training on a treadmill for 42 days at 70-80% of the lactate threshold intensity. Fecal samples were obtained before and 48 h after acute exercise (incremental exercise test). Bacterial populations were characterized by sequencing the V4 region of the 16S rRNA gene using the MiSeq Illumina platform, and 5,224,389 sequences were obtained from 48 samples. The results showed that, overall, the two most abundant phyla were Firmicutes (50.22%) followed by Verrucomicrobia (15.13%). The taxa with the highest relative abundances were unclassified Clostridiales (17.06%) and "5 genus incertae sedis" from the phylum Verrucomicrobia (12.98%). There was a decrease in the phylum Chlamydiae and in the genus Mycobacterium after the second incremental exercise test. Intense exercise changed the community's structure and aerobic conditioning was associated with changes in the composition and structure of the intestinal bacterial population of fillies. The intra-group comparison showed that chromium or L-carnitine induced moderate changes in the fecal microbiota of fillies, but the microbiota did not differ from the control group, which was exercised with no supplementation. Fecal pH correlated positively with Simpson's index, while plasma pH correlated negatively. Our results show that exercise and aerobic conditioning can change in the microbiota and provide a basis for further studies enrolling a larger number of horses at different fitness levels to better understand the effects of exercise and training on the intestinal microbiota of horses.

Asymptomatic encephalitis in calves experimentally infected with bovine herpesvirus-5.

This study demonstrated that bovine herpesvirus 5 (BoHV)-5 infected calves can develop encephalitis and remain asymptomatic. Seven calves were infected intranasally and monitored for 30 days. Cerebrospinal fluid (CSF) analysis was performed from the onset of neurological signs. Multiple sections of brain and the trigeminal ganglion were submitted to histopathology. Virus detection (PCR and isolation) was performed on CSF and tissues. Four calves developed signs of neurologic disease and died. Three calves remained asymptomatic and were euthanized 30 days post-infection. Cerebrospinal fluid mononuclear pleocytosis occurred in symptomatic and asymptomatic calves. BoHV-5 was isolated and viral DNA was detected in multiple areas of the encephalon of all calves. The viral DNA was detected in the CSF of 2 calves showing neurological signs. Histologically, inflammation was noted in the brain of all calves and confirmed that the encephalitis caused by BoHV-5 may be mild and asymptomatic.

Transferência de imunidade passiva em bezerros das raças Nelore e Limousin e proteinograma sérico nos primeiros quatro meses de vida / Passive transfer of immunity in Nelore and Limousin calves and serum proteinogram in the first four months of life

To study the passive transfer of immunity, 90 healthy calves, 45 Nelore and 45 Limousin, were grouped (n=15) according to their dam's parity: first, second or third or more calvings. Blood samples were draw from each calf with 24 to 36 hours of life and 15, 30, 60, 90, and 120 days. The total serum (TSP) and plasma (TPP) proteins, gamaglutamiltransferase activity (GGT), serum albumin, alpha, beta and gammaglobulin by electrophoresis in agarose gel and IgG estimated by the zinc sulphate turbidity test were determined. Two-way-ANOVA was used to compare the data in the first age. The age behaviour was analysed through repeated measures ANOVA. Correlations were established between the variables. The passive transfer of immunity was successful in both breeds and the parity of the dam had no effect on the calves' serum gammaglobulin concentration. The levels of gammaglobulins were higher at the end of the first day of life, and decreased until 60 days. From there on, the increase due to the active production of antibodies was precocious in taurine calves and slower in Zebu calves. At the end of the first day of life, the gammaglobulin was correlated with IgG (r=0,859), TPP (r=0,807), TSP (r=0,811) and GGT (r=0,399). The variation of serum proteins followed the normal pattern throughout the first four months of life, with little differences between taurine and Zebu calves.

Com o objetivo de investigar alguns aspectos relacionados à transferência de imunidade passiva em bovinos de corte, foram selecionados 90 bezerros aparentemente sadios, 45 da raça Nelore e 45 da raça Limousin, distribuídos em 3 grupos (com 15 bezerros cada) de acordo com o número de parições de suas mães: primeira cria; segunda cria; e terceira ou mais crias. Amostras de sangue foram colhidas de cada bezerro entre 24 e 36 horas de vida e com 15, 30, 60, 90 e 120 dias. Determinaram-se as concentrações de proteína total no soro (PT) e no plasma (PPT), a atividade sérica da gamaglutamiltransferase (GGT), e as concentrações séricas de albumina, alfa, beta e gamaglobulinas por eletroforese em gel de agarose e de IgG estimada por meio do método de turvação pelo sulfato de zinco. Empregou-se a análise de variância bifatorial para as variáveis mensuradas na primeira colheita. O comportamento das variáveis em função da idade foi estudado por meio da análise de variância de medidas repetidas. Correlações foram estabelecidas entre as variáveis. A transferência de imunidade passiva foi bem sucedida nos bezerros de ambas as raças e o número de parições das mães não interferiu no processo. As concentrações mais elevadas de gamaglobulinas ao término do primeiro dia de vida declinaram até valores mínimos aos 60 dias. A partir dessa idade, a elevação conseqüente à produção ativa de anticorpos foi mais precoce nos bezerros taurinos e mais lenta nos zebuínos. A gamaglobulina, ao término do primeiro dia de vida, correlacionou-se com as seguintes variáveis: IgG (r=0,859), PPT (r=0,807), PT (r=0,811) e GGT (r=0,399). O fator etário exerceu efeito sobre todas as variáveis mensuradas. As variações das proteínas séricas obedeceram a um padrão de comportamento fisiológico ao longo dos quatro primeiros meses de vida, de forma geral, não distinto em taurinos e zebuínos.

Metabolismo oxidativo dos neutrófilos de bezerros das raças Nelore e Limousin nos primeiros quatro meses de vida / Neutrophil oxidative burst in Nelore and Limousin calves in the first four months of life

To study some defence mechanisms of beef calves kept on range conditions, 90 healthy calves, 45 Nelore and 45 Limousin, were bled at 24 to 36 hours of life, 15, 30, 60, 90 and, 120 days. Leukogram and neutrophil oxidative burst through the non-stimulated and the stimulated NBT tests were determined. The parasitemia with Anaplasma marginale, Babesia bigemina and B. bovis was investigated. Repeated measure ANOVA was used to analyse the age effect on leukocyte counts. Variation of positive neutrophil throughout the age was analysed through Krushkal-Wallis test. Comparisons among breeds were done using the Mann-Whithney test. The white blood cell count increased with age in both breeds, with a decrease in neutrophil and an increase in lymphocyte counts. The neutrophil lymphocyte ratio had been inverted before 15 days of life. The neutrophil oxidative burst was less effective in the newborn calves increasing with age. Limousin calves' neutrophils had higher capacity to reduce NBT in advanced ages. The observed differences between taurine and Zebu calves had no effects on calves' health and could be atributed to Anaplasma marginale natural and asynptomatic infection.

Com o objetivo de investigar alguns mecanismos de defesa em bezerros de raças de corte criadas a campo, foram utilizados 90 animais recém-nascidos aparentemente sadios, 45 da raça Nelore e 45 da raça Limousin. Amostras de sangue foram colhidas de cada bezerro entre 24 e 36 horas de vida e aos 15, 30, 60, 90 e 120 dias. Determinaram-se o leucograma e o metabolismo oxidativo dos neutrófilos por meio do teste da redução do tetrazólio de nitroazul (NBT), provas não estimulada (NBT-NE) e estimulada (NBT-E). Investigou-se a taxa de parasitemia determinada por Anaplasma marginale, Babesia bigemina e B. bovis. Utilizou-se a análise de variância de medidas repetidas para estudar o comportamento das variáveis hematológicas com o avançar da idade. O teste de Krushkal-Wallis foi empregado para caracterizar a variação da porcentagem de neutrófilos reativos relacionada à idade. Comparações entre as raças foram realizadas em cada idade por meio do teste de Mann-Whithney. A contagem total de leucócitos aumentou com a idade nas duas raças estudadas. Observou-se diminuição do número de neutrófilos e aumento do de linfócitos, ocorrendo sua inversão antes dos 15 dias de idade. A capacidade oxidativa dos neutrófilos foi menor nos bezerros recém-nascidos e aumentou com o avançar da idade. Os neutrófilos dos bezerros Limousin apresentaram maior capacidade de redução do NBT do que os dos bezerros Nelore, notadamente após o segundo mês de vida. Essa diferença não provocou reflexos sobre a saúde dos animais e pode ser resultado da infecção natural e assintomática com o Anaplasma marginale.