Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Broodstock nutritional programming differentially affects the hepatic transcriptome and genome-wide DNA methylome of farmed gilthead sea bream (Sparus aurata) depending on genetic background.

BACKGROUND: Broodstock nutritional programming improves the offspring utilization of plant-based diets in gilthead sea bream through changes in hepatic metabolism. Attention was initially focused on fatty acid desaturases, but it can involve a wide range of processes that remain largely unexplored. How all this can be driven by a different genetic background is hardly underlined, and the present study aimed to assess how broodstock nutrition affects differentially the transcriptome and genome-wide DNA methylome of reference and genetically selected fish within the PROGENSA® selection program. RESULTS: After the stimulus phase with a low fish oil diet, two offspring subsets of each genetic background received a control or a FUTURE-based diet. This highlighted a different hepatic transcriptome (RNA-seq) and genome-wide DNA methylation (MBD-seq) pattern depending on the genetic background. The number of differentially expressed transcripts following the challenge phase varied from 323 in reference fish to 2,009 in genetically selected fish. The number of discriminant transcripts, and associated enriched functions, were also markedly higher in selected fish. Moreover, correlation analysis depicted a hyper-methylated and down-regulated gene expression state in selected fish with the FUTURE diet, whereas the opposite pattern appeared in reference fish. After filtering for highly represented functions in selected fish, 115 epigenetic markers were retrieved in this group. Among them, lipid metabolism genes (23) were the most reactive following ordering by fold-change in expression, rendering a final list of 10 top markers with a key role on hepatic lipogenesis and fatty acid metabolism (cd36, pitpna, cidea, fasn, g6pd, lipt1, scd1a, acsbg2, acsl14, acsbg2). CONCLUSIONS: Gene expression profiles and methylation signatures were dependent on genetic background in our experimental model. Such assumption affected the magnitude, but also the type and direction of change. Thus, the resulting epigenetic clock of reference fish might depict an older phenotype with a lower methylation for the epigenetically responsive genes with a negative methylation-expression pattern. Therefore, epigenetic markers will be specific of each genetic lineage, serving the broodstock programming in our selected fish to prevent and mitigate later in life the risk of hepatic steatosis through changes in hepatic lipogenesis and fatty acid metabolism.

The impact of replacing barley by dehydrated orange pulp in finishing pig diets on performance, carcass quality, and gaseous emissions from slurry.

Using agricultural by-products such as dehydrated orange pulp (DOP) in animal feeds is of interest to increase pig sector sustainability. With this aim, an assay was carried out to assess the effects of increasing inclusion levels of DOP in pig diets regarding animal performance, carcass quality, and environmental impact. Four experimental diets were designed, a control diet (T1) and three more diets with increasing levels of DOP with 80, 160, and 240 g/kg of DOP for diets T2, T3, and T4, respectively. One hundred and sixty growing pigs were used in the experiment. Growth performance (average daily gain, ADG; average daily feed intake, ADFI and feed conversion ratio, FCR) and in vivo backfat thickness (BF) and loin depth (LD) gain were recorded during the finishing phase (from 70 to 130 kg BW). Faecal samples were incubated for bacteria enumeration. At slaughter, carcass characteristics and meat quality traits were measured, and subcutaneous fat was sampled to analyse the fatty acid (FA) profile. Additionally, the slurry excreted by the animals was measured, characterised and subjected to a gaseous emission assay during its storage. The final BW and overall ADFI, ADG and FCR were similar among treatments. In vivo final LD and BF gain decreased (P ≤ 0.10) as the inclusion level of DOP increased. No differences were observed in carcass characteristics with the inclusion of DOP, except carcass weight that decreased linearly (P = 0.05) with DOP. Regarding the FA profile of the subcutaneous fat, the ratio of total monounsaturated to saturated FA increased with the inclusion level of DOP. Neither slurry excretion and characterisation nor bacterial counts from faeces showed any significant difference among treatments. The inclusion of DOP led to greater CH4 emissions in mg per L of slurry and hour, whereas these differences disappeared when expressed in mg per animal and day. In all, it has been demonstrated that the inclusion of DOP up to 240 mg/kg in pig diets had minor effects on growth performance, carcass quality traits or gaseous emissions from slurry, favouring the circular economy strategy and pig sector sustainability.

Rumen biohydrogenation and milk fatty acid profile in dairy ewes divergent for feed efficiency.

A sustainable increase in livestock production would require selection for improved feed efficiency, but the mechanisms underlying this trait and explaining its large individual variation in dairy ruminants remain unclear. This study was conducted in lactating ewes to test the hypothesis that rumen biohydrogenation (BH) would differ between high- and low-efficiency animals, and these differences would be reflected in rumen fatty acid (FA) profile and affect milk FA composition. A second aim was to identify differences in FA that may serve as biomarkers of feed efficiency. Data of daily feed intake and milk yield and composition, as well as body weight, were collected individually over a 3-wk period in 40 ewes. The difference between the mean actual and predicted feed intake (estimated through metabolizable energy requirements for maintenance, production, and body weight change) over the period was used as the feed efficiency index (FEI) to select 8 of the highest feed efficiency (H-FE) and 8 of the lowest feed efficiency (L-FE) animals. In addition, residual feed intake (RFI) was estimated as the residual term from the regression of feed intake on various energy sinks. Rumen and milk FA composition were characterized by using gas chromatography, and results were analyzed using a statistical model that included the fixed effect of the group (H-FE vs. L-FE). The FEI averaged -0.29 ± 0.046 and 0.81 ± 0.084 in H-FE and L-FE, respectively, whereas RFI averaged -0.16 ± 0.084 and 0.18 ± 0.082, respectively. The correlation coefficient between both metrics was 0.69. Feed intake was similar in both groups, but H-FE showed greater milk yield, with increases in lactose content and yield, and in milk protein and fat production. Results from rumen FA profiles included a lower proportion of 18:2n-6, cis-9 18:1, and of several of their BH metabolites, and a greater concentration of 18:0, which may indicate that the apparent BH would be more complete in more efficient sheep. Milk FA analysis suggested that the greater fat yield in the H-FE group was mostly explained by increased de novo FA synthesis, whereas their milk would have lower proportions of cis-9 18:1 and C20 to 22n-6 polyunsaturated FA than L-FE. Stepwise multiple linear regression suggested that milk C20 to 22n-6 PUFA might be convenient biomarkers to discriminate more efficient dairy sheep. Further research is needed to validate these findings (e.g., under different dietary conditions).

Genotyping and molecular characterization of antimicrobial resistance in thermophilic Campylobacter isolated from poultry breeders and their progeny in Eastern Spain.

Thermophilic Campylobacter spp. are recognized as a major cause of acute bacterial diarrhea in humans, with broiler meat being the most common source of human infection. Antibiotic therapy is usually necessary for severe or prolonged infections, especially in immunocompromised populations such as young or elderly individuals. However, different studies have demonstrated a close association between antibiotic use in animal production and antimicrobial resistance (AMR) in humans. In this sense, there is social pressure to reduce antibiotic administration and find adequate alternatives to control the presence of bacterial infections in farms. However, there is a lack of information related to Campylobacter AMR dynamics through the entire production system from breeders to their progeny. It is unknown if resistance genes are a result of adaptation through chromosomal mutation or through horizontal gene transfer, instead of vertical transmission of DNA from the parent to their progeny. Thus, the main objectives of this study were to assess the main AMR rates present in a poultry production system, to study the relationship between Campylobacter AMR profiles from breeders and their progeny, and to study the presence and distribution of antibiotic resistance genes in poultry production. Regarding AMR rates, ciprofloxacin was classified as extremely high, followed by nalidixic acid and tetracyclines that were classified as very high. Moreover, this study demonstrated a relationship between the AMR patterns and genes found from Campylobacter strains isolated in breeders and those present in their progeny.

Partially defatted olive cake in finishing pig diets: implications on performance, faecal microbiota, carcass quality, slurry composition and gas emission.

One of the key factors to improve swine production sustainability is the use of agro-industrial by-products in feeds, such as olive by-products. However, it is necessary to assess its effects on the overall production process, including the animal and the environment. With this aim, an experiment was conducted to determine the effects of including a partially defatted olive cake (PDOC) in pig diets on growth performance, faecal microbiota, carcass quality and gas emission from the slurry. Two finishing diets were formulated, a control (C) diet and a diet with PDOC included at 120 g/kg. Eighty finishing male pigs Duroc-Danbred × (Landrace × Large White) of 60.4 ± 7.00 kg BW were divided between these two treatments. During the finishing period (60 to 110 kg BW, 55 days) average daily gain, average daily feed intake and feed conversion ratio were recorded. Faecal samples from the rectum of 16 animals per treatment were incubated for bacteria enumeration. At the end of finishing period, backfat thickness and loin depth (LD) were measured. Animals were slaughtered to obtain carcass weight and carcass composition parameters, and subcutaneous fat was sampled to analyse the fatty acid (FA) profile. In addition greenhouse gas and ammonia emissions were measured during pig slurry storage using the methodology of dynamic flux chambers. An initial slurry characterisation and biochemical methane potential (B0) were also determined. No significant differences between treatments were found in performance, carcass quality and microbial counts with the exception of LD, which was lower in PDOC compared with C animals (45.5 v. 47.5 mm, SEM: 0.62; P = 0.020). The FA profile of the subcutaneous fat did not differ between treatments, but the monounsaturated FA (MUFA) concentration was higher and the polyunsaturated FA was lower in the animals fed PDOC (50.9 v. 48.3, SEM: 0.48, P < 0.001; 17.6 v. 19.3, SEM: 0.30, P < 0.001 in mg/100 g of Total FA, for PDOC and C animals, respectively). The initial pig slurry characterisation only showed differences in ADF concentration that was higher (P < 0.05) in the slurry from PDOC treatment. Regarding gas emission, slurries from both treatments emitted similar amounts of ammonia (NH3), carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O), as well as B0 values. The results obtained suggest that PDOC may be included in balanced pig diets at rates of up to 120 g/kg without negative effects on performance, carcass quality, gut microflora and slurry gas emission, while improving the MUFA concentration of subcutaneous fat.

Rumen bacterial community responses to DPA, EPA and DHA in cattle and sheep: A comparative in vitro study.

The role of marine lipids as modulators of ruminal biohydrogenation of dietary unsaturated fatty acids may be explained by the effects of their n-3 polyunsaturated fatty acids (PUFA) on the bacterial community. However, the impact of individual PUFA has barely been examined, and it is uncertain which bacteria are truly involved in biohydrogenation. In addition, despite interspecies differences in rumen bacterial composition, we are not aware of any direct comparison of bovine and ovine responses to dietary PUFA. Therefore, rumen fluid from cannulated cattle and sheep were used as inocula to examine in vitro the effect of 20:5n-3 (EPA), 22:5n-3 (DPA), and 22:6n-3 (DHA) on the bacterial community. Amplicon 16 S rRNA sequencing suggested that EPA and DHA had a greater contribution to the action of marine lipids than DPA both in cattle and sheep. Certain effects were exclusive to each ruminant species, which underlines the complexity of rumen microbial responses to dietary fatty acids. Based on changes in bacterial abundance, Barnesiella, Prevotella, Paraprevotella, Hallela, Anaerovorax, Succiniclasticum, Ruminococcus and Ruminobacter may be involved in the ruminal response in biohydrogenation to the addition of marine lipids, but further research is necessary to confirm their actual role in ruminal lipid metabolism.

Milk fat depression in dairy ewes fed fish oil: Might differences in rumen biohydrogenation, fermentation, or bacterial community explain the individual variation?

Dairy ewes show large individual variation in the extent of diet-induced milk fat depression (MFD) but reasons behind this variability remain uncertain. Previous results offered no convincing support for these differences being related to relevant changes in the milk fatty acid (FA) profile, including potentially antilipogenic FA, or in the transcript abundance of candidate genes involved in mammary lipogenesis. Therefore, we hypothesized that alterations in the processes of rumen biohydrogenation and fermentation, as well as in the bacterial community structure, might account for individual variation in fish oil-induced MFD severity. To test this explanation, 15 ewes received a total mixed ration without lipid supplementation (control; n = 5) or supplemented with 20 g of fish oil/kg of dry matter [10 animals divided into those showing a strong (RESPON+; -25.4%; n = 5) or a mild (RESPON-; -7.7%; n = 5) decrease in milk fat concentration] for 5 wk. Rumen fermentation parameters, biohydrogenation metabolites, and bacterial structure and diversity were analyzed in rumen samples collected before and after treatments. Although the fish oil supplementation increased the concentration of demonstrated or putative antilipogenic FA (e.g., cis-9 16:1, cis-11 18:1, or trans-10,cis-12 CLA), surprisingly, none of them differed significantly in relation to the extent of MFD (i.e., between RESPON- and RESPON+), and this was the case only for a few minor FA (e.g., cis-6+7 16:1 or 17:0 anteiso). Changes in total volatile FA, acetate, and propionate concentrations were associated with MFD severity, with higher decreases in more susceptible animals. Individual responses were not related to shifts in rumen bacterial structure but some terminal restriction fragments compatible with Clostridiales, Ruminococcaceae, Lachnospiraceae, and Succiniclasticum showed greater abundances in RESPON-, whereas some others that may correspond to Prevotella, Mogibacterium, and Quinella-related spp. were more abundant in RESPON+. Overall, the results suggest that individual variation in MFD severity in dairy ewes fed fish oil cannot be fully explained by differences in the processes of rumen biohydrogenation and fermentation or in the bacterial community, and further research would be necessary to elucidate the large variability in the responsiveness to MFD-inducing marine lipids.

In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.

The modulation of milk fat nutritional quality through fish oil supplementation seems to be largely explained by the action of n-3 very long chain polyunsaturated fatty acids (PUFA) on ruminal biohydrogenation (BH) of C18 fatty acids (FA). However, relationships among this action, disappearance of those PUFA in the rumen, and potential detrimental consequences on ruminal fermentation remain uncertain. This study compared the effect of 20:5n-3 (eicosapentaenoic acid; EPA), 22:5n-3 (docosapentaenoic acid; DPA), and 22:6n-3 (docosahexaenoic acid; DHA) on rumen fermentation and BH of C18 FA and was conducted simultaneously in cows and sheep to provide novel insights into interspecies differences. The trial was performed in vitro using batch cultures of rumen microorganisms with inocula collected from cannulated cows and ewes. The PUFA were added at a dose of 2% incubated dry matter, and treatment effects on ruminal C18 FA concentrations, PUFA disappearances, and fermentation parameters (gas production, ammonia and volatile FA concentrations, and dry matter and neutral detergent fiber disappearances) were examined after 24 h of incubation. A principal component analysis suggested that responses to PUFA treatments explained most of the variability; those of ruminant species were of lower relevance. Overall, EPA and DHA were equally effective for inhibiting the saturation of trans-11 18:1 to 18:0 and had a similar influence on ruminal fermentation in cows and sheep (e.g., reductions in gas production and acetate:propionate ratio). Nevertheless, DHA further promoted alternative BH pathways that lead to trans-10 18:1 accumulation, and EPA seemed to have specific effects on 18:3n-3 metabolism. Only minor variations attributable to DPA were observed in the studied parameters, suggesting a low contribution of this FA to the action of marine lipids. Although most changes due to the added PUFA were comparable in bovine and ovine, there were also relevant specificities, such as a stronger inhibition of 18:0 formation in cows and a greater increase in 18:3n-3 metabolites in sheep. No direct relationship between in vitro disappearance of the incubated PUFA and effect on BH (in particular, inhibition of the last step) was found in either cows or ewes, calling into question a putative link between extent of disappearance and toxicity for microbiota. Conversely, an association between the influence of these PUFA on ruminal lipid metabolism and fermentation may exist in both species. In vivo verification of these findings would be advisable.

mRNA abundance of genes involved in mammary lipogenesis during fish oil- or trans-10,cis-12 CLA-induced milk fat depression in dairy ewes.

Milk fat depression (MFD) caused by trans-10,cis-12 18:2 is known to be mediated in cows and ewes by downregulation of mammary lipogenic genes. However, transcriptional mechanisms underlying marine lipid-induced MFD have not been well defined yet and the few available studies in ovine are not consistent. This trial was conducted to directly compare changes in animal performance, milk fatty acid composition, and particularly mammary mRNA abundance of candidate lipogenic genes and transcription factors in response to the inclusion of fish oil or trans-10,cis-12 18:2 in the dairy sheep diet. To meet this objective, 12 lactating Assaf ewes (on average, 64 days in milk, producing 1.72 kg of milk/d with 5.17% of fat) were divided into 3 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil (FO treatment) or 1% dry matter of a commercial product rich in trans-10,cis-12 18:2 (CLA treatment) for 39 d. Measurements and samplings were conducted before starting the treatments and at the end of the trial. Milk samples were used for RNA extraction from somatic cells. Feed intake was not affected by lipid supplements, and as designed, reductions in milk fat concentration (-31%) were similar in the 2 treatments, although the unpredicted increase in milk production with FO counteracted the anticipated reduction in milk fat yield. Nevertheless, this did not preclude the detection of FO-induced decreases in the mRNA abundance of candidate lipogenic genes [e.g., acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid synthase (FASN), and lipin 1 (LPIN1)], thus supporting the hypothesis that transcriptional regulation would be a relevant component of this type of MFD in sheep. Expected CLA-induced downregulation of some genes, such as FASN or sterol regulatory element binding transcription factor 1 (SREBF1), could not be detected in our samples, which might be related, at least in part, to high inter-individual variation and relatively advanced lactation stage (on average 102-103 d in milk on d 38 and 39). Overall, direct comparison of the effects of dietary FO and CLA on transcript abundance of candidate lipogenic genes and transcription factors suggest that there might be relevant differences in the transcriptional control mechanisms underlying the MFD induced by each kind of supplement (i.e., fish oil or CLA).

Comparison of feed intake, digestion and rumen function among domestic ruminant species grazing in upland vegetation communities.

This study aimed to compare feed intake, digestion, rumen fermentation parameters and bacterial community of 5 beef cows, 12 crossed ewes and 12 goats grazing together in spring-early summer on heather-gorse vegetation communities with an adjacent area of improved pasture. Organic matter intake (OMI) and digestibility (OMD) were estimated using alkane markers. Ruminal fluid samples were collected for measuring fermentation parameters, and studying the bacterial community using terminal restriction fragment length polymorphism (T-RFLP). Spot samples of urine were taken to determine purine derivative (PD) and creatinine concentrations to estimate microbial protein synthesis in the rumen. Herbaceous species were the main dietary component in all animal species. Cattle had higher (p < 0.05) daily OMI (g/kg LW0.75 ) and OMD, whereas sheep and goats showed similar values. The highest ammonia concentration was observed in sheep. Total VFA, acetate and butyrate concentrations were not influenced by animal species, while propionate concentrations in goats were 1.8 times lower (p < 0.05) than in sheep. Acetate:propionate ratio was greater (p < 0.05) in goats, whereas cattle excreted more allantoin (p < 0.05). Estimated supply of microbial N was higher in cows (p < 0.01), whereas the efficiency of microbial protein synthesis was lower (p < 0.01) in this animal species. Hierarchical clustering analysis indicated a clear effect of animal species on rumen bacterial structure. Differences among animal species were also observed in the relative frequency of several T-RFs. Certain T-RFs compatible with Lachnospiraceae, Proteobacteria and Clostridiales species were not found in goats, while these animals showed high relative frequencies of some fragments compatible with the Ruminococcaceae family that were not detected in sheep and cattle. Results suggest a close relationship between animals' grazing behaviour and rumen bacterial structure and its function. Goats seem to show a greater specialization of their microbial populations to deal with the greater fibrous and tannin content of their diet.

Role of Carnoy's solution in the treatment of keratocystic odontogenic tumor: A systematic review.

INTRODUCTION AND OBJECTIVE: The keratocystic odontogenic tumor is a benign but aggressive neoplasm. As enucleation alone obtains high recurrence rates, some adjuvant treatments such as Carnoy's solution have been proposed. The aim of this study is to evaluate the reduction of recurrences with the use of Carnoy's solution as adjuvant in the treatment of keratocystic odontogenic tumors. MATERIAL AND METHODS: An electronic search in Pubmed (MEDLINE), ScienceDirect and Cochrane databases was conducted with the key words "odontogenic keratocyst", "keratocystic odontogenic tumor", "carnoy's solution", "treatment" and "enucleation". The inclusion criteria were clinical studies using Carnoy's solution as adjuvant for the treatment of keratocystic odontogenic tumors, published in English, including at least 10 patients. Articles with an unclear reporting of the treatment applied, nonhuman studies, case reports and lesions associated to Gorlin-Goltz syndrome were excluded. RESULTS: All the studies included were case series. The recurrence rate of enucleation ranged from 0% to 58.8%. With the only use of Carnoy's solution as adjuvant treatment to the enucleation, recurrences varied from 0% to 100%. The use of ≥ 2 adjuvant treatments reduced the range between 0% and 7.9%. CONCLUSIONS: The use of Carnoy's solution as adjuvant therapy for the treatment of keratocystic odontogenic tumor has a grade C recommendation.

Increased regional gray matter atrophy and enhanced functional connectivity in male multiple sclerosis patients.

Evidence suggests that sex/gender is an important factor for understanding multiple sclerosis (MS) and that some of its neuropathological consequences might manifest earlier in males. In the present study, we assessed gray matter (GM) volume and functional connectivity (FC) in a sample of female and male MS patients (MSp) and female and male healthy controls (HCs). As compared to female MSp, male MSp showed decreased GM volume in the bilateral frontal areas and increased FC between different brain regions. Because both sets of changes correlated significantly and no differences in cognitive performance were observed, we suggest that the FC increase observed in male MSp acts as a compensatory mechanism for their more extensive GM loss and that it promotes a functional convergence between male- and female-MSp.

Comparison of ruminal lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil.

Direct comparison of cow and goat performance and milk fatty acid responses to diets known to induce milk fat depression (MFD) in the bovine reveals relevant species-by-diet interactions in ruminal lipid metabolism. Thus, this study was conducted to infer potential mechanisms responsible for differences in the rumen microbial biohydrogenation (BH) due to diet and ruminant species. To meet this objective, 12 cows and 15 goats were fed a basal diet (control), a similar diet supplemented with 2.2% fish oil (FO), or a diet containing 5.3% sunflower oil and additional starch (+38%; SOS) according to a 3 × 3 Latin square design with 25-d experimental periods. On the last day of each period, fatty acid composition (by gas chromatography) and bacterial community (by terminal-RFLP), as well as fermentation characteristics, were measured in rumen fluid samples. Results showed significant differences in the response of cows and goats to dietary treatments, although variations in some fermentation parameters (e.g., decreases in the acetate-to-propionate ratio due to FO or SOS) were similar in both species. Main alterations in ruminal BH pathways potentially responsible for MFD on the SOS diet (i.e., the shift from trans-11 to trans-10 18:1 and related increases in trans-10,cis-12 18:2) tended to be more pronounced in cows, which is consistent with an associated MFD only in this species. However, changes linked to FO-induced MFD (e.g., decreases in 18:0 and increases in total trans-18:1) were stronger in caprine rumen fluid, which may explain their unexpected susceptibility (although less marked than in bovine) to the negative effect of FO on milk fat content. Altogether, these results suggest that distinct ruminal mechanisms lead to each type of diet-induced MFD and confirm a pronounced interaction with species. With regard to microbiota, differences between cows and goats in the composition of the rumen bacterial community might be behind the disparity in the microorganisms affected by the experimental diets (e.g., Ruminococcaceae, Lachnospiraceae, and Succinivibrionaceae in the bovine, and Pseudobutryrivibrio, Clostridium cluster IV, Prevotella, and Veillonellaceae in the caprine), which hindered the assignation of bacterial populations to particular BH steps or pathways. Furthermore, most relevant variations in microbial groups corresponded to as yet uncultured bacteria and suggest that these microorganisms may play a predominant role in the ruminal lipid metabolism in both cows and goats.

Solvation and surface effects on polymorph stabilities at the nanoscale.

We explore the effects of particle size and solvent environment on the thermodynamic stability of two pairs of polymorphs subjected to ball-mill neat grinding (NG) and liquid assisted grinding (LAG). Two systems were studied: (i) forms I and II of a 1 : 1 theophylline : benzamide cocrystal and (ii) forms A and B of an aromatic disulfide compound. For both systems, the most stable-bulk polymorph converted to the metastable-bulk polymorph upon NG. LAG experiments yielded different outcomes depending on the amount of solvent used. This was further investigated by performing carefully controlled LAG experiments with increasing µL amounts of solvents of different nature. With these experiments, we were able to monitor form A to B and form I to II conversions as a function of solvent concentration and derive polymorph equilibrium curves. The concentration required for a switch in polymorphic outcome was found to be dependent on solvent nature. We propose that these experiments demonstrate a switch in thermodynamic stability of the polymorphs in the milling jar. Form B, the stable-bulk polymorph, has less stable surfaces than form A, thus becoming metastable at the nanoscale when surface effects become important. Ex situ diffraction and electron microscopy data confirm crystal sizes in the order of tens of nanometers after the ball mill grinding experiments reach equilibrium. DFT-d computations of the polymorph particles stabilities support these findings and were used to calculate cross-over sizes of forms A and B as a function of solvent. Attachment energies and surface stabilities of the various crystalline faces exposed were found to be very sensitive to the solvent environment. Our findings suggest that surface effects are significant in polymorphism at the nanoscale and that the outcomes of equilibrium ball-mill NG and LAG experiments are in general controlled by thermodynamics.

Dietary sunflower oil modulates milk fatty acid composition without major changes in adipose and mammary tissue fatty acid profile or related gene mRNA abundance in sheep.

There are very few studies in ruminants characterizing mammary and adipose tissue (AT) expression of genes and gene networks for diets causing variations in milk fatty acid (FA) composition without altering milk fat secretion, and even less complementing this information with data on tissue FA profiles. This work was conducted in sheep in order to investigate the response of the mammary gland and the subcutaneous and perirenal AT, in terms of FA profile and mRNA abundance of genes involved in lipid metabolism, to a diet known to modify milk FA composition. Ten lactating Assaf ewes were randomly assigned to two treatments consisting of a total mixed ration based on alfalfa hay and a concentrate (60 : 40) supplemented with 0 (control diet) or 25 (SO diet) g of sunflower oil/kg of diet dry matter for 7 weeks. Milk composition, including FA profile, was analysed after 48 days on treatments. On day 49, the animals were euthanized and tissue samples were collected to analyse FA and mRNA abundance of 16 candidate genes. Feeding SO did not affect animal performance but modified milk FA composition. Major changes included decreases in the concentration of FA derived from de novo synthesis (e.g. 12:0, 14:0 and 16:0) and increases in that of long-chain FA (e.g. 18:0, c9-18:1, trans-18:1 isomers and c9,t11-CLA); however, they were not accompanied by significant variations in the mRNA abundance of the studied lipogenic genes (i.e. ACACA, FASN, LPL, CD36, FABP3, SCD1 and SCD5) and transcription factors (SREBF1 and PPARG), or in the constituent FA of mammary tissue. Regarding the FA composition of AT, the little influence of SO did not appear to be linked to changes in gene mRNA abundance (decreases of GPAM and SREBF1 in both tissues, and of PPARG in the subcutaneous depot). Similarly, the great variation between AT (higher contents of saturated FA and trans-18:1 isomers in the perirenal, and of cis-18:1, c9,t11-CLA and n-3 PUFA in the subcutaneous AT) could not be related to differences in gene mRNA abundance due to tissue site (higher LPL and CD36, and lower SREBF1 in perirenal than in subcutaneous AT). Overall, these results suggest a marginal contribution of gene expression to the nutritional regulation of lipid metabolism in these tissues, at least with the examined diets and after 7 weeks on treatments. It cannot be ruled out, however, that the response to SO is mediated by other genes or post-transcriptional mechanisms.

Pseudoictus en el código ictus. Frecuencia, diagnóstico, tratamiento y destino / Stroke mimics in stroke code activations: frequency, diagnoses, treatment, and discharge destination

Objetivos: Conocer la frecuencia, diagnóstico, tratamiento trombolítico y destino de los pseudoictus (PI) atendidos como código ictus (CI), y valorar el impacto de incluir a mayores de 80 años. Método: Estudio prospectivo de una serie consecutiva de pacientes atendidos como CI, durante 50 meses, en los que se empleó tomografía computarizada craneal como prueba de neuroimagen. Se compara el primer periodo (2008-2010) en el que se siguieron los criterios SITS-MOST, con el segundo periodo (2010-2012) que se amplía la ventana temporal y se suprime el límite superior de edad. El diagnóstico se agrupa en 2 categorías: ictus (transitorio o establecido, que incluye infartos cerebrales, ataques isquémicos transitorios y hemorragias intracerebrales) y PI. Resultados: Se activaron 1.450 CI, de los que 288 se diagnosticaron como PI: 19,9% (IC 95%: 17,7-21,9%). Éstos presentaron menor edad y mayor frecuencia de mujeres. En el segundo periodo, aumentaron los mayores de 80 años (6,0% vs 22,6%, p < 0,001) y los PI disminuyeron del 23,8% al 16,2% (p 0,04). En mayores de 80 años, los PI son menos frecuentes (14,5% vs 20,4% p = 0,004). Los diagnósticos más frecuentes entre los PI son: crisis epiléptica 25,6% (IC 95%: 18,2-28,3%), síncope 16,8% (12,1-21,5%) y encefalopatía por enfermedad sistémica 16,4% (11,7-21,1%). Se trombolisaron 3 PI (1,0% IC95%: 0,2-3,0%) sin complicaciones hemorrágicas, que representan el 1,2% de los 257 trombolisados. El 46% (36,1-48,1%) de PI ingresaron en el hospital, y un 47,4% fue seguido por neurología. Conclusiones: El diagnóstico de PI es frecuente y básicamente clínico, donde una valoración neurológica experta es recomendable. La inclusión de mayores de 80 años se asoció a una reducción de PI (AU)

Objectives: To determine the frequency, of stroke mimics in stroke code activations, to analyze diagnoses, use of thrombolytic therapy and the discharge destinations of these patients; and to estimate the impact on these variables when patients over the age of 80 years are included. Methods: Prospective study of consecutive patients attended after stroke code activation; the study period was 50 months and the diagnostic image used was cranial computed tomography (CT). We compared the first period (2008-2010), in which we applied the SITS-MOST (Safe Implementation of Thrombolysis in Stroke Monitoring Study) criteria, to a second period (2010-2012), in which the time window for treatment was enlarged and there was no upper age limit for applying the criteria. Patients were grouped in 2 categories: stroke (transient or established, including cerebral infarction, transient ischemic attacks, and intracerebral hemorrhage) and pseudo-stroke (conditions mimicking stroke). Results: Stroke code was activated 1450 times; 288 cases were diagnosed as stroke mimics (19.9%; 95% CI, 17.7%-21.9%). This group was younger and included more women. In the second period, in which more patients over 80 years of age were attended (6.0% in the first period vs. 22.6% in the second, P<.001), the percentage of cases diagnosed as stroke mimics decreased (23.8% in the first period, 16.2% in the second; P<.001). Mimics were less frequent in patients over the age of 80 years (14.5% vs. 20.4% in younger patients, P=.004). The most frequent diagnoses in patients with conditions mimicking stroke were epileptic seizures (15.6%; 95% CI, 18.2%-28.3%), syncope (16.8%; 95% CI, 12.1%-21.5%), and encephalopathy due to systemic disease (16.4%; 95% CI, 11.7%-21.1%). Thrombolytic therapy was used in 3 patients with mimics (1%; 95% CI, 0.2%-3.0%) without hemorrhagic complications, which occurred in 1.2% of the 257 patients under this therapy. Forty-six percent of the patients with mimics were hospitalized (95% CI, 36.1%-48.1%); 47.4% were evaluated by a neurologist. Conclusions: Stroke mimics are common and diagnosis is essentially clinical; evaluation by a neurologist is advisable. The frequency of stroke mimics was lower when patients over the age of 80 years were considered (AU)

Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition.

Oral administration of cobalt acetate alters milk fatty acid composition, consistent with an inhibition of stearoyl-coenzyme A desaturase in lactating ewes.

Previous investigations have shown that cobalt (Co) modifies milk fat composition in cattle, consistent with an inhibition of stearoyl-coenzyme A desaturase (SCD) activity, but it remains unclear whether other ruminant species are also affected. The present study examined the effects of oral administration of Co acetate on intake, rumen function, and milk production and fatty acid (FA) composition in sheep. Twenty lactating Assaf ewes were allocated into 1 of 4 groups and used in a continuous randomized block design that involved a 15-d adaptation, a 6-d treatment, and a 10-d posttreatment period. During the treatment period, animals received an oral drench supplying 0 (control), 3 (Co3), 6 (Co6), and 9 (Co9) mg of Co/kg of BW per day, administered in 3 equal doses at 8-h intervals. Cobalt acetate had no influence on intake or milk fat and protein concentrations, whereas treatments Co6 and Co9 tended to lower milk yield. Results on rumen parameters showed no effects on rumen fermentation, FA composition, or bacterial community structure. Administration of Co acetate decreased milk concentrations of FA containing a cis-9 double bond and SCD product:substrate ratios, consistent with an inhibition of SCD activity in the ovine mammary gland. Temporal changes in milk fat composition indicated that the effects of treatments were evident within 3d of dosing, with further changes being apparent after 6d and reverting to pretreatment values by d 6 after administration. Effect on milk FA composition did not differ substantially in response to incremental doses of Co acetate. On average, Co decreased milk cis-9 10:1/10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, and cis-9,trans-11 18:2/trans-11 18:1 concentration ratios by 30, 32, 38, 33, 21, 24, and 25%, respectively. Changes in milk fat cis-9 10:1, cis-9 12:1, and cis-9 14:1 concentrations to Co treatment indicated that 51% of cis-9 18:1 and cis-9,trans-11 18:2 secreted in milk originated from Δ(9)-desaturation. In conclusion, results demonstrated the potential of oral Co administration for the estimation of endogenous synthesis of FA containing a cis-9 double bond in the mammary gland of lactating ruminants. Indirect comparisons suggest that the effects of Co differ between sheep and cattle.

Task-load manipulation in the Symbol Digit Modalities Test: an alternative measure of information processing speed.

OBJECTIVE: To evaluate the utility of an oral fMRI-adapted version of the Symbol Digit Modalities Test (SDMT) to assess information processing speed (IPS) using three different interstimulus intervals (ISI). METHODS: Nineteen right-handed healthy controls performed the adapted version of the SDMT, consisting of a block design that had a total of 6 control/activation block pairs with 3 different ISIs (1.5, 2 and 2.5s) presented in two different runs: in ascending and descending orders. The brain activation patterns during different ISIs were assessed by effective functional connectivity analysis based on independent component analysis. RESULTS: As expected, all conditions yielded activations in the fronto-parietal networks (FPNs) related to attention processes. Shorter ISIs (1.5 and 2s) not only yielded greater patterns of connectivity within fronto-parietal and occipital regions such as the FPN and fronto-occipital network (FON), but also recruited more functional networks overall. Task performance at the shortest ISI was negatively correlated with connectivity at the FPN and activity of the pre-supplementary motor area extending to the cingulate gyrus. CONCLUSION: Increasing IPS demands due to shorter ISIs resulted in an increased level and number of functional networks required, increased connectivity within the FPN and FON, and enhancement of the prefrontal cortex. IPS does not arise from activity of a single b area but from affective information transfer among distant cortical regions of the frontal and parietal cortices. This adapted version of the SDMT may be useful for studying alterations of IPS in clinical and nonclinical populations.