The Effect of By-Pass Linseed Oil Supplementation on the Maternal Antioxidant System during the Embryo-Maternal Recognition Period in Ewes.

This study analyzed the effects of dietary supplementation with by-pass linseed oil (LO; rich in α-linolenic acid) on maternal antioxidant systems at Days 14 and 16 of pregnancy in Sarda ewes. This trial used sixteen dry ewes. Eight ewes (CT group) were fed with a control diet without LO, and eight ewes (LO group) were fed with a diet supplemented with LO (10.8 g of α-linolenic acid/ewe/day). Both diets had similar crude protein and energy levels. The experiment included 10 days of an adaptation period and 31 days of a supplementation period. This supplementation period was divided into Period -2 (from Day -15 to -8), Period -1 (from Day -7 to -1; before synchronized mating period/Day 0), Period +1 (from Day +1 to + 7 after mating), and Period +2 (from Day +8 to +15 after mating). Estrous synchronization was induced in all the ewes using an intravaginal sponge (45 mg fluorgestone acetate) for 14 days and equine chorionic gonadotropin (350 UI/ewe) at the end of the treatment. On Days 14 (CT, N = 4; LO, N = 4) and 16 (CT, N = 4; LO, N = 4) after mating, the ewes were slaughtered. Samples of plasma, uterine, and luteal tissues were collected. Thiols, total antioxidant activity (TEAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were measured. On Day 16, thiol and TEAC in luteal tissues were higher in the LO group when compared with the control one (p < 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p < 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p < 0.001). On Day 16, uterine MDA content was lower for the LO group (p < 0.001). No differences were found between groups at the plasmatic level. However, the by-pass LO supplementation enhanced the analyzed antioxidant parameters in luteal and uterine tissues. In conclusion, these results demonstrate that by-pass LO supplementation exerted a positive effect on antioxidative defenses on maternal structures during the embryo-maternal recognition period in ewes. Thus, this could contribute to improving the maternal environment during the embryo-maternal recognition period in mammals.

Incorporation of fresh leaves of wormwood (Artemisia herba alba) and/or rosemary (Rosmarinus officinalis) in the diet of rams: Effect on testicular function, sexual behavior, and blood parameters.

This study aimed to investigate the effect of wormwood and rosemary supplementation on some reproductive traits of Barbarine rams. The experiment lasted 2 months. Twenty-four adult rams were divided into four groups (n = 6) balanced for the weight (53.3 ± 1.2 kg body weight [BW] ± SD). All rams received 1200 g of straw and 600 g of barley. Control rams (C) without aromatic medicinal plant (AMP), while experimental rams received 20 g of fresh rosemary leaves (R), 20 g of fresh wormwood leaves (A), and 10 g of fresh rosemary leaves plus 10 g of fresh wormwood leaves (RA). The results revealed that the live weight of all rams increased (p < .05) in the RA group compared to the C, A, and R groups. Scrotal circumference increased in the R rams when compared to the controls rams (p < .05). For sperm parameters we showed that the A rams had higher sperm concentrations (p < .05). But, the sperm volume decreased in the R rams (p > .05). However, when the rams received rosemary plus wormwood, their sperm volume increased (p > .05). The sperm mass motility was higher for the A, R and AR rams in comparison to the C rams (p = .05). On the other hand, biochemical analysis of the seminal fluid showed no effect of diets on calcium and total proteins concentration. But the measurement of glucose and seminal insulin showed a decrease (p < .05) in these two biochemical markers in group A rams and a decrease (p < .05) in insulin without modification of the glucose concentration in R rams. Blood glucose and insulin decreased in the animals on AMP diet compared to the other groups (p < .05) while aspartate aminotransferase (AST) increased (p < .05). Rosemary leaves (R and RA groups) increased (p < .05) plasma cortisol compared to the other groups. It can be concluded that the addition of Rosmarinus officinalis and/or Artemisia herba alba in ram diet can have a positive effect on the reproductive function by increasing the concentration and motility of sperm, plasma testosterone, and sexual behavior.

Does the timing of pasture allocation affect rumen and plasma metabolites and ghrelin, insulin and cortisol profile in dairy ewes?

A study was undertaken to assess the impact of the timing of grazing on rumen and plasma metabolites and some metabolic hormones in lactating dairy sheep allocated to an Italian ryegrass (Lolium multiflorum Lam) pasture in spring for 4 h/d. Twenty-four mid lactation Sarda ewes stratified for milk yield, body weight, and body condition score, were divided into four homogeneous groups randomly allocated to the treatments (2 replicate groups per treatment). Treatments were morning (AM, from 08:00 to 12:00) and afternoon pasture allocation (PM, from 15:30 to 19:30). Samples of rumen liquor (day 39) and blood plasma (days 17 and 34 of the experimental period) were collected before and after the grazing sessions. Moreover, on days 11 and 35, grazing time was assessed by direct observation and herbage intake measured by the double weighing procedure. Grazing time was longer in PM than AM ewes (P < 0.001) but herbage intake was undifferentiated between groups. The intake of water-soluble carbohydrates at pasture was higher in PM than AM ewes (P < 0.05). The post-grazing propionic and butyric acid concentration, as measured on day 39, were higher in PM than AM ewes (P < 0.05). The basal level of glucose on day 34 and insulin (on both sampling days) were higher in PM than AM (P < 0.05). The opposite trend was detected for non-esterified fatty acids (P < 0.05, day 34) and urea (both days). Pasture allocation in the afternoon rather than in the morning decreased plasma concentration of ghrelin (P < 0.001) and cortisol (P < 0.001), with a smoothed trend on day 34 in the latter variable. To conclude, postponing the pasture allocation to afternoon increased the intake of WSC, favoring a glucogenic pattern of rumen fermentation and a rise of glucose and insulin levels in blood, although these effects were not consistent across the whole experimental period. Moreover, the afternoon grazing decreased the level of cortisol and ghrelin, suggesting a higher satiation-relaxing effect.

Dairy Sheep Grazing Management and Pasture Botanical Composition Affect Milk Macro and Micro Components: A Methodological Approach to Assess the Main Managerial Factors at Farm Level.

The fatty acid profile, vitamins A and E, cholesterol, antioxidant power colour and the phenols profile of Sarda sheep milk from 11 commercial sheep flocks managed under permanent grassland were investigated. In each farm, the structural and managerial data and milk samples were collected during four periods (sampling dates, SD): January, March, May, and July. Data from the milk composition (fat, protein, casein, lactose, and somatic cell count), 68 fatty acids, 7 phenols, 1 total gallocatechin equivalent, ferric reducing antioxidant power, vitamins A and E, cholesterol, degree of antioxidant protection, and the colour (b *, a * and L *) were analyzed by multivariate factorial analysis using a principal component analysis approach. A proc mixed model for repeated measurement to point out the studied factors affecting significant macro and micro milk composition was also used. Only the first five components were detailed in this paper, with approximately 70% of the explained variance detected. PC1 presented the highest positive loadings for milk lactose, de novo FA synthesis and the BH intermediate, whereas OBCFA had negative loadings values. The PC2, LCFA, UFA, MUFA, vitamins E, and DAP showed positive loadings values, while SFA had a negative value. The PC3 showed a high positive loading for total phenols and non-flavonoids. PC4 presented a high positive loading for the milk macro-composition and negative values for n-3 FAs. The PC5 is characterized by high positive loadings for the a * and L * colour parameters whereas negative loadings were detected for the milk flavonoids content. These preliminary results could help to establish future threshold values for the biomarkers in milk sourced from grazing dairy sheep in natural, permanent pasture-based diets.

Impact of Pasture-Based Diets on the Untargeted Metabolomics Profile of Sarda Sheep Milk.

In this work, untargeted metabolomics was used to shed light on the impact of different pasture-based diets on the chemical profile of Sarda sheep milk. The study considered 11 dairy sheep farms located in Sardinia, and milk samples were collected in 4 different periods, namely January, March, May, and July 2019, when all sheep had 58, 98, 138, and 178 days in milk, respectively. The animal diet composition was based on the intake of grazed herbage in natural pasture, hay, and concentrate. Overall, the combination of two comprehensive databases on food, namely the Milk Composition Database and Phenol-Explorer, allowed the putative identification of 406 metabolites, with a significant (p < 0.01) enrichment of several metabolite classes, namely amino acids and peptides, monosaccharides, fatty acids, phenylacetic acids, benzoic acids, cinnamic acids, and flavonoids. The multivariate statistical approach based on supervised orthogonal projections to latent structures (OPLS-DA) allowed us to predict the chemical profile of sheep milk samples as a function of the high vs no fresh herbage intake, while the prediction model was not significant when considering both hay and concentrate intake. Among the discriminant markers of the herbage intake, we found five phenolic metabolites (such as hippuric and coumaric acids), together with lutein and cresol (belonging to carotenoids and their metabolites). Additionally, a high discriminant power was outlined for lipid derivatives followed by sugars, amino acids, and peptides. Finally, a pathway analysis revealed that the herbage intake affected mainly five biochemical pathways in milk, namely galactose metabolism, phenylalanine metabolism, alpha-linolenic acid metabolism, linoleic acid metabolism, and aromatic amino acids involved in protein synthesis (namely tyrosine, phenylalanine, and tryptophan).

Untargeted lipidomics of ovine milk to analyse the influence of different diet regimens.

In this work we report a lipidomics approach to study the effects of two diet systems on the composition of ovine milk. Milk from two groups of Sarda sheep grazing on 40% (P40) and 60% (P60) of pasture were analyzed by a UHPLC-QTOF-MS analytical platform and data submitted to multivariate statistical analysis. Pairwise partial least square discriminant analysis of the lipid profile of the data was carried out to classify samples and to find discriminant lipids. The two dietary groups were characterized by differences in triacylglycerols, phosphocholines and phosphatidylethanolamines levels. Discriminants of the P40 group were TG and PC containing in their backbone saturated medium chain FA thus suggesting greater de novo fatty synthesis in the mammary gland. On the other hand, the P60 group was characterized by TG and PC formed by unsaturated long chain FA originating from the diet or from lipid mobilization.

Can FT-Mid-Infrared Spectroscopy of Milk Samples Discriminate Different Dietary Regimens of Sheep Grazing With Restricted Access Time?

Milk obtained from sheep grazing natural pastures and some forage crops may be worth a plus value as compared to milk obtained from stall-fed sheep, due to their apparently higher content of beneficial fatty acids (FAs). Fourier transformed mid-infrared (FT-MIR) analysis of FA can help distinguish milk from different areas and diverse feeding systems. The objective was to discriminate milk from sheep and milk from dairy sheep rotationally grazing Italian ryegrass or berseem clover for 2, 4, or 6 h/day. To test this hypothesis, a data-mining study was undertaken using a database of 1,230 individual milk spectra. Data were elaborated by principal component analysis (PCA) and analyzed by linear discriminant analysis (LDA) with or without the use of genetic algorithm (GA) as a variable selection tool with the primary aim to discriminate grazed forages (grass vs. legume), access time (2, 4, or 6 h/day), grazing day (first vs. last grazing day during the 7-day grazing period), and the milking time (morning vs. afternoon milking). The best-fitting discriminant models of FT-MIR spectra were able to correctly predict 100% of the samples differing for the pasture forage, 91.9% of the samples differing for grazing day, and 97.1% of the samples regarding their milking time. The access time (AT) to pasture was correctly predicted by the model in 60.3% of the samples, and the classification ability was improved to 77.0% when considering only the 2 and 6 h/day classes.

A Note on the Tracing of Herbage Contribution to Grazing Sheep Diet Using Milk and Feces Biomarkers.

Milk from grazing ruminants is usually rich in beneficial components for human health, but distinguishing milks sourced from grazing is difficult, and this hinders the valuing of the grazing benefit. This study aimed at evaluating the ability of milk biomarkers (1) to trace milks sourced from sheep submitted to different access times (ATs) to pasture and (2) to estimate sheep herbage dry matter intake (HDMI, g DM ewe-1 d-1) and herbage percentage (HP, % DM) in sheep diet. Animal data derive from a published experiment in which six replicated groups of mid-lactation Sarda sheep had ATs of 2, 4, or 6 h d-1 to a ryegrass pasture. Sheep HDMI and HP of each group were measured on four dates in April 2013. Group milk was sampled, and milk fatty acids (FAs) and n-alkanes were determined by gas chromatography. The latter markers were also measured in feces samples bulked by group. The data (N = 24 records) were submitted to Linear Discriminant Analysis (LDA) aimed at distinguishing the AT to pasture based on biomarkers previously selected by Genetic Algorithms (GA). Partial Least Square Regression (PLSR) models were used to estimate HDMI and HP using biomarkers selected by GA. Based on one milk alkane and six milk FAs as biomarkers, estimates of the AT using GA-LDA were 95.8% accurate. The estimation of HDMI by GA-PLSR based on five milk FAs was moderately precise [explained variance = 75.2%; percentage of the residual mean square error of cross-validation over the mean value (RMSECV%) = 15.0%]. The estimation of HP by GA-PLSR based on 1 milk alkane and 10 FAs was precise (explained variance = 80.8%; RMSECV% = 7.4%). To conclude, these preliminary results suggest that milks sourced from sheep flocks with AT to pasture differentiated by 2 h in the range 2-6 h d-1 can be precisely discriminated using milk biomarkers. The contribution of herbage to sheep diet can also be precisely estimated.

Extensive Ruminant Production Systems and Milk Quality with Emphasis on Unsaturated Fatty Acids, Volatile Compounds, Antioxidant Protection Degree and Phenol Content.

Dairy products from grazing ruminant have numerous positive effects on human health thanks to their higher content essential fatty acids, vitamins, and polyphenols. Compared to livestock fed a conventional maize silage- and/or grain-based diet, grass-fed livestock produce milk with higher levels of n-3 fatty acids, vitamins A, E, carotenoids, and phenols. The effect is even more pronounced if animals are grazing on legume/forbs-rich grasslands. This review argues, based on the available literature, about the effect of grazing ruminant on milk and cheese quality, including the hedonistic aspects, pointing out the link between territory and dairy products quality (Protected Designation Origin; Protected Geografic Origin; namely PDO and PGI labels). Moreover, it points out the main plant biomarkers which can be used to discriminate grazing sourced from stall-fed sourced milk and dairy products. Overall milk and cheese sourced from grazing animals (cows, sheep and goat) showed higher levels (compared to stall system) of FA, vitamins, phenols, putatively beneficial for consumers' health. FA and plant secondary metabolites can also affect flavor and some nutritional and technological features of dairy products such as their antioxidant protection degree. This would favour a fair pricing of dairy products sourced from grazing systems and the persistence of viable and sustainable extensive production systems.

Goats' Feeding Supplementation with Acacia farnesiana Pods and Their Relationship with Milk Composition: Fatty Acids, Polyphenols, and Antioxidant Activity.

BACKGROUND: Research efforts have focused on the evaluation of the bioactive quality of animal products (milk, cheese, meat, and other by-products) contrasting various feeding strategies coming from different ecological zones. The study aimed to describe the fatty acids (FA), polyphenols (P), bioactive compounds (BC), and antioxidant activity (AA) of goat's milk. METHODS: Dairy goats were fed with five systems: (1) Grazing; (2) conventional diet (CD); (3) CD + 10% of Acacia farnesiana (AF) pods; (4) CD + 20% AF; and (5) CD + 30% AF. The fatty acid profile, health promoting and thrombogenic indexes were calculated. Milk extracts were evaluated by HPLC to determent phenolic compounds (gallic, caffeic, chlorogenic, and ferulic acids, catechin, epicatechin, and quercetin). Antioxidant activity of goat's milk extract was evaluated using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), oxygen radical absorbance capacity (ORAC), and the ferric reducing antioxidant power (FRAP) assays. RESULTS: Conventional diet showed the highest content of polyunsaturated fatty acids while grazing showed the best n-6:n-3 and the linoleic:alpha linolenic acid ratio. Similarly, grazing and AF boosted the polyphenol content. CONCLUSIONS: Acacia farnesiana inclusion in the goats' diets increased the presence of bioactive compounds and the antioxidant activity while diminishing the cholesterol content of goat's milk.

Circulating Concentrations of Key Regulators of Nitric Oxide Production in Undernourished Sheep Carrying Single and Multiple Fetuses.

The aim of this study was to investigate the blood concentrations of L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine, which are regulators of nitric oxide (NO) synthesis, in single, twin, and triplet pregnancies in ewes undergoing either a dietary energy restriction or receiving 100% of their energy requirements. From day 24 to 100 of pregnancy, the ewes were fed ryegrass hay and two different iso-proteic concentrates fulfilling either 100% of ewes' energy requirements (control group; n = 30, 14 singleton pregnancies, 12 twin pregnancies, and 4 triplet pregnancies) or only 45% (feed-restricted group; n = 29; 11 singleton pregnancies, 15 twin pregnancies, and 3 triplet pregnancies). Blood samples were collected monthly to measure, by capillary electrophoresis, the circulating concentrations of arginine, ADMA, homoarginine, SDMA, and of other amino acids not involved in NO synthesis to rule out possible direct effects of diet restriction on their concentrations. No differences between groups were observed in the circulating concentrations of most of the amino acids investigated. L-homoarginine increased markedly in both groups during pregnancy (p < 0.001). SDMA (p < 0.01), L-arginine, and ADMA concentrations were higher in feed-restricted ewes than in controls. The L-arginine/ADMA ratio, an indicator of NO production by NOS, decreased towards term without differences between groups. The ADMA/SDMA ratio, an index of the ADMA degrading enzyme activity, was higher in controls than in feed-restricted ewes (p < 0.001). Obtained results show that circulating concentrations of L-arginine, of its metabolites, and the ratio between NO synthesis boosters and inhibitors are altered in energy-restricted ewes, and that these alterations are more marked in ewes carrying multiple fetuses.

The influence of plant polyphenols on lipolysis and biohydrogenation in dried forages at different phenological stages: in vitro study.

BACKGROUND: It is known that forage legumes show a higher transfer efficiency of polyunsaturated fatty acids (PUFA) to ruminant dairy products in comparison with grasses. Legumes are usually characterised by moderate levels of plant secondary metabolites, which can have an effect on lipolysis and biohydrogenation in the rumen. An in vitro study was carried out to compare two species with different plant phenol compositions, Vicia sativa (VS, common vetch, cv. Jose) and Trifolium incarnatum (TI, crimson clover, cv. Viterbo) cut at the vegetative (Veg) and reproductive (Rep) stages, on lipolysis and PUFA biohydrogenation in the rumen. RESULTS: The study showed that forage species and phenological stage affected the levels of bound phenols (BP) and tannic polyphenols (TP). VS was characterised by a higher level of TP than TI at both Veg and Rep stages, whereas BP levels were low in both forages. BP and TP had a negative effect on lipolysis and biohydrogenation, but TP showed a greater negative correlation than BP for both forages. CONCLUSION: These results showed that lipolysis and biohydrogenation of PUFA could be affected by plant phenols, particularly TP.

Determination of poly(ethylene glycol)-binding to browse foliage, as an assay of tannin, by near-infrared reflectance spectroscopy.

Nutritionists are interested in functional assays of tannins that do not require time-consuming and expensive extraction, such as the (14)C-labeled poly(ethylene glycol) (PEG)-binding (PEG-b) assay. This paper reports the application of near-infrared (NIR) spectroscopy to determine the percentage of PEG binding, in place of the (14)C-labeled PEG-b assay of tannin, in Mediterranean woodland vegetation. Calibration was done with 53 samples from 14 species and was validated on 25 samples from 10 species. PEG-b ranged between 1.4 and 20.7% in the samples. The calibration obtained by using the modified partial least-squares (MPLS) method, with all wavelengths in the 1100-2500 nm range combined, and the validation were reasonably linear (R (2) = 0.96 and 0.91, respectively). The accuracies, estimated from the standard errors of cross-validation and prediction, were +/-1.6 and +/-1.7% PEG-b, respectively. The NIRS-aided procedure proposed here can serve as an accurate, inexpensive, time-saving, and environment-friendly functional assay of tannin in Mediterranean browse.