Andrea Minuti

Nutrition and Ageing

The world elderly population is rapidly increasing. This demographic change represents a new challenge for the society and demands for a multisectorial intervention to promote a long, healthy, and active life span. Between the factors that contribute in fostering a long healthy life, the nutritional regime plays a central role and is recognized as a major factor in the onset of chronic diseases. A better understanding of the interaction between nutrition and ageing is essential to unravel the mechanisms responsible for these positive/negative effects and to identify diet components promoting the quality of life in the old age and to contribute to the prevention of late-life disabilities. At Università Cattolica del Sacro Cuore, the research activity in food science is focusing on four main objectives: food quality, food safety, functional foods and diet balancing. These objectives are the target of multidisciplinary ongoing and future research activities for a better understanding of the link between diet and ageing. Briefly, the different activities are addressed to the study of the following subjects: the most relevant factors affecting food choices and habits of old aged persons; the effects of long term low dose supplementation of conjugated linoleic acid in mouse; the use of low glycemic index and high resistant starch foods to prevent diabetes and obesity; the adjuvant effect of food bacteria for vaccination; the role of food ingredients in disease; the immunosuppression effect of mycotoxins, and its relevance in ageing people; the production of sustainable and natural antioxidant ingredients to encourage a healthy diet. Our research projects emphasize an holistic and integrated approach that, by bringing together complementary research groups, can combine the collective expertise and thus provide a comprehensive assessment of the role of nutrition in healthy ageing people.

Abundance of ruminal bacteria, epithelial gene expression, and systemic biomarkers of metabolism and inflammation are altered during the peripartal period in dairy cows

Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and β-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from -14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). Overall, these multitier changes revealed important features associated with the transition into lactation. Alterations in ruminal epithelium gene expression could be driven by nutrient intake-induced changes in microbes; microbial metabolism; and the systemic metabolic, hormonal, and immune changes. Understanding causes and mechanisms driving the interaction among ruminal bacteria and host immunometabolic responses merits further study.

Rumination time around calving: An early signal to detect cows at greater risk of disease

The main objective of this experiment was to evaluate the use of rumination time (RT) during the peripartum period as a tool for early disease detection. The study was carried out in an experimental freestall barn and involved 23 Italian Friesian cows (9 primiparous and 14 multiparous). The RT was continuously recorded by using an automatic system (Hr-Tag, SCR Engineers Ltd., Netanya, Israel), and data were summarized in 2-h intervals. Blood samples were collected from 30 d before calving to 42 d in milk (DIM) to assess biochemical indicators related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. The liver functionality index, which includes some negative acute-phase proteins and related parameters (albumin, cholesterol, and bilirubin), was used to evaluate the severity of inflammatory conditions occurring around calving. The cows were retrospectively categorized according to RT observed between 3 and 6 DIM into those with the lowest (L) and highest (H) RT. The average RT before calving (-20 to -2d) was 479 min/d (range 264 to 599), reached a minimum value at calving (30% of RT before calving), and was nearly stable after 15 DIM (on average 452 min/d). Milk yield in early lactation (on average 26.8 kg/d) was positively correlated with RT (r = 0.33). After calving, compared with H cows, the L cows had higher values of haptoglobin (0.61 and 0.34 g/L at 10 DIM in L and H, respectively) for a longer time, had a greater increase in total bilirubin (9.5 and 5.7 μmol/L at 5 DIM in L and H), had greater reductions of albumin (31.2 and 33.5 g/L at 10 DIM in L and H) and paraoxonase (54 and 76 U/ml at 10 DIM in L and H), and had a slower increase of total cholesterol (2.7 and 3.2 mmol/L at 20 DIM in L and H). Furthermore, a lower average value of liver functionality index was observed in L (-6.97) compared with H (-1.91) cows. These results suggest that severe inflammation around parturition is associated with a slower increase of RT after calving. Furthermore, more than 90% of the cows in the L group had clinical diseases in early lactation compared with 42% of the H cows. Overall, our results demonstrate the utility of monitoring RT around calving, and in particular during the first week of lactation, as a way to identify in a timely fashion those cows at a greater risk of developing a disease in early lactation.

Experimental acute rumen acidosis in sheep: Consequences on clinical, rumen, and gastrointestinal permeability conditions and blood chemistry

Acute acidosis was induced in sheep, and gastrointestinal permeability was assessed by using lactulose as a permeability marker. Metabolism was evaluated by monitoring blood metabolites. Four rams (72.5 ± 4.6 kg BW) were used in a 2 × 2 changeover design experiment. The experimental period lasted 96 h from -24 to 72 h. After 24 h of fasting (from -24 to 0 h) for both controls and acidosis-induced rams (ACID), 0.5 kg of wheat flour was orally dosed at 0 and 12 h of the experimental period to ACID, while the basal diet (grass hay, ad libitum) was restored to control. At 24 h, a lactulose solution (30 g of lactulose in 200 mL of water) was orally administered. Blood samples were collected at -24, 0, 24, 48, and 72 h of the experimental periods for the analysis of metabolic profiles and during the 10 h after lactulose dosage to monitor lactulose changes in blood. In addition, rumen and fecal samples were collected at 24 h of the experimental period. The acidotic challenge markedly reduced (P < 0.01) rumen pH and VFA but increased rumen d- and l-lactic acid (P < 0.01). Concurrently, a decrease of fecal pH and VFA occurred in ACID (P < 0.01), together with an abrupt increase (P < 0.01) of lactate and fecal alkaline phosphatase. Blood lactulose was significantly increased in ACID peaking 2 h after lactulose dosage. Blood glucose, β-hydroxybutyrate, Ca, K, Mg, and alkaline phosphatase showed a significant reduction (P < 0.05) at 24 h, whereas urea and NEFA declined (P < 0.05) from 48 to 72 h. A strong inflammatory acute phase response with oxidative stress in ACID group was observed from 24 to 72 h; higher values of haptoglobin (P < 0.01) were measured from 24 to 72 h and of ceruloplasmin from 48 (P < 0.05) to 72 h (P < 0.01). Among the negative acute phase reactants, plasma albumin, cholesterol, paraoxonase, and Zn concentration also decreased (P < 0.05) in ACID at different time points between 24 and 72 h after acidotic challenge start. A rise (P < 0.05) of reactive oxygen metabolites and a drop of vitamin E (P < 0.01) between 24 and 72 h were indicative of oxidative stress in ACID. The perturbation of these blood metabolites suggests that acute acidosis was effectively induced by our model. The increase of lactulose in blood in ACID indicates that gastrointestinal permeability for the marker increased and the large increment after 2 h from dosage suggests that most of the passage occurred through the rumen or abomasal walls.

Relation of inflammation and liver function with the plasma cortisol response to adrenocorticotropin in early lactating dairy cows

In this study we examined the relationship between cortisol and inflammatory status in early lactating dairy cows after a stimulation test of the adrenal cortex. Twenty-four cows were grouped into quartiles (6 cows per each quartile) in accordance with the liver activity index (based on plasma concentration of negative acute phase proteins in early lactation); the quartiles were lower (LO; cows with the lowest liver functionality), intermediate lower, intermediate upper, and upper (UP; cows with the highest liver functionality). Each cow was injected i.v. with 20 µg of a synthetic analog of ACTH at 35 d in milk (DIM). Blood samples were taken to assess inflammatory status, and at 0, 30, and 60 min after ACTH challenge to measure total cortisol. The free cortisol fraction was analyzed in the LO and UP quartiles and the bound cortisol fraction was estimated as the difference between total and free cortisol. The LO, in comparison with the other quartiles, suffered a more severe inflammatory status, with the highest values of haptoglobin, reactive oxygen metabolites, and total nitric oxide metabolites and the lowest concentration of direct or indirect markers of negative acute phase proteins. The cows in the LO quartile had the highest values of plasma nonesterified fatty acids and β-hydroxybutyrate at 7 DIM, suggesting a more severe body lipid mobilization. The LO quartile cows showed the highest frequency of health problems and the lowest milk yield in the first 35 DIM. Thirty minutes after the ACTH treatment, the concentration of total cortisol was lower in LO in comparison to other groups. Similarly, the bound cortisol fraction was lower in LO versus UP. The adrenal response appeared inversely related with health status after calving (e.g., lower in LO cows, experiencing the most severe inflammatory status). The lower increase in cortisol after the ACTH challenge in cows with greater inflammation (LO quartile) seems a consequence of the lower availability of cortisol-binding globulin synthetized by the liver, but other mechanisms can be involved (e.g., rate of cortisol production, secretion, and metabolic clearance). Our data provide evidence that inflammation and metabolic changes reduce the concentration of circulating plasma cortisol during an acute stress. Hence, the acute phase response in dairy cows should be taken into account to interpret the results obtained from stimulation tests of the adrenal cortex.

Short-term modifications in the distal gut microbiota of weaning mice induced by a high-fat diet.

The gut microbiota has been shown to be involved in host energy homeostasis and diet-induced metabolic disorders. To gain insight into the relationships among diet, microbiota and the host, we evaluated the effects of a high-fat (HF) diet on the gut bacterial community in weaning mice. C57BL/6 mice were fed either a control diet or a diet enriched with soy oil for 1 and 2 weeks. Administration of the HF diet caused an increase in plasma total cholesterol levels, while no significant differences in body weight gain were observed between the two diets. Denaturing gradient gel electrophoresis (DGGE) profiles indicated considerable variations in the caecal microbial communities of mice on the HF diet, as compared with controls. Two DGGE bands with reduced intensities in HF-fed mice were identified as representing Lactobacillus gasseri and an uncultured Bacteroides species, whereas a band of increased intensity was identified as representing a Clostridium populeti-related species upon sequencing. Quantitative real-time PCR confirmed a statistically significant 1-log decrease in L. gasseri cell numbers after HF feeding, and revealed a significantly lower level of Bifidobacterium spp. in the control groups after 1 and 2 weeks compared with that in the HF groups. These alterations of intestinal microbiota were not associated with caecum inflammation, as assessed by histological analysis. The observed shifts of specific bacterial populations within the gut may represent an early consequence of increased dietary fat.

Assessment of immune response in periparturient dairy cows using ex vivo whole blood stimulation assay with lipopolysaccharides and carrageenan skin test

The transition period is known to be the most critical phase in the life of high yielding dairy cow. Changes in the immune functions have been observed during the transition period which may account for the onset of clinical and subclinical (e.g. inflammatory response) problems at calving or at the beginning of lactation however this relationship has not yet been adequately investigated. Thus, to establish the potential of the periparturient dairy cows immune system to respond to stimuli, two challenges [an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test (CST)] were performed in addition to characterizing the metabolic and inflammatory profile. The WBA was performed using 0, 0.01 and 5 μg LPS/mL on whole blood and CST was administered by subcutaneous injection of 0.7 mL solution containing 4.2mg of carrageenan to the shoulder region of the cows. These tests were performed on 10 Holstein-Friesian cows at -45 ± 2, -20 ± 2, -3, 3, 7, 28 ± 2 days from parturition (DFP). Cows were also monitored for health status, body condition score, milk yield. The results demonstrate a higher production of IL-1β and IL-6 from leukocytes after LPS stimulation around calving (from -3 to 3 DFP) compared to -45 DFP (P < 0.05). Moreover, IL-6 (but not IL-1β) was able to reach close to the maximum response at the lower stimulus intensity (0.01 μg LPS/mL), maintaining a higher response over a longer time in early lactation. The release of higher levels of IL-6 in the transition period, with low LPS dose, suggests its crucial role in the regulation of inflammatory response around calving. The response of cows to CST decreased a few days before calving (-3 DFP) compared with response at -45 and 28 DFP (P<0.05), and remained low in the first week of lactation. This result suggests the reduction of the functionality of some vascular factors, which decreases diapedesis. Overall, the WBA and CST tests confirm changes in immunocompetence around calving. These tests are able to better describe the changes of the innate immune response at a local and systemic level, mainly when combined with conventional metabolic and inflammatory indices.

Impact of cystic fibrosis disease on archaea and bacteria composition of gut microbiota

&NA; Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10–22 years of age, and compared the findings with age‐matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non‐delF508 mutations. We used PCR‐DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate‐reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate‐reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H2‐consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non‐microbial routes.

In Vitro Rumen Fermentation Characteristics of Some Naturally Occurring and Synthetic Sugars

Soluble sugars are thought to play an important role in the fermentation processes of the rumen but their actual fermentation rate has not been fully assessed. Some sugars are also used as markers to assess gut permeability in monogastrics but their use in ruminants can be compromised by the hydrolytic activity of rumen microflora. This study aimed to evaluate the fermentability of some naturally occurring and synthetic soluble sugars. The synthetic soluble sugars were included to verify their possible use as markers for studies of gut permeability in ruminants. In vitro gas and volatile fatty acid (VFA) production from glucose, fructose, xylose, galactose, sucrose, lactose, arabinose mannitol, lactulose and sucralose were measured in a 24 h-incubation trial using ruminal fluid from heifers adapted or not-adapted to additional sugars in the diet, and with caecal content as inocula. Gas production from the same sugars was further evaluated in a 72 h-incubation trial with not-adapted rumen fluid only. Gas and VFA production were not affected by feeding additional sugars, but significant effects of inocula (ruminal vs caecal), sugars and their interaction were observed. Caecal inoculum produced less gas but higher VFA than ruminal inocula. Fructose and glucose had the highest rates of gas production (10.57% h–1 and 10.42% h–1, respectively), and lactulose and mannitol the lowest (3.47% h–1 and 4.63% h–1, respectively) when fermented with ruminal fluid. Sucralose seemed to have a negative effect on microbial fermentations. Our results indicate that lactulose and mannitol might largely escape rumen fermentation, suggesting their possible use as markers to test gut permeability also in ruminants. This needs to be verified in vivo.

Immune system, inflammation and nutrition in dairy cattle

Good health is essential for good performance and the welfare of dairy cows, and nutrition is an important component of good health. Health is influenced by the interaction between the innate adaptive components of the immune system and other factors, such as the local and systemic inflammatory response, which can sometimes be more harmful than useful. Therefore, for dairy cows, particularly those in the periparturient period, it is important to avoid, or reduce as much as possible, any kind of infectious, parasitic or metabolic disease and the associated inflammation. Such inflammation can impair cow performance by lowering milk yield, dry matter intake, fertility and energy efficiency, and can reduce liver function. Good nutrition is essential in maintaining a functional immune system, while also avoiding other causes of inflammation, such as tissue damage, and digestive and metabolic syndrome-related disorders. Provision of appropriate nutrients, such as antioxidants, omega-3 polyunsaturated fatty acids, conjugated linoleic acid and vitamin D can have anti-inflammatory effects. In the future, ways to reduce inflammation while maintaining a good immune defence must be developed and the susceptibility of the cow to diseases and inflammation evaluated. Ideally, we would be able to selectively breed for cows with a lower susceptibility to both diseases and inflammation.