Feeding Chicory–Plantain Silage and/or Se Yeast Does Not Improve Streptococcus uberis-Induced Subclinical Mastitis in Lactating Sheep
by
, , , and
Hunter R. Ford
1
,
Joseph Klopfenstein
2,
Serkan Ates
1,
Sebastiano Busato
1,
Erminio Trevisi
3
and
Massimo Bionaz
1,*
1
Department of Animal and Rangeland Sciences, Oregon State University, Corvallis, OR 97331, USA
2
Carlson College of Veterinary Science, Oregon State University, Corvallis, OR 97331, USA
3
Department of Animal Sciences, Food and Nutrition, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
*
Author to whom correspondence should be addressed.
Dairy 2025, 6(4), 40; https://doi.org/10.3390/dairy6040040
Submission received: 11 April 2025
/
Revised: 22 June 2025
/
Accepted: 21 July 2025
/
Published: 29 July 2025
(This article belongs to the Section Dairy Animal Nutrition and Welfare)
Abstract
The objective of this study was to evaluate the effects of feeding a combination of chicory–plantain silage and supplementing Se yeast on the response of early-lactating ewes to induce subclinical mastitis. Polypay ewes (n = 32) were fed either chicory–plantain silage or grass silage and supplemented with 3.6 mg Se yeast/ewe/day for approximately 2 months prior to the infusion of S. uberis into both mammary glands (i.e., intramammary infection or IMI). The ewes had a typical subclinical mastitis response with an 8-fold increase in milk somatic cell count within 24 h post-IMI, a decrease in milk yield, and changes in all milk components measured. The ewes experienced a mild systemic inflammation post-IMI as determined by an increase in rectal temperature and decrease in feed and water intake and, in blood, by an increase in the concentration of ceruloplasmin, haptoglobin, and myeloperoxidase and a decrease in paraoxonase, Zn, advanced oxidation protein products, and hematocrit with no effect on pro-inflammatory cytokines. No effect of silage type, likely due to a low concentration of secondary compounds, or Se supplementation was detected in response to IMI. In summary, the subclinical mastitis model used was effective in mounting an inflammatory response, although this was mild; however, feeding chicory–plantain silage with a low concentration of secondary compounds and supplementing Se yeast had no significant effect on the response of ewes to mammary infection.
1. Introduction
The cost of each case of clinical mastitis in dairy cows in the United States has been estimated to be $444 [1], with an average cost of $131 per cow per year, as proposed by a review of the literature [2]. Mastitis is also a significant disease in dairy sheep, with approximately 80% of cases due to Staphylococcus aureus [3]. Like in dairy cattle, mastitis represents a substantial economic burden for dairy sheep, leading to an estimated 2.6–43.1% loss in milk yield depending on the severity of intramammary infection [4]. Further complicating the issue of mastitis in sheep are cases of subclinical mastitis where physical symptoms, such as a swollen or inflamed udder and visible changes in milk quality, are not apparent. While in dairy cattle, milk somatic cell count (SCC) > 200,000 cells/mL is commonly used to diagnose cases of subclinical mastitis [5], there is a lack of consensus in sheep [6], making diagnosing and treating these cases difficult. While animals with subclinical mastitis may appear healthy, they suffer from reduced milk production and elevated SCC, significantly impacting farm profitability [7]. It has been estimated that subclinical mastitis costs > $1 billion in the US alone [8].
While antibiotics are still the most common form of mastitis treatment, their use can lead to bacterial resistance to antibiotics [9], emphasizing the need for alternative therapies for treating and preventing mastitis. Several alternatives to antibiotics exist to prevent and treat mastitis, including the use of anti-inflammatory drugs, herbal treatments, antimicrobial peptides therapy, bacteriophages, and vaccinations, among others, as previously reviewed [10].
One alternative method for preventing or treating mastitis currently being explored is through diet. In a recent investigation, we observed lower SCC in the mid-lactation cows grazing in a chicory- and plantain-dominated pasture compared to cows grazing a more traditional grass-clover pasture [11]. While the specific compounds present in chicory that could be generating this effect, including sesquiterpene lactones, hydroxycinnamic acids, flavonoids, anthocyanins, and coumarins, have not been investigated in ruminants, research utilizing other animal models has demonstrated a wide range of therapeutic benefits in chicory, including antioxidant, anti-inflammatory, and antimicrobial properties [12]. Plantain has primarily been investigated for its potential to reduce the environmental impact of livestock [13]; however, limited work has been carried out to elucidate its effects on animal health. Like chicory, plantain is also known to contain a high amount of bioactive compounds, primarily aucubin and acetoside [11], that, in addition to improving rumen nitrogen efficiency [14], can suppress NF-κB-mediated inflammatory responses [15].
Given the important role that the immune system plays in preventing and eliminating intramammary infections (IMI) [16], improving the immune system that protects the mammary gland may be effective at limiting or preventing mastitis. Selenium, a critical micromineral for selenoproteins, has been extensively studied for its ability to improve immune function [17] and the antioxidant status via increased glutathione peroxidase activity [18]. Prior investigations on sheep have shown that supplementation of Se yeast (25 mg Se/week) can restore immune function in foot rot-affected animals [19]. Furthermore, the supplementation of Se (0.1 mg Se/kg body weight) in dairy cows reduced the duration of mastitis symptoms by up to 46% [20] limiting the economic impact of intramammary infections.
To expand upon previous investigations on mastitis, the objectives of this study were to investigate the effect of feeding a 50%/50% mixture of chicory and plantain silage in combination with a supplementation of Se yeast to lactating ewes subjected to IMI. Based on previous findings, it was hypothesized that sheep fed chicory + plantain silage would improve the response to intramammary infection compared to ewes fed a standard grass silage, with further improvement among ewes supplemented with Se yeast.
2. Materials and Methods
2.1. Animals
Experimental procedures in this investigation were approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon State University (protocol # 2019-0042). Thirty-two Polypay ewes, approximately one month into lactation, from the Oregon State University Sheep Center, were transitioned from a previous study [21]. Details of animal management were previously described [21]. Briefly, ewes (≥2 lactation; 69.6 ± 8.1 kg BW) were housed in individual 1.5 m × 1.5 m pens at the HOGG Animal Metabolism Laboratory Building (HAMB) on the Oregon State University campus. The pens provided animals with continuous access to water and were bedded with wheat straw that was replaced every 3–4 days or as needed.
2.2. Diets and Experimental Design
Before the start of this investigation, ewes were fed diets composed of either chicory silage or grass silage and supplemented with 3.6 mg/ewe/day of Se yeast (DiaMune, Diamond V) or a placebo from approx. 30 days before parturition to 30 days post-lambing [21]. For the present study, ewes receiving grass silage continued to receive the same feeding treatment. In contrast, ewes receiving chicory silage were fed with a 50/50 chicory/plantain bale silage (donated by Don Wirth, Saddle Butte Ag, Inc. Tangent, OR, USA). Diets are available in Table 1. The bromological analysis (Appendix A.1, Table A1) was performed by a commercial laboratory (Cumberland Valley Analytical Services, Waynesboro, PA, USA). The results of secondary compounds analysis (plus details of the protocol) of the three silages are available in Appendix A.2 (Table A2, Table A3 and Table A4 and Figure A1). Diets were formulated to ensure they were isocaloric and isonitrogenous. The experiment involved animals in 4 groups (n = 8/group; Figure 1): CPCT (chicory + plantain silage without supplementation of Se yeast); CPSY (chicory + plantain silage with supplementation of Se yeast); GCT (grass silage without supplementation of Se yeast); and GSY (grass silage with supplementation of Se yeast). Four animals were later excluded due to unrelated clinical mastitis, resulting in final group sizes of n = 7 (CPCT), n = 6 (CPSY), n = 8 (GCT), and n = 7 (GSY).
Ewes were fed a fixed amount of grain and silage ad libitum, and residual feed was weighed to calculate feed intake. During the experiment, the amount of grain provided was 1.5% of the body weight of each of the ewes, while the amount of silage provided was approximately 110% of the amount of silage consumed the previous day. Ewes were fed 60% of the allocated feed at approximately 9 AM after they were milked, with the rest of the feed being fed at approximately 5 PM. Diets were formulated to meet the recommendations for lactating sheep [22]. Before milking, teats were treated with a pre-dip 0.5% iodine solution and cleaned using a disposable commercial paper towel. The same solution was used to perform a post-dip.
2.3. Intramammary Infection
After 4 days of adaptation to milking and diet (day 0), an infusion of 2 × 107 cfu of Streptococcus uberis 0140J [23] in 10 mL sterile saline solution in both glands was performed in all ewes immediately after the morning milking in accordance with a previously characterized model developed by Addis et al. [24]. The Strep. uberis was grown and provided as 2 × 107 cfu aliquots in cell culture freezing vials by the Oregon Veterinary Diagnostic Laboratory, Carlson College of Veterinary Medicine. Corvallis, OR, USA. The teats were cleaned with the pre-dip solution and with a sterile gauze and sprayed with 70% ethanol before IMI. This was performed using a 10 mL syringe with a sterile teat infusion cannula (Jorgensen Laboratories, Inc., Loveland, CO, USA). The gland was thoroughly massaged to spread the inoculum after infusion.
2.4. Dry Matter Intake, Water Intake, Milk Yield, Milk Quality, and Rectal Temperature
Feed intake (as dry matter intake or DMI) and water intake were measured as previously described [21]. Ewes were with lambs until 5 days before the experiment started, when the lambs were weaned, and ewes started to be milked. Ewes were milked in the morning (~7 AM) and the evening (~4 PM) before feeding. Ewes were milked using a Quadruple Goat Milker Twin Bucket (Model TKKC 4-2PS, Melasty, Bursa, Turkey) and fed approximately 100 g of the control grain containing no additional Se to ease the stress of milking.
Milk yield was measured at each milking by using a graded measuring cup. Milk samples were collected during both AM and PM on days −2, −1, 0, 1, 3, 5 and 9 relative to IMI in 60 mL cupped vials (cat# CPP03EDM-CL, Capitol Plastic Products, Amsterdam, NY, USA) containing 1 pill of Broad Spectrum Microtabs II™ (Cat# 3038-50, Weber Scientific Inc., Trenton, NJ, USA) for assessment of milk components using a LactoScope™ FT-A and SomaScope Smart (both from Perkin Elmers, Waltham, MA, USA). The LactoScope was calibrated using 14 calibration milk samples from Dr. David Barbano at Cornell University before starting the experiment. Milk samples were preserved at 4 °C and measured, after warming at 42 °C in a water bath, within 14 h of collection. Rectal temperature was measured at each milking using commercial digital rectal thermometers.
2.5. Milk Bacteria Sampling and Culture
Bacteria count from milk samples was evaluated at day 0 before glands were infused with Strep. uberis and on day 4 post-infection, following a previous method [25]. Briefly, milk obtained for evaluation was collected aseptically in sterile Olympus 1.7 mL microtubes (#24-282S, Genesee Scientific Corporation, El Cajon, CA, USA). For total bacteria counts, milk was agitated gently, then a 1 mL sample was obtained with a 1 mL calibrated lab pipette. Milk was then placed on Petrifilm (3M Company, Maplewood, MN, USA) aerobic culture plates according to the manufacturer’s instructions. After incubation at 37 °C for 48 h, bacterial colonies were counted and recorded. The detailed protocol is available in Appendix A.3.
2.6. Blood Collection and Analysis
Blood was collected via jugular venipuncture into vacutainer tubes (Becton Dickinson and Company, Cat# 366480) on days −1, 0, 1, 3, 5, and 9 relative to IMI and processed as previously described [21]. The plasma was analyzed for parameters associated with metabolism, liver and kidney function, antioxidant health, inflammation, and immune status using a clinical auto-analyzer (ILAB 650, Instrumentation Laboratory, USA Spa, Werfen Co., Milan, Italy) as previously described [21,26].
2.7. Phagocytosis Assay
The leukocyte phagocytosis assay was performed using pHrodo Green S. aureus BioParticles Phagocytosis Kit for Flow Cytometry (Cat# P35382, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol except for the addition of DAPI to visualize nucleated cells. Flow cytometry was performed using a CytoFlex Flow Cytometer (Beckman Coulter, Brea, CA, USA), and cell populations were manually gated using the PB450 and APC filters.
2.8. Migration Assay
Cellular migration was assayed using a commercially available kit (Cat# CBA-104, Cell Biolabs Inc., San Diego, CA, USA). Briefly, 100 µL of whole blood collected in EDTA vacutainer tubes was added to a migration plate containing 150 µL of endotoxin-activated sheep plasma as the chemoattractant. The latter was obtained according to Jahan et al. [27] by treating each mL of whole blood from sheep collected in heparinized vacutainer tubes with 5 µg LPS from E. Coli EH100 (cat#IAX100010M001, Adipogen, San Diego, CA, USA) for 4 h at 37 °C. The plasma was immediately obtained after centrifugation at 8500× g for 10 min at 4 °C and stored at −80 °C in 1 mL aliquots. Following 24 h incubation, cells were detached from the migration plate using the provided cell detachment solution. Following detachment, the solution was mixed with the provided buffers and dye solution, incubated, and added to a black 96-well plate for fluorescence measurement. As blank, RPMI media was used instead of the LPS-activated plasma. Fluorescence was measured using a Biotek Synergy H1 Plate Reader at 480 nm/520 nm, and data were corrected for the blank and by the known number of neutrophils as determined by the complete blood count. This correction was performed by taking the fluorescence data for each sample and dividing it by the number of neutrophils/mL for the corresponding sample.
2.9. Statistical Analysis
The power analysis (G*Power 3.1.9.7) using the log2 somatic cell count data from a prior experiment, where the same mastitis model was used in goats [23], revealed that 8 animals per group provided 80% power to determine successful intramammary infection. For most parameters, data were analyzed using the PROC GLIMMIX procedure in SAS v9.4 (SAS Institute, Cary, NC, USA) with forage (F), selenium supplementation (Se), and time (T) and their interactions as the fixed effects and with ewe as the random effect. The TYPE = AR(1) covariance structure was used as it has the lowest Akaike’s information criterion. The model used was:
where y = response variable; mean = constant common to all observations; Forage = effect of the forage; Se = effect of selenium; Time = effect of the time; Forage × Se = effect of the interaction of forage and Se; Se × Time = effect of the interaction of Se and time; Forage × Time = effect of the interaction of forage and time; Forage × Se × Time = effect of the interaction of forage, Se, and Time; ewe = is the random effect of animal (considering repeated measures using a variance-covariance matrix AR(1)—autoregressive of first-order); and error = random error. Significance was declared at p ≤ 0.05 and a tendency when p > 0.05 and p ≤ 0.10.
y = mean + silage + se + Time + Forage × Se + Se × Time + Forage × Time + Se × Forage × Time + ewe + error
Milk bacteria frequencies were assessed using the PROC FREQ procedure in SAS v9.4 with F, Se, and T as the categorical variables, with significance declared at Chi-Square ≤ 0.05.
3. Results
Animals (1 for CPCT, 2 for CPSY, and 1 for GCT) were removed from the study due to the presence of clinical mastitis that was independent of the Strep. uberis infused.
3.1. Characterization of the Silages
All the silages used had a pH higher than ideal (i.e., >4.2), while the grass silage had high ammonia (>10%); the lactic acid was <8% in all silages (Appendix A.1, Table A1).
Few samples of the silages were analyzed for secondary compounds with the concentration of flavonoid compounds in the chicory being similar to that of grass silage, with a rapid decrease after opening the bale (each bale was opened approx. every 5 days), except for resveratrol, which was > 2 times higher in chicory than grass silage and persisted after opening the bale (Appendix A.2, Figure A1). The sequiterpene lactone and other secondary compounds were higher in chicory compared to plantain, with higher values in fresh cut forage compared to the silage of the same forages, with several secondary compounds almost undetectable in the silage, except for germacranolide, which had a similar value between all tested forages, both fresh cut and silage (Appendix A.2, Figure A1).
3.2. Overall Response to Intramammary Infection (IMI)
The intramammary infection model used was effective in increasing the concentration of SCC in milk after 8 h post-IMI, with a slight but significant increase in rectal temperature that persisted until the end of the 10-day trial (Figure 2). There was no increase in bacteria count after IMI; however, supplementation with Se increased the bacterial count (p = 0.03) but only when considering the sheep receiving grass silage (Se × F with p = 0.02). Cultured milk had a higher frequency of various bacteria after IMI compared to prior IMI (Chi-Square p = 0.003; Figure 3), but the type of forage or Se supplementation did not affect the type of bacteria in the milk. There was a numerical (p = 0.11) increase in the frequency of Strep. uberis after IMI but not in the GCT group, so the frequency of Strep. uberis was higher (Chi-square p = 0.03) in animals supplemented vs. non-supplemented with Se. The frequency of Staph aureus after IMI was also increased (Chi-square p = 0.02).
Feed intake, milk yield, blood urea, and water intake were decreased after IMI. DMI and urea were significantly lower than baseline only 1 day after IMI (Figure 4). Most of milk components measured were affected by IMI, with a decrease in lactose, SNF, total and other solids, C18:0, and medium chain fatty acids and an increase in proportion of protein, fat, C16:0, C18:1, de novo and mixed fatty acids, acetone, β-hydroxybutyrate (BHBA), and various nitrogen components (i.e., NPN and MUN) (Figure 5 and Figure 6). Most of the parameters related to milk fat were affected only during the first 120 h (5 days) post-IMI.
Parameters in blood were also affected by IMI (Figure 7 and Figure 8). Total protein, globulin, albumin, haptoglobin, ceruloplasmin, interleukin 6 (IL6), myeloperoxidase, ROM, and ROM/FRAP (ferric reducing ability of the plasma) ratio increased starting at 3-day post-IMI and remained high compared to pre-IMI until the end of the experiment. The concentration of paraoxonase and the albumin–globulin ratio decreased after IMI until the end of the experiment. The concentration of zinc and AOPP (advanced oxidation protein products) decreased temporarily after IMI. Nitric oxide (NOx) and nitrate (NO3) increased significantly after IMI but briefly, only in animals supplemented with Se yeast.
3.3. Response of Ewes Fed Chicory-Plantain and Se Yeast
Rectal temperature was overall lower in animals supplemented vs. not supplemented with Se yeast (Table 2). The type of silage had a tendency (p = 0.07) to affect DMI, with ewes fed chicory + plantain silage having less DMI than ewes fed grass silage, especially after IMI, but milk yield and SCC were not affected by silage type or Se yeast (Table 2 and Figure 4). The significant Se × T effect for SCC was due to a higher SCC before IMI in animals receiving Se (Figure 2). The count of bacteria in milk was overall higher in animals supplemented with Se compared to non-supplemented animals, although this was due to a higher value in the GSY group compared to the other groups, already prior to IMI (Figure 3 and Table 2).
Among milk components (Table 2), compared to ewes fed grass silage, ewes fed chicory + plantain silage had higher levels of milk protein, butterfat (due to higher de novo-derived fatty acids), and total solids, with a lower level of medium-chain fatty acids (Table 2). Se yeast-supplemented ewes had lower milk fat due to decreased C16:0 and de novo-derived fatty acids (Table 2). A significant interaction F × Se was observed for preformed fatty acids, especially C18:0 and C18:1, and acetone, where a decrease in animals fed chicory–plantain silage and an increase in animals fed grass silage when supplemented with Se yeast were observed (Figure 5 and Figure 6).
The type of silage offered and Se yeast supplementation had minor effects on blood parameters (Table 3). Ewes fed chicory + plantain silage had lower blood levels of IL6 and higher levels of BHBA and FRAP. Ewes supplemented with Se had higher blood NEFA–albumin ratio, driven in part by the tendency for these ewes to have higher levels of NEFA (p = 0.08) and lower levels of albumin (p = 0.09) in comparison to ewes that were not supplemented with selenium.
The interaction between forage type and Se yeast was significant on a few blood parameters (Table 3 and Figure 7 and Figure 8). Globulin level was higher in animals supplemented vs. non-supplemented with Se prior to IMI, but no difference was observed post-IMI. Ceruloplasmin had a similar pattern in all groups (i.e., increased after IMI), except for the GSY, which was numerically higher before IMI compared to the other groups and was unaffected by IMI. The concentration of IL6 was lower overall in ewes fed chicory–plantain vs. grass silage and increased after IMI only in the latter. The concentration of ROM was numerically higher before IMI, and ROM/FRAP increased faster after IMI in ewes fed grass vs. chicory–plantain silage.
Few parameters of the complete blood count were affected by the IMI. Among those, the number of lymphocytes increased after IMI, but mainly due to an increase observed in animals supplemented with Se yeast, while the hematocrit and related parameters such as red blood cells, hemoglobin, and mean corpuscular hemoglobin were decreased by IMI (Figure 9). Migration of neutrophils was reduced while phagocytosis of PMN was increased after IMI (Figure 10).
Except for the significant Se×T for lymphocytes and MCH, few CBC parameters were affected by forage or Se supplementation (Table 4 and Figure 9). Overall, forage or Se supplementation did not affect leukocyte phagocytosis. Neutrophil migration was highest amongst ewes fed chicory + plantain vs. grass silage before IMI; however, a similar level of migration was observed after IMI (Figure 10).
4. Discussion
4.1. Characterization of the IMI Model
The data presented in this study indicate successful establishment of IMI as demonstrated by a rapid and consistent increase in SCC in milk and the increase and maintenance of a higher rectal temperature compared to prior IMI until the end of the experiment without eliciting a large systemic response. This confirms the original method developed by Addis et al. [24] is working correctly. The 40% reduction in milk production in response to IMI observed in the present study is consistent with other studies assessing subclinical mastitis in sheep [28] and goats [23].
The effects of IMI on the main components of milk, including the increase in % protein and the decrease in % lactose, were also consistent with the data obtained in goats after IMI induced using the same bacteria [23] and dairy cows after intramammary infusion with E. coli [29]. Different than IMI in goats [23] but similar to IMI in dairy cows [29], we observed an increase in % milk fat. Among fat components, our data indicated that IMI negatively affected the proportion of long-chain fatty acids but had a positive effect on the proportion of de novo synthesized and short-chain fatty acids, confirming prior field observation in dairy cows [30], suggesting at the least a maintenance of milk fat synthesis using acetic acid and butyrate by the mammary gland [31].
As observed in the goat model of subclinical IMI [23], a mild systemic inflammatory response was observed with parameters following an expected pattern, such as an increase in concentration of positive acute phase proteins haptoglobin and ceruloplasmin and oxidative stress, as observed by the increase in ROM, and a decrease in concentration of paraoxonase [23], typical of an inflammatory response [32]. An exception was albumin, where a peculiar increase after IMI was observed in our study, but not in the study where subclinical IMI was induced in goats [23]. Albumin generally has a very long half-life in circulation, and the replacement of albumin is low, thus, an increase in plasma can be the only consequence of acute dehydration. The sheep after IMI drank less water, which could have caused dehydration, which is also supported by an increased concentration of urea in milk after IMI, as previously observed [33]. It is unclear what the reason is for the decreased water intake.
In our study, we did not observe a change in NEFA, glucose, and BHBA in blood plasma as was observed in prior studies [23,29]. The latter indicates a likely peculiar difference in the response to IMI of sheep compared to goats and dairy cows.
Despite the well-established subclinical IMI model, the bacteria count in milk was not significantly different between prior and after IMI in our study. Also, around 50% of the glands did not have any significant growth when milk was cultured, and we expected to detect Streptococcus in milk from all glands after IMI; despite a significant increase after IMI of those bacteria, only up to 25% of the glands had Streptococcus without any case in the GCT group.
Between 10 and 40% of the milk collected before IMI had an agar growth of Staphylococcus; however, this did not induce any infection as evidenced by the lack of an increase in milk SCC and blood inflammatory markers in most sheep. Most milk from the mammary glands after IMI had growth in the culture of Staph. Aureus. The latter is very diffuse in glands of lactating ewes and is a significant cause of IMI in sheep farms [34]. However, the result might be explained by contamination during the milk collection and/or our animals may have experienced an IMI due to contamination during the injection of Strep. uberis. Although we attempted to maximize sterility during the intramammary infusion of Strep. uberis, the milking parlor we used was not an ideal place to keep sterility. In any case, our purpose was not to study specifically the IMI induced by Strep. uberis, but a general IMI, which was obtained. The inconsistency in the type of bacteria found, including the ones that were not detected as there was no growth in the agar, could have affected the uniformity of the response. However, all the animals had a very consistent increase in SCC and a general inflammatory response.
In addition, our data revealed higher bacteria count and SCC in the milk of GSY vs. the other groups before IMI. The same group also had higher ceruloplasmin levels before IMI, indicating a possible mild inflammation due to a pre-existing subclinical IMI. The same group also presented a few cases of yeast in milk and was the only group with no effect on the concentration of ceruloplasmin in blood post-IMI. It is unclear why feeding grass silage in combination with Se yeast could have increased the presence of bacteria and/or a mild intramammary infection in the mammary gland of the ewes.
4.2. The Type of Silage Had a Minimal Effect on the Response to IMI
Chicory and plantain forages have gained increased attention recently due to their high concentration of bioactive compounds, and can provide additional benefits that serve to improve animal health and reduce the environmental impact of livestock production [12,13,35]. In a previous study, mid-lactation cows grazing pastures rich in chicory and plantain were found to have lower milk SCC than cows grazing grass or legume-dominated pastures, indicating that forb pastures may be able to improve animal health, particularly at the level of the mammary gland [11]. In the current study, no differences in milk SCC were found between ewes fed chicory + plantain silage and grass silage, somewhat in contrast to the previous finding.
An important difference between the previous study on grazing cattle and the present investigation was the utilization of silage instead of fresh forages in a pasture setting. It is worth noting that the ensilability of chicory and plantain is often challenging due to several factors, including low dry matter and water-soluble carbohydrate content, as well as the presence of high levels of bioactive compounds that may inhibit microbial activity [36,37]. The chicory variety used in this study typically contains high concentrations of total phenolics and condensed tannins (2.1–4.3%) [38,39]. While studies on the effects of ensilement on the secondary compound profile of chicory and plantain are limited, prior studies in other forage varieties have observed changes in secondary compound concentrations following the silage process. In one study, silage made from Paulownia leaves had higher concentrations of phenolic acids and flavonoids than the fresh plant material [40], whereas another study examining sainfoin and Sulla found decreases in polyphenols and proanthocyanins and a lower antioxidant activity in both silages when compared to fresh forages [41]. As demonstrated by Martens et al. [42], the effects of ensilement on secondary compound concentrations are highly species-dependent, even without considering the impact of different ensilement methods or the properties of the forages used to produce the silage. The chicory silage in our experiment had lower concentration of phytochemicals compared to the fresh chicory. Furthermore, although we did not conduct an extensive analysis of secondary compounds in our silages, our results indicated that the flavonoid content of chicory decreased over time after the silage was opened for feeding. This may help explain the lack of response observed in animals fed forb silages in the current study, in contrast to the positive effects reported when animals graze fresh chicory [11]. Further work is necessary to determine if the different results obtained in this study, compared to previous studies using chicory and/or plantain silage, can be attributed to changes in secondary compound concentrations due to the ensilement process or were driven by other factors.
While feeding chicory + plantain silage did not have a significant effect on the response of the ewes to IMI in terms of milk production or quality, the reduced plasma levels of IL-6 and higher FRAP may indicate that these forages can improve the basal antioxidant function and inflammatory status. Prior studies have shown that serum levels of IL6 are elevated in cows with subclinical mastitis [43,44]. High levels of IL6 have been associated with poor outcomes in humans with sepsis [45]. IL6 is one of the primary pro-inflammatory cytokines controlling the acute phase reaction [46], although it acts in a hermetic fashion, where high doses induce inflammation and low doses decrease inflammation [47]. In our experimental model, only ewes fed grass silage had an increase in IL6 after IMI. Despite the increase in IL6, the ewes fed grass silage did not experience a higher or lower inflammation compared to the ewes fed chicory–plantain silage. Thus, the importance of the response of the IL6 level to IMI is unclear. A reduction in FRAP has also been found among goats suffering from subclinical mastitis [48]; however, in our study, FRAP was not affected by IMI, but the ROM/FRAP ratio was better in ewes fed chicory–plantain vs. grass silage, indicating a possible better antioxidative status of the former [49]. The role of FRAP on mastitis appears somewhat unclear, as FRAP in the plasma of dairy cows with subclinical mastitis from Staph. aureus was lower when a spontaneous healing was observed vs. cows that did not have a spontaneous healing [50].
Overall, the data do not support a significant effect of chicory–plantain silage in improving the response to mastitis.
4.3. Se Supplementation Does Not Affect the Response to IMI
Selenium has been extensively studied for its apparent benefits on immune and antioxidant function. Blood selenium levels have been negatively associated with somatic cell count [51], an effect that may be mediated by Se-sensitive long non-coding RNAs that modulate antioxidant and inflammatory gene networks [52]. Supplementation of 0.2 ppm of Se yeast has also been shown to reduce the presence of mastitis pathogens in healthy quarters of dairy cows [53]. Selenium deficiency has been associated with a higher frequency and severity of mastitis and poorer immune function [54]. It has been proposed that supplementing dairy animals with higher than recommended organic Se in the diet could improve the health of the mammary gland [55]. Oral provision of 0.25 mg/d during pregnancy and two injections of 5 mg of sodium selenite did not improve the resistance to mastitis in Indian crossbred cows [56].
In our study, we did not observe any significant effects of a high dose (3.6 mg/d) of Se yeast supplementation on any of the immune or antioxidant parameters measured, except for a larger increase in the number of circulating leukocytes after IMI. It is unclear the reason for our observation; however, a mild effect of Se level in blood on subclinical mastitis has been observed in dairy cows [51] and sheep [57]. In our study, ewes without supplementation of Se yeast were not deficient in Se, as the basal diet contained inorganic Se [21]. Thus, our study does not support an improved response to sub-clinical mastitis with supplementation of Se above the recommendation.
4.4. Limitations
Although our research confirmed that the subclinical model used is robust, the lack of effect on helping to address the IMI by either chicory and plantain silage and Se yeast (or their combination) detected may be limited by the low number of animals used. The statistical power needed to observe differences was calculated after we obtained the results using the one-way ANOVA Proc Power of SAS, considering all time points after the IMI. The analysis was performed for milk components, milk yield, and blood parameters. The analysis revealed that for a few parameters (albumin, glucose, and milk protein) 8 animals/groups would suffice to have an 80% power with an α = 0.05, other parameters required 12 animals/group, such as ceruloplasmin and myeloperoxidase; however, other parameters required a larger number of animals/group, including haptoglobin. Additionally, the presence of other bacterial species, like S. aureus, further complicates the interpretation of the results, as other infections may alter the response of the ewes to IMI. As such, a greater number of animals would also provide flexibility in removing animals dealing with non-Strep. uberis infections from the study and clarifying the results. The quality of the silage provided was also not very good, and there was a low concentration of secondary compounds. Both could explain the lack of effect of the chicory and plantain silages on the response of IMI.
5. Conclusions
Despite previous work supporting the positive effects of chicory/plantain forages and Se supplementation on udder health and animal immune and antioxidant response, no differences were observed in the response to intramammary infection in the present trial. Multiple factors may have contributed to this outcome, such as the utilization of silage with low levels of secondary compounds as opposed to fresh forage, as well as the fact that none of the ewes were in a state of Se deficiency before this experiment was conducted. As such, future investigations are necessary to evaluate the effect of ensilement on these forages’ anti-inflammatory and antioxidant characteristics. Additionally, a better understanding of optimal Se levels for lactating sheep will provide insight into how Se supplementation and dietary changes can be utilized to promote improved mammary health.
Author Contributions
Conceptualization, M.B. and S.A.; methodology, H.R.F., J.K., S.B. and E.T.; validation H.R.F., J.K., S.B. and E.T.; formal analysis, H.R.F., J.K., S.B. and E.T.; investigation, H.R.F., J.K. and M.B.; resources, J.K., E.T. and M.B.; data curation, E.T. and M.B.; writing—original draft preparation, H.R.F. and M.B.; writing—review and editing, J.K., S.A., S.B., E.T. and M.B.; visualization, H.R.F. and M.B.; supervision, M.B.; project administration, M.B.; funding acquisition, J.K., S.A. and M.B. All authors have read and agreed to the published version of the manuscript.
Funding
The experiment was funded by the USDA NIFA ORE (project #ORE00170) to M.B., S.A. and J.K. The USDA NIFA multistate project # NE-1748 financially supported M.B.
Institutional Review Board Statement
Experimental procedures in this investigation were approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon State University (protocol # 2019-0042).
Data Availability Statement
The raw data supporting the conclusions of this article will be made available by the authors on request.
Acknowledgments
The Se yeast DiaMune was a donation from Diamond V, provided by Ilkyu Yoon. The authors thank the following people for their great help during the experiment: Benjamin Grismer for managing the animals and helping through all the aspects of the experiment, including feeding, milking, and collecting samples; Maria Gracia Puerto-Hernandez for the measurement of water intake and milk analysis; Daniella Hasan for helping collect blood samples and the following undergraduate students for helping managing the animals, feeding them, milking, and helping collecting samples: Kate Ness, Magdalena Ibarra, Samantha Heney, Jula VanDerZant, Tyler Quinones, Mariangel Torres, Caitlynn Dougherty, Sarah Kemp, Alyssa Thibodeau, Corinna Cauchy, Callan Stowell, Michele McCann, Carolyn Pearce, Mustafa Bozkus, Shelby Park, and Paige Tafoya.
Conflicts of Interest
The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript:
| AOPP | Advanced oxidation protein products |
| BHBA | β-hydroxybutyric acid |
| FRAP | Ferric-reducing ability of the plasma |
| IL6 | Interleukin 6 |
| IMI | Intramammary infection |
| NEFA | Non-esterified fatty acid |
| NO | Nitric Oxide |
| SCC | Milk somatic cells |
Appendix A
Appendix A.1. Bromological Analysis of the Silages
Table A1.
Bromological Analysis of the Silages.
| Unit | Grass | Chicory | Plantain | |
|---|---|---|---|---|
| Dry matter | 45.5 | 38.2 | 54.4 | |
| pH | 4.43 | 4.37 | 4.74 | |
| Ammonia | % DM | 1.71 | 0.74 | 0.70 |
| Ammonia | % CP | 15.0 | 8.54 | 5.92 |
| Total VFA | % DM | 6.10 | 7.38 | 6.30 |
| Lactic acid | % VFA | 82.0 | 78.6 | 92.1 |
| Lactic acid | % DM | 5.0 | 5.8 | 5.8 |
| Acetic acid | % DM | 1.10 | 1.58 | 0.50 |
| Propionic acid | % DM | ND | ND | ND |
| Butyric acid | % DM | ND | ND | ND |
| Iso-butyric acid | % DM | ND | ND | ND |
| Alcohols | ND | ND | ND | |
| Acetates | ND | ND | ND | |
| Lactates | ND | ND | ND |
ND = not detectable.
Appendix A.2. Protocol and Results of Secondary Compounds in Silages
The analysis was performed in the laboratory of Dr. van Breemen at Oregon State University by Dr. Ruth Muchiri.
Sample Preparation
Chicory and grass ground samples were stored at −80 °C prior to processing. 500 mg of chicory or grass sample (total of 10 samples) was transferred into 10 mL glass tube. 5 mL of methanol was added and vortexed for 2 min. The sample was centrifuged at 2000× g rpm and 4 °C for 2 min. The supernatant was transferred to 50 mL volumetric flask. The methanol extraction was repeated 8 additional times, and the supernatant was pooled. Methanol was added to bring the volume up to 50 mL. An aliquot of the supernatant (500 µL) was transferred to a 0.22 µm PVDF filter and centrifuged at 5000× g rpm and 4 °C for 3 min. A 50 µL aliquot of the filtrate was transferred to a glass HPLC vial in which 25 µL of biochanin A internal standard (500 ng/mL final concentration) was added. The sample was further diluted with 425 µL of deionized water to obtain 1 mg/mL extract. The samples were analyzed using a Shimadzu 8060 triple quadrupole mass spectrometer. For analysis using high-resolution mass spectrometry, a 10 mg/mL sample was prepared using the same filtrate (internal standard was 1 mg/mL).
Table A2.
LC-MS/MS Method for flavonoids analysis.
| System: | Shimadzu LC Nexera and Shimadzu MS QQQ 8060 | |
| Column: | Waters Cortecs C18 1.6 µm particle size | |
| 2.1 mm I.D. × 50 mm L | ||
| Guard Column: | C18 | |
| Oven Temperature: | 40 °C | |
| Flow Rate: | 0.4 mL/min | |
| Pressure: | 7300 psi initial condition | |
| Solvents: | (A) H2O + 0.01% formic acid | |
| (B) Acetonitrile + 0.01% formic acid | ||
| Gradient: | 0.00 min | 10% B |
| 0.10 min | 10% B | |
| 1.00 min | 30% B | |
| 1.75 min | 90% B | |
| 2.00 min | 90% B | |
| 2.01 min | 10% B | |
| 3.00 min | end | |
| Injection: | 5 µL | |
| Elution Time: | ||
| 1.09 min | cathechin | |
| 1.26 min | epicatechin | |
| 1.53 min | taxifolin | |
| 1.72 min | resveratrol | |
| 1.80 min | quercetin | |
| 1.91 min | kaempferol | |
| 1.93 min | isorhamnetin | |
| 2.10 min | biochanin A | |
| MS Runtime: | 0.00 min–3.00 min | |
| divert valve 0.50 min | ||
| SRM Transitions: | ||
| catechin (–) | Q: 289.30 → 245.20 m/z (CE: +15 eV) 15 ms | |
| q: 289.30 → 203.20 m/z (CE: +20 eV) 15 ms | ||
| epicatechin (–) | Q: 289.30 → 245.20 m/z (CE: +15 eV) 15 ms | |
| q: 289.30 → 203.20 m/z (CE: +20 eV) 15 ms | ||
| taxifolin (–) | Q: 303.30 → 285.20 m/z (CE: +12 eV) 15 ms | |
| q: 303.30 → 125.20 m/z (CE: +22 eV) 15 ms | ||
| resveratrol (+) | Q: 229.30 → 107.20 m/z (CE: −21 eV) 15 ms | |
| q: 229.30 → 135.20 m/z (CE: −14 eV) 15 ms | ||
| quercetin (–) | Q: 301.30 → 151.20 m/z (CE: +22 eV) 15 ms | |
| q: 301.30 → 179.20 m/z (CE: +19 eV) 15 ms | ||
| kaempferol (+) | Q: 287.30 → 153.20 m/z (CE: −31 eV) 15 ms | |
| q: 287.30 → 121.20 m/z (CE: −30 eV) 15 ms | ||
| isorhamnetin (+) | Q: 317.30 → 302.20 m/z (CE: −24 eV) 15 ms | |
| q: 317.30 → 153.20 m/z (CE: −34 eV) 15 ms | ||
| biochanin A (+) | Q: 285.30 → 213.20 m/z (CE: −39 eV) 15 ms | |
| q: 285.30 → 253.20 m/z (CE: −17 eV) 15 ms | ||
Table A3.
LC-MS/MS Method for flavonoid conjugates (derivatives) analysis.
| System: | Shimadzu LC Nexera and Shimadzu MS QQQ 8060 | |
| Column: | Waters Cortecs C18 1.6 µm particle size | |
| 2.1 mm I.D. × 50 mm L | ||
| Guard Column: | C18 | |
| Oven Temperature: | 40 °C | |
| Flow Rate: | 0.4 mL/min | |
| Pressure: | 8000 psi initial condition | |
| Solvents: | (A) H2O + 0.1% formic acid | |
| (B) Acetonitrile + 0.1% formic acid | ||
| Gradient: | 0.00 min | 5% B |
| 0.15 min | 5% B | |
| 1.75 min | 45% B | |
| 2.75 min | 90% B | |
| 3.00 min | 90% B | |
| 3.01 min | 5% B | |
| 4.00 min | end | |
| Injection: | 5 µL | |
| Elution Time: | ||
| 1.56 min | cyanidin-3-O-glucoside | |
| 1.65 min | peonidin-3-O-glucoside | |
| 1.66 min | malvidin-3-O-glucoside | |
| 1.85 min | peonidin-3-O-acetylglucoside | |
| 1.86 min | malvidin-3-O-acetylglucoside | |
| 1.90 min | malvidin-3-O-caffeoylglucoside | |
| 1.92 min | cyanidin-3-O-p-coumarylglucoside | |
| 1.99 min | peonidin-3-O-p-coumarylglucoside | |
| 2.00 min | malvidin-3-O-p-coumarylglucoside | |
| 2.82 min | biochanin A | |
| MS Runtime: | 0.00 min–4.00 min | |
| divert valve 0.50 min | ||
| SRM Transitions: | ||
| *cyanidin-3-O-glucoside (+) | Q: 449.30 → 287.20 m/z (CE: −25 eV) 15 ms | |
| q: 449.30 → 137.20 m/z (CE: −50 eV) 15 ms | ||
| *peonidin-3-O-glucoside (+) | Q: 463.30 → 301.20 m/z (CE: −25 eV) 15 ms | |
| q: 463.30 → 286.20 m/z (CE: −43 eV) 15 ms | ||
| *malvidin-3-O-glucoside (+) | Q: 493.30 → 331.20 m/z (CE: −25 eV) 15 ms | |
| q: 493.30 → 315.20 m/z (CE: −50 eV) 15 ms | ||
| *peonidin-3-O-acetylglucoside (+) | Q: 505.30 → 301.20 m/z (CE: −25 eV) 15 ms | |
| *malvidin-3-O-acetylglucoside (+) | Q: 535.30 → 331.20 m/z (CE: −25 eV) 15 ms | |
| *malvidin-3-O-caffeoylglucoside (+) | Q: 655.30 → 331.20 m/z (CE: −25 eV) 15 ms | |
| *cyanidin-3-O-p-coumarylglucoside (+) | Q: 595.30 → 287.20 m/z (CE: −25 eV) 15 ms | |
| *peonidin-3-O-p-coumarylglucoside (+) | Q: 609.30 → 301.20 m/z (CE: −25 eV) 15 ms | |
| *malvidin-3-O-p-coumarylglucoside (+) | Q: 639.30 → 331.20 m/z (CE: −25 eV) 15 ms | |
| *biochanin A (+) | Q: 285.30 → 213.20 m/z (CE: −39 eV) 15 ms | |
| q: 285.30 → 253.20 m/z (CE: −17 eV) 15 ms | ||
* For ionization potential, +2 kV was used instead of default settings. The positively charged anthocyanins do not require ionization. The low potential energy is used to maintain the Taylor cone of the electrospray ionization.
Table A4.
LC-MS-DDA Method for flavonoid conjugates (derivatives) analysis.
| System: | Shimadzu LC Nexera and Shimadzu MS Q-ToF 9030 | |
| Column: | Waters Cortecs C18 2.7 µm particle size | |
| 2.1 mm I.D. × 50 mm L | ||
| Guard Column: | C18 | |
| Oven Temperature: | 35 °C | |
| Flow Rate: | 0.3 mL/min | |
| Pressure: | 2500 psi initial condition | |
| Solvents: | (A) H2O + 0.1% formic acid | |
| (B) Acetonitrile + 0.1% formic acid | ||
| Gradient: | ||
| 0.00 min | 10% B | |
| 0.10 min | 10% B | |
| 1.00 min | 30% B | |
| 13.0 min | 90% B | |
| 14.0 min | 10% B | |
| 16.0 min | end | |
The MS was set to acquire data in both scanning mode and data-dependent acquisition (DDA), in which peaks with defined intensity (1000) were fragmented to give product ions within the same analysis. Both positive and negative electrospray ionization modes were used for the analysis. The MS scan range was set at m/z 100–900.
- Data analysis:
The analysis of each pair of chromatograms (chicory numbered 1, 2, or 3) was carried out using the Online XCMS (Scripps Institute, La Jolla, CA, USA). The retention time and assignment of peaks to their corresponding m/z values were determined following literature reports on the secondary compound profile of chicory plants. Additionally, the XCMS metabolomics software identified some of the secondary compounds using algorithms and data from Metlin database. To minimize false positives, data from 3 chromatograms from chicory samples labeled Feb1-1, Feb 1-2, Feb 1-3 were aligned, and each m/z value identified by XCMS was manually inspected to ensure the peaks were not artifacts.
The single reaction monitoring (SRM) data acquired using 8060 triple quadruple was analyzed using LabSolutions V5.2.
- Results
Figure A1.
Secondary compounds detected in the silages used.
Appendix A.3. Protocol for the Bacterial Analysis in Milk
Bacterial Count Protocol from Milk Samples of Infected Ewes
- Samples were plated on MacConkey (Gram-negative specific) and blood agar with esculin (sheep blood, supports general growth).
- Plates were placed in a humidified incubator at 37 °C.
- Agar plates were observed for growth at 24 and 48 h after inoculation.
- If no growth on blood or MacConkey agars was observed after 48 h, the sample was identified as “No Growth”.
- If growth occurred on MacConkey agar after 24 h, colonies were identified as Gram-negative organisms. Identification was by colony morphology on blood and MacConkey agar.
- If growth occurred on blood agar but not on MacConkey agar at 24 or 48 h, colonies were identified as Gram-positive organisms.
- Initial identification was based on colony morphology on blood agar.
- Organisms were identified as Staphylococcus or Streptococcus organisms using the catalase test and esculin fermentation on the plate:
- i.
- Catalase positive = Staphylococcus;
- ii.
- Catalase negative = Streptococcus.
- Staphylococcal organisms were further speciated using the coagulase test and colony morphology:
- i.
- Coagulase positive = Staphylococcus aureus;
- ii.
- Coagulase negative = “Staphylococcus species”.
- If 2–3 species were present on the blood agar, the sample was classified as mixed. If more than 3 species occurred on the blood agar, the sample was classified as contaminated.
- evaluated by Gram-stain and microscopic evaluation.
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Figure 1.
Experimental design with treatments and a timeline for the collection of samples.
Figure 2.
Somatic cell count in milk and rectal temperature in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line; time 0 from IMI). Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05). For a tendency, the p-value is reported. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI.
Figure 2.
Somatic cell count in milk and rectal temperature in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line; time 0 from IMI). Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05). For a tendency, the p-value is reported. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI.
Figure 3.
Frequency of different bacterium types and yeast and mean bacteria count in milk of ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection was induced in both mammary glands using Strep. uberis. Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05).
Figure 3.
Frequency of different bacterium types and yeast and mean bacteria count in milk of ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection was induced in both mammary glands using Strep. uberis. Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05).
Figure 4.
Dry matter intake (DMI), milk yield, water intake, and blood urea in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05). For a tendency, the p-value is reported. * denotes significant (p < 0.05) difference compared to −1 day relative to IMI; f = denotes significant (p < 0.05) difference between the two forages at specific time points; s = denotes significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 4.
Dry matter intake (DMI), milk yield, water intake, and blood urea in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Indicated in the graph as T (Time), Se supplementation (Se), and/or F (type of forage), and their interactions when significant (p < 0.05). For a tendency, the p-value is reported. * denotes significant (p < 0.05) difference compared to −1 day relative to IMI; f = denotes significant (p < 0.05) difference between the two forages at specific time points; s = denotes significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 5.
Trend of the non-fat milk components in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. A significant (p < 0.05) Forage×Time was detected for % of protein. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI; f = denotes significant (p < 0.05) difference between the two forages at specific time points.
Figure 5.
Trend of the non-fat milk components in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. A significant (p < 0.05) Forage×Time was detected for % of protein. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI; f = denotes significant (p < 0.05) difference between the two forages at specific time points.
Figure 6.
Trend of fat-related milk components in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI.
Figure 6.
Trend of fat-related milk components in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. * denotes significant (p < 0.05) difference compared to −24 h relative to IMI.
Figure 7.
Trend of blood parameters related to inflammation in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Forage (F), Se×Time (Se*T), and Forage × Time (F*T). * denotes significant (p < 0.05) difference compared to time −1 relative to IMI. s = denotes a significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 7.
Trend of blood parameters related to inflammation in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Forage (F), Se×Time (Se*T), and Forage × Time (F*T). * denotes significant (p < 0.05) difference compared to time −1 relative to IMI. s = denotes a significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 8.
Trend of blood parameters related to oxidative stress in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Se × Time (Se*T) and Forage × Time (F*T). * denotes significant (p < 0.05) difference compared to time −1 relative to IMI. f = denotes a significant (p < 0.05) difference between the two forages at specific time points.
Figure 8.
Trend of blood parameters related to oxidative stress in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Se × Time (Se*T) and Forage × Time (F*T). * denotes significant (p < 0.05) difference compared to time −1 relative to IMI. f = denotes a significant (p < 0.05) difference between the two forages at specific time points.
Figure 9.
Trend of blood parameters related to complete blood counts in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Se supplementation × Time (Se*T). * denotes significant (p < 0.05) difference relative to time −1 relative to IMI. s = denotes a significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 9.
Trend of blood parameters related to complete blood counts in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis (dotted line). Time was significant (p < 0.05) in all the reported parameters. Few parameters were significantly (p < 0.05) affected by Se supplementation × Time (Se*T). * denotes significant (p < 0.05) difference relative to time −1 relative to IMI. s = denotes a significant (p < 0.05) difference between the two doses of Se at specific time points.
Figure 10.
Trend of leukocyte phagocytosis and neutrophil migration in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis. Indicated in the graph as T (Time), Se supplementation (Se), and/or type of forage (F), and their interactions when significant (p < 0.05). Different letters denote significant (p < 0.05) differences between groups and time.
Figure 10.
Trend of leukocyte phagocytosis and neutrophil migration in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented with Se yeast (SY) or not (CT). An intramammary infection (IMI) was induced in both mammary glands using Strep. uberis. Indicated in the graph as T (Time), Se supplementation (Se), and/or type of forage (F), and their interactions when significant (p < 0.05). Different letters denote significant (p < 0.05) differences between groups and time.
Table 1.
Diet composition and chemical characteristics for each of the treatment groups.
| Treatment Group 1 | ||||
|---|---|---|---|---|
| GCT | GSY | CPCT | CPSY | |
| Ingredients, % DM | ||||
| Grass silage | 50.0 | 50.0 | 0.00 | 0.00 |
| Chicory silage | 0.00 | 0.00 | 25.0 | 25.0 |
| Plantain silage | 0.00 | 0.00 | 25.0 | 25.0 |
| Corn | 16.5 | 16.5 | 20.7 | 20.7 |
| Barley | 20.5 | 20.5 | 14.8 | 14.8 |
| Soybean meal | 11.4 | 11.4 | 13.2 | 13.2 |
| Alfalfa meal | 0.62 | 0.00 | 0.62 | 0.00 |
| Se yeast | 0.00 | 0.62 | 0.00 | 0.62 |
| CaCO3 | 0.75 | 0.75 | 0.40 | 0.40 |
| NaCl | 0.20 | 0.20 | 0.20 | 0.20 |
| Mineral mix 2 | 0.10 | 0.10 | 0.10 | 0.10 |
| Chemical composition * | ||||
| CP% | 14.7 | 14.7 | 14.7 | 14.7 |
| NDF% | 34.9 | 34.9 | 30.7 | 30.7 |
| NFC% | 42.5 | 42.5 | 44.2 | 44.2 |
| Ca% | 0.57 | 0.57 | 0.82 | 0.82 |
| P% | 0.26 | 0.26 | 0.34 | 0.34 |
| Ca/P | 2.19 | 2.19 | 2.41 | 2.41 |
| ME Mcal/kg DM | 2.69 | 2.69 | 2.47 | 2.47 |
1 GCT = grass silage without Se yeast; GSY = grass silage with Se yeast; CPCT = chicory + plantain silage without Se yeast; CPSY = chicory + plantain silage with Se yeast. 2 Composition reported (min or min-max): Ca = 8.0–9.6%; p = 6%; NaCl = 34–39%; Mg = 2.7%; Cu = 0–40 ppm; Se = 90 ppm; Vitamin A = 160,000 IU/lb. * calculated; CP = crude protein; NDF = neutral detergent fiber; NFC = non-fiber carbohydrates; ME = metabolizable energy.
Table 2.
Rectal temperature, feed intake, and milk yield and quality in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
Table 2.
Rectal temperature, feed intake, and milk yield and quality in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
| Parameter 1 | Unit | Chicory | Grass | p-Value 2 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CPCT | CPSY | GCT | GSY | SEM 3 | F | Se | F*Se | F*T | Se*T | F*Se*T | ||
| Rectal Temp. | °C | 39.2 | 39.0 | 39.2 | 39.1 | 0.07 | 0.28 | 0.01 | 0.45 | 0.09 | 0.76 | 0.05 |
| DMI | g/d | 1879 | 1997 | 2226 | 2221 | 163 | 0.07 | 0.71 | 0.69 | 0.02 | 0.46 | 0.99 |
| Milk Yield | mL/d | 470 | 561 | 649 | 557 | 64.8 | 0.19 | 1.00 | 0.17 | 0.66 | 0.52 | 0.32 |
| Water intake | L/d | 4.03 | 2.10 | 3.32 | 2.48 | 0.56 | 0.75 | 0.01 | 0.31 | 0.31 | 0.55 | 0.74 |
| Bacteria count | log2/mL | 4.20 b | 4.36 b | 2.04 c | 5.88 a | 0.69 | 0.79 | <0.01 | <0.01 | 0.18 | 0.53 | 0.70 |
| SCC | log2(×103) | 11.0 | 11.0 | 10.7 | 11.3 | 0.28 | 0.80 | 0.35 | 0.26 | 0.44 | 0.03 | 0.31 |
| Lactose | % | 3.24 | 3.43 | 3.44 | 3.29 | 0.22 | 0.90 | 0.93 | 0.43 | 0.79 | 0.76 | 0.47 |
| Protein | % | 6.14 | 5.69 | 5.46 | 5.63 | 0.19 | 0.05 | 0.44 | 0.09 | 0.00 | 0.18 | 0.50 |
| Fat | % | 7.11 | 6.26 | 6.33 | 5.94 | 0.29 | 0.05 | 0.03 | 0.10 | 0.30 | 0.64 | 0.87 |
| Solids | % | 17.9 | 16.4 | 16.2 | 16.0 | 0.54 | 0.05 | 0.09 | 0.18 | 0.11 | 0.64 | 0.95 |
| SNF | % | 10.6 | 10.3 | 10.1 | 10.1 | 0.26 | 0.14 | 0.59 | 0.53 | 0.11 | 0.76 | 0.83 |
| Other Solids | % | 4.45 | 4.61 | 4.60 | 4.45 | 0.24 | 0.98 | 0.98 | 0.50 | 0.78 | 0.80 | 0.53 |
| NPN | mg/dL | 26.9 | 25.0 | 26.9 | 26.6 | 1.44 | 0.56 | 0.44 | 0.54 | 0.18 | 0.41 | 0.76 |
| MUN | mg/dL | 21.5 | 21.3 | 23.3 | 23.3 | 1.34 | 0.24 | 0.51 | 0.83 | 0.45 | 0.59 | 0.84 |
| C16:0 | % | 2.45 | 2.03 | 2.14 | 1.97 | 0.15 | 0.19 | 0.04 | 0.36 | 0.29 | 0.63 | 0.81 |
| C18:0 | % | 0.38 a | 0.30 ab | 0.28 b | 0.33 ab | 0.03 | 0.22 | 0.58 | 0.03 | 0.08 | 0.74 | 0.74 |
| C18:1 | % | 0.45 a | 0.15 b | 0.24 b | 0.32 ab | 0.09 | 0.77 | 0.16 | 0.02 | 0.08 | 1.00 | 0.19 |
| De novo FA | % | 2.94 | 2.62 | 2.55 | 2.31 | 0.14 | 0.01 | 0.05 | 0.76 | 0.31 | 0.60 | 0.66 |
| Mixed FA | % | 2.51 | 2.07 | 2.20 | 2.08 | 0.15 | 0.21 | 0.04 | 0.38 | 0.29 | 0.62 | 0.79 |
| Preformed FA | % | 1.61 a | 1.18 b | 1.17 b | 1.37 ab | 0.10 | 0.21 | 0.22 | <0.01 | 0.28 | 0.83 | 0.58 |
| Unsaturated FA | % | 0.12 | 0.11 | 0.11 | 0.14 | 0.02 | 0.60 | 0.58 | 0.16 | 0.69 | 0.58 | 0.39 |
| Acetone | % | 0.20 ab | 0.13 b | 0.14 b | 0.24 a | 0.04 | 0.43 | 0.69 | 0.03 | 0.29 | 0.34 | 0.17 |
| BHBA | % | 0.33 | 0.28 | 0.29 | 0.36 | 0.05 | 0.73 | 0.78 | 0.17 | 0.27 | 0.13 | 0.45 |
| De novo FA | % FA | 41.6 b | 44.8 a | 43.3 ab | 40.7 b | 1.15 | 0.28 | 0.77 | 0.01 | 0.11 | 0.91 | 0.36 |
| Mixed FA | % FA | 34.7 | 34.6 | 37.2 | 35.3 | 1.11 | 0.15 | 0.35 | 0.40 | 0.30 | 0.94 | 0.75 |
| Preformed FA | % FA | 23.7 a | 20.5 ab | 19.8 b | 24.0 a | 1.40 | 0.86 | 0.69 | 0.01 | 0.33 | 0.75 | 0.18 |
| Medium Chain FA | % FA | 13.1 | 13.1 | 13.2 | 13.4 | 0.13 | 0.05 | 0.35 | 0.27 | 0.33 | 0.60 | 0.37 |
1 DMI = dry matter intake; SCC = somatic cell count; NPN = non-protein nitrogen; MUN = milk urea nitrogen; BHBA = β-hydroxybutyrate; FA = fatty acids. 2 Overall statistical effect of: F = silage type; Se = Se supplementation; T = time; and their interactions. All the parameters were significantly affected by time. Letters are reported to identify a statistical difference (p < 0.05) between groups when a Forage × Se (F*Se) interaction is significant (p ≤ 0.05). 3 Higher SEM.
Table 3.
Parameters in blood plasma in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
Table 3.
Parameters in blood plasma in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
| Parameter 1 | Unit | Chicory | Grass | p-Value 2 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CPCT | CPSY | GCT | GSY | SEM 3 | F | Se | Time | F*Se | F*T | Se*T | F*Se*T | ||
| Metabolism | |||||||||||||
| Glucose | mM | 4.27 | 4.24 | 4.19 | 4.21 | 0.10 | 0.57 | 0.92 | 0.07 | 0.81 | 0.99 | 0.21 | 0.85 |
| Cholesterol | mM | 1.78 | 1.79 | 2.15 | 1.75 | 0.22 | 0.44 | 0.35 | 0.19 | 0.32 | 0.21 | 0.58 | 0.07 |
| BHBA | mM | 0.51 | 0.56 | 0.43 | 0.45 | 0.05 | 0.05 | 0.39 | 0.83 | 0.69 | 0.82 | 0.21 | 0.69 |
| NEFA | mM | 0.31 | 0.35 | 0.31 | 0.34 | 0.01 | 0.80 | 0.08 | 0.22 | 0.66 | 0.47 | 0.25 | 0.97 |
| Total Protein | g/L | 68.4 a | 63.0 b | 63.1 b | 67.1 ab | 1.54 | 0.69 | 0.64 | <0.01 | <0.01 | 0.42 | 0.06 | 0.20 |
| Urea | mM | 6.75 | 6.79 | 7.67 | 7.05 | 0.46 | 0.18 | 0.51 | <0.01 | 0.45 | 0.43 | 0.87 | 0.04 |
| Ca | mM | 2.40 | 2.35 | 2.37 | 2.42 | 0.04 | 0.65 | 0.97 | 0.09 | 0.23 | 0.32 | 0.68 | 0.51 |
| NEFA–Albumin | 0.67 | 0.87 | 0.69 | 0.78 | 0.06 | 0.53 | 0.01 | 0.21 | 0.34 | 0.32 | 0.58 | 0.49 | |
| Acute phase indexes/inflammation/liver status | |||||||||||||
| Albumin | g/L | 30.5 | 29.3 | 30.4 | 29.1 | 0.70 | 0.79 | 0.09 | 0.03 | 0.95 | 0.15 | 0.38 | 0.36 |
| Haptoglobin | g/L | 0.54 | 0.49 | 0.44 | 0.67 | 0.14 | 0.80 | 0.51 | <0.01 | 0.31 | 0.17 | 0.63 | 0.73 |
| Ceruloplasmin | µM | 2.84 | 3.49 | 3.48 | 3.13 | 0.52 | 0.78 | 0.76 | <0.01 | 0.32 | 0.05 | 0.15 | 0.05 |
| Paraoxonase | U/L | 170 | 178 | 189 | 192 | 17.1 | 0.32 | 0.73 | <0.01 | 0.88 | 0.23 | 0.61 | 0.37 |
| Zn | mM | 10.0 | 10.0 | 10.3 | 11.1 | 0.68 | 0.28 | 0.54 | <0.01 | 0.52 | 0.34 | 0.92 | 0.68 |
| IL6 | pg/mL | 390 | 699 | 1317 | 1590 | 422 | 0.03 | 0.47 | 0.19 | 0.96 | 0.05 | 0.46 | 0.69 |
| IL1B | pg/mL | 396 | 165 | 55 | 156 | 173 | 0.33 | 0.72 | 0.37 | 0.36 | 0.28 | 0.49 | 0.42 |
| Bilirubin | µM | 1.13 | 0.82 | 0.94 | 0.89 | 0.16 | 0.41 | 0.57 | 0.13 | 0.80 | 0.22 | 0.76 | 0.30 |
| GGT | U/L | 74.3 a | 63.6 b | 61.5 b | 71.9 ab | 4.08 | 0.57 | 0.97 | 0.09 | 0.01 | 0.36 | 0.93 | 0.06 |
| Immune system | |||||||||||||
| Globulin | g/L | 37.7 a | 33.7 ab | 32.8 b | 38.0 a | 1.63 | 0.85 | 0.70 | <0.01 | 0.01 | 0.19 | 0.05 | 0.13 |
| Albumin–Globulin | 0.81 ab | 0.90 ab | 0.94 a | 0.78 b | 0.05 | 0.97 | 0.41 | <0.01 | 0.01 | 0.07 | 0.07 | 0.23 | |
| Myeloperoxidase | U/L | 494 | 480 | 478 | 538 | 32.5 | 0.51 | 0.46 | <0.01 | 0.24 | 0.26 | 0.10 | 0.52 |
| Oxidative status | |||||||||||||
| AOPP | µM | 61.7 | 62.8 | 61.8 | 63.1 | 4.73 | 0.96 | 0.79 | 0.01 | 0.99 | 0.89 | 0.19 | 0.62 |
| ROM | mg H2O2/dL | 16.3 | 19.6 | 19.8 | 18.9 | 2.33 | 0.55 | 0.59 | <0.01 | 0.36 | 0.04 | 0.11 | 0.11 |
| FRAP | µM | 108 | 111 | 99 | 106 | 2.79 | 0.02 | 0.12 | 0.08 | 0.41 | 0.37 | 0.31 | 0.77 |
| ROM/FRAP | 0.15 | 0.18 | 0.20 | 0.18 | 0.02 | 0.25 | 0.92 | <0.01 | 0.28 | 0.02 | 0.25 | 0.19 | |
| NOX | µM | 50.3 | 52.8 | 50.9 | 51.4 | 1.88 | 0.83 | 0.43 | 0.42 | 0.57 | 0.74 | 0.02 | 0.54 |
| NO2 | µM | 14.9 | 14.3 | 15.2 | 16.2 | 0.91 | 0.23 | 0.82 | 0.31 | 0.35 | 0.08 | 0.40 | 0.49 |
| NO3 | µM | 35.4 | 38.5 | 35.7 | 35.2 | 1.95 | 0.43 | 0.49 | 0.80 | 0.33 | 0.57 | <0.01 | 0.28 |
1 BHBA = β-hydroxybutyrate; NEFA = non-esterified fatty acids; IL6 = interleukin 6; IL1β = interleukin 1 beta; GGT = γ glutamyl transferase; AOPP = advanced oxidation protein products; ROM = reactive oxygen metabolites; FRAP = ferric reducing ability of the plasma; nitric oxide (NOX), nitrite (NO2-) and nitrate. 2 Overall statistical effect of: F = silage type; Se = Se supplementation; T = time; and their interactions. Letters are reported to identify a statistical difference (p < 0.05) between groups when a Forage × Se (F*Se) interaction is significant (p ≤ 0.05). 3 Higher SEM.
Table 4.
Complete blood count, phagocytosis, and migration of leukocytes in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
Table 4.
Complete blood count, phagocytosis, and migration of leukocytes in ewes fed chicory + plantain (CP) or grass (G) silage and either supplemented (SY) or not (CT) with Se yeast. An intramammary infection was induced in both mammary glands using Strep. uberis.
| Parameter 1 | Unit | Chicory | Grass | p-Value 2 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CPCT | CPSY | GCT | GSY | SEM 3 | F | Se | Time | F*Se | F*T | Se*T | F*Se*T | ||
| WBC | 103/mL | 10.8 | 12.1 | 9.2 | 12.5 | 1.31 | 0.76 | 0.28 | 0.25 | 0.11 | 0.11 | 0.09 | 0.96 |
| Lymphocytes | 103/mL | 6.20 | 6.07 | 5.42 | 7.62 | 0.65 | 0.53 | 0.10 | 0.04 | 0.06 | 0.55 | 0.03 | 1.00 |
| Monocytes | 103/mL | 0.054 | 0.051 | 0.046 | 0.062 | 0.006 | 0.82 | 0.28 | 0.21 | 0.10 | 0.12 | 0.14 | 0.93 |
| Neutrophils | 103/mL | 4.40 | 4.00 | 3.73 | 4.58 | 0.69 | 0.94 | 0.73 | 0.28 | 0.33 | 0.23 | 0.59 | 0.87 |
| Lymphocytes | % | 58.5 | 61.4 | 59.7 | 62.3 | 2.82 | 0.68 | 0.29 | 0.09 | 0.94 | 0.58 | 0.87 | 0.89 |
| Monocytes | % | 0.50 | 0.51 | 0.50 | 0.50 | 0.01 | 0.36 | 0.84 | 0.62 | 0.62 | 0.29 | 0.32 | 0.94 |
| Neutrophils | % | 41.0 | 38.1 | 39.8 | 37.2 | 2.82 | 0.68 | 0.30 | 0.09 | 0.94 | 0.58 | 0.86 | 0.89 |
| RBC | 106/µL | 8.43 | 8.59 | 8.57 | 8.48 | 0.32 | 0.95 | 0.89 | <0.01 | 0.67 | 0.32 | 0.26 | 0.49 |
| HGB | g/dL | 6.04 | 6.21 | 6.22 | 6.16 | 0.24 | 0.78 | 0.82 | <0.01 | 0.63 | 0.58 | 0.59 | 0.70 |
| HCT | g/dL | 27.7 | 28.1 | 27.2 | 27.0 | 1.06 | 0.39 | 0.94 | <0.01 | 0.76 | 0.36 | 0.41 | 0.48 |
| MCV | fL | 33.0 | 32.6 | 31.8 | 31.7 | 0.41 | 0.01 | 0.66 | 0.32 | 0.70 | 0.61 | 0.78 | 0.49 |
| MCH | fmol | 0.72 | 0.72 | 0.72 | 0.73 | 0.01 | 0.38 | 0.66 | 0.03 | 0.79 | 0.44 | 0.02 | 0.14 |
| MCHC | g/dL | 21.8 | 22.1 | 22.8 | 22.8 | 0.22 | 0.00 | 0.58 | 0.02 | 0.36 | 0.80 | 0.15 | 0.66 |
| RDW | fL | 28.6 | 27.9 | 27.7 | 27.4 | 0.40 | 0.06 | 0.19 | 0.17 | 0.57 | 0.53 | 0.36 | 0.63 |
| RDW | % | 21.4 | 21.1 | 21.5 | 21.4 | 0.31 | 0.43 | 0.49 | 0.08 | 0.81 | 0.53 | 0.19 | 0.30 |
| PLT | 103/dL | 151 | 102 | 115 | 190 | 42.3 | 0.51 | 0.74 | 0.10 | 0.12 | 0.51 | 0.08 | 0.49 |
| PCT | % | 0.09 | 0.06 | 0.06 | 0.11 | 0.02 | 0.61 | 0.68 | 0.09 | 0.08 | 0.29 | 0.07 | 0.31 |
| MPV | fL | 6.16 | 5.91 | 5.72 | 5.88 | 0.16 | 0.12 | 0.76 | 0.07 | 0.17 | 0.40 | 0.21 | 0.95 |
| PDW | fL | 7.91 a | 7.20 ab | 6.31 b | 7.19 ab | 0.35 | 0.02 | 0.79 | 0.20 | 0.02 | 0.08 | 0.91 | 0.97 |
| PDW | % | 30.5 a | 28.9 ab | 27.5 b | 29.4 ab | 0.71 | 0.06 | 0.85 | 0.18 | 0.01 | 0.08 | 0.69 | 0.97 |
| PMN phagoc | % | 61.3 | 55.3 | 53.1 | 58.6 | 5.38 | 0.63 | 0.96 | 0.04 | 0.27 | 0.60 | 0.49 | 0.84 |
| Monoc phagoc | % | 19.4 | 14.9 | 13.0 | 18.1 | 3.21 | 0.62 | 0.92 | 0.58 | 0.14 | 0.72 | 0.99 | 0.98 |
| Migration | RFU | 92,960 | 80,502 | 66,035 | 68,464 | 8820 | 0.02 | 0.53 | <0.01 | 0.36 | 0.06 | 0.58 | 0.82 |
| Migration/PMN | 2657 | 2281 | 1572 | 1809 | 304 | <0.01 | 0.80 | <0.01 | 0.27 | <0.01 | 0.34 | 0.34 | |
1 RBC = red blood cells; HGB = hemoglobin; HCT = hematocrit; MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = MCH concentration; RDW = red cell distribution width; PLT = platelet; PCT = plateletcrit; MPV = mean platelet volume; PDW = platelet distribution width. 2 Overall statistical effect of: F = silage type; Se = Se supplementation; T = time; and their interactions. Letters are reported to identify a statistical difference (p < 0.05) between groups when a Forage × Se (F*Se) interaction is significant (p ≤ 0.05). 3 Higher SEM.
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MDPI and ACS Style
Ford, H.R.; Klopfenstein, J.; Ates, S.; Busato, S.; Trevisi, E.; Bionaz, M. Feeding Chicory–Plantain Silage and/or Se Yeast Does Not Improve Streptococcus uberis-Induced Subclinical Mastitis in Lactating Sheep. Dairy 2025, 6, 40. https://doi.org/10.3390/dairy6040040
AMA Style
Ford HR, Klopfenstein J, Ates S, Busato S, Trevisi E, Bionaz M. Feeding Chicory–Plantain Silage and/or Se Yeast Does Not Improve Streptococcus uberis-Induced Subclinical Mastitis in Lactating Sheep. Dairy. 2025; 6(4):40. https://doi.org/10.3390/dairy6040040
Chicago/Turabian StyleFord, Hunter R., Joseph Klopfenstein, Serkan Ates, Sebastiano Busato, Erminio Trevisi, and Massimo Bionaz. 2025. "Feeding Chicory–Plantain Silage and/or Se Yeast Does Not Improve Streptococcus uberis-Induced Subclinical Mastitis in Lactating Sheep" Dairy 6, no. 4: 40. https://doi.org/10.3390/dairy6040040
APA StyleFord, H. R., Klopfenstein, J., Ates, S., Busato, S., Trevisi, E., & Bionaz, M. (2025). Feeding Chicory–Plantain Silage and/or Se Yeast Does Not Improve Streptococcus uberis-Induced Subclinical Mastitis in Lactating Sheep. Dairy, 6(4), 40. https://doi.org/10.3390/dairy6040040
