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Effects of increasing dietary zinc sulfate fed to gestating ewes: II. Milk somatic cell count, microbial populations, and fatty acid composition

      ABSTRACT

      Objective

      The objective of the research was to evaluate the effects of increasing dietary Zn sulfate concentration for primiparous gestating ewes on subsequent milk SCC, intramammary microbial identifications, and fatty acid composition.

      Materials and Methods

      Commercial white-face (WF; n = 27) and black-face (BF; n = 24) ewes (age ≈18 mo; BW = 87.48 ± 8.37 kg) were sorted into breed-type groups and within groups ranked by BW, and then, they were randomly divided into 3 dietary supplement treatment groups: CON (n = 13; 40 mg/kg Zn; ≈1 × NASEM recommendations), Zn500 (n = 21; 500 mg/kg Zn; ≈4 × NASEM recommendations), and Zn1000 (n = 17; 1,000 mg/kg Zn; ≈7 × NASEM recommendations). Treatments were administered in Zn-fortified pelleted alfalfa (0.45 kg/ewe per day) and fed from 87.5 ± 8.9 d of gestation until parturition. Milk traits collected at parturition (d 1 of lactation), ≈30 d of lactation, and lamb weaning (≈90 d of lactation) were assessed as repeated measures with fixed effects of treatment, breed type, and litter size.

      Results and Discussion

      The treatment × breed type interaction affected ewe logSCC (P = 0.01), and within Zn500, BF had greater logSCC than WF ewes (5.90 ± 0.08 vs. 5.46 ± 0.08; P < 0.01). However, breed types did not differ between CON and Zn1000 treatments (P ≥ 0.92). Ewe logSCC was greatest (P < 0.01) at weaning (6.03 ± 0.06), intermediate at parturition (5.72 ± 0.06; d 1), and least at d 30 of lactation (5.21 ± 0.06). Intramammary infections were common in milk samples collected at parturition (77%) and weaning (47%) based on culture-based microbial identifications. The most frequently identified species included Bacillus spp. and Staphylococcus spp. Black-face ewes had greater concentrations of C16:1 (1.78 mg/100 vs. 1.39 mg/100 mg of fatty acid per 100 mg of total fatty acids), C17:1 (0.86 mg/100 vs. 0.76 mg/100 mg of fatty acid per 100 mg of total fatty acids), and C20:4 (0.28 mg/100 vs. 0.24 mg/100 mg of fatty acid per 100 mg of total fatty acids; P ≤ 0.04) than WF ewes.

      Implications and Applications

      Including Zn in diets beyond NASEM recommendations from mid to late gestation had no effect on ewe milk SCC, microbial pathogens identified, or fatty acid composition. However, findings indicated there may be important breed differences in dietary Zn utilization and requirements affecting intramammary inflammation.

      Key words

      INTRODUCTION

      In extensively and semi-extensively managed sheep populations in the western United States, forage Zn concentrations are lowest during the periods that coincide with breeding, gestation, and early lactation (
      • Julian A.A.M.
      • Scasta J.D.
      • Stam B.R.
      • Sebade B.M.
      • Page C.M.
      • Springer B.E.
      • Renner W.T.
      • Cunningham-Hollinger H.
      • Stewart W.C.
      Mineral element concentrations of common grass and shrub species on sheep winter range in Wyoming: Insights for mineral supplementation strategies..
      ). Furthermore, it is estimated that 30 to 50% of extensively managed sheep operations in the western United States fail to consistently provide supplemental mineral (
      • Page C.M.
      • Murphy T.W.
      • Van Emon M.L.
      • Bowman J.G.P.
      • Wyffels S.A.
      • Stewart W.C.
      Blood serum mineral element concentrations of weaned Montana ram lambs and their relationship with water quality characteristics..
      ;
      • Stewart W.C.
      • Murphy T.W.
      • Page C.M.
      • Rule D.C.
      • Taylor J.B.
      • Austin K.
      • Pankey C.
      Effect of increasing dietary zinc sulfate fed to primiparous ewes: I. Effects on serum metabolites, mineral transfer efficiency, and animal performance..
      ). The companion research to the present found that feeding Zn to gestating primiparous ewes above

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2007. Nutrient Requirement of Small Ruminants. 7th ed. The National Academies Press.

      recommendations did not improve indices of lamb performance but increased wool production and exerted differential effects in serum mineral element concentrations and progeny’s neonatal concentrations between fine-wool and meat-type ewes (
      • Stewart W.C.
      • Murphy T.W.
      • Page C.M.
      • Rule D.C.
      • Taylor J.B.
      • Austin K.
      • Pankey C.
      Effect of increasing dietary zinc sulfate fed to primiparous ewes: I. Effects on serum metabolites, mineral transfer efficiency, and animal performance..
      ). Zinc’s essential role in proper immune system function, and specifically in indices of mammary health, has been observed in dairy cattle when included at recommended dietary levels (
      • Whitaker D.A.
      • Eayres H.F.
      • Aitchison K.
      • Kelly J.M.
      No effect of a dietary zinc proteinate on clinical mastitis, infection rate, recovery rate and somatic cell count in dairy cows..
      ;
      • Cope C.M.
      • Mackenzie A.M.
      • Wilde D.
      • Sinclair L.A.
      Effects of level and form of dietary zinc on dairy cow performance and health..
      ). Additionally, Zn has been shown to influence de novo fatty acid synthesis in dairy cattle (
      • Wiking L.
      • Larsen T.
      • Sehested J.
      Transfer of dietary zinc and fat to milk—Evaluation of milk fat quality, milk fat precursors, and mastitis indicators..
      ) and alter lipid membrane integrity by increasing unsaturated and longer chain fatty acid concentrations (
      • Hermansen J.E.
      • Larsen T.
      • Andersen J.O.
      Does zinc play a role in the resistance of milk to spontaneous lipolysis?.
      ). Still, the effect of Zn supplementation above recommended

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2007. Nutrient Requirement of Small Ruminants. 7th ed. The National Academies Press.

      levels on ewe mammary health and milk composition in extensive and semi-extensive sheep production is not well understood. Therefore, the objective of the current research was to determine the effects of increasing Zn sulfate (ZnSO4) concentrations from mid to late gestation on subsequent milk SCC, mastitis microbial pathogens, and fatty acid composition.

      MATERIALS AND METHODS

      The experiment was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Wyoming (#A-3216-01).

      Study Design and Treatment Diet Administration

      The experimental design was described in detail by
      • Stewart W.C.
      • Murphy T.W.
      • Page C.M.
      • Rule D.C.
      • Taylor J.B.
      • Austin K.
      • Pankey C.
      Effect of increasing dietary zinc sulfate fed to primiparous ewes: I. Effects on serum metabolites, mineral transfer efficiency, and animal performance..
      . Briefly, a total of 78 ewes (age ≈18 mo; BW 87.48 ± 8.37 kg) of Rambouillet and Hampshire × Suffolk background [white face (WF) and black face (BF), respectively] were used in the experiment. Ewes were sorted into breed type groups and, within groups, ranked by BW and randomly divided into 3 dietary supplement treatment groups along the rank: CON (n = 27, 40 mg/kg Zn, ≈1 × NASEM recommendations), Zn500 (n = 29, 500 mg/kg Zn, ≈4 × NASEM recommendations), and Zn1000 (n = 24, 1,000 mg/kg Zn, ≈7 × NASEM recommendations). Each ewe received 0.45 kg/d of their respective Zn-fortified alfalfa pellet, which was dispensed from 2 mobile feed apparatuses (Super SmartFeeder; C-Lock Inc.) each with 4 feed pans. Therefore, ewes were managed in one contemporary group but could only gain access to their treatment group’s feed pans, and ewe was the experimental unit. Ewes were acclimated to the Super SmartFeeder for 10 d in a 100 × 20 m enclosure with the CON alfalfa pellet. Total consumption of each treatment throughout the trial was used to determine mean daily intake of Zn and was as follows: CON = 104 mg/d; Zn500 = 462 mg/d; Zn1000 = 772 mg/d. Thus, according to

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2007. Nutrient Requirement of Small Ruminants. 7th ed. The National Academies Press.

      factorial estimated daily Zn recommendations, diets met 100, 400, and 700% for CON, Zn500, and Zn1000 treatment groups, respectively.

      Animal Management

      Ewes were exposed to rams of their breed type in multiple-sire mating groups for 51 d, started the trial at 87.5 ± 8.85 d of gestation, and remained on their treatment until parturition. This corresponded to an average trial period of 61.3 ± 8.62 d. At the beginning of the trial (d 0–42), ewes were managed on 19.2-ha hay meadow pasture regrowth with meadow brome (Bromus riparius), timothy (Phleum pretense), and creeping foxtail (Alopecurus arundinaceus) as predominant grass species. Site selection emphasized a forage source deficient in Zn (17 mg/kg Zn; DM basis; Table 1). Ewes were allowed to graze until d 42 of the trial (132.7 ± 8.62 d gestation) and then moved to a 40 × 40 m housed facility where they were offered ad libitum access to meadow hay (21 mg/kg Zn; Table 1). All ewes were subject to the same management after parturition.
      Table 1Chemical composition of zinc-fortified pelleted diets, meadow hay regrowth, and meadow hay
      ItemTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Basal dietary component
      Ewes were managed on meadow hay regrowth (d 0–42) with meadow brome (Bromus riparius), timothy (Phleum pretense), and creeping foxtail (Alopecurus arundinaceus) as the predominant grass species. From d 42 (≈d 133 of gestation) to parturition ewes were managed in a 40 × 40 m housed facility where they were offered ab libitum access to meadow hay.
      CONZn500Zn1000Meadow

      hay regrowth
      Meadow hay
      Nutrient composition
       DM, %92.993.293.437.586.6
       CP, %20.420.920.26.811.2
       NDF, %38.537.238.670.754.2
       ADF, %31.930.534.046.533.4
      Mineral composition
       Ca, %1.241.291.210.820.59
       P, %0.280.310.280.130.23
       K, %3.033.133.140.820.59
       S, %0.320.370.380.140.22
       Mg, %0.330.340.310.180.21
       Na, %0.110.120.120.070.05
       Fe, mg/kg375.0396.0330.0469.0173.0
       Mn, mg/kg79.0109.081.091.053.0
       Cu, mg/kg14.69.012.42.98.0
       Zn, mg/kg32.4551.5998.319.324.5
       Mo, mg/kg3.263.543.301.591.23
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 Ewes were managed on meadow hay regrowth (d 0–42) with meadow brome (Bromus riparius), timothy (Phleum pretense), and creeping foxtail (Alopecurus arundinaceus) as the predominant grass species. From d 42 (≈d 133 of gestation) to parturition ewes were managed in a 40 × 40 m housed facility where they were offered ab libitum access to meadow hay.

      Data Collection

      Milk Collection and SCC.

      Colostrum was collected between 12 and 18 h after parturition (d 1), and milk was collected at ≈30 d (mid-lactation) and ≈90 d (lamb weaning) after parturition. Before each sampling, teats were scrubbed using a 70% ethanol wipe, and ewes were manually milked with a gloved hand. One sample of approximately 20 mL of milk was collected from each udder half and combined into a single 50-mL conical tube and used for SCC analysis. An additional subsample was collected and frozen at −20°C until further analyzation for microbial identification or fatty acid composition. Samples for SCC testing were preserved with 8 mg of Bronopol and 0.3 mg of Natamycin (Microtabs II; D & F Control Systems Inc.) and put on ice and shipped to a participating laboratory (Montana State University) for analysis within 3 d of collection. Somatic cell count was quantified on a LactiCyte HD (Page & Pedersen International Ltd.) according to the manufacturer’s protocol.

      Microbial Analysis.

      Ewe colostrum and milk samples were analyzed for microbial identification. The thawed sample (1 mL) was pipetted into 1.5-mL microcentrifuge tubes and centrifuged at 5,000 × g for 5 min at room temperature to separate fat, supernatant liquid, and milk pellet. Fat and the majority of the supernatant were discarded, and the pellet was resuspended in the remaining supernatant (≈0.1 mL) via vortexing. Resuspended pellets were streaked onto compartmentalized plates (X-plate, Thermo Fischer Scientific Inc.) containing 1 each of 4 microbiological growth media: MacConkey agar (HiMedia Laboratories Pvt. Ltd.); Trypticase soy agar (TSA; Becton, Dickinson and Company); TSA + 5% sheep blood agar (Hardy Diagnostics); and Sabouraud dextrose agar (Neogen Corporation) using a 10-μL inoculating loop. These media were chosen to select for the following targets: MacConkey agar suppresses the growth of Gram-positive bacteria, thereby favoring growth of Gram-negative and enteric bacilli; TSA is a general-purpose, nonselective medium for recovery of most bacteria present; TSA + 5% sheep blood media contains added nutrients and growth factors to improve recovery of fastidious bacteria in comparison to the base medium TSA; and Sabouraud dextrose agar is used to support the growth of fungi. Plates were incubated aerobically at 37°C for 18 to 24 h. The number of colonies and colony morphologies were recorded following the standard incubation period; however, plates that did not display microbial growth were reincubated for an additional 24 h to ensure that growth of slow growing and fastidious bacteria was captured by the procedure (
      • Dohoo I.R.
      • Smith J.
      • Andersen S.
      • Kelton D.F.
      • Godden S.
      Diagnosing intramammary infections: Evaluation of definitions based on a single milk sample..
      ). Culture-positive plates were considered indicative of an intramammary infection.
      Confirmation of all isolates was carried out via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The direct colony transfer method was applied, which involves transfer of purified colonies in triplicate with a sterile 1-μL inoculating loop onto a 48-well steel-target plate for microbial identification. Subsequently, 1 μl of Matrix A CHCA (α-cyano-4-hycroxycinnamic acid) was added to the center of each well before identification was completed by using an Agena MassARRAY instrument (Agena Bioscience) and a spectrum match factor library. If none of the replicates yielded a successful match, samples were reanalyzed in triplicate.
      Multiplex-PCR was used in parallel as a culture-independent method for microbial identification. Frozen milk samples containing sufficient volume for DNA extraction following culturing methods were subjected to microbial identification using a Thermo Scientific PathoProof Complete-16 kit with the Primer Mix 4 for Applied Biosystems 7500 and 7500 Fast instruments. This test kit identifies 15 possible taxa commonly isolated from cases of bovine mastitis, including Corynebacterium bovis, Enterococcus spp., Escherichia coli, Klebsiella oxytoca and Klebsiellapneumoniae, Mycoplasma bovis, Mycoplasma spp., Prototheca spp., Serratia marcescens, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella pyogenes and Peptoniphilus indolicus, and many common yeasts. The kit also screened for the β-lactamase penicillin resistance gene in staphylococci, including in Staph. aureus and all major coagulase-negative staphylococci. Four PCR reactions were run for these milk samples (Eurofins DQCI LLC.).

      Colostrum Fatty Acid Composition.

      Colostrum fatty acid composition was determined by GLC. Frozen colostrum samples were thawed, weighed, and freeze-dried (Labconco Freeze Dryer 5, model 75070). For each analysis, 1 mL of a 15 mg/mL internal standard solution of 13:0 (glyceryl-tritridecanoate, Sigma Chemical Co.) in chloroform was dried under nitrogen, and a 100-mg sample of freeze-dried colostrum was added. Fatty acid methyl esters were prepared using direct transesterification with an alkaline catalyst as described by
      • Murrieta C.M.
      • Hess B.W.
      • Rule D.C.
      Comparison of acidic and alkaline catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis on conjugated linoleic acid..
      . Ewe colostrum conjugated linoleic acid content was quantified by gas chromatography (GC), and all samples were analyzed in duplicate according to methods described by
      • Moutsioulis A.A.
      • Rule D.C.
      • Murrieta C.M.
      • Bauman D.E.
      • Lock A.L.
      • Barbano D.M.
      • Carey G.B.
      Human breast milk enrichment in conjugated linoleic acid after consumption of a conjugated linoleic acid-rich food product: A pilot study..
      . Fatty acid methyl esters were separated using an Agilent 6890 GC (Agilent Technologies Inc.) equipped with a 100 mm × 0.25 mm fused silica capillary column (SP-2560, 0.2 µm film thickness, Supelco) and flame ionization detector. Fatty acid methyl ester peaks were identified by comparing retention times with fatty acid methyl esters standards (Nu-Check Prep Inc. and Matreya Inc.). Fatty acid methyl esters were evaluated using ChemStation software (Agilent Technologies, Inc.).

      Statistical Analyses

      Colostrum fatty acid concentrations (n = 42) were analyzed in the MIXED procedure of SAS (v9.4; SAS Institute Inc.) with fixed effects of treatment (CON, Zn500, or Zn1000), breed type (BF or WF), litter size (1 or 2+), and all 2-way interactions. Milk SCC were log10 transformed (logSCC) and then analyzed as repeated measures with all fixed effects described above in addition to day of lactation (d 1, ≈30, or ≈90) and all 2-way interactions. A compound symmetric covariance structure was determined to be the most parsimonious by Akaike’s Information Criterion.
      Binomial proportions and associated 95% confidence intervals of microbial species were analyzed within treatment and stage of lactation using the binom package of R (

      Dorai-Raj, S. 2014. binom: Binomial Confidence Intervals for Several Parameterizations. Accessed Jun. 18, 2021. https://CRAN.R-project.org/package=binom.

      ;

      RStudio Team. 2021. RStudio: Integrated Development Environment for R. RStudio PBC. http://www.rstudio.com/.

      ). Culture- and PCR-status for each microbial species were modeled as binary variables (0 = negative, 1 = positive) in the GLIMMIX procedure and analyzed with fixed effects of treatment, breed type, and day of lactation (d ≈30 or ≈90) and the random effect of ewe. To ensure model convergence, 2-way interaction terms were removed if no statistical tendency was detected (P > 0.10).

      RESULTS

      Ten ewes were removed from the analyses because >15% of their cumulative supplement intake came from another treatment (CON = 6; Zn500 = 3; Zn1000 = 3) and 17 ewes were removed because they were not pregnant (CON = 8; Zn500 = 5; Zn1000 = 4). In total, data from 13, 21, and 17 CON, Zn500, and Zn1000 ewes remained for analyses, respectively.

      SCC

      There was a treatment × breed type interaction for logSCC (P = 0.01) where BF had greater logSCC than WF within Zn500 (5.90 ± 0.08 vs. 5.46 ± 0.08; P < 0.01), but breed types did not differ within CON and Zn1000 treatments (P ≥ 0.92; Figure 1). Least squares means for the main effects of ewe liter size and lactation period on logSCC are presented in Table 2. Ewe logSCC was greatest at weaning (6.03 ± 0.06), intermediate shortly after parturition (5.72 ± 0.06), and least at mid-lactation (5.21 ± 0.06; P ≤ 0.01). There was no difference in logSCC between ewes bearing singles or multiples (P = 0.57).
      Figure 1
      Figure 1Least squares means for the interactive effect of treatment and breed type on log10 transformed SCC (LogSCC). CON = 104 mg of Zn/d; Zn500 = 462 mg of Zn/d; Zn1000 = 772 mg of Zn/d; Black Face = purebred and crossbred black-face breeds; White Face = purebred and crossbred white-face breeds. a,bMeans within a treatment with no common letter are different (P < 0.01). The error bars indicate SE.
      Table 2Least squares means (± SE) for the main effects of ewe treatment, litter size, and lactation period on log10 transformed SCC (logSCC; n = 51)
      EffectLevel
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d; BF = purebred and crossbred black-face breeds, WF = purebred and crossbred white-face breeds; 1 = single bearing, 2+ = multiple bearing.
      logSCC
      TreatmentCON5.65 ± 0.07
      Zn5005.68 ± 0.05
      Zn10005.62 ± 0.06
      Breed typeBF5.74 ± 0.05
      WF5.57 ± 0.05
      No. of lambs born15.67 ± 0.07
      2+5.63 ± 0.04
      Lactation period, dEarly (d 1)5.72 ± 0.06b
      Mid-lactation (d 30)5.21 ± 0.06c
      Weaning (d ≈90)6.03 ± 0.06a
      a–cMeans within a column and effect with no common superscript are different (P < 0.01).
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d; BF = purebred and crossbred black-face breeds, WF = purebred and crossbred white-face breeds; 1 = single bearing, 2+ = multiple bearing.

      Culture and PCR Microbial Results

      Binomial proportions of microbial taxa identified via culture-based isolation and MALDI-TOF MS confirmation occurring at an overall proportion ≥5% within and across treatment group are presented in Tables 3 and 4. During early lactation, 77% (n = 23/30) of milk samples were positive by culture, and the most commonly identified bacteria were Bacillus altitudinis (35%), Bacillus amyloliquefaciens (26%), Bacillus licheniformis (30%), Bacillus subtilis (9%), Staphylococcus auricularis (17%), Staphylococcus epidermidis (13%), and Staphylococcus lugdunensis (9%). At weaning, fewer milk samples were culture positive (47%, n = 14/30), but a greater number of species were confirmed by MALDI-TOF MS at frequencies ≥5%. These microorganisms included Achromobacter xylosoxidans (7%), B. altitudinis (43%), B. amyloliquefaciens (14%), B. licheniformis (29%), Bacillus pumilus (7%), Cryptococcus neoformans (a fungus, 7%), Clostridium tertium (7%), Enterobacter pulveris (7%), Legionella pneumophila (7%), Pantoea ananatis (7%), Psychrobacter immobilis (14%), Staph. aureus (7%), Staph. auricularis (14%) and Staph. epidermidis (7%; Table 4). The main effects of ewe breed type and treatment were not significant in the analysis of culture-status (P > 0.51). However, culture positivity was greater in early lactation than at weaning (77 vs. 47%, respectively; P = 0.02).
      Table 3Number of samples collected in early lactation (18–24 h postpartum) within and across treatment and estimated species frequency (95% CI in brackets) within culture-positive samples determined by culture or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods
      ItemTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Overall
      CONZn500Zn1000
      No. of samples1091130
      No. positive (%)7 (70)7 (77)9 (82)23 (77)
      Species
      Only species that occurred at an overall frequency ≥0.05 are presented.
      Unidentified
      Includes samples that were culture positive but not identified by MALDI-TOF MS.
      0.22 [0.03, 0.60]0.09 [0.01, 0.28]
      Bacillus altitudinis0.14 [ 0.00, 0.58]0.57 [0.18, 0.90]0.33 [0.07, 0.70]0.35 [0.16, 0.57]
      Bacillus amyloliquefaciens0.57 [0.18, 0.90]0.29 [0.04, 0.71]0.26 [0.10, 0.48]
      Bacillus licheniformis0.43 [0.10, 0.82]0.14 [0.00, 0.58]0.33 [0.07, 0.70]0.30 [0.13, 0.53]
      Bacillus subtilis0.29 [0.04, 0.71]0.09 [0.01, 0.28]
      Staphylococcus auricularis0.43 [0.10, 0.82]0.11 [0.00, 0.48]0.17 [0.05, 0.39]
      Staphylococcus epidermidis0.14 [ 0.00, 0.58]0.22 [0.03, 0.60]0.13 [0.03, 0.34]
      Staphylococcus lugdunensis0.14 [0.00, 0.58]0.11 [0.00, 0.48]0.09 [0.01, 0.28]
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 Only species that occurred at an overall frequency ≥0.05 are presented.
      3 Includes samples that were culture positive but not identified by MALDI-TOF MS.
      Table 4Number of samples collected at weaning (≈90 d postpartum) within and across treatment and estimated species frequency (95% CI in brackets) within culture-positive samples determined by culture or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods
      ItemTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Overall
      CONZn500Zn1000
      No. of samples1091130
      No. positive (%)4 (40)3 (33)7 (64)14 (47)
      Species
      Only species that occurred at an overall frequency ≥0.05 are presented.
      Unidentified
      Includes samples that were culture positive but not identified by MALDI-TOF MS.
      0.29 [ 0.04, 0.71]0.14 [0.02, 0.43]
      Achromobacter xylosoxidans0.33 [0.01, 0.91]0.07 [0.00, 0.34]
      Bacillus altitudinis0.25 [0.01, 0.81]0.33 [0.01, 0.91]0.57 [0.18, 0.90]0.43 [0.18, 0.71]
      Bacillus amyloliquefaciens0.33 [0.01, 0.91]0.14 [0.00, 0.58]0.14 [0.02, 0.43]
      Bacillus licheniformis0.50 [0.07, 0.93]0.33 [0.01, 0.91]0.14 [0.00, 0.58]0.29 [0.08, 0.58]
      Bacillus pumilus0.14 [0.00, 0.58]0.07 [0.00, 0.34]
      Cryptococcus neoformans0.25 [0.01, 0.81]0.07 [0.00, 0.34]
      Clostridium tertium0.14 [0.00, 0.58]0.07 [0.00, 0.34]
      Enterobacter pulveris0.25 [0.01, 0.81]0.07 [0.00, 0.34]
      Legionella pneumophila0.14 [0.00, 0.58]0.07 [0.00, 0.34]
      Pantoea ananatis0.25 [0.01, 0.81]0.07 [0.00, 0.34]
      Psychrobacter immobilis0.25 [0.01, 0.81]0.33 [0.01, 0.91]0.14 [0.02, 0.43]
      Staphylococcus aureus0.14 [0.00, 0.58]0.07 [0.00, 0.34]
      Staphylococcus auricularis0.25 [0.01, 0.81]0.14 [0.00, 0.58]0.14 [0.02, 0.43]
      Staphylococcus epidermidis0.25 [0.01, 0.81]0.07 [0.00, 0.34]
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 Only species that occurred at an overall frequency ≥0.05 are presented.
      3 Includes samples that were culture positive but not identified by MALDI-TOF MS.
      Overall, sample positivity determined via PCR was considerably lower than observed for culture and MALDI-TOF MS. A total of 5 and 3 of the 16 microorganisms included in the panel were detected in early lactation (Table 5) and at weaning (Table 6), respectively. During early lactation, 17% (n = 5/30) of the milk samples contained at least one of the panel targets, including T. pyogenes or P. indolicus (40%), Staph. aureus (20%), Staphylococcus spp. (60%), yeasts (20%), and the β-lactamase penicillin resistance gene in staphylococci (20%). At weaning, only 2 (7%) of the milk samples were PCR positive and included Staph. aureus (50%), Staphylococcus spp. (100%), and the β-lactamase penicillin resistance gene in staphylococci (50%). Ewe breed type, treatment, and day of lactation did not affect PCR status (P > 0.19).
      Table 5Number of samples collected in early lactation (18–24 h postpartum) within and across treatment and estimated species frequency (95% CI in brackets) determined by PCR methods
      ItemTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Overall
      CONZn500Zn1000
      No. of samples1091130
      No. positive (%)1 (10)1 (11)3 (27)5 (17)
      Test screen item
      All identifications are presented.
      Trueperella pyogenes or

      Peptoniphilus indolicus
      1.00 [0.03, 1.00]0.33 [0.01, 0.91]0.40 [0.05, 0.85]
      β-Lactamase gene0.33 [0.01, 0.91]0.20 [0.01, 0.72]
      Staphylococcus aureus1.00 [0.03, 1.00]0.20 [0.01, 0.72]
      Staphylococcus spp.1.00 [0.29, 1.00]0.60 [0.15, 0.95]
      Yeasts0.33 [0.01, 0.91]0.20 [0.01, 0.72]
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 All identifications are presented.
      Table 6Number of samples collected at weaning (≈90 d postpartum) within and across and treatment and estimated species frequency (95% CI in brackets) determined by PCR methods
      ItemTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Overall
      CONZn500Zn1000
      No. of samples1091130
      No. positive (%)1 (10)0 (0)1 (9)2 (7)
      Test screen item
      All identifications are presented.
      β-Lactamase gene1.00 [0.03, 1.00]0.50 [0.01, 0.99]
      Staphylococcus aureus1.00 [0.03, 1.00]0.50 [0.01, 0.99]
      Staphylococcus spp.1.00 [0.03, 1.00]1.00 [0.03, 1.00]1.00 [0.16, 1.00]
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 All identifications are presented.

      Colostrum Fatty Acid Composition

      Least squares means for model main effects on colostrum fatty acid concentrations are displayed in Table 7. There was no effect of treatment on any colostrum fatty acid concentration (P ≥ 0.21). However, BF ewes had greater concentrations of palmitoleic acid (C16:1; 1.78 vs. 1.39 mg of fatty acid/100 mg of total fatty acids), heptadecenoic acid (C17:1; 0.86 vs. 0.76 mg of fatty acid/100 mg of total fatty acids), and arachidonic acid (C20:4; 0.28 vs. 0.24 mg of fatty acid/100 mg of total fatty acids; P ≤ 0.04) compared with WF ewes.
      Table 7Least squares means (± SE) for the main effects of treatment and ewe breed type on milk fatty acid concentration (mg/100 mg of fatty acids) at 12 to 18 h after parturition (n = 47)
      Fatty acidTreatment
      CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      Breed type
      BF = purebred and crossbred black-face breeds, WF = purebred and crossbred white-face breeds.
      CONZn500Zn1000BFWF
      C8:00.63 ± 0.070.56 ± 0.060.62 ± 0.070.63 ± 0.050.59 ± 0.05
      C10:02.42 ± 0.292.16 ± 0.242.40 ± 0.272.42 ± 0.212.26 ± 0.22
      C12:02.25 ± 0.152.08 ± 0.132.32 ± 0.142.18 ± 0.112.26 ± 0.12
      C14:09.94 ± 0.449.14 ± 0.369.76 ± 0.419.43 ± 0.339.80 ± 0.34
      C15:00.95 ± 0.080.96 ± 0.060.93 ± 0.070.97 ± 0.060.92 ± 0.06
      C16:029.40 ± 0.9627.96 ± 0.7928.19 ± 0.8928.74 ± 0.7128.29 ± 0.74
      C16:11.62 ± 0.091.51 ± 0.071.63 ± 0.081.78 ± 0.07a1.39 ± 0.07b
      C17:01.14 ± 0.041.22 ± 0.031.18 ± 0.031.18 ± 0.031.18 ± 0.03
      C17:10.80 ± 0.040.82 ± 0.030.83 ± 0.040.86 ± 0.03a0.78 ± 0.03b
      C18:06.67 ± 0.427.36 ± 0.356.88 ± 0.396.70 ± 0.317.24 ± 0.32
      C18:134.70 ± 1.5536.62 ± 1.2835.76 ± 1.4435.28 ± 1.1536.11 ± 1.19
      C18:22.76 ± 0.072.89 ± 0.052.77 ± 0.062.77 ± 0.052.85 ± 0.05
      C18:2 cis-9,trans-111.00 ± 0.040.99 ± 0.041.04 ± 0.041.00 ± 0.031.02 ± 0.03
      C18:31.23 ± 0.061.18 ± 0.051.23 ± 0.051.22 ± 0.041.22 ± 0.04
      C20:40.26 ± 0.020.25 ± 0.010.26 ± 0.020.28 ± 0.01a0.23 ± 0.01b
      a,bMeans within an effect with no common superscript are different (P < 0.01).
      1 CON = 104 mg of Zn/d, Zn500 = 462 mg of Zn/d, Zn1000 = 772 mg of Zn/d.
      2 BF = purebred and crossbred black-face breeds, WF = purebred and crossbred white-face breeds.

      DISCUSSION

      Zinc is key in maintaining skin integrity and mammary tissue through its role in cellular division and protein synthesis (

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI Publishing.

      ). Specific to mammary health, Zn plays a role in keratin synthesis in the teat canal, providing protection against bacterial infection with regeneration in between milking and nursing events and may be one mechanism by which Zn decreases mastitis susceptibility (
      • Capuco A.V.
      • Wood D.L.
      • Bright S.A.
      • Miller R.H.
      • Bitman J.
      Regeneration of teat canal keratin in lactating dairy cows..
      ;

      Spain, J. N. 1994. The role of zinc proteinates and a theory for a mode of action. Pages 125–132 in Tissue Integrity—A Key Defense Against Mastitis. T. P. Lyon, ed. Nottingham University Press.

      ;

      Suttle, N. F. 2010. Mineral Nutrition of Livestock. 4th ed. CABI Publishing.

      ). The beneficial effects of Zn on udder health have been well documented in dairy cattle (
      • Wedekind K.J.
      • Hortin A.E.
      • Baker D.H.
      Methodology for assessing zinc bioavailability: Efficacy estimates for zinc-methionine, zinc sulfate, and zinc oxide..
      ;

      Spain, J. N. 1994. The role of zinc proteinates and a theory for a mode of action. Pages 125–132 in Tissue Integrity—A Key Defense Against Mastitis. T. P. Lyon, ed. Nottingham University Press.

      ;
      • Kellogg D.W.
      • Tomlinson D.J.
      • Socha M.T.
      • Johnson A.B.
      Effects of zinc methionine complex on milk production and somatic cell count of dairy cows: Twelve-trial summary..
      ;
      • Pechová A.
      • Pavlata L.
      • Lokajová E.
      Zinc supplementation and somatic cell count in milk of dairy cows..
      ;
      • Cope C.M.
      • Mackenzie A.M.
      • Wilde D.
      • Sinclair L.A.
      Effects of level and form of dietary zinc on dairy cow performance and health..
      ). Similarly,
      • Saianda I.
      • Bettencourt C.
      • Queiroga C.
      • Ferreira-Dias G.
      • Vilela C.
      Effect of dietary zinc upon in vitro bacterial adherence to ovine mammary epithelium..
      reported a significant reduction in bacterial adherence to the mammary epithelium in dairy ewes supplemented with 30 mg/kg Zn DM compared with unsupplemented ewes. Greater research emphasis has been placed on Zn deficiency compared with feeding dietary Zn more than recommended levels (1, 4, and 7 × NASEM) such as those in the current study.
      • Cope C.M.
      • Mackenzie A.M.
      • Wilde D.
      • Sinclair L.A.
      Effects of level and form of dietary zinc on dairy cow performance and health..
      observed a decrease in dairy cow milk SCC when they were fed either an inorganic (ZnO) or organic form (chelated) of Zn at recommended dietary concentrations compared with approximately 66% of the daily recommendation. In contrast,
      • Pechová A.
      • Pavlata L.
      • Lokajová E.
      Zinc supplementation and somatic cell count in milk of dairy cows..
      fed dairy cattle ≈4 times the recommended dietary Zn concentration, and although authors observed no difference in milk Zn concentrations, they reported a reduction in SCC by 45% in mo 3 of dietary treatment during lactation. Adequacy of Zn status pre- and postpartum has been associated with a decrease in SCC in dairy cattle and sheep (
      • Davidov I.
      • Radinović M.
      • Erdelian M.
      • Cincović M.R.
      • Stančić I.
      • Belić B.
      Relations between blood zinc concentrations and udder health in dairy cows..
      ;
      • Murphy T.W.
      • Stewart W.C.
      • Taylor J.B.
      Factors affecting ewe somatic cell count and its relationship with lamb weaning weight in extensively managed flocks..
      ). Although treatments were not applied into lactation and Zn concentrations of colostrum and milk were not quantified in the current study, successful Zn fortification of the milk up to 30 d into lactation has been observed when feeding ZnSO4 to meat-type ewes ≈1 and ≈4 times NASEM recommended levels (
      • Page C.M.
      • Murphy T.W.
      • Taylor J.B.
      • Julian A.A.
      • Whaley J.R.
      • Woodruff K.L.
      • Hummel G.L.
      • Demarco C.F.
      • Laverell D.M.
      • Cunningham-Hollinger H.C.
      • Rule D.C.
      • Stewart W.C.
      Effects of dietary Zn on ewe milk minerals and somatic cell count..
      ). Zinc requirements increase ≈41% from breeding to late gestation but only ≈5% from late gestation to early lactation (

      NASEM (National Academies of Sciences, Engineering, and Medicine). 2007. Nutrient Requirement of Small Ruminants. 7th ed. The National Academies Press.

      ), which are unlikely to be met in semi-extensive sheep production systems of the western United States.
      Results from the present study found a significant treatment × breed type effect on logSCC. When fed additional dietary Zn concentrations (≈4 times NASEM), BF ewes had greater logSCC than WF ewes, an effect not observed when dietary Zn levels were fed at ≈1 and 7 times NASEM recommended concentrations. This finding points to the importance of understanding the production factors that contribute to how much dietary Zn is required (BW, gain, fetal weight, milk production, and wool) and how requirements may differ based on breed type.
      • Snowder G.D.
      • Glimp H.A.
      Influence of breed, number of suckling lambs, and stage of lactation on ewe milk production and lamb growth under range conditions..
      observed 13 to 17% greater milk production from Suffolk-type ewes compared with Rambouillet, Polypay, and Columbia ewes, which would translate to greater Zn demands when extrapolated to the lactational Zn demands in BF type ewes in the current study. In part 1 of this study (
      • Stewart W.C.
      • Murphy T.W.
      • Page C.M.
      • Rule D.C.
      • Taylor J.B.
      • Austin K.
      • Pankey C.
      Effect of increasing dietary zinc sulfate fed to primiparous ewes: I. Effects on serum metabolites, mineral transfer efficiency, and animal performance..
      ), BF and WF ewes did not differ in serum Zn concentrations at the time of lambing, but WF lambs had ≈28% greater serum Zn concentration and 60% greater calculated transfer efficiency than BF lambs. Our study observed increases in SCC and microbial positivity rates based on the day of lactation, with larger increases during early lactation in comparison with mid-lactation, and weaning had the greatest SCC. This may not be surprising as
      • Huntley S.J.
      • Cooper S.
      • Bradley A.J.
      • Green L.E.
      A cohort study of the associations between udder conformation, milk somatic cell count, and lamb weight in suckler ewes..
      and
      • Paape M.J.
      • Wiggans G.R.
      • Bannerman D.D.
      • Thomas D.L.
      • Sanders A.H.
      • Contreras A.
      • Moroni P.
      • Miller R.H.
      Monitoring goat and sheep milk somatic cell counts..
      both reported a decrease in ewe SCC in the second month of lactation, hypothesizing that increased milk production caused the decrease by dilution.
      The combination of SCC and microbiological analyses from milk in the current study provide a more holistic investigation into the effects of dietary Zn on ewe mammary health and immune response. Bacteria were commonly isolated from milk samples collected during early lactation (d 1; 77%) and weaning (≈90 d; 47%), and the most commonly identified isolates included Bacillus spp. and Staphylococcus spp., coinciding with other mastitis research in sheep (
      • Zadoks R.N.
      • Tassi R.
      • Martin E.
      • Holopainen J.
      • McCallum S.
      • Gibbons J.
      • Ballingall K.T.
      Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk-Sheep are not small cows..
      ;
      • Knuth R.M.
      • Stewart W.C.
      • Taylor J.B.
      • Bisha B.
      • Yeoman C.J.
      • Van Emon M.L.
      • Murphy T.W.
      Relationships among intramammary health, udder and teat characteristics, and productivity of extensively managed ewes..
      ;
      • Van den Crommenacker-Konings L.W.J.H.
      • Van Dam P.
      • Everts R.
      • Shittu A.
      • Nielen M.
      • Lam T.J.G.M.
      • Koop G.
      Dynamics of intramammary infections in suckler ewes during early lactation..
      ). Though bacilli are commonly present in soil, both culture-dependent (
      • Watkins G.H.
      • Burriel A.R.
      • Jones J.E.T.
      A field investigation of subclinical mastitis in sheep in southern England..
      ;
      • Al-Majali A.M.
      • Jawabreh S.
      Period prevalence and etiology of subclinical mastitis in Awassi sheep in southern Jordan..
      ;
      • Arsenault J.
      • Dubreuil P.
      • Higgins R.
      • Bélanger D.
      Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada..
      ;
      • Spanu C.
      • Berger Y.M.
      • Thomas D.L.
      • Ruegg P.L.
      Impact of intramammary antimicrobial dry treatment and teat sanitation on somatic cell count and intramammary infection in dairy ewes..
      ) and culture-independent (
      • Bhatt V.D.
      • Ahir V.B.
      • Koringa P.G.
      • Jakhesara S.J.
      • Rank D.N.
      • Nauriyal D.S.
      • Kunjadia A.P.
      • Joshi C.G.
      Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing..
      ;
      • Braem G.
      • De Vliegher S.
      • Verbist B.
      • Piessens V.
      • Van Coillie E.
      • De Vuyst L.
      • Leroy F.
      Unraveling the microbiota of teat apices of clinically healthy lactating dairy cows, with special emphasis on coagulase-negative staphylococci..
      ;
      • Bonsaglia E.C.R.
      • Gomes M.S.
      • Canisso I.F.
      • Zhou Z.
      • Lima S.F.
      • Rall V.L.M.
      • Oikonomou G.
      • Bicalho R.C.
      • Lima F.S.
      Milk microbiome and bacterial load following dry cow therapy without antibiotics in dairy cows with healthy mammary gland..
      ) techniques have identified the genus as a member of the milk and teat microbial communities. A clear link between Bacillus spp. and ovine milk has been established (
      • Fotou K.
      • Tzora A.
      • Voidarou Ch.
      • Alexopoulos A.
      • Plessas S.
      • Avgeris I.
      • Bezirtzoglou E.
      • Akrida-Demertzi K.
      • Demertzis P.G.
      Isolation of microbial pathogens of subclinical mastitis from raw sheep’s milk of Epirus (Greece) and their role in its hygiene..
      ). Staphylococcus aureus are commonly found on the skin and in the environment and have been isolated and identified as a major mastitis-causing pathogen in cases of ovine subclinical mastitis (
      • Watson D.L.
      • Franklin N.A.
      • Davies H.I.
      • Kettlewells P.
      • Frost A.J.
      Survey of intramammary infections in ewes on the New England Tableland of New South Wales..
      ;
      • Arsenault J.
      • Dubreuil P.
      • Higgins R.
      • Bélanger D.
      Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada..
      ). Coagulase-negative staphylococci such as Staph. epidermidis and Staph. lugdunensis are also common members of the skin and mucus membrane microbial communities. Furthermore, both have been previously isolated in cases of ovine subclinical mastitis and are a group of mastitis-causing pathogens more common in nondairy systems (
      • Arsenault J.
      • Dubreuil P.
      • Higgins R.
      • Bélanger D.
      Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada..
      ). Together, these studies illustrate the pathogenic-nature of bacilli and staphylococci in relationship to mammary health. This has notable repercussions, because certain bacterial species including E. coli, Staph. aureus, and streptococci typically cause clinical infections, whereas the majorityof mastitis cases present in a subclinical state with no visible signs and can be the result of infection from numerous bacteria species (i.e., Staphylococcus spp., including Staph. aureus, and CNS;
      • Rovai M.
      • Caja G.
      • Salama A.A.K.
      • Jubert A.
      • Lázaro B.
      • Lázaro M.
      • Leitner G.
      Identifying the major bacteria causing intramammary infections in individual milk samples of sheep and goats using traditional bacteria culturing and real-time polymerase chain reaction..
      ). In the present study, samples were first collected at 12 to 18 h after parturition, so there may be some remaining colostrum properties compared with milk collected at weaning. The inherent differences (e.g., fat and protein content, mineral concentrations, enzyme activity, and so on) discernable between milk and colostrum composition could contribute to the nutrient availability supporting growth of mastitis-related microorganisms, therefore partially accounting for the differences observed in subclinical mastitis prevalence at these time points. Culture-based methods were not always on concordance with the results of molecular detection. This is to be expected, because multiple microbial species can be isolated and identified using cultural isolation and mass spectrometry–based identification, but PCR is limited to detecting targets that are part of the testing panel. The ability of PCR to detect nonviable organisms (
      • Taponen S.
      • Salmikivi L.
      • Simojoki H.
      • Koskinen M.T.
      • Pyörälä S.
      Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing..
      ), and the perceived increased sensitivity compared with culture (
      • Cederlöf S.E.
      • Toft N.
      • Aalbaek B.
      • Klaas I.C.
      Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off..
      ;
      • Keane O.M.
      • Budd K.E.
      • Flynn J.
      • McCoy F.
      Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture..
      ), partially accounts for such differences. However, PCR results must be used cautiously, because interpretation may be complicated when more than one target species is present in samples or false-positive results are recorded in absence of an inflammatory response (
      • Koskinen M.T.
      • Wellenberg G.J.
      • Sampimon O.C.
      • Holopainen J.
      • Rothkamp A.
      • Salmikivi L.
      • van Haeringen W.A.
      • Lam T.J.G.M.
      • Pyörälä S.
      Field comparison of real-time polymerase chain reaction and bacterial culture for identification of bovine mastitis bacteria..
      ).
      Mild Zn deficiency during the periods of late gestation and early lactation may impair immune function as Zn is a key nutrient in both innate and adaptive immunity (
      • Ibs K.H.
      • Rink L.
      Zinc-altered immune function..
      ;
      • Zhou X.
      • Wang H.
      • Lian S.
      • Wang J.
      • Wu R.
      Effect of copper, zinc, and selenium on the formation of bovine neutrophil extracellular traps..
      ). Furthermore, Zn can exhibit antibacterial activity (
      • Etaiw S.E.H.
      • Abd El-Aziz D.M.
      • Abd El-Zaher E.H.
      • Ali E.A.
      Synthesis, spectral, antimicrobial and antitumor assessment of Schiff base derived from 2-aminobenzothiazole and its transition metal complexes..
      ) and may help maintain epithelial cell integrity (
      • Saianda I.
      • Bettencourt C.
      • Queiroga C.
      • Ferreira-Dias G.
      • Vilela C.
      Effect of dietary zinc upon in vitro bacterial adherence to ovine mammary epithelium..
      ). Together, these properties suggest that dietary Zn supplementation could serve as a tool to control etiological agents of mastitis. However, the present study fell short of supporting a link between the status of culture or mastitis status and increasing dietary Zn concentrations.
      • Starke I.C.
      • Zentek J.
      • Vahjen W.
      Ex vivo-growth response of porcine small intestinal bacterial communities to pharmacological doses of dietary zinc oxide..
      identified pronounced reductions of Enterobacteriaceae, E. coli, and Lactobacillus in the intestines of weaned piglets supplemented with zinc oxide, though duration of the reduction was dependent on the bacterium. Because Zn has antibacterial properties, researchers have investigated the clinical mastitis resistome and have revealed Zn-resistant Shigella and Enterobacter spp. (
      • Hoque M.N.
      • Istiaq A.
      • Clement R.A.
      • Sultana M.
      • Crandall K.A.
      • Siddiki A.Z.
      • Hossain M.A.
      Metagenomic deep sequencing reveals association of microbiome signature with functional biases in bovine mastitis..
      ). This raises concern that even if dietary zinc supplementation could reduce the prevalence and effects of subclinical mastitis, Zn-resistant bacterial populations may dampen the benefits of supranutritional Zn supplementation. Despite whether the beneficial effects of Zn reside in its nutraceutical or antibacterial properties, indices of mammary health were unaltered in the current study.
      Others have evaluated effects of differing dietary Zn concentrations on milk fatty acid production in dairy cattle.
      • Wiking L.
      • Larsen T.
      • Sehested J.
      Transfer of dietary zinc and fat to milk—Evaluation of milk fat quality, milk fat precursors, and mastitis indicators..
      reported that milk production and fatty acid composition were unaffected by dietary Zn treatment (39 vs. 83 mg of Zn/kg of DM).
      • Hermansen J.E.
      • Larsen T.
      • Andersen J.O.
      Does zinc play a role in the resistance of milk to spontaneous lipolysis?.
      also observed no change in free fatty acids (FFA) between dietary Zn concentrations immediately after milking, although Zn treatment (90 mg of Zn/kg of DM) decreased FFA compared with control (34 mg of Zn/kg of DM) cows after 72 h of chilling. In the present study there was no Zn treatment effect on milk fatty acid concentrations. However, interesting breed effects were observed as BF ewes had greater concentrations of palmitoleic, heptadecenoic, and arachidonic acids than WF ewes. White-face ewes had numerically greater amounts of linoleic acid, the precursor for arachidonic acid, which was 22% greater in BF ewes. Breed differences in de novo fatty acid synthesis could explain this difference with BF ewes converting a greater percentage of linoleic acid to arachidonic acid. Although few studies have focused on milk FFA differences between breeds,
      • Signorelli F.
      • Contarini G.
      • Annicchiarico G.
      • Napolitano F.
      • Orru L.
      • Catillo G.
      • Haenlein G.F.W.
      • Moioli B.
      Breed differences in sheep milk fatty acid profiles: Opportunities for sustainable use of animal genetic resources..
      reported differences in milk FFA composition including heptadecenoic acid (C16:1) between 3 native Italian sheep breeds. These breed differences in FFA milk composition may also contribute modestly to preweaning growth differences of BF versus WF lambs. Overall fatty acid composition in the current study is similar to concentrations reported by
      • Casoli C.
      • Duranti E.
      • Morbidini L.
      • Panella F.
      • Vizioli V.
      Quantitative and compositional variations of Massese sheep milk by parity and stage of lactation..
      with palmitic, oleic, stearic, and myristic acids making up the highest percentages in the fatty acid profile of Massese ewes. In the current study, colostrum fatty acid composition was only measured at lambing. However, future research may look at the effects of Zn fed throughout lactation on temporal changes in FFA composition, as others have reported changes in ewe milk fatty acid composition throughout lactation (
      • Casoli C.
      • Duranti E.
      • Morbidini L.
      • Panella F.
      • Vizioli V.
      Quantitative and compositional variations of Massese sheep milk by parity and stage of lactation..
      ;
      • Nudda A.
      • McGuire M.A.
      • Battacone G.
      • Pulina G.
      Seasonal variation in conjugated linoleic acid and vaccenic acid in milk fat of sheep and its transfer to cheese and ricotta..
      ).
      In conclusion, supplementing primiparous ewes with ZnSO4 during late gestation to increase overall dietary Zn intake up to 7-fold the current NASEM recommendations did not affect SCC, positivity rates of mastitis-related microorganisms, or colostrum fatty acid profiles. Nevertheless, the observed elevated SCC and the established frequency of positive culture and PCR-positive samples indicate the need for preventative mastitis interventions in nondairy sheep, particularly around the time of parturition. Differential biological responses to Zn treatment by breed type in part 1 of this research and the current SCC data may indicate a differing triage of Zn utilization for production parameters (i.e., milk production, BW, wool growth, and so on;
      • Stewart W.C.
      • Murphy T.W.
      • Page C.M.
      • Rule D.C.
      • Taylor J.B.
      • Austin K.
      • Pankey C.
      Effect of increasing dietary zinc sulfate fed to primiparous ewes: I. Effects on serum metabolites, mineral transfer efficiency, and animal performance..
      ).

      APPLICATIONS

      Including ZnSO4 above NASEM recommendations during late gestation had little effect on altering milk SCC and colostrum fatty acid profile of semi-extensively managed, primiparous ewes. However, positive microbial culture results suggest subclinical mastitis to be more prevalent during early lactation than at weaning, and etiological agents include bacilli and staphylococci. Therefore, future research is warranted to investigate additional management methods that reduce bacterial load and target mechanisms by which pathogenic bacteria infect the mammary gland. Vast breed diversity and specialized sheep production systems internationally may benefit from precision mineral supplementation programs. Breed-type differences in SCC when fed Zn at ≈4 × NASEM concentrations indicate the nuance of breed and related production levels when implementing mineral supplementation strategies for pregnant ewes.

      ACKNOWLEDGMENTS

      The authors thank the University of Wyoming Agriculture Experiment Station, Kalli Koepke (University of Wyoming assistant farm manager—sheep unit manager), and employees at the Sheep Unit for helping with animal care and management. This work was in part supported by Hatch-Multistate “Increased Efficiency of Sheep Production” project accession no. 1025808 from the USDA National Institute of Food and Agriculture.

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