Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20-100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.

Prolonged feeding of high-concentrate diet remodels the hindgut microbiome and modulates nutrient degradation in the rumen and the total gastrointestinal tract of cows.

The aims of this research were to evaluate how prolonged feeding of a high-concentrate diet affects the ruminal degradation kinetics of fiber and starch, and to evaluate the effects of the high-concentrate diet on apparent total-tract nutrient digestibility in dairy cows. We also investigated the dysbiotic effects and the remodeling of the hindgut microbiome with prolonged high-concentrate feeding. Nine Holstein cows were used in 2 experimental periods; in each period, cows were first fed a 100% forage diet (Forage) for 1 week, followed by stepwise adaptation during one week to a high-concentrate diet (HC; 65% concentrate), which was then fed for 4 consecutive weeks. The kinetics of in situ ruminal degradability of grass silage (DM and NDF), corn grain and wheat grain (DM and starch) as well as the apparent total-tract nutrient digestibility were evaluated in the Forage feeding and in wk 4 on HC. Whereas the hindgut microbiome and fermentation profile were evaluated on a weekly basis. Regarding the in situ ruminal degradability due to grain type, the rate of degradation of the potentially degradable fraction of the grain and the effective rumen degradability of wheat grain were greater compared with corn grain. The in situ ruminal degradability of NDF decreased with the HC diet. However, the apparent total-tract digestibility of crude protein, fat, starch, NDF, ADF and NFC increased with HC compared with Forage feeding. In addition, the HC diet increased the concentration of short-chain fatty acids in the hindgut, lowering fecal pH by 0.6 units, which correlated positively with microbial α diversity. This resulted in lower α diversity with HC; however, α diversity (number of ASVs) showed recovery in wk 3 and 4 on HC; in addition, microbial ß diversity did not change from wk 2 on HC onwards. Two microbial enterotypes were identified: one for the Forage diet with abundance of Akkermansia and Anaerosporobacter, and another enterotype for the HC diet with enrichment in Bifidobacterium and Butyrivibrio. Overall, results show that major microbial shifts and hindgut dysbiosis occurred in wk 1 on HC. However, the hindgut microbial diversity of cows adapted after 3 weeks of consuming the starch-rich ration. Thus, feeding HC diet impaired fiber degradation in the rumen, but increased apparent total-tract nutrient digestibility. Likely, the forage diet contained less digestible NDF than the HC diet due to greater inclusion of forages with lower NDF digestibility and lower inclusion of more digestible non-forage NDF. Results also suggest that the adaptation of the hindgut microbial diversity of cows observed 3 weeks after the diet transition likely contributed to enhance total-tract nutrient digestibility.

Bovine rumen epithelial miRNA-mRNA dynamics reveals post-transcriptional regulation of gene expression upon transition to high-grain feeding and phytogenic supplementation.

The rumen epithelium has a pivotal role in nutrient uptake and host health. This study aimed to explore the role of microRNAs (miRNAs) in the epithelial transcriptome during diet transition from forage to high-grain feeding and the modulation through supplementation with a phytogenic feed additive. Rumen biopsies were collected from 9 ruminally-cannulated non-lactating Holstein cows fed a baseline forage diet (FD) and then transitioned to high-grain feeding (HG; 65% concentrate on a dry matter basis). Cows were randomly allocated into a control group (CON, n = 5) and a group supplemented with a phytogenic feed additive (PHY, n = 4). MiRNA and mRNA sequencing was performed in parallel and transcripts were analyzed for differential expression, pathway enrichment analysis, and miRNA-mRNA interaction networks. We identified 527 miRNAs shared by all samples of the rumen epithelium, from which, bta-miR-21-5p, bta-miR-143 and bta-miR-24-3p were the most expressed. Six miRNAs were differentially expressed between CON and PHY and 8 miRNAs between FD and HG feeding, which were mainly associated with fat metabolism. Transcriptome analysis identified 9481 differentially expressed genes (DEGs) between FD and HG, whereas PHY supplementation resulted in 5 DEGs. DEGs were mainly involved in epithelium development and morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with tricarboxylic acid and short chain fatty acid (SCFA) metabolism were enriched in DEGs between diets. MiRNA target prediction and anti-correlation analysis was used to construct networks and identify DEGs targeted by DE miRNAs responsive to diet or PHY. This study allowed the identification of potential miRNA regulation mechanisms of gene expression during transition from FD to HG feeding and phytogenic supplementation, evidencing a direct role of miRNAs in host responses to nutrition.

Changes in fermentation profile of the reticulorumen and hindgut, and nutrient digestion in dry cows fed concentrate-rich diets supplemented with a phytogenic feed additive.

This study evaluated the effects of duration of high-concentrate feeding on ruminal and fecal fermentation profile, as well as selected systemic health biomarkers in nonlactating cows supplemented with or without a phytogenic feed additive (PHY). In addition, ruminal degradation kinetics and total-tract nutrient digestibility were evaluated when feeding either only forage or a high-concentrate diet. Nine nonlactating, cannulated Holstein cows were used in a crossover design. Each period included 1 wk of forage feeding (wk 0), diet transition, and 4 wk on the high-concentrate diet (1, 2, 3 and wk 4; 65% dry matter basis). Cows received PHY or not (control). Compared with wk 0, from wk 1 onward, cows on high concentrate showed greater reticular, ruminal, and fecal total volatile fatty acids (VFA), with a greater level of VFA in the rumen than in the hindgut. However, ruminal fermentation was modulated differently by PHY, which showed increased total VFA in wk 1 and increased butyrate in wk 2 in the particle-associated fluid of rumen. In the hindgut, PHY increased propionate in wk 3. Cows fed a high-concentrate diet from wk 1 and onward also showed greater ruminal lactate, as well as lower ruminal and fecal pH, independent of PHY. In addition, compared with cows in wk 1 on a high-concentrate diet, cows in wk 4 had a greater total VFA in free fluid of the rumen and lower fecal pH. Compared with cows at wk 0, cows at wk 1 on high concentrate onward showed greater serum amyloid A and greater activity of glutamate dehydrogenase. In contrast, the high-concentrate diet decreased in situ ruminal degradability of grass silage but increased degradability of corn grain as well as total-tract nutrient digestibility, with total-tract neutral detergent fiber digestibility being greater for cows on the PHY treatment. Overall, from the start of high-concentrate feeding, gut fermentation increased, but differently according to location or PHY, with a stronger build-up of VFA in the rumen compared with the hindgut. In addition, a longer duration on high concentrate exacerbated gut acidification. The enhancing effects of PHY on total VFA and butyrate in particle-associated fluid of the rumen suggest beneficial effects of PHY on particle-associated bacteria, likely contributing to the increased neutral detergent fiber digestibility. The greater production of ruminal butyrate with PHY may be beneficial for the host, given the health benefits of this acid, but more research is needed to elucidate the effects on gut microbiota and the effects of increased butyrate in nonlactating dairy cows.

Short-term screening of multiple phytogenic compounds for their potential to modulate chewing behavior, ruminal fermentation profile, and pH in cattle fed grain-rich diets.

In cattle, proper rumen functioning and digestion are intimately linked to chewing behavior. Yet, high grain feeding impairs chewing activity, increasing the risk of subacute ruminal acidosis and dysfermentation. This study aimed to screen 9 different phytogenic compounds for their potential to modulate chewing activity, meal size, rumino-reticular short-chain fatty acids (SCFA), and pH during consumption in a first daily meal and shortly thereafter in cattle fed a grain-rich diet. Treatments were control (total mixed ration without phytogenic) or addition of a phytogenic compound at a low or high dose. Phytogenic compounds and doses (all in mg/kg) were angelica root (6.6 and 66), capsaicin (10 and 100), gentian root (6.6 and 66), garlic oil (0.3 and 3), ginger extract (40 and 400), L-menthol (6.7 and 67), mint oil (15.3 and 153), thyme oil (9.4 and 94), and thymol (5 and 50), for the low and high groups, respectively. Before the start of the screening experiment, cows were fed to reach subacute ruminal acidosis conditions, confirmed with the time of ruminal pH <5.8 being 655 ± 148.2 min/d. During the screening experiment, the treatments were offered in a controlled meal (2.5 kg of DM for 4 h) as part of the daily diet with 65% concentrate. Each treatment was tested in 4 of the 9 cannulated Holstein cows using an incomplete Latin square design. Ruminal and reticular fluids were sampled before and after each treatment, and data collected before the meal were used as covariates. Chewing and ruminal pH were monitored during the treatment, followed by 2 h of complete feed restriction, and then 4 h of ad libitum feed intake without phytogenic. Data showed that supplementation of angelica root tended to linearly increase rumination time immediately after the first meal when feed was restricted (27.3, 41.9, and 42.6 ± 5.99 min for control, low and high groups, respectively). Capsaicin increased eating time (43.6, 49.4, and 66.4 ± 4.93 min) during consumption but did not affect ruminal total SCFA or mean ruminal pH. Garlic oil reduced the concentration of reticular total SCFA (75.7, 71.3, and 60.1 mM) and tended to decrease ruminal acetate-to-propionate ratio (2.50, 1.78, and 1.87 ± 0.177) with no effect on ruminal pH. The L-menthol affected reticular total SCFA quadratically (76.1, 64.9, and 81.0 ± 4.22%), and ruminal pH responded quadratically when feed was reintroduced ad libitum (6.0, 6.3, and 6.1 ± 0.07). Mint oil did not affect chewing or total SCFA during consumption, but the low dose increased ruminal pH (6.5, 6.7, and 6.5 ± 0.08). Thyme oil tended to lower the severity of ruminal acidosis. Overall, phytogenic compounds demonstrated distinct dose-dependent effects to beneficially influence chewing behavior, modulate fermentation, and mitigate ruminal acidosis in dairy cows under a high-grain challenge diet.

Dynamic changes in salivation, salivary composition, and rumen fermentation associated with duration of high-grain feeding in cows.

Salivary secretions are essential for the regulation of digestive processes, as well as rumen and cow health. This research evaluated the effects of the duration of high-grain feeding, and of the time relative to a meal, on salivation, saliva properties, feed bolus characteristics, chewing activity, ruminal and reticular volatile fatty acids, as well as salivary and ruminal pH. Nine nonlactating cannulated Holstein cows were sampled at 1 and 23 d after transition to a 65% grain diet (short term and long term, respectively). Both before and after a controlled meal (2.5 kg of dry matter, offered over 4 h), unstimulated saliva was taken orally for composition analysis. Stimulated salivation and feed boli characteristics were evaluated by collection of ingesta from cardia during 30 min. Chewing and ruminal pH were measured during the controlled meal and for a total of 6 h thereafter. Results from unstimulated saliva showed no effect of the duration of high-grain feeding on bicarbonate, phosphate, total proteins, mucins, lysozyme, and buffer capacity, but increased osmolality at the long term. Lysozyme activity did not differ with high-grain feeding duration, but tended to be lower after the meal. In contrast to short-term-fed cows, the long-term-fed cows increased both meal consumption and feed bolus size, but decreased chewing and feed ensalivation (5.2 vs. 4.6 ± 0.50 g of saliva/g of dry matter), and had lower pH of the stimulated saliva (7.00 vs. 6.67 ± 0.076). These cows also had decreased chewing index (66.5 vs. 45.4 min/kg of neutral detergent fiber), and despite the increase in stimulated saliva buffer capacity (0.027 vs. 0.039 ± 0.006), mean ruminal pH decreased (6.31 vs. 6.11 ± 0.065) during ad libitum feeding. Both in the rumen and reticulum, the concentration of total volatile fatty acids was lower and propionate proportion was higher at the long term. Linear regression analyses revealed a positive influence of the flow rates of salivary bicarbonate and phosphate on ruminal pH during the short term. For every 1-mol increment in the flow of bicarbonate or phosphate, ruminal pH increased by 0.062 or 0.439 units, respectively. Overall, salivary buffers are key determinants of ruminal pH regulation, especially during short-term grain feeding. However, in the long term, ruminal pH drop during ad libitum feeding was stronger, and this effect seems to be exacerbated by increased feed bolus size, accompanied by reductions in feed ensalivation, stimulated saliva pH, and chewing index.

Patients' perceptions of implant therapy and maintenance: A questionnaire-based survey.

OBJECTIVES: To evaluate patients' perceptions regarding implant treatment and maintenance. MATERIAL AND METHODS: A semi-structured questionnaire was developed focusing on 4 main domains: (a) information regarding possible complications received before implant therapy; (b) information received after treatment regarding maintenance; (c) self-performed daily home care; and (d) implant maintenance received at the dental office. Adult patients, restored with implant-supported prosthesis by at least 6 months, were recruited between June 2016 and December 2017. RESULTS: The study population consisted of 522 patients with a mean age of 61 years. The majority of the participants referred to have received information about the need of periodical check-ups (91.6%). However, only 58.9% declared to have been informed about complications and failures before treatment commencement. Even though 91.2% of the participants declared to have received instruction about cleaning measures around implants, less than half of the sample reported to have been asked to try the cleaning tools at the office (40.4%). Even though patients' awareness about implant treatment and maintenance resulted to be low in a consistent part of the sample, patients' satisfaction level about information received regarding implant care was high (Visual Analogue Scale 8.18). CONCLUSIONS: Most patients appeared to be informed about the importance of specific oral hygiene measurements and recall programs. However, an unsatisfactory level of knowledge about implant-related complications was evidenced. Improvements in time and energy employed by dentists and hygienist in communication with patients are needed, in order to enhance health outcomes and compliance both to the treatment and to the maintenance.

Evaluation and comparison of histologic changes and implant survival in extraction sites immediately grafted with two different xenografts: A randomized clinical pilot study.

OBJECTIVES: The purpose of this prospective, single-center randomized pilot study was to histologically evaluate and compare vital bone development in premolar and molar-extraction sites grafted with two different bovine-derived xenografts. The secondary outcome of interest was implant survival in the grafted sites. MATERIALS AND METHODS: Adult patients in need of at least two tooth extractions were enrolled. A paired design was used; each patient received at least one of each type of graft at different sites. Each extraction site was randomized to one of two xenograft treatment groups. A resorbable membrane was always placed, and primary intention soft tissue closure was achieved. Four months later, implants were placed and a trephine drill was used to remove bone cores for histologic and histomorphometric analysis. RESULTS: Sixteen patients with 40 extraction sites were enrolled; 20 sites were grafted with one type of xenograft and 20 with another. Mean patient age was 53.5 years, and 65% of patients were male. Evaluation of bone core samples taken from grafted sites showed no significant difference in the mean value of percentage of new bone formation between the different grafted sites (33.4% and 32.4%, p = .76). Cumulative implant survival was 97.5% at the 24-month follow-up visit. CONCLUSION: Within the limitations of this pilot study, no statistically significant differences in new bone growth between sites grafted with two different types of xenograft were found. Both graft materials promoted the formation of new bone and provided osseous support for implant placement after socket grafting.

Healing at implants placed in bone of different morphology: an experimental study in dogs.

OBJECTIVE: To study osseointegration at implants installed using a standard bed preparation in sites of different bone morphology. MATERIAL AND METHODS: In six Labrador dogs, all mandibular premolars and first molars were extracted. After 4 months of healing, flaps were elevated, and two recipient sites were prepared in each side of the mandible, one in the second premolar and the other in the molar regions. Bone morphology and final insertion torque were evaluated. Healing abutments were applied, and the flaps were sutured to allow a non-submerged healing. After 4 months, the animals were sacrificed and ground sections were obtained for histomorphometric analyses. RESULTS: At the premolar sites, bone morphology Class 2 and at the molar regions Class 3 or 4 were identified. The final insertion torque was 50-55 Ncm at the premolar and 30-35 Ncm at the molar sites. Mean osseointegration in percentage reached 61.5 ± 11.5% and 63.3 ± 10.1% at the premolar and molar sites, respectively. Mineralized bone density evaluated from the implant surface up to a distance of about 0.6 mm lateral to the implant surface was 63.0 ± 7.4% and 65.4 ± 17.7% at the premolar and molar sites, respectively. CONCLUSIONS: Similar implant bed preparations performed at premolar and molar sites with different bone morphology, yielding insertion torque values of about 30-35 and 50-55 Ncm, respectively, did not affect osseointegration after 4 months at non-submerged implants.

Marginal healing using Polyetheretherketone as healing abutments: an experimental study in dogs.

OBJECTIVE: To evaluate the marginal soft and hard tissue healing at titanium and Polyetheretherketone (PEEK) healing implant abutments over a 4-month period. MATERIAL AND METHODS: In six Labrador dogs, all mandibular premolars and first molars were extracted. After 4 months of healing, flaps were elevated, and two implants were installed at each side of the mandible, one in the premolar and the other in the molar regions. Four different types of healing abutments were positioned on the top of each implant: (i) titanium (Ti); (ii) PEEK material bonded to a base made of titanium (Ti-P), randomly positioned in the premolar region; (iii) PEEK, pristine (P); and (iv) PEEK, roughened (P-R), randomly positioned in the molar region. The flaps were sutured to allow a non-submerged healing, and after 4 months, the animals were sacrificed and ground sections obtained for histological evaluation. RESULTS: A higher resorption of the buccal bone crest was observed at the PEEK bonded to a base made of titanium abutments (1.0 ± 0.3 mm) compared to those made of titanium (0.3 ± 0.4 mm). However, similar dimensions of the peri-implant mucosa and similar locations of the soft tissues in relation to the implant shoulder were observed. No statistically significant differences were seen in the outcomes when the pristine PEEK was compared with the roughened PEEK abutments. The mean apical extension of the junctional epithelium did not exceed the implant shoulder at any of the abutment types used. CONCLUSIONS: The coronal level of the hard and soft tissues allows the conclusion that the use of PEEK as healing abutments may be indicated.

Morphometric evaluation of the early stages of healing at cortical and marrow compartments at titanium implants: an experimental study in the dog.

OBJECTIVE: To study the early sequential stages of tissue composition in the cortical and marrow compartments of the alveolar bone crest at implants with a moderately rough surface. MATERIALS AND METHODS: Three month after tooth extraction in 12 Labrador dogs, full-thickness flaps were elevated in the edentulous region of the right side of the mandible and one implant was installed. The flaps were sutured to allow a fully submerged healing. The timing of the installations in the left side of the mandible and of sacrifices were scheduled in such a way to obtained biopsies representing the healing after 5, 10, 20, and 30 days. Ground sections (n = 6 per each healing period) were prepared, and the percentages of osteoid/new bone, old bone, new soft tissues (provisional matrix and primitive marrow), mature bone marrow, vessels, and other tissues (bone debris/particles and clot) were evaluated laterally to the implant surface up to a distance of about 0.4 mm from it. RESULTS: Osteoid/new bone was found after 5 days at percentages of 10.8 ± 4.3% at the marrow and 0.6 ± 0.6% at the cortical compartments. After 30 days, these percentages increased up to 56.4 ± 4.0% and 23.3 ± 6.1%, respectively. Old parent bone was resorbed between 5 and 30 days from 28.7 ± 10.9% to 14.9 ± 3.4% at the marrow (~48% of resorption) and from 81.2 ± 9.4% to 67.6 ± 5.6% at the cortical (~17% of resorption) compartments. All differences were statistically significant. CONCLUSION: Bone apposition to an implant surface followed a significantly different pattern in the compact and the marrow compartments around the implants. While in the compact compartments, bone apposition had to develop through the BMUs following resorption, it developed in very dense layers through an early apposition in the marrow compartments.

Influence of immediate loading on healing of implants installed with different insertion torques--an experimental study in dogs.

OBJECTIVE: To evaluate the influence of different insertion torques on healing of implants loaded immediately or left unloaded. MATERIAL AND METHODS: In six Labrador dogs, all mandibular premolars and molars were extracted. After 4 months of healing, flaps were elevated, and two implant sites were prepared at each side of the mandible. The distal sites were prepared conventionally while the mesial sites were underprepared by 0.3 mm. As a consequence, different final insertion torques of about 30 Ncm at the distal and >70 Ncm at the mesial sites were recorded. Healing abutments were applied to the left and transmucosal abutments to the right side. Flaps were sutured, crown preparation of the upper right second and third premolars was performed, and impressions were taken. Within 24 h, crowns were cemented both to implants and teeth in the right side of the mouth. After 4 months, the animals were sacrificed and ground sections obtained for histological evaluation. RESULTS: A higher buccal bony crestal resorption and a more apical position of the coronal level of osseointegration were found at the loaded compared with the unloaded sites. MBIC% and percentages of peri-implant mineralized tissue (MB%) were higher at the loaded compared with the unloaded sites. Moreover, a higher MBIC% was found at the lower compared with the higher final insertion torque. CONCLUSIONS: Immediate loading does not seem to have a negative effect on osseointegration. High torque values for the immediate loading procedures were not necessary. Probably, low torque values, were sufficient to obtain primary stability and hence may provide better osseointegration than high torque value.

Healing of implants installed in over- or under-prepared sites--an experimental study in dogs.

OBJECTIVE: To study bone healing at implants installed with different insertion torques. MATERIAL AND METHODS: In six Labrador dogs, all mandibular premolars and first molars were extracted. After 4 months of healing, flaps were elevated, and two implant sites were prepared at each side of the mandible. In the right side of the mandible, the distal sites were prepared conventionally, while the mesial sites were over-prepared by 0.2 mm. As a consequence, a final insertion torque of ~30 Ncm at the distal and a minimal insertion torque close to 0 Ncm at the mesial sites were obtained. In the left sides of the mandible, however, the recipient sites were underprepared by 0.3 mm resulting in an insertion torque of ≥ 70 Ncm at both implants. Cover screws were applied, and flaps sutured to fully submerge the experimental sites. After 4 months, the animals were sacrificed and ground sections obtained for histological evaluation. RESULTS: The mineralized bone-to-implant contact was in the range of 55.2-62.1%, displaying the highest value at implants with ~30 Ncm insertion torque and the lowest value at the implant sites with close to 0 Ncm insertion torque. No statistically significant differences were revealed. Bone density was in the range of 43.4-54.9%, yielding the highest value at implants with ≥ 70 Ncm insertion torque and the lowest at the implant sites with close to 0 Ncm insertion torque. The difference between the sites of ~30 Ncm and the corresponding ≥ 70 Ncm insertion torque reached statistical significance. CONCLUSIONS: Similar amounts of osseointegration were obtained irrespective of the insertion torque applied. Moreover, implants installed in sites with close to 0 Ncm insertion torque may properly osseointegrate as well.

The platform switching concept revisited. An experimental study in dogs.

OBJECTIVE: To evaluate the influence of the configuration of the marginal aspect of implants placed immediately into extraction sockets on peri-implant hard tissue adaptation. MATERIAL AND METHODS: In 6 Labrador dogs, endodontic treatments of the mesial roots of 1 M1 were performed and the distal roots were removed. 2 P2 was extracted as well. Implants were immediately placed in the center of the distal alveoli. Cylindrical straight implants were installed in the right side of the mandible (Control), while, in the left side, implants with a reduced diameter in the coronal portion, yielding an indentation in the surface continuity (Test), were installed. Cover screws were affixed, and the flaps were sutured to allow non-submerged healing. After 4 months of healing, histological slides were obtained for assessments. RESULTS: A buccal resorption of 1.58 ± 1.28 and 1.90 ± 1.93 mm at the control and of 0.26 ± 0.90 and 0.14 ± 0.66 mm at the test sites was observed at the premolar and molar regions, respectively. The buccal coronal level of osseointegration was located apically to the margin of the smooth/rough surface border by 2.40 ± 0.90 and 3.70 ± 0.87 mm at the control sites and 1.19 ± 0.45 and 2.16 ± 0.96 mm at the test sites at the premolar and molar sites, respectively. All differences yielded statistical significance. CONCLUSIONS: The use of implants with a reduced diameter in their coronal aspect may contribute to preservation of the buccal bony crest in a more coronal level compared with conventional implants. Thus, the study confirmed the efficacy of the "platform switching" concept.

Duration of Zearalenone Exposure Has Implications on Health Parameters of Lactating Cows.

There is a limited research focus on evaluating the detrimental effects of prolonged zearalenone (ZEN) intake on dairy cows' health under controlled conditions. This experiment was conducted to evaluate whether the length of exposure to a ZEN-contaminated total mixed ration (TMR) at a level of 9.45 mg per day can negatively influence animal health parameters, such as milk composition, rumen and fecal fermentation, and the chewing activity of lactating dairy cows. For this experiment, we used 18 lactating Simmental cows that were fed a diet of 60% forage and 40% concentrate (on dry matter basis) for 26 consecutive days. The first 4 days were for adaptation prior to the first sampling day (day 0). The sampling events took place on day 0 (baseline) without ZEN, followed by day 1, day 7, day 14, and day 21 (with toxin). Dry matter intake (DMI) and ruminating chews per minute increased on the third week of ZEN inclusion; meanwhile, ruminating, eating, and drinking times were not affected. Most milk composition variables were also unaffected. Rumen fluid osmolality increased on day 21 and total short-chain fatty acids (SCFA) of ruminal fluid decreased on day 7. Fecal SCFA increased on day 21 and the acetate-to-propionate ratio increased from day 1 onwards, showing the influence of toxin intake. Animal health parameters, like heart rate, respiratory rate, and body temperature, were negatively influenced by ZEN intake, all increasing consistently on days 4 and 6, 9 and 12, and 16 and 18, respectively. The liver enzyme glutamate dehydrogenase decreased in response to ZEN intake on day 7. A total daily ZEN intake at the level of 9.45 mg did not show detrimental effects on DMI. Nevertheless, certain health parameters were negatively affected, including body temperature, respiratory rate, and heart rate, starting from the 7th day of ZEN intake, with additional signs of possible loss of water balance on the last sampling day.

Dynamic interplay of immune response, metabolome, and microbiota in cows during high-grain feeding: insights from multi-omics analysis.

This study explores the dynamics of immune gene expression, ruminal metabolome, and gut microbiota in cows due to the duration of high-grain feeding, shedding light on host response and microbial dynamics in parallel. Cows consumed forage for a week, then gradually transitioned to a high-grain diet, which they consumed for 4 weeks. Immune response was evaluated in ruminal papillae by expression of genes related to the nuclear factor-kappaB (NFkB) pathway and correlated with the microbiota. Rumen metabolome was evaluated with high-performance liquid chromatography coupled with mass spectrometry and anion-exchange chromatography. Rumen and fecal microbiota were evaluated with 16S rRNA gene amplicon sequencing. In the rumen, expression of inflammation-associated genes increased with the duration on high grain, indicating activation of pro-inflammatory cascades; microbial diversity decreased with a high-grain diet but stabilized after week 3 on high grain. Changes in microbial relative abundance and metabolite enrichment were observed throughout the 4 weeks on high grain, with increments in propionogenic taxa (i.e., Succinivibrionaceae). Metabolite enrichment analysis showed that at the start of high-grain feeding, simple carbohydrates were enriched; then, these were substituted by their fermentation products. There were correlations between certain ruminal bacterial taxa (i.e., Ruminococcaceae UCG-005) and expression of genes of the NFkB pathway, suggesting the influence of these taxa on host immune response. In feces, microbial diversity and several Ruminococcaceae members initially declined but recovered by weeks 3 and 4. Overall, despite the stabilization of microbial diversity, changes in microbial relative abundance and proinflammatory genes were observed throughout high-grain feeding, suggesting that cows need more than 4 weeks to fully adjust once consuming a high-grain diet.IMPORTANCEDespite the stepwise diet transition typically assumed to serve for animal adaptation, expression of signaling receptors, mediators, and downstream targets of nuclear factor-kappaB pathway were found throughout the 4 weeks on high grain, which correlated with changes in the rumen microbial profile. In addition, although microbial diversity recovered in the feces and stabilized in the rumen in week 3 on high grain, we observed changes in microbial relative abundance throughout the 4 weeks on high grain, suggesting that cows need more than 4 weeks to adjust once consuming this diet. Findings are particularly important to consider when planning experiments involving dietary changes.

Integrated microbiota-host-metabolome approaches reveal adaptive ruminal changes to prolonged high-grain feeding and phytogenic supplementation in cattle.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment.

Diet and phytogenic supplementation substantially modulate the salivary proteome in dairy cows.

Phytogenic compounds may influence salivation or salivary properties. However, their effects on the bovine salivary proteome have not been evaluated. We investigated changes in the bovine salivary proteome due to transition from forage to high-concentrate diet, with and without supplementation with a phytogenic feed additive. Eight non-lactating cows were fed forage, then transitioned to a 65% concentrate diet (DM basis) over a week. Cows were control (n = 4, CON) or supplemented with a phytogenic feed additive (n = 4, PHY). Proteomic analysis was conducted using liquid chromatography coupled with mass spectrometry. We identified 1233 proteins; 878 were bovine proteins, 189 corresponded to bacteria, and 166 were plant proteins. Between forage and high-concentrate, 139 proteins were differentially abundant (P < 0.05), with 48 proteins having a log2FC difference > |2|. The salivary proteome reflected shifts in processes involving nutrient utilization, body tissue accretion, and immune response. Between PHY and CON, 195 proteins were differently abundant (P < 0.05), with 37 having a log2FC difference > |2|; 86 proteins were increased by PHY, including proteins involved in smell recognition. Many differentially abundant proteins correlated (r > |0.70|) with salivary bicarbonate, total mucins or pH. Results provide novel insights into the bovine salivary proteome using a non-invasive approach, and the association of specific proteins with major salivary properties influencing rumen homeostasis. SIGNIFICANCE: Phytogenic compounds may stimulate salivation due to their olfactory properties, but their effects on the salivary proteome have not been investigated. We investigated the effect of high-concentrate diets and supplementation with a phytogenic additive on the salivary proteome of cows. We show that analysis of cows' saliva can be a non-invasive approach to detect effects occurring not only in the gut, but also systemically including indications for gut health and immune response. Thus, results provide unique insights into the bovine salivary proteome, and will have a crucial contribution to further understand animal response in terms of nutrient utilization and immune activity due to the change from forage to a high-energy diet. Additionally, our findings reveal changes due to supplementation with a phytogenic feed additive with regard to health and olfactory stimulation. Furthermore, findings suggest an association between salivary proteins and other components like bicarbonate content.

Supplementing a Phytogenic Feed Additive Modulates the Risk of Subacute Rumen Acidosis, Rumen Fermentation and Systemic Inflammation in Cattle Fed Acidogenic Diets.

Feeding with high-concentrate diets increases the risk of subacute ruminal acidosis (SARA). This experiment was conducted to evaluate whether supplementing a phytogenic feed additive based on L-menthol, thymol, eugenol, mint oil (Mentha arvensis) and cloves powder (Syzygium aromaticum) (PHY) can amend the ruminal fermentation profile, modulate the risk of SARA and reduce inflammation in cattle. The experiment was designed as a crossover design with nine non-lactating Holstein cows, and was conducted in two experimental runs. In each run, cows were fed a 100% forage diet one week (wk 0), and were then transitioned stepwise over one week (0 to 65% concentrate, wk adapt.) to a high concentrate diet that was fed for 4 weeks. Animals were fed diets either with PHY or without (CON). The PHY group had an increased ruminal pH compared to CON, reduced time to pH < 5.8 in wk 3, which tended to decrease further in wk 4, reduced the ruminal concentration of D-lactate, and tended to decrease total lactate (wk 3). In wk 2, PHY increased acetate, butyrate, isobutyrate, isovalerate, and the acetate to propionate ratio compared to CON. Phytogenic supplementation reduced inflammation compared to CON in wk 3. Overall, PHY had beneficial effects on ruminal fermentation, reduced inflammation, and modulated the risk of SARA starting from wk 3 of supplementation.

Mandibular Molar Uprighting Using Skeletal Anchorage: A Novel Approach.

BACKGROUND: The management of eruption anomalies affecting second molars, although quite uncommon, may represent a real challenge for the clinician. The aim of this study is to present a novel technique that combines the surgical and orthodontic approach in order to obtain the retrieval of impacted second molars through a complex distalizing movement and skeletal anchorage. METHODS: Nineteen consecutive patients with impacted second molars were treated according to this technique, which involved extraction of the third molar followed by the placement of a distally positioned screw, and the subsequent use of a coil spring to connect the screw to an eyelet directly bonded on the second molar. In selected cases, it proved to be necessary to reposition the second molar through traditional orthodontics. All of the parameters were recorded: the time required for completing the treatment, the initial angle between the second molar and the adjacent tooth, and treatment related-complications. RESULTS: Twenty impacted second molars were correctly repositioned. The mean initial angle of the second molar was 55.7° (SD 22.6°, min 13.3°, max 104.8°). The mean treatment time was 10 months. There were no major complications. CONCLUSIONS: Given that periodontal tissues were minimally affected, the temporary anchorage device was fully controlled, and there was no unwanted movement of adjacent teeth, the presented technique not only proved effective but also minimally invasive.