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Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation

Open AccessPublished:November 15, 2022DOI:https://doi.org/10.3168/jds.2022-22248

      ABSTRACT

      Small ruminants are susceptible to milk fat depression (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide individual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the ruminal responses of goats and sheep with varying degrees of MFD extent to improve our understanding of this complex syndrome. Our specific aims were to attempt to elucidate whether pre-existing variations in ruminal fermentation and biohydrogenation determine a higher tolerance or susceptibility to MFD, and whether the severity of MFD depends exclusively on the response to the diet. The trial was conducted with 25 does and 23 ewes fed a basal diet without lipid supplementation for 3 wk (control period). Then, 2% fish oil (FO) was added to the same diet for 5 additional weeks (MFD period). Based on the extent of the elicited MFD (i.e., the percentage variation between milk fat concentrations recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON) animals were selected within each species. On the last day of each period, ruminal fluid samples were collected to examine fermentation parameters and fatty acid profiles. In general, the individual degree of MFD in sheep and goats did not seem to be predetermined by traits related to ruminal fermentation and biohydrogenation, including fatty acids that may serve as biomarkers of microorganisms. Regarding differences in the response to FO, the results suggest no link between MFD susceptibility and concentration of biohydrogenation intermediates such as trans-10-containing C18, C20, and C22 metabolites. The explanation for individual responses based on a shortage of ruminal acetate and 18:0 for mammary uptake also seems to be dismissed, based on the lack of variation in these compounds between RESPON+ and RESPON−. However, the concentration of unsaturated fatty acids provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3) was higher in the rumen of RESPON+ than RESPON− ewes and does. Thus, although further research is needed, the extent of biohydrogenation of these fatty acids might be associated with tolerance or susceptibility to MFD.

      Key words

      INTRODUCTION

      Over the last decade, several reports have contributed to the elucidation of interspecies differences in susceptibility to diet-induced milk fat depression (MFD) in dairy ruminants (
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ;
      • Dewanckele L.
      • Toral P.G.
      • Vlaeminck B.
      • Fievez V.
      Invited review: Role of rumen biohydrogenation intermediates and rumen microbes in diet-induced milk fat depression: An update.
      ). In contrast to the earlier perception that small ruminants are resistant to this syndrome (
      • Chilliard Y.
      • Glasser F.
      • Ferlay A.
      • Bernard L.
      • Rouel J.
      • Doreau M.
      Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.
      ;
      • Sanz Sampelayo M.R.
      • Chilliard Y.
      • Schmidely P.
      • Boza J.
      Influence of type of diet on the fat constituents of goat and sheep milk.
      ), the development of MFD has been confirmed when sheep and goats are fed supplemental fish oil (FO) or marine microalgae to modulate the milk fatty acid (FA) profile (
      • Toral P.G.
      • Hervás G.
      • Carreño D.
      • Frutos P.
      Does supplemental 18:0 alleviate fish oil-induced milk fat depression in dairy ewes?.
      ;
      • Bernard L.
      • Toral P.G.
      • Chilliard Y.
      Comparison of mammary lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil.
      ). However, much less is known about intraspecies variation in response to MFD-inducing diets (i.e., individual differences in the degree of reduction in milk fat concentration and yield when animals consume the same diet;
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Individual variation of the extent of milk fat depression in dairy ewes fed fish oil: Milk fatty acid profile and mRNA abundance of candidate genes involved in mammary lipogenesis.
      ;
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ).
      According to some studies, individual variation in the extent of MFD might be predetermined by differences in certain traits (e.g., the milk production level or the milk fat concentration), but the information on this is scarce and inconsistent (
      • Baldin M.
      • Zanton G.I.
      • Harvatine K.J.
      Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression.
      ;
      • Dewanckele L.
      • Jing L.
      • Stefanska B.
      • Vlaeminck B.
      • Jeyanathan J.
      • Van Straalen W.M.
      • Koopmans A.
      • Fievez V.
      Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.
      ;
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ). Some such pre-exiting traits may be related to ruminal processes. Ruminal fermentation provides substrates for milk fat synthesis, and biohydrogenation (BH) provides not only substrates for milk fat synthesis but also bioactive C18 FA with antilipogenic activity (
      • Bauman D.E.
      • Harvatine K.J.
      • Lock A.L.
      Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis.
      ;
      • Enjalbert F.
      • Combes S.
      • Zened A.
      • Meynadier A.
      Rumen microbiota and dietary fat: a mutual shaping.
      ;
      • Dewanckele L.
      • Toral P.G.
      • Vlaeminck B.
      • Fievez V.
      Invited review: Role of rumen biohydrogenation intermediates and rumen microbes in diet-induced milk fat depression: An update.
      ). Therefore, individual differences in ruminal function might account for individual differences in the response to MFD-inducing diets.
      However, in a recent study in sheep and goats (
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ), we found no solid relationship between MFD severity and milk concentrations of antilipogenic C18 FA formed in the rumen (e.g., trans-10 18:1, trans-10,cis-12 CLA, or trans-9,cis-11 CLA;
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ). In that study (
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ), we were also interested in certain C20–22 BH intermediates because they had been suggested as candidate milk fat inhibitors (
      • Kairenius P.
      • Leskinen H.
      • Toivonen V.
      • Muetzel S.
      • Ahvenjärvi S.
      • Vanhatalo A.
      • Huhtanen P.
      • Wallace R.J.
      • Shingfield K.J.
      Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows.
      ;
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ). Nevertheless, their very low proportion in milk precluded their quantification, which might be overcome by analyzing their concentration in ruminal samples. Moreover,
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      showed a possible link between milk cis-9 16:1 concentration and the extent of MFD. Yet, the endogenous synthesis of this putative antilipogenic FA (
      • Burns T.A.
      • Duckett S.K.
      • Pratt S.L.
      • Jenkins T.C.
      Supplemental palmitoleic (C16:1 cis-9) acid reduces lipogenesis and desaturation in bovine adipocyte cultures.
      ;
      • Bernard L.
      • Leroux C.
      • Chilliard Y.
      Expression and nutritional regulation of stearoyl-CoA desaturase genes in the ruminant mammary gland: Relationship with milk fatty acid composition.
      ;
      • Duckett S.K.
      • Volpi-Lagreca G.
      • Alende M.
      • Long N.M.
      Palmitoleic acid reduces intramuscular lipid and restores insulin sensitivity in obese sheep.
      ) precluded firm conclusions about its actual origin (i.e., from the diet or from mammary Δ9-desaturation) and involvement in MFD susceptibility.
      For these reasons, we propose that individual differences derived from ruminal processes might be more easily detected in ruminal fluid than in milk. It must be considered that the milk FA profile is only examined in fat that has been successfully secreted, which may not be an accurate representation of FA leaving the rumen and reaching the mammary gland. On this basis, this study was conducted to examine (1) whether the individual responses of dairy goats and sheep to FO, in terms of the extent of MFD, are predetermined by variations in ruminal fermentation and BH under normal (i.e., non-MFD) conditions, and (2) whether individual differences in these ruminal processes contribute to explain tolerance or susceptibility to diet-induced MFD. An investigation of variations similarly detected in both ruminant species is expected to strengthen our knowledge of the mechanisms underlying MFD syndrome.

      MATERIALS AND METHODS

      Ethics Statements

      All procedures involving animals were completed in accordance with European Union and Spanish regulations [Council Directive 2010/63/EU (EU, 2010) and Royal Decree 53/2013 (
      • BOE (Boletín Oficial del Estado)
      Royal Decree 53/2013, of 8 February, on the protection of animals used for experimental purposes.
      )] and granted prior approval by the Research Ethics Committees of the Instituto de Ganadería de Montaña, the Spanish National Research Council (CSIC), and the Junta de Castilla y León (Spain).

      Animals and Experimental Treatments

      This assay is part of a larger study conducted to provide insight into MFD in small ruminants. A detailed description of the experimental design and composition of the diets was reported in a previous article (
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ). In brief, 25 Murciano-Granadina does (30.5 ± 3.6 kg of BW; 40.1 ± 5.9 DIM; 1.49 ± 0.06 kg of milk/d) and 23 Assaf ewes (64.5 ± 9.5 kg of BW; 38.3 ± 4.7 DIM; 1.11 ± 0.07 kg of milk/d) were housed in individual pens and milked once daily (0830 h). They were fed a TMR composed of dehydrated alfalfa, whole corn and barley grains, soybean meal, sugar beep pulp and molasses, and a vitamin-mineral supplement (50:50 forage:concentrate ratio; 184 g of CP and 276 g of NDF per kilogram of DM) without lipid supplementation for 3 wk (control period). Then, this basal TMR was supplemented with 20 g of FO (providing 66 g of 20:5n-3 and 204 g of 22:6n-3 per kilogram of total FA) per kilogram of diet DM for 5 additional weeks (MFD period). At the end of this second period, based on the extent of the elicited MFD (i.e., the percentage of variation between milk fat concentrations recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON−) animals were selected within each species (10 does and 10 ewes in total). The mean reductions in milk fat concentration were 25.4% in RESPON+ and 7.3% in RESPON− animals (
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ; Supplemental Figure S1, https://digital.csic.es/handle/10261/272926 ,
      • Della Badia A.
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Dataset: Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation. Digital CSIC.
      ).

      Rumen Sampling Procedure

      On the last day of each period, animals were given free access to the TMR for 1 h after milking. Orts were then removed and, 3 h later, individual samples of ruminal fluid (approximately 150 mL) were obtained using an oral stomach probe (
      • Ramos-Morales E.
      • Arco-Pérez A.
      • Martín-García A.I.
      • Yáñez-Ruiz D.R.
      • Frutos P.
      • Hervás G.
      Use of stomach tubing as an alternative to rumen cannulation to study ruminal fermentation and microbiota in sheep and goats.
      ). The fluid was immediately strained through a nylon membrane (400 μm; Fisher Scientific S.L.). Then, 3 mL of ruminal fluid were acidified with 3 mL of 0.2 M HCl for ammonia analysis, and 0.8 mL was deproteinized with 0.5 mL of 20 g of metaphosphoric acid/L and 4 g of crotonic acid/L in 0.5 M HCl for VFA determinations. These samples were stored at −30°C until laboratory analyses. Aliquots of ruminal fluid (approximately 50 mL) were also collected for FA analysis, immediately frozen at −80°C, freeze-dried, and stored again at −80°C.

      Chemical Analyses

      The ruminal fluid concentration of ammonia was determined using a colorimetric method (
      • Reardon J.
      • Foreman J.A.
      • Searcy R.L.
      New reactants for the colorimetric determination of ammonia.
      ) and that of VFA by GC, using crotonic acid as an internal standard (
      • Ottenstein D.M.
      • Bartley D.A.
      Separation of free acids C2–C5 in dilute aqueous solution column technology.
      ), both in centrifuged samples.
      Total lipids were extracted twice from 200 mg of freeze-dried ruminal digesta samples using 4 mL of a hexane-isopropanol mixture (3:2, vol/vol) following adjustment of the digesta pH to 2 using 2 M HCl (
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Äröla A.
      • Nurmela K.V.V.
      • Huhtanen P.
      • Griinari J.M.
      Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows.
      ) and the addition of cis-12 13:1 (10–1301–9, Larodan Fine Chemicals AB) as an internal standard. Organic extracts were combined and dried under nitrogen at 50°C. Lipids dissolved in 2 mL of hexane were converted to FAME using a sequential base-acid catalyzed transesterification procedure with freshly prepared 0.5 M sodium methoxide in methanol for 5 min at 20°C followed by reaction with 1% (vol/vol) sulfuric acid in methanol at 50°C for 30 min (
      • Toral P.G.
      • Hervás G.
      • Carreño D.
      • Leskinen H.
      • Belenguer A.
      • Shingfield K.J.
      • Frutos P.
      In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.
      ). Methyl esters were separated and quantified using a gas chromatograph (Agilent 7890A GC System) equipped with a flame-ionization detector (FID) and a 100-m fused silica capillary column (0.25 mm i.d., 0.2-µm film thickness; CP-SIL 88, CP7489, Varian Ibérica S.A.), with hydrogen as the fuel and carrier gas. The total FAME profile in a 2-µL sample volume at a split ratio of 1:20 was determined using a temperature gradient program, and isomers of 18:1 were resolved in a separate analysis under isothermal conditions at 170°C (
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Äröla A.
      • Nurmela K.V.V.
      • Huhtanen P.
      • Griinari J.M.
      Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows.
      ). Overlapping trans-10,cis-15 + trans-11,cis-15 18:2, and cis-9,trans-11 + trans-7,cis-9 + trans-8,cis-10 CLA were further resolved using a 100-m ionic liquid coated capillary column (SLB-IL111, Sigma-Aldrich) and the temperature gradient program employed by
      • de la Fuente M.A.
      • Rodríguez-Pino V.
      • Juárez M.
      Use of an extremely polar 100-m column in combination with a cyanoalkyl polysiloxane column to complement the study of milk fats with different fatty acid profiles.
      , with hydrogen as fuel and carrier gas. All peaks were identified by comparison of their retention times with those of commercially available FAME standards (GLC463, U-37-M, U-43-M, U-45-M, and U-64-M, Nu-Chek Prep.; 18919-1AMP Supelco, L6031, L8404, and O5632, Sigma-Aldrich; and 11-1600-8, 20-2024-1, 20-2210-9, 20-2305-1-4, 21-1211-7, 21-1413-7, 21-1614-7, 21-1615-7, and BR mixtures 2 and 3, Larodan Fine Chemical AB), cross-referencing chromatograms reported in the literature (e.g.,
      • Shingfield K.J.
      • Ahvenjärvi S.
      • Toivonen V.
      • Äröla A.
      • Nurmela K.V.V.
      • Huhtanen P.
      • Griinari J.M.
      Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows.
      ;
      • de la Fuente M.A.
      • Rodríguez-Pino V.
      • Juárez M.
      Use of an extremely polar 100-m column in combination with a cyanoalkyl polysiloxane column to complement the study of milk fats with different fatty acid profiles.
      ), and comparison with reference samples for which the FA composition was determined based on GC-FID analysis of FAME and GC-MS analysis of corresponding 4,4-dimethyloxazoline derivatives (
      • Toral P.G.
      • Hervás G.
      • Carreño D.
      • Leskinen H.
      • Belenguer A.
      • Shingfield K.J.
      • Frutos P.
      In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.
      ,
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ).

      Statistical Analysis

      Statistical analyses were performed using the MIXED procedure of the SAS software package (version 9.4, SAS Institute Inc.) and focused on the following 2 aims of the study: (1) to examine pre-existing variations in ruminal fermentation and BH between RESPON+ and RESPON− goats and sheep in the control period (i.e., under non-MFD conditions), and (2) to examine the ruminal responses of these goats and sheep to FO supplementation (i.e., to explain the tolerance or susceptibility to diet-induced MFD). All differences, both in the control period and in the response to FO supplementation, were analyzed by 2-way ANOVA according to the following model:
      Yijk = μ + Spi + Resj + Sp × Resij + ξijk,


      where Yijk is the individual value of each dependent variable; μ, the overall mean; Spi, the fixed effect of the species (Sp; i = caprine vs. ovine), Resj, the fixed effect of the response (Res; j = RESPON− vs. RESPON+); Sp × Resij, their interaction; and ξijk, the residual error. Means were separated through the pairwise differences (pdiff) option of the least squares means (lsmeans) statement of the MIXED procedure and adjusted for multiple comparisons using a Bonferroni correction. Differences were declared significant at P < 0.05 and considered a trend toward significance at 0.05 ≤ P < 0.10. Least squares means are reported.

      RESULTS

      Table 1 reports ruminal fermentation parameters, whereas the FA profile is displayed in 3 tables: SFA (Table 2), MUFA (Table 3), and PUFA (Table 4). Each table reports pre-existing variations during the control period, as well as the difference in the response to FO supplementation (ΔMFD). These topics will be described in 2 independent subsections.
      Table 1Ruminal concentrations of ammonia (mg/L) and total VFA (mmol/L), molar proportions (mol/100 mol) of VFA, and acetate:propionate ratio in dairy sheep and goats with a mild (RESPON−) or strong (RESPON+) response to a diet inducing milk fat depression (MFD)
      VariableItem
      Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      GoatsSheepSED
      SED = standard error of the difference.
      P
      Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      RESPON−RESPON+RESPON−RESPON+SpResSp × Res
      AmmoniaControl64.6141.4123.8156.761.90.3550.1810.582
      ΔMFD130.125.3−21.8−24.954.30.0110.1370.159
      Total VFAControl106.292.1108.1103.310.40.3850.2180.532
      ΔMFD−28.6−16.52.5628.39.59<0.0010.0130.329
      Molar proportions
       AcetateControl65.966.365.065.60.900.2380.9090.419
      ΔMFD−1.86−2.40−0.64−2.861.2070.6610.1270.341
       PropionateControl17.115.916.717.51.610.5840.8570.382
      ΔMFD−2.11−0.971.884.502.0310.0050.2090.613
       ButyrateControl14.115.114.113.01.280.2720.9640.254
      ΔMFD2.832.02−1.17−0.201.6400.0160.9430.456
       IsobutyrateControl0.8820.8041.061.340.2650.0760.5940.351
      ΔMFD0.400.43−0.09−0.550.247<0.0010.2400.177
       ValerateControl1.030.971.101.260.1330.0700.5580.258
      ΔMFD0.080.18−0.01−0.030.1410.1710.7160.537
       IsovalerateControl0.760.671.041.550.4050.0590.4830.308
      ΔMFD0.720.75−0.01−0.800.352<0.0010.1490.118
       CaproateControl0.250.230.310.260.0760.4080.4700.799
      ΔMFD−0.05−0.010.03−0.070.0680.7760.4930.179
       Acetate:propionate ratioControl3.874.333.983.790.3750.4280.6250.233
      ΔMFD0.440.05−0.48−0.910.4390.0080.2080.944
      1 Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      2 SED = standard error of the difference.
      3 Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      Table 2Ruminal SFA profile (g/100 g fatty acids) in dairy sheep and goats with a mild (RESPON−) or strong (RESPON+) response to a diet inducing milk fat depression (MFD)
      Other SFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926; Della Badia et al., 2022). OCFA = odd-chain fatty acids; BCFA = branched-chain fatty acids.
      VariableItem
      Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      GoatsSheepSED
      SED = standard error of the difference.
      P
      Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      RESPON−RESPON+RESPON−RESPON+SpResSp × Res
      12:0Control0.110.0840.170.160.015<0.0010.1230.652
      ΔMFD−0.046−0.015−0.059−0.0320.0260.4230.1330.905
      anteiso-13:0Control0.0050.0050.0120.0140.002<0.0010.6540.705
      ΔMFD0.0020.002−0.005−0.0070.003<0.0010.6470.701
      iso-13:0Control0.0370.0430.0660.0610.0110.0060.9500.483
      ΔMFD0.0110.005−0.019−0.0260.0110.0020.2290.786
      14:0Control0.780.580.810.800.1080.1190.1930.220
      ΔMFD0.550.790.370.340.1490.0080.3110.212
      iso-14:0Control0.120.140.180.160.0350.1530.8500.494
      ΔMFD−0.033−0.044−0.067−0.0910.0280.0600.4010.736
      15:0Control0.640.640.951.070.1500.0030.5990.578
      ΔMFD0.150.17−0.090−0.340.1340.0010.2510.153
      anteiso-15:0Control0.550.530.890.890.089<0.0010.8980.807
      ΔMFD0.0760.074−0.37−0.410.091<0.0010.7530.780
      iso-15:0Control0.380.370.490.490.0520.0060.7760.898
      ΔMFD0.0830.049−0.12−0.180.063<0.0010.3210.785
      16:0Control15.816.718.417.60.9490.0200.8910.208
      ΔMFD4.683.130.391.531.0440.0010.7880.088
      iso-16:0Control0.300.420.520.390.0820.1140.9090.050
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      ΔMFD0.066−0.078−0.25−0.210.0920.0030.4290.176
      17:0Control0.540.550.720.810.0790.0010.4180.504
      ΔMFD0.230.240.096−0.0460.0870.0040.2960.233
      anteiso-17:0Control0.380.440.590.570.0770.0080.7410.519
      ΔMFD0.210.077−0.23−0.250.081<0.0010.1950.313
      iso-17:0
      Contains a 16:1 isomer of indeterminate double bond position as minor component.
      Control0.320.370.540.550.1040.0150.6750.778
      ΔMFD0.480.310.14−0.0070.104<0.0010.0490.882
      7-methyl-hexadec-7 -enoate 17:0Control0.0180.0140.0210.0220.0040.0640.5910.362
      ΔMFD0.280.340.300.350.0400.5550.0920.910
      18:0Control45.844.744.347.33.4790.8650.7190.428
      ΔMFD−40.9−39.2−37.6−41.23.6370.7990.7100.321
      10-O-18:0Control0.0480.0250.0480.0310.0190.8240.1580.840
      ΔMFD1.461.772.151.690.3370.2210.7390.124
      iso-18:0Control0.0070.0050.0110.0110.0030.0640.5890.672
      ΔMFD0.064
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.068
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.073
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.055
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0060.5840.0790.014
      Σ OCFAControl1.831.732.462.720.213<0.0010.5930.239
      ΔMFD1.531.690.500.0840.248<0.0010.4840.119
      Σ BCFAControl2.532.783.943.780.279<0.0010.8000.314
      ΔMFD1.560.98−0.51−0.940.291<0.0010.0270.710
      a,b Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      1 Other SFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926;
      • Della Badia A.
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Dataset: Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation. Digital CSIC.
      ). OCFA = odd-chain fatty acids; BCFA = branched-chain fatty acids.
      2 Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      3 SED = standard error of the difference.
      4 Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      5 In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      6 Contains a 16:1 isomer of indeterminate double bond position as minor component.
      Table 3Ruminal MUFA profile (g/100 g fatty acids) in dairy sheep and goats with a mild (RESPON−) or strong (RESPON+) response to a diet inducing milk fat depression (MFD)
      Other MUFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926; Della Badia et al., 2022).
      VariableItem
      Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      GoatsSheepSED
      SED = standard error of the difference.
      P
      Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      RESPON−RESPON+RESPON−RESPON+SpResSp × Res
      cis-7 16:1Control0.250.140.230.280.0970.4050.6310.278
      ΔMFD0.290.420.210.140.1080.0310.6880.198
      cis-9 16:1Control0.0910.0720.0830.0960.0160.4790.8000.182
      ΔMFD0.630.750.530.840.1160.9760.0210.249
      cis-11 16:1Control0.0060.0050.0090.0090.0020.0190.8750.526
      ΔMFD0.0340.0390.0260.0340.0050.0960.0780.640
      cis-13 16:1Control0.0230.0080.0060.0140.0090.3620.5510.075
      ΔMFD0.0140.0230.0240.0070.0120.7460.6740.145
      trans-5 16:1Control0.0200.0210.0300.0250.0030.0100.3810.172
      ΔMFD0.0250.0190.0010.0080.005<0.0010.8590.091
      trans-6 + 7 + 8 16:1Control0.150.0920.0760.0930.0340.1540.4130.143
      ΔMFD0.400.400.280.300.0650.0340.8440.801
      trans-9 16:1Control0.0080.0060.0150.0130.0020.0010.2130.969
      ΔMFD0.110.110.130.130.0120.0650.7200.669
      cis-9 17:1Control0.0290.0210.0410.0310.0070.0480.0970.846
      ΔMFD−0.008−0.005−0.022−0.0060.0110.3390.2360.397
      cis-9 18:1Control6.206.795.054.400.9610.0190.9680.375
      ΔMFD0.028
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      −0.51
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.94
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      3.50
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.778<0.0010.0850.013
      cis-11 18:1Control0.370.350.400.390.0640.4470.7470.866
      ΔMFD0.921.040.941.150.1320.4830.0920.613
      cis-12 18:1Control0.450.470.370.450.0790.3210.3650.618
      ΔMFD−0.27−0.29−0.13−0.260.0820.1430.2180.347
      Σ cis-18:1Control7.337.876.055.501.0920.0310.9980.493
      ΔMFD0.82
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.46
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      2.07
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      4.69
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.8940.0010.0930.032
      trans-9 18:1Control0.370.430.270.280.0530.0040.3950.557
      ΔMFD0.540.501.030.670.1410.0050.0670.121
      trans-10 18:1Control0.710.720.460.510.1030.0060.6780.822
      ΔMFD0.511.111.392.500.9790.1200.2330.715
      trans-11 18:1Control4.424.953.683.240.6480.0170.9160.304
      ΔMFD21.618.419.517.41.630.1960.0330.649
      trans-12 18:1Control0.840.860.550.610.1290.0100.6420.830
      ΔMFD0.840.831.771.110.2540.0040.0820.089
      trans-13 + 14 18:1Control1.281.240.831.010.1810.0160.5750.382
      ΔMFD−0.0050.0791.230.340.3380.0060.1110.059
      Σ trans-18:1Control9.319.916.916.921.210.0060.7250.736
      ΔMFD23.721.226.522.72.240.2010.0640.697
      cis-7 + trans-13 20:1Control0.0100.0080.0080.0090.0020.5790.6220.147
      ΔMFD0.0930.0690.0850.0390.0210.2030.0270.454
      cis-11 20:1
      Coelutes with trans-15 + 16 20:1.
      Control0.120.110.110.110.0180.6730.5850.908
      ΔMFD0.630.710.630.720.0580.9820.0570.925
      cis-13 + trans-17Control0.0080.0060.0090.0090.0010.0590.4110.411
      20:1ΔMFD0.0620.0720.0560.0710.0090.5740.0680.754
      cis-17 20:1Control
      ΔMFD0.036
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.039
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.092
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.034
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0090.0010.001<0.001
      trans-9 + 10 20:1Control
      ΔMFD0.0810.0610.0400.0200.011<0.0010.0230.967
      trans-11 20:1Control0.0140.0120.0180.0180.0050.1600.8370.680
      ΔMFD0.0210.0290.0530.0260.0110.0830.2390.035
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      trans-12 20:1Control
      ΔMFD0.026
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.037
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.073
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.037
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0120.0190.1740.017
      cis-13 22:1Control0.0270.0150.0090.0270.0140.7600.7690.147
      ΔMFD0.130.170.0390.260.0650.9590.0110.076
      cis-19 22:1Control
      ΔMFD0.013
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.012
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.016
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.007
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0020.8000.0090.031
      a,b Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      1 Other MUFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926;
      • Della Badia A.
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Dataset: Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation. Digital CSIC.
      ).
      2 Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      3 SED = standard error of the difference.
      4 Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      5 Coelutes with trans-15 + 16 20:1.
      6 In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      Table 4Ruminal PUFA profile (g/100 g fatty acids) in dairy sheep and goats with a mild (RESPON−) or strong (RESPON+) response to a diet inducing milk fat depression (MFD)
      Other PUFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926; Della Badia et al., 2022).
      Variable
      Except for n-3 and n-6 PUFA, the tentative geometry of double bonds was inferred from the retention times and elution order of FAME during GC analysis (Toral et al., 2018).
      Item
      Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      GoatsSheepSED
      SED = standard error of the difference.
      P
      Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      RESPON−RESPON+RESPON−RESPON+SpResSp × Res
      cis-9,cis-12 18:2Control8.498.477.716.211.430.1530.4650.474
      ΔMFD−5.29−5.34−4.78−2.671.450.1420.3320.306
      cis-11,cis-15 + cis-10, cis-15 18:2Control0.0340.0310.0290.0260.0060.2920.4910.968
      ΔMFD0.0100.0160.0180.0370.0070.0070.0220.191
      cis-9,trans-12 18:2Control0.0310.0290.0390.0300.0060.3070.2190.492
      ΔMFD0.0150.0170.0110.0250.0050.6660.0410.120
      trans-10,cis-15 18:2Control0.0110.0100.0280.0270.005<0.0010.8230.994
      ΔMFD0.170.370.0300.150.1490.1050.1440.721
      trans-11,cis-15 18:2Control0.220.210.230.170.0530.7820.3590.472
      ΔMFD0.510.500.320.740.1540.8000.0850.069
      trans-9,trans-12 18:2Control0.0160.0120.0110.0110.0030.1500.3210.343
      ΔMFD0.0320.0620.0570.0900.0240.1380.0850.919
      cis-9,trans-11 CLA
      Contains trans-8,cis-10 CLA as minor component, representing on average 5.87 ± 1.14% of the peak in the control period. In the MFD period, trans-8,cis-10 CLA could not be resolved due to coelution with a 20:2 isomer of indeterminate double bond position. No traces of trans-7 cis-9 CLA were detected in Control or MFD periods.
      Control0.130.250.110.110.0570.0730.1620.131
      ΔMFD−0.020−0.1130.0020.0290.0490.0310.3550.100
      trans-9,cis-11 CLAControl0.0370.0170.0110.0380.0120.8000.6700.015
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      ΔMFD−0.0030.0130.011−0.0150.0110.3570.5290.016
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      trans-10,cis-12 CLAControl0.0220.0210.0180.0260.0080.9440.5010.431
      ΔMFD−0.010−0.0090.003−0.0090.0080.2600.2830.264
      trans-11,cis-13 CLAControl0.0080.0090.0120.0120.0020.0630.8480.783
      ΔMFD0.029
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.035
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.084
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.037
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0130.0070.0370.011
      trans-12,trans-14 CLA
      Coelutes with a 20:2 isomer of indeterminate double bound position.
      Control0.0050.0060.0100.0160.0060.06850.4310.549
      ΔMFD0.057
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.057
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.099
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.025
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0100.492<0.001<0.001
      cis-6,cis-9,cis-12 18:3Control0.013
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.008
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.018
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.020
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.001<0.0010.2220.005
      ΔMFD−0.0020.006−0.006−0.0070.002<0.0010.0270.005
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      cis-9,cis-12,cis-15 18:3
      Contains cis-9 + trans-14 20:1 as minor components.
      Control1.341.051.631.330.2680.1600.1380.979
      ΔMFD−0.46−0.19−0.95−0.430.2540.0570.0440.502
      trans-9,trans-12,cis-15 + cis-9, cis-12,trans-15 18:3Control0.0230.0190.0280.0230.0040.1140.1100.789
      ΔMFD−0.007−0.004−0.014−0.0060.0040.1900.0940.396
      cis-11,cis-14 20:2Control0.0500.0230.0140.0150.0110.0140.1360.092
      ΔMFD0.0840.120.130.120.0170.1260.3130.073
      trans-9,cis-17 + trans-14, cis-17 20:2Control
      ΔMFD0.0080.0110.0170.0080.0030.1540.2070.016
      In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      trans-10,cis-17 20:2Control
      ΔMFD0.036
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.039
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.078
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.038
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0070.0010.001<0.001
      trans-13,cis-17 20:2Control
      ΔMFD0.022
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.033
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.093
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.036
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0120.0010.0170.001
      trans-10,trans-16 20:2Control
      ΔMFD0.070
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.077
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.16
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.085
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.014<0.0010.0030.001
      trans-11,trans-17 + trans-12, trans-17 20:2Control
      ΔMFD0.006
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.006
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.021
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.009
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.002<0.0010.0010.001
      cis-11,trans-14,cis-17 20:3Control
      ΔMFD0.017
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.018
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.025
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.017
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0030.0960.0680.035
      trans-10,trans-14,trans-17 20:3Control
      ΔMFD0.0390.0380.0520.0350.0070.3420.0880.099
      cis-5,cis-8,cis-11,cis-14, cis-17 20:5Control
      ΔMFD0.400.560.330.740.1350.5740.0080.229
      trans-15,cis-19 22:2Control
      ΔMFD0.0350.0280.0650.0350.0090.0090.0090.078
      trans-11,trans-17 + trans-13, trans-18 22:2Control
      ΔMFD0.049
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.058
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.085
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.058
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0110.0340.2610.033
      cis-11,cis-16, cis-19 + trans-13, cis-16,trans-19 22:3Control
      ΔMFD0.064
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.063
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.17
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.10
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.021<0.0010.0300.036
      cis-13,cis-16,trans-17 22:3
      Coelutes with cis-16,cis-19 22:2.
      Control
      ΔMFD0.030
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.035
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.070
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.025
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0110.0740.0220.005
      trans-10,trans-14,cis-19 + trans-12, trans-15,cis-19 22:3Control
      ΔMFD0.020
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.029
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.074
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.024
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0130.0140.0340.004
      trans-11,trans-14,trans-17 22:3Control
      ΔMFD0.011
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.017
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.033
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.018
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0040.0010.0710.001
      cis-10,cis-13,cis-16,cis-19 22:4Control
      ΔMFD0.40
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.50
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.72
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.48
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0560.0020.0990.001
      trans-9,cis-13,cis-16,cis-19Control
       + trans-8,cis-13,cis-16,cis-19 22:4ΔMFD0.150.150.210.150.0240.1040.0680.080
      trans-10,trans-13,cis-16,cis-19 22:4Control
      ΔMFD0.073
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.077
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.20
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.057
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0170.001<0.001<0.001
      cis-7,cis-10,cis-13,cis-16, cis-19 22:5Control
      ΔMFD0.580.701.391.490.2650.0010.5580.980
      cis-4,cis-7,cis-10,trans-14, cis-19 22:5Control
      ΔMFD0.11
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.073
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.046
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.14
      Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      0.0320.9060.2110.008
      cis-4,cis-7,cis-10,trans-14, trans-17 22:5Control
      ΔMFD0.0790.0810.0080.0850.0260.0880.0510.058
      cis-4,cis-7,cis-10,cis-13,cis-16, cis-19 22:6Control
      ΔMFD1.642.350.682.840.6670.6310.0080.146
      a–c Within a row, different superscripts indicate significant differences (P < 0.05) due to the effect of Sp × Res.
      1 Other PUFA are reported in Supplemental Table S1 (http://hdl.handle.net/10261/272926;
      • Della Badia A.
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Dataset: Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation. Digital CSIC.
      ).
      2 Except for n-3 and n-6 PUFA, the tentative geometry of double bonds was inferred from the retention times and elution order of FAME during GC analysis (
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ).
      3 Control = data obtained when animals were fed a TMR without lipid supplementation; ΔMFD = difference between the data obtained after diet supplementation with 20 g of fish oil/kg of DM (to induce MFD) and those previously recorded in the control period.
      4 SED = standard error of the difference.
      5 Probability of significant effects due to species (Sp), response (Res), and their interaction (Sp × Res).
      6 Contains trans-8,cis-10 CLA as minor component, representing on average 5.87 ± 1.14% of the peak in the control period. In the MFD period, trans-8,cis-10 CLA could not be resolved due to coelution with a 20:2 isomer of indeterminate double bond position. No traces of trans-7 cis-9 CLA were detected in Control or MFD periods.
      7 In the pairwise analysis, no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction.
      8 Coelutes with a 20:2 isomer of indeterminate double bound position.
      9 Contains cis-9 + trans-14 20:1 as minor components.
      10 Coelutes with cis-16,cis-19 22:2.

      Pre-Existing Variations (Control Period)

      No differences between the least and most responsive goats and sheep were observed in ammonia and VFA concentration and molar proportions during the control period (P > 0.10). Similarly, ruminal SFA and MUFA profiles showed almost no pre-existing variation between the RESPON− and RESPON+ groups, except for the tendency toward a lower accumulation of cis-9 17:1 in animals that displayed more severe MFD (P = 0.097; Table 3). Regarding PUFA (Table 4), the minor 18:3n-6 was more abundant in RESPON− than RESPON+ goats (P = 0.005 for the interaction Sp × Res), but other C18 and C20-C22 PUFA showed comparable concentrations in ewes and does.
      It was not the aim of our study to examine interspecies differences. In brief, variations in fermentation were scant: only the concentrations of isobutyrate, valerate, and isovalerate tended to be greater in sheep than in goats (P < 0.10; Table 1). Ewes also showed a higher proportion (P < 0.05) of most SFA (e.g., 16:0 and odd- and branched-chain FA; Table 2), 16:1 isomers (Table 3), and some BH intermediates of 18:3n-3 (e.g., trans-10,cis-15 and trans-11,cis-13 18:2; Table 4). However, goats had greater percentages of 18:1 isomers (P < 0.05; Table 3) or cis-9,trans-11 CLA (P = 0.073; Table 4).

      Differences in the Response to FO Supplementation

      Total VFA concentrations showed differences due to both Sp and Res (P < 0.001 and P = 0.013, respectively), but the interaction Sp × Res was not significant (P = 0.329; Table 1). Thus, ΔMFD values were always lower in goats than in sheep, and in RESPON− than in RESPON+ animals.
      Overall, 3 major types of response to FO supplementation were observed in ruminal FA concentrations: increases in both species were the most prevalent, followed by decreases in both species and, finally, by few inconsistent changes in goats and sheep.
      Beginning with FA that increased in abundance in both species during the MFD period, no difference due to susceptibility to MFD (P > 0.10) was detected for some ruminal metabolites that were previously associated with MFD, such as 10-O-18:0 (Table 2), trans-10 18:1, and trans-10,cis-15 18:2 (Table 3, Table 4, respectively, and Supplemental Figure S2; https://digital.csic.es/handle/10261/272926 ;
      • Della Badia A.
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Dataset: Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation. Digital CSIC.
      ). However, increases in FA directly supplied with the FO were significantly greater (P < 0.05) or tended to be greater (P < 0.10) in RESPON+ than RESPON− groups of both sheep and goats. These included the cis-9 and cis-11 16:1, cis-11 18:1, cis-11 20:1, and cis-13 22:1 MUFA (Table 3), and the 20:5n-3 and 22:6n-3 PUFA (Table 4). For a graphic representation of variations in these FA, please see Supplemental Figure S1. Furthermore, RESPON+ animals showed the greatest increments in 7-methyl-hexadec-7-enoate (P = 0.092; Table 2), cis-11,cis-15, trans-9,trans-12, and cis-9,trans-12 18:2 (P < 0.05; Table 4), and cis-4,cis-7,cis-10,trans-14,trans-17 22:5 (P = 0.051; Table 4) in both species, and in cis-4,cis-7,cis-10,trans-14,cis-19 22:5 in ewes (P = 0.008 for the interaction Sp × Res; Table 4). By contrast, FA with more pronounced increases in RESPON− animals were, for example, iso-17:0 (P = 0.049; Table 2), trans-9 + 10 20:1 and trans-11 18:1 (P < 0.05; Table 3). Several polyunsaturated C20–22 metabolites were also more abundant in RESPON−, particularly in sheep (P < 0.05 for the interaction Sp × Res), including some trans-10-containing FA, such as trans-10,cis-17 and trans-10,trans-16 20:2, trans-10,trans-14,cis-19 22:3 and trans-10,trans-13,cis-16,cis-19 22:4 (Table 4).
      Fewer FA showed negative responses to the FO supply in both species. Among them, decreases in 18:0 and cis-9,cis-12 18:2 were similar in the RESPON− and RESPON+ groups (P > 0.10; Table 2, Table 4, respectively). In fact, significant differences due to the response to MFD were only observed in cis-9,cis-12,cis-15 18:3, which decreased to a greater extent in RESPON− animals (P = 0.044; Table 4 and Supplemental Figure S2). A similar trend was found for trans-9,trans-12,cis-15 18:3 (P = 0.094; Table 4).
      Ruminal FA showing inconsistent changes (i.e., either increases or decreases depending on the species) during the MFD period included trans-10,cis-12 CLA and trans-9,cis-11 CLA, but no effect of the response was detected (P > 0.10; Table 4). Although the interaction Sp × Res was significant for the latter CLA isomer (P = 0.016; Table 4), no significant differences were found after adjustment for multiple comparisons using a Bonferroni correction. The abundance of cis-9 18:1 was increased in some groups, with a greater increment in RESPON+ sheep (P = 0.013 for the interaction Sp × Res; Table 3).
      Finally, some interspecies differences in the MFD period included opposite changes (P < 0.05) in the concentration of ammonia, in the molar proportions of butyrate, isobutyrate and isovalerate, and in the acetate:propionate ratio in goats (increases) and ewes (decreases). On the contrary, total VFA and the molar proportion of propionate increased in sheep and decreased in goats (P < 0.05; Table 1). Goats showed stronger responses to FO (P < 0.05) in the concentrations of 14:0 and 16:0 (Table 2) and most 16:1 isomers (Table 3). By contrast, changes in most 18:1 isomers (Table 3), and in cis-9,cis-12,cis-15 18:3, and C20–22 PUFA (Table 4) were more dramatic in sheep (P < 0.05). All individual FA were detected in the 2 species, and no unique metabolites were identified in goats or sheep.

      DISCUSSION

      The biohydrogenation theory proposes that diet-induced MFD is caused by alterations in ruminal FA metabolism leading to the production of antilipogenic BH intermediates (
      • Bauman D.E.
      • Griinari J.M.
      Regulation and nutritional manipulation of milk fat: Low-fat milk syndrome.
      ). However, we are still uncertain as to whether a higher tolerance or susceptibility to MFD is determined by certain pre-existing traits or whether the severity of MFD depends only on the response to the diet (
      • Baldin M.
      • Zanton G.I.
      • Harvatine K.J.
      Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression.
      ;
      • Dewanckele L.
      • Jing L.
      • Stefanska B.
      • Vlaeminck B.
      • Jeyanathan J.
      • Van Straalen W.M.
      • Koopmans A.
      • Fievez V.
      Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.
      ;
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ). In this study, we examined the ruminal response of goats and sheep with varying extents of MFD to attempt to answer that question and improve our understanding of this complex syndrome.

      Pre-Existing Variations (Control Period)

      In dairy cows, production has been associated with individual responses to MFD (although inconsistently, because animals with both high and low production have been suggested as more sensitive to the syndrome;
      • Bradford B.J.
      • Allen M.S.
      Milk fat responses to a change in diet fermentability vary by production level in dairy cattle.
      ;
      • Baldin M.
      • Zanton G.I.
      • Harvatine K.J.
      Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression.
      ;
      • Dewanckele L.
      • Jing L.
      • Stefanska B.
      • Vlaeminck B.
      • Jeyanathan J.
      • Van Straalen W.M.
      • Koopmans A.
      • Fievez V.
      Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.
      ). Our 2 previous reports on this subject (
      • Frutos P.
      • Toral P.G.
      • Hervás G.
      Individual variation of the extent of milk fat depression in dairy ewes fed fish oil: Milk fatty acid profile and mRNA abundance of candidate genes involved in mammary lipogenesis.
      ;
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ) agree that sheep and goats with a lower energy balance before consuming the MFD-inducing diet displayed greater decreases in milk fat concentration and yield. Those individual variations in energy balance were not accompanied by differences in DMI (
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ), leaving room for speculation about a putative role of the digestive or metabolic utilization of the diet. In the present study, however, ruminal fermentation parameters indicative of energy metabolism (i.e., VFA or the acetate:propionate ratio) could not be related to MFD susceptibility.
      Between-animal differences in the response to diet might also be predetermined by the rumen microbial composition (
      • Weimer P.J.
      • Stevenson D.M.
      • Mertens D.R.
      Shifts in bacterial community composition in the rumen of lactating dairy cows under milk fat-depressing conditions.
      ). Concentrations of odd- and branched-chain FA may serve as potential markers of microorganisms (
      • Fievez V.
      • Colman E.
      • Castro-Montoya J.M.
      • Stefanov I.
      • Vlaeminck B.
      Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update.
      ), although functional redundancy of the microbiota and endogenous FA synthesis might represent limitations (
      • Fievez V.
      • Colman E.
      • Castro-Montoya J.M.
      • Stefanov I.
      • Vlaeminck B.
      Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update.
      ;
      • Weimer P.J.
      Redundancy, resilience, and host specificity of the ruminal microbiota: implications for engineering improved ruminal fermentations.
      ). In the control period, MFD-tolerant and MFD-susceptible animals showed similar proportions of odd- and branched-chain FA, except for the difference in cis-9 17:1. Nonetheless, this FA appears to have no known role as a biomarker of specific microbial populations (
      • Fievez V.
      • Colman E.
      • Castro-Montoya J.M.
      • Stefanov I.
      • Vlaeminck B.
      Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update.
      ) or as a regulator of mammary lipogenesis in ruminants (
      • Shingfield K.J.
      • Griinari J.M.
      Role of biohydrogenation intermediates in milk fat depression.
      ;
      • Dewanckele L.
      • Toral P.G.
      • Vlaeminck B.
      • Fievez V.
      Invited review: Role of rumen biohydrogenation intermediates and rumen microbes in diet-induced milk fat depression: An update.
      ). Therefore, this observation could be a chance finding, without a cause-effect relationship with MFD. Another FA that differed between RESPON− and RESPON+ animals, 18:3n-6, was only affected in goats, which also refuted a major role in the MFD susceptibility.
      Concerning BH, despite the comprehensive FA profile of ruminal fluid analyzed, it was not possible to find any trend in BH explaining the individual susceptibility to MFD. Overall, traits related to ruminal fermentation and BH did not seem to predetermine the individual degree of MFD in sheep and goats. More conclusive results might be obtained through a meta-analysis of more trials, which would help to increase the statistical power compared with single experiments.
      Regarding interspecies differences, the scant variations between goats and sheep suggest only a minor effect of the species in animals fed a standard hay-based TMR. Such small differences in ruminal fermentation and BH might derive from interspecies variations in the microbial composition, which would be supported by divergences in odd- and branched-chain FA (
      • Fievez V.
      • Colman E.
      • Castro-Montoya J.M.
      • Stefanov I.
      • Vlaeminck B.
      Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update.
      ).

      Differences in the Response to FO Supplementation

      Starting with ruminal fermentation, the hypothesis relating the deficiency in ruminal acetate and the reduction in milk fat has been reconsidered in recent years (
      • Maxin G.
      • Glasser F.
      • Hurtaud C.
      • Peyraud J.L.
      • Rulquin H.
      Combined effects of trans-10,cis-12 conjugated linoleic acid, propionate, and acetate on milk fat yield and composition in dairy cows.
      ;
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ). Nevertheless, the present results suggest no link between this VFA and susceptibility to FO-induced MFD. Some studies report that marine lipids often decrease acetate and total VFA concentrations in the rumen (e.g.,
      • Fievez V.
      • Boeckaert C.
      • Vlaeminck B.
      • Mestdagh J.
      • Demeyer D.
      In vitro examination of DHA-edible micro-algae: 2. Effect on rumen methane production and apparent degradability of hay.
      ;
      • Zhu H.
      • Fievez V.
      • Mao S.
      • He W.
      • Zhu W.
      Dose and time response of ruminally infused algae on rumen fermentation characteristics, biohydrogenation and Butyrivibrio group bacteria in goats.
      ), and we observed in a previous trial with dairy ewes (
      • Frutos P.
      • Toral P.G.
      • Belenguer A.
      • Hervás G.
      Milk fat depression in dairy ewes fed fish oil: Might differences in rumen biohydrogenation, fermentation, or bacterial community explain the individual variation?.
      ) that these reductions were related to MFD severity. However, inconsistent changes in sheep and goats were found in the present study, which would rule out a clear relationship with MFD susceptibility.
      Regarding BH, no variation in ruminal trans-10,cis-12 CLA was detected (Supplemental Figure S2), which would also downplay the role of this CLA isomer in determining the individual response to FO consumption, consistent with available information in dairy sheep and goats (
      • Frutos P.
      • Toral P.G.
      • Belenguer A.
      • Hervás G.
      Milk fat depression in dairy ewes fed fish oil: Might differences in rumen biohydrogenation, fermentation, or bacterial community explain the individual variation?.
      ;
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      ). By contrast, the milk FA profiles of RESPON− and RESPON+ animals reported previously by
      • Della Badia A.
      • Hervás G.
      • Toral P.G.
      • Frutos P.
      Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.
      drew attention to trans-10 18:1 and trans-10,cis-15 18:2 as potential determinants of the extent of MFD. A similar relationship with trans-10 18:1 may exist in dairy cows (
      • Baldin M.
      • Zanton G.I.
      • Harvatine K.J.
      Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression.
      ;
      • Dewanckele L.
      • Jing L.
      • Stefanska B.
      • Vlaeminck B.
      • Jeyanathan J.
      • Van Straalen W.M.
      • Koopmans A.
      • Fievez V.
      Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.
      ), although the few studies that examined the biological effects of both trans-10 FA in cattle were inconclusive (
      • Lock A.L.
      • Tyburczy C.
      • Dwyer D.A.
      • Harvatine K.J.
      • Destaillats F.
      • Mouloungui Z.
      • Candy L.
      • Bauman D.E.
      Trans-10 octadecenoic acid does not reduce milk fat synthesis in dairy cows.
      ;
      • Kadegowda A.K.G.
      • Bionaz M.
      • Piperova L.S.
      • Erdman R.A.
      • Loor J.J.
      Peroxisome proliferator-activated receptor-gamma activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents.
      ;
      • Shingfield K.J.
      • Sæbø A.
      • Sæbø P.-C.
      • Toivonen V.
      • Griinari J.M.
      Effect of abomasal infusions of a mixture of octadecenoic acids on milk fat synthesis in lactating cows.
      ;
      • Vahmani P.
      • Meadus W.J.
      • Rolland D.C.
      • Duff P.
      • Dugan M.E.R.
      Trans10,cis15 18:2 isolated from beef fat does not have the same anti-adipogenic properties as trans10,cis12–18:2 in 3T3–L1 adipocytes.
      ). In the present trial, ruminal concentrations of trans-10 18:1 and trans-10,cis-15 18:2 showed numerical variations between RESPON− and RESPON+ animals, both in goats and in sheep, but the differences did not reach the required level of significance (Supplemental Figure S2). This is not surprising because trans-10 FA usually show higher individual variations than other BH intermediates, in particular when diet-induced increases take place (e.g.,
      • Bernard L.
      • Leroux C.
      • Rouel J.
      • Delavaud C.
      • Shingfield K.J.
      • Chilliard Y.
      Effect of extruded linseeds alone or in combination with fish oil on intake, milk production, plasma metabolite concentrations and milk fatty acid composition in lactating goats.
      ;
      • Baldin M.
      • Zanton G.I.
      • Harvatine K.J.
      Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression.
      ;
      • Frutos P.
      • Toral P.G.
      • Belenguer A.
      • Hervás G.
      Milk fat depression in dairy ewes fed fish oil: Might differences in rumen biohydrogenation, fermentation, or bacterial community explain the individual variation?.
      ).
      The BH of very-long-chain PUFA from FO also leads to the production of other trans-10 isomers with a putative role in MFD (
      • Kairenius P.
      • Leskinen H.
      • Toivonen V.
      • Muetzel S.
      • Ahvenjärvi S.
      • Vanhatalo A.
      • Huhtanen P.
      • Wallace R.J.
      • Shingfield K.J.
      Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows.
      ;
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ). In general, the very low amounts of most C20–22 BH intermediates would hinder their determination in milk samples, but not that in ruminal fluid. Specifically, digesta samples were collected 4 h after the morning feeding, when ruminal processes were very active and accumulation of BH metabolites was probably favored (
      • Aldai N.
      • Delmonte P.
      • Alves S.P.
      • Bessa R.J.B.
      • Kramer J.K.G.
      Evidence for the initial steps of DHA biohydrogenation by mixed ruminal microorganisms from sheep involves formation of conjugated fatty acids.
      ;
      • Baldin M.
      • Rico D.E.
      • Green M.H.
      • Harvatine K.J.
      Technical note: An in vivo method to determine kinetics of unsaturated fatty acid biohydrogenation in the rumen.
      ). In any event, ruminal concentrations of trans-10-containing C20–22 FA observed in this trial were not associated with the individual MFD susceptibility of sheep and goats.
      Neither could changes in ruminal 18:0 be related to MFD severity, even though its large reductions during FO-induced MFD suggested a role in the syndrome and motivated an extension of the biohydrogenation theory some years ago (
      • Loor J.J.
      • Doreau M.
      • Chardigny J.M.
      • Ollier A.
      • Sebedio J.L.
      • Chilliard Y.
      Effects of ruminal or duodenal supply of fish oil on milk fat secretion and profiles of trans-fatty acids and conjugated linoleic acid isomers in dairy cows fed maize silage.
      ;
      • Shingfield K.J.
      • Griinari J.M.
      Role of biohydrogenation intermediates in milk fat depression.
      ;
      • Gama M.A.S.
      • Garnsworthy P.C.
      • Griinari J.M.
      • Leme P.R.
      • Rodrigues P.H.M.
      • Souza L.W.O.
      • Lanna D.P.D.
      Diet-induced milk fat depression: Association with changes in milk fatty acid composition and fluidity of milk fat.
      ). The shortage of ruminal 18:0 for mammary uptake and Δ9-desaturation was presumed to impair the capacity to achieve adequate milk fat fluidity for efficient secretion, but this hypothesis was then challenged as a major determinant of MFD (
      • Toral P.G.
      • Hervás G.
      • Carreño D.
      • Frutos P.
      Does supplemental 18:0 alleviate fish oil-induced milk fat depression in dairy ewes?.
      ). The present results also dismiss it as an explanation for the individual response to dietary FO.
      Interestingly, most UFA provided by FO (i.e., cis-9 and cis-11 16:1, cis-11 18:1, cis-11 20:1, cis-13 22:1, 20:5n-3, and 22:6n-3) were more abundant in the ruminal fluid of sheep and goats suffering more severe MFD (Supplemental Figure S1). Duodenal infusion of FO has been shown to cause MFD in cows (
      • Loor J.J.
      • Doreau M.
      • Chardigny J.M.
      • Ollier A.
      • Sebedio J.L.
      • Chilliard Y.
      Effects of ruminal or duodenal supply of fish oil on milk fat secretion and profiles of trans-fatty acids and conjugated linoleic acid isomers in dairy cows fed maize silage.
      ;
      • Dallaire M.P.
      • Taga H.
      • Ma L.
      • Corl B.A.
      • Gervais R.
      • Lebeuf Y.
      • Richard F.J.
      • Chouinard P.Y.
      Effects of abomasal infusion of conjugated linoleic acids, Sterculia foetida oil, and fish oil on production performance and the extent of fatty acid Δ9-desaturation in dairy cows.
      ), which could be attributed to a direct antilipogenic effect of certain FA from this lipid supplement (
      • Kadegowda A.K.G.
      • Bionaz M.
      • Piperova L.S.
      • Erdman R.A.
      • Loor J.J.
      Peroxisome proliferator-activated receptor-gamma activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents.
      ;
      • Burns T.A.
      • Duckett S.K.
      • Pratt S.L.
      • Jenkins T.C.
      Supplemental palmitoleic (C16:1 cis-9) acid reduces lipogenesis and desaturation in bovine adipocyte cultures.
      ;
      • Duckett S.K.
      • Volpi-Lagreca G.
      • Alende M.
      • Long N.M.
      Palmitoleic acid reduces intramuscular lipid and restores insulin sensitivity in obese sheep.
      ). In particular, 20:5n-3 was reported to downregulate the expression of lipoprotein lipase (LPL) and sterol regulatory element-binding transcription factor 1 (SREBF1) in mammary epithelial cell cultures (
      • Kadegowda A.K.G.
      • Bionaz M.
      • Piperova L.S.
      • Erdman R.A.
      • Loor J.J.
      Peroxisome proliferator-activated receptor-gamma activation and long-chain fatty acids alter lipogenic gene networks in bovine mammary epithelial cells to various extents.
      ). The first gene is involved in preformed FA uptake by mammary epithelial cells, whereas the transcription factor has a central role in the regulation of mammary lipogenesis, and both of them are often downregulated during MFD (
      • Bauman D.E.
      • Harvatine K.J.
      • Lock A.L.
      Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis.
      ;
      • Shingfield K.J.
      • Bonnet M.
      • Scollan N.D.
      Recent developments in altering the fatty acid composition of ruminant-derived foods.
      ).
      In vitro studies suggested that cis-9 16:1 and cis-11 18:1 also impair lipogenesis in bovine adipocytes (
      • Burns T.A.
      • Duckett S.K.
      • Pratt S.L.
      • Jenkins T.C.
      Supplemental palmitoleic (C16:1 cis-9) acid reduces lipogenesis and desaturation in bovine adipocyte cultures.
      ,
      • Burns T.A.
      • Kadegowda A.K.G.
      • Duckett S.K.
      • Pratt S.L.
      • Jenkins T.C.
      Palmitoleic (16:1 cis-9) and cis-vaccenic (18:1 cis-11) acid alter lipogenesis in bovine adipocyte cultures.
      ). Likewise, in vivo administration of an oil rich in cis-9 16:1 reduced the intramuscular lipid percentage in sheep (
      • Duckett S.K.
      • Volpi-Lagreca G.
      • Alende M.
      • Long N.M.
      Palmitoleic acid reduces intramuscular lipid and restores insulin sensitivity in obese sheep.
      ,
      • Duckett S.K.
      • Furusho-Garcia I.
      • Rico J.E.
      • McFadden J.W.
      Flaxseed oil or n-7 fatty acid-enhanced fish oil supplementation alters fatty acid composition, plasma insulin and serum ceramide concentrations, and gene expression in lambs.
      ), but duodenal infusion of a similar lipid source did not impair milk fat synthesis in cows (
      • Plante-Dubé M.
      • Picard C.
      • Gilbert I.
      • Robert C.
      • Fievez V.
      • Vlaeminck B.
      • Belleannée C.
      • Gervais R.
      • Chouinard P.Y.
      Effects of a dietary supplement enriched in palmitoleic acid on fatty acid composition of follicular fluid, granulosa cell metabolism, and oocyte developmental capacity in early lactation dairy cows.
      ). Therefore, further research in dairy ruminants is needed to characterize the biological effects of cis-9 16:1, which has raised interest in human nutrition due to its identification as a lipokine (i.e., a FA that regulates systemic metabolism;
      • de Souza C.O.
      • Vannice G.K.
      • Rosa Neto J.C.
      • Calder P.C.
      Is palmitoleic acid a plausible nonpharmacological strategy to prevent or control chronic metabolic and inflammatory disorders?.
      ). Additional studies are also required to examine the link between MFD severity and other FA from FO (such as cis-11 16:1, which was previously correlated with milk fat concentration;
      • Bernard L.
      • Leroux C.
      • Rouel J.
      • Delavaud C.
      • Shingfield K.J.
      • Chilliard Y.
      Effect of extruded linseeds alone or in combination with fish oil on intake, milk production, plasma metabolite concentrations and milk fatty acid composition in lactating goats.
      ,
      • Bernard L.
      • Toral P.G.
      • Chilliard Y.
      Comparison of mammary lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil.
      ) and to clarify if they exert inhibitory effects or just co-vary with milk fat due to FO intake.
      Overall, the association between the MFD susceptibility and ruminal concentrations of presumably antilipogenic UFA provided by FO suggests lower BH extent in the rumen of RESPON+ goats and sheep. However, this seems to disagree with the greater reduction in ruminal cis-9,cis-12,cis-15 18:3 in these more responsive animals. This apparent contradiction may be explained by the specificity of bacterial enzymes involved in FA metabolism (
      • Wallace R.J.
      • McKain N.
      • Shingfield K.J.
      • Devillard E.
      Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria.
      ;
      • Enjalbert F.
      • Combes S.
      • Zened A.
      • Meynadier A.
      Rumen microbiota and dietary fat: a mutual shaping.
      ). In this regard, dietary marine lipids are known to favor the ruminal disappearance of cis-9,cis-12,cis-15 18:3 and cis-9,cis-12 18:2 (
      • Kim E.J.
      • Huws S.A.
      • Lee M.R.F.
      • Wood J.D.
      • Muetzel S.M.
      • Wallace R.J.
      • Scollan N.D.
      Fish oil increases the duodenal flow of long chain polyunsaturated fatty acids and trans-11 18:1 and decreases 18:0 in steers via changes in the rumen bacterial community.
      ;
      • Zhu H.
      • Fievez V.
      • Mao S.
      • He W.
      • Zhu W.
      Dose and time response of ruminally infused algae on rumen fermentation characteristics, biohydrogenation and Butyrivibrio group bacteria in goats.
      ;
      • Kairenius P.
      • Leskinen H.
      • Toivonen V.
      • Muetzel S.
      • Ahvenjärvi S.
      • Vanhatalo A.
      • Huhtanen P.
      • Wallace R.J.
      • Shingfield K.J.
      Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows.
      ), the most abundant PUFA in plants and therefore in standard ruminant diets. Their BH process seems mostly initiated by cis-12 isomerase activity (
      • Wallace R.J.
      • McKain N.
      • Shingfield K.J.
      • Devillard E.
      Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria.
      ;
      • Honkanen A.M.
      • Griinari J.M.
      • Vanhatalo A.
      • Ahvenjärvi S.
      • Toivonen V.
      • Shingfield K.J.
      Characterization of the disappearance and formation of biohydrogenation intermediates during incubations of linoleic acid with rumen fluid in vitro.
      ), which may be mediated by a higher abundance or activity of bacteria in RESPON+ animals. However, these microbial populations would be irrelevant in the initial BH of all the above-mentioned FA derived from FO (i.e., cis-9 16:1, cis-11 16:1, cis-11 18:1, cis-11 20:1, cis-13 22:1, 20:5n-3, and 22:6n-3) because none of them contains a cis-12 double bond. Therefore, bacteria showing greater isomerase or hydrogenase activity on double bonds other than cis-12 (e.g., cis-4, cis-5, cis-9, or cis-11;
      • Honkanen A.M.
      • Griinari J.M.
      • Vanhatalo A.
      • Ahvenjärvi S.
      • Toivonen V.
      • Shingfield K.J.
      Characterization of the disappearance and formation of biohydrogenation intermediates during incubations of linoleic acid with rumen fluid in vitro.
      ;
      • Aldai N.
      • Delmonte P.
      • Alves S.P.
      • Bessa R.J.B.
      • Kramer J.K.G.
      Evidence for the initial steps of DHA biohydrogenation by mixed ruminal microorganisms from sheep involves formation of conjugated fatty acids.
      ;
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ) could be less abundant in RESPON+ animals and may explain the apparent inconsistency in the extent of BH of UFA from vegetable or marine lipids.
      Finally, differences between ruminant species were more evident in the MFD than in the control period. Again, interspecies variation in the ruminal microbial community might be at the core of these differences, as suggested by changes in odd- and branched-chain FA (
      • Fievez V.
      • Colman E.
      • Castro-Montoya J.M.
      • Stefanov I.
      • Vlaeminck B.
      Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update.
      ). As previously reported for sheep and cattle (
      • Toral P.G.
      • Hervás G.
      • Carreño D.
      • Leskinen H.
      • Belenguer A.
      • Shingfield K.J.
      • Frutos P.
      In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.
      ,
      • Toral P.G.
      • Hervás G.
      • Leskinen H.
      • Shingfield K.J.
      • Frutos P.
      In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.
      ), the same FA were found in ewes and does, indicating that BH followed similar pathways in both species, although an influence on BH kinetics existed (e.g., a slower process for C20–22 PUFA in sheep than in goats).

      CONCLUSIONS

      The individual responses of dairy goats and sheep to FO-induced MFD would not be predetermined by variations in ruminal fermentation and BH. Based on the commonality of the responses in both ruminant species, the tolerance or susceptibility to MFD may depend predominantly on individual differences in the extent of BH of certain potentially antilipogenic UFA provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3). Further research is needed to establish the actual biological activity of these FA in dairy ruminants.

      ACKNOWLEDGMENTS

      This work was supported by the Spanish Research State Agency (Agencia Estatal de Investigación) and the European Regional Development Fund (projects AGL2017-87812-R, MINECO/AEI/FEDER, EU and PID2020-113441RB-I00, MCIN/AEI). A. Della Badia benefited from an FPI predoctoral contract, co-funded by the European Social Fund (PRE2018-086174, MCIU/AEI/FSE, EU). The authors thank L. Barrios (Biostatistics Department, CSIC; Madrid, Spain) for her helpful assistance with statistical analysis. The authors have not stated any conflicts of interest.