Supplementing the Diet of Dairy Goats with Dried Orange Pulp throughout Lactation: II Effect on Milk Fatty Acids Profile, Phenolic Compounds, Fat-Soluble Vitamins and Antioxidant Capacity

animals Article Supplementing the Diet of Dairy Goats with Dried Orange Pulp throughout Lactation: II Effect on Milk Fatty Acids Profile, Phenolic Compounds, Fat-Soluble Vitamins and Antioxidant Capacity Manuel Delgado-Pertíñez 1, Ignacio Martín-García2, Yolanda Mena 1, Luis Ángel Zarazaga 3 and JoséLuis Guzmán3,*   Citation: Delgado-Pertíñez, M.; Martín-García, I.; Mena, Y.; Zarazaga, L.Á.; Guzmán, J.L. Supplementing the Diet of Dairy Goats with Dried Orange Pulp throughout Lactation: II Effect on Milk Fatty Acids Profile, Phenolic Compounds, Fat-Soluble Vitamins and Antioxidant Capacity. Animals 2021,11, 2421. https:// doi.org/10.3390/ani11082421 Academic Editor: Igino Andrighetto Received: 6 July 2021 Accepted: 13 August 2021 Published: 17 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Departamento de Agronomía, Escuela Técnica Superior de Ingeniería Agronómica, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain; [email protected] (M.D.-P.); [email protected] (Y.M.) 2Estación Experimental del Zaidín (CSIC), Profesor Albareda 1, 18008 Granada, Spain; [email protected] 3Departamento de Ciencias Agroforestales, Escuela Técnica Superior de Ingeniería, Universidad de Huelva, “Campus de Excelencia Internacional Agroalimentario, ceiA3”, Campus Universitario de la Rábida, Carretera Huelva-Palos de la Frontera s/n, 21819 Palos de la Frontera, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-959-217-711 Simple Summary: Spain is a major global producer of both goats and oranges. The orange juice industry produces high levels of organic waste that could be used as alternative feedstock for ruminants, enhancing dairy farm sustainability by lowering feed costs and reducing the environmental impact. An example of such organic waste is dried orange pulp (DOP), which has been proven beneficial as a 40% or 80% replacement for cereal in the diet of goats at an early lactation stage; therefore, it is pertinent to study these by-products over a longer period, such as the complete lactation cycle of 180 days. This study evaluated the Payoya dairy breed and the resultant milk’s antioxidant composition and fatty acid (FA) content in terms of saturated, monounsaturated, and polyunsaturated FAs (SFA, MUFA, and PUFA, respectively). The levels of vitamin E, total phenolic compounds, and antioxidant capacity in milk increased as the percentage of DOP replacing cereals increased. Moreover, the inclusion of DOP improved the nutritional value of the milk for human health (according to the thrombogenicity index, MUFA/SFA, and PUFA/SFA ratios), especially at the end of lactation, leading to the conclusion that DOP might be an appropriate alternative to cereals in the diets of goats. Abstract: Although dried orange pulp (DOP) as a short-term dietary supplementation has been proven an effective substitute for cereals in goat diets–without impairing milk quality–there have been no studies considering its use over the full lactation period. This study evaluated replacing cereal with DOP in goat diets for the full 180-day lactation period on milk’s fatty acid (FA) and antioxidant composition. Payoya goats were assigned to three diet groups: a control group consuming a commercial concentrate with alfalfa hay as forage; a DOP40 or DOP80 group, wherein 40% or 80% of the cereal in the concentrate was replaced by DOP. The α -tocopherol and phenolic compounds levels and the antioxidant capacity in the milk increased as the DOP percentage increased. Including DOP might improve the FA indices of milk in the context of human health, especially when included at the end of lactation because it contributes to reducing the thrombogenicity index and increasing both the monounsaturated/saturated FA and polyunsaturated/saturated FA indices and the amounts of indispensables α -C18:3 n-3 and C18:2 n-6 cis. Ultimately, DOP presents a plausible alternative to cereals in the diet of goats throughout lactation to improve the nutritional milk quality, especially the healthy antioxidant capacity. Keywords: alternative feedstuffs; orange by-product; Payoya breed; fatty acid profile; phenolic compounds; fat-soluble vitamins; full lactation Animals 2021,11, 2421. https://doi.org/10.3390/ani11082421 https://www.mdpi.com/journal/animals Animals 2021,11, 2421 2 of 17 1. Introduction The role of the human diet in reducing the progression of chronic disease has become increasingly important [ 1 ], with a diet’s fatty acid (FA) profile being considered an important health determinant [ 2 ]. Saturated FA (SFA) intake is generally recognized as harmful, while higher consumption of monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) has been reported as associated with a reduced risk of cardiovascular disease (CVD) [ 3 ]. Hence, the Mediterranean Diet Score (MDS) features the MUFA/SFA ratio in its index, and the Alternative Healthy Eating Index (AHEI) features the PUFA/SFA ratio [ 1 ]. Linoleic acid (C18:2) and alfa-linolenic acid ( α -C18:3 n-3) have been demonstrated to be indispensable because they cannot be synthesized by humans, and acceptable intake has been defined for both types of FA [ 1 ]. Additionally, a relationship between human health and the n-6/n-3 ratio has been reported, with a value of less than four proposed to reduce the risk of CVD [ 4 ]. Ulbricht and Southgate [ 5 ] proposed two indices which might better characterize the diet’s atherogenic (refers to the FA aggregation forming a plaque in the arteries) and thrombogenic (refers to the tendency to form clots in the blood vessels) potential. The type of dietary fat consumed might contribute to both of these processes; C14:0 and C16:0 have great roles both in atherogenesis and thrombogenesis, while C12:0 has a greater role in atherogenesis and C18:0 has a greater role in thrombogenesis. Minor FAs, such as conjugated linoleic acid (CLA), have been reported to have anticancer effects [ 6 ] or protect against CVD [ 7 ]. Finally, foods containing natural antioxidants (such as vitamins A, C and E, carotenoids, phenolic compounds) have become popular because antioxidants can neutralize free radicals and their deleterious effects in both humans and animals, as well as negating the oxidation processes which can result in a deterioration of nutritional quality [8,9]. Recent research has demonstrated the benefits of full-fat dairy consumption [ 10 ]; in general, evidence suggests that milk has a neutral effect on CVD. Furthermore, milk and other dairy products contain natural antioxidants, such as fat-soluble vitamins (such as A and E), carotenoids, sulfur (containing amino acids), enzyme systems, oligosaccharides, peptides, phosphate, zinc, and selenium [ 11 ]. Milk might also contain appreciable amounts of phenolic compounds [ 12 , 13 ], which present antioxidant activity too [ 12 ]. In general, the content of FAs and the bioactive molecules in milk change in quantity and quality mainly according to diet-related factors [14,15]. The use of local resources, especially when they are recovered from by-products, might significantly enhance dairy farm sustainability. Organic waste from the orange juice industry in Spain is substantial, given that Spain is the leading orange producer in Europe (3.9 million tonnes in 2018) [ 16 ]. An example of such organic waste is dried orange pulp (DOP), a by-product that represents up to 15% w/wof the input. In the context of animal feed, it can be used as a source of both energy and, importantly, bioactive compounds, including phenolic compounds and vitamin E [ 17 – 19 ], compounds that have health-related properties, especially based on their antioxidant activity [ 8 , 9 ]. However, few studies have considered the substitution of cereals with DOP in goat diets or the effect this might have on the resultant milk’s FA and antioxidant properties [ 20 , 21 ]. In this experimental study’s previous paper [ 21 ], which evaluated the effects of replacing cereal with DOP in the diet of goats during the early lactation phase, diet had no significant effect on FA content. However, the resultant milk’s α -tocopherol, total phenolic compound (TPC), and total antioxidant capacity (TAC) increased. Accordingly, this study was designed to evaluate the effects of replacing cereal with DOP in the diet of dairy goats for the complete 180-day lactation on the FA and antioxidant composition of their milk. 2. Materials and Methods 2.1. Experimental Diets, Goats, and Experimental Procedure The experiment was performed with lactating Payoya goats at the University of Huelva’s (Huelva, Spain) experimental farm over the duration of their 180-day lactation. Animals 2021,11, 2421 3 of 17 Animals were handled in accordance with the Spanish guidelines for experimental animal protection (Royal Decree 53/2013). DOP pellets were prepared using orange juice residues, following a conventional industrial process (Cítricos del Andévalo, S.A., Huelva, Spain) as described by Guzmán et al. [22]. As previously described [ 21 ], forty-four primiparous goats were assigned to three diet treatments, with each group housed in a communal pen: the control (CD, n= 14) was given commercial concentrate plus alfalfa hay as forage; the DOP40 (n= 16) group was based on the CD but with 40% of the cereals in the concentrate replaced with DOP; finally, the DOP80 (n= 14) group was based on the CD but with 80% of the cereals in the concentrate replaced with DOP. The groups were balanced according to the live weight (LW) (37.0 ± 1.26, 38.0 ± 1.33 and 37.5 ± 2.12 kg for the Control, DOP40, and DOP80 groups, respectively) and body condition scores (BCS) (2.76 ± 0.09, 2.51 ± 0.06, and 2.55 ± 0.08 for the Control, DOP40 and DOP80 groups, respectively) of the goats. After delivery, the animals were fed the experimental diet adapted for early-to-mid stages of lactation; approaching the late stage, over 300 g of concentrate were removed from the diet. The ingredients and the nutritive value of the rations are shown in Table 1; they were designed (isoenergetic and isoproteic) using the Feed Ration Balancer (Format Solutions) software, version 2.0 (2017; Cargill, Inc., Wayzata, MN, USA). Table 1. Ration ingredients and proximate and fatty acid (FA) composition of the experimental diets throughout the lactation period. Items Earlyand Mid-Lactation Experimental Diets 1Late-Lactation Experimental Diets 1 Control DOP40 DOP80 Control DOP40 DOP80 Ration ingredients, % DM basis Alfalfa hay 17.4 17.5 17.6 20.2 20.3 20.4 Concentrate Dehydrated orange pulp (pellets) 0 20.0 40.0 0.00 19.4 38.6 Grain oats 22.1 13.2 4.38 21.4 12.8 4.24 Grain barley 8.53 5.11 1.70 8.28 4.96 1.65 Grain corn 19.3 11.6 3.89 18.8 11.3 3.77 Soy flour, 44% 7.31 10.2 13.0 7.09 9.92 12.6 Sunflower pellets, 28% 12.8 12.5 13.8 12.5 12.1 13.5 Grain peas 10.3 8.12 4.05 10.0 7.87 3.93 Salt 0.41 0.41 0.41 0.39 0.39 0.39 Stabilized lard 0.41 0.00 0.00 0.39 0.00 0.00 Vitamins and minerals 1.31 1.31 1.32 1.01 1.01 1.02 Proximate composition and nutritive value, % DM DM, % 87.2 87.6 88.1 87.1 87.1 88.1 Crude protein 17.8 16.6 18.4 20.9 18.7 18.3 Neutral detergent fiber 24.3 22.6 25.4 29.8 26.6 28.3 Acid detergent fiber 12.3 15.9 19.0 14.7 15.2 16.8 Acid detergent lignin 4.14 4.19 5.45 3.09 3.13 3.43 Ether extract 3.42 2.31 1.71 2.63 1.85 1.43 Ash 4.82 6.44 7.50 6.50 7.47 8.64 Gross energy, kcal/g DM 4.56 4.44 4.42 4.37 4.31 4.25 Forage unit for lactation, UFL/kg 0.98 0.98 0.97 0.98 0.98 0.96 Protein digestible in the intestine (PDI) 10.5 10.9 11.3 10.4 10.4 11.4 Total phenolic compounds, g gallic acid equivalents/kg DM 5.29 6.74 8.57 5.68 7.07 8.67 Total antioxidant capacity, mmol trolox equivalents/kg DM 14.9 20.4 26.3 11.4 19.4 31.5 Animals 2021,11, 2421 4 of 17 Table 1. Cont. Items Earlyand Mid-Lactation Experimental Diets 1Late-Lactation Experimental Diets 1 Control DOP40 DOP80 Control DOP40 DOP80 α-tocopherol, mg/kg DM 3.18 26.5 70.1 - - - FA composition, % total FA C8:0–C14:0 1.10 2.99 4.58 3.88 4.22 6.11 C16:0 23.7 23.6 27.7 23.3 25.1 27.6 C16:1 0.77 0.62 0.64 0.57 0.42 0.69 C18:0 9.02 9.83 11.4 8.90 8.98 11.9 C18:1 n-9 cis 26.0 23.6 20.6 26.9 20.7 15.5 C18:2 n-6 cis 37.0 35.3 30.2 34.2 36.3 31.7 C18:3 n-6 0.14 0.19 0.47 0.29 0.20 0.38 C18:3 n-3 2.22 3.85 4.38 1.93 4.00 6.08 ∑SFA 33.8 36.5 43.7 36.1 38.3 45.6 ∑MUFA 26.8 24.2 21.2 27.5 21.1 16.2 ∑PUFA 39.4 39.3 35.1 36.4 40.5 38.2 ∑n-6 37.2 35.5 30.7 34.5 36.5 32.1 ∑n-3 2.22 3.85 4.38 1.93 4.00 6.08 n6/n3 16.7 9.22 7.01 17.8 9.13 5.28 1 The control group’s diet was based on a commercial concentrate, with alfalfa hay as forage; for the DOP40 and DOP80 groups, 40% and 80% of the concentrate’s cereal was replaced with DOP. The goats were fed once daily—in the mornings—and food intake for each group was calculated daily by subtracting the orts from the amount of food offered every day. For forage, the average daily consumption per animal was 0.4 kg; for concentrate, the average daily consumption per animal was either 1.88 kg (early lactation, up to 60 d, see Guzmán et al. [ 21 ]) or 1.81 kg (mid-lactation, up to 120 d) or 1.57 kg (late lactation, up to 180 d). After weaning the kids at 28–32 days old, the average total intake per goat was calculated for each group for early (for CD, DOP40 and DOP80, respectively: DM was 2.04, 2.00 and 1.98 kg/d; crude protein was 0.36, 0.33 and 0.36 kg/d; gross energy was 9.28, 8.90 and 8.74 Mcal/d), mid (for CD, DOP40 and DOP80, respectively: DM was 1.92, 1.92 and 1.89 kg/d; crude protein was 0.34, 0.32 and 0.35 kg/d; gross energy was 8.76, 8.53 and 8.35 Mcal/d) and late lactation (for CD, DOP40 and DOP80, respectively: DM was 1.78, 1.76 and 1.75 kg/d, crude protein was 0.32, 0.29 and 0.32 kg/d, and gross energy was 8.14, 7.83 and 7.75 Mcal/d). After weaning the kids, the animals began being milked once a day (at 09:00 a.m.) in a 12-stall Casse system milking parlor. Coinciding with the test-day milk yield recordings, representative samples from each animal (50 mL aliquots placed in plastic bottles) were taken from the volumetric flask at early lactation (55 ± 5 [ 21 ]), mid-lactation (120 ± 5), and late lactation (180 ± 5) days post-partum. The aliquots were frozen at − 20 ◦ C until analysis, except for the samples for vitamin analysis, which were frozen at −80 ◦C. 2.2. Feed and Milk Chemical Analyses A sample of diets (hay and concentrates) at each lactation stage was prepared for analysis by mixing equal amounts of subsamples collected throughout the lactation and storing them at 4 ◦ C. Before analysis, the samples were dried and ground using a Wiley mill with a 1-mm screen. This sampling approach was previously described by Guzmán et al. [ 21 ], with AOAC [ 23 ] methods being used to determine the dry matter, ash, N content, and ether extract; the analyses of neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were conducted according to Van Soest et al. [ 24 ], the gross energy (GE) content being determined using an adiabatic calorimeter, according to the manufacturer’s instructions, the forage unit for lactation (UFL) and protein digestible in the small intestine (PDI) being calculated using the Feed Ration Balancer software, the TAC being analyzed by the DPPH (2,2-diphenyl-l-picrylhydrazyl) assay as described by Shin et al. [ 25 ], the TPC content being estimated according to the procedure described by Seiquer et al. [ 26 ], with some modifications, and, finally, the methods described by Animals 2021,11, 2421 5 of 17 Delgado-Pertíñez et al. [ 27 ] and Gutiérrez-Peña et al. [ 28 ] being used to determine the FA profile and vitamin E ( α -tocopherol) content. The details for the analyses of TAC, TPC, FA, and vitamin E in feed samples can be found in the work of Guzmán et al. [21]. For the milk samples—as previously described by Guzmán et al. [ 21 ]—fat content was estimated using near-infrared spectroscopy (NIR). Fat extraction of milk and the direct methylation of FAs were performed in a single-step method developed by Sukhija and Palmquist [ 29 ] and revised by Juárez et al. [ 30 ] according to the work of DelgadoPertíñez et al. [ 27 ] and Gutiérrez-Peña et al. [ 28 ]. Separation and quantification of FA methyl esters (FAMEs) were carried out using a gas chromatograph (Agilent 6890N Network GS System, Agilent, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and automatic sample injector HP 7683, and fitted with an HP-88 J&W fused silica capillary column (100 m, 0.25 mm i.d., 0.2µ m film thickness; Agilent Technologies Spain, S.L., Madrid, Spain). Nonanoic acid methyl ester (C9:0 ME) was used as an internal standard (Sigma Aldrich Co., Madrid, Spain). Individual FAs were identified by comparing their retention times with those of the authenticated standard FA mix Supelco 37 (Sigma, Madrid, Spain). The CLA (conjugated linoleic acid) isomers (cis9, trans11, and trans10, cis12) were identified by comparing retention times with those of another authenticated standard (Matreya, LLC, Pleasant Gap, PA, USA). Fat-soluble vitamin (A and E) measurement of milk was based on procedures developed Herrero-Barbudo et al. [ 31 ] and ChauveauDuriot et al. [ 32 ] and modified by Gutiérrez-Peña et al. [ 28 ]. A chromatographic analysis was carried out on an Acquity UPLC, with a fluorometric detector, isocratic pump, PDA, and 150 × 2.1 mm Acquity UPLC HSS T3 1.8µ m column (Waters, Saint-Quentin-enYvelines, France). Tocopherols and retinol were positively identified by comparing their retention times with those of high purity standards of the measured substances (all-transretinol, α -tocopherol, β -tocopherol, and γ -tocopherol; Sigma, Madrid, Spain). Other standards of high purity (retinyl acetate, retinyl palmitate, and tocopheryl acetate; Sigma) were used as internal standards. TAC was determined using the ABTS (2,2 0 -azino-bis [3-ethylbenzothiazoline-6-sulphonic acid]) procedure developed by Fellegrini et al. [ 33 ] and modified by Delgado-Pertíñez et al. [ 27 ]. The milk sample was added to 1 mL of ABTS solution and incubated at 25 ◦ C for 10 min. Scavenging of the ABTS+ radical was monitored by the absorbance decrease at 730 nm. The water-soluble vitamin E analog Trolox was used as a standard, and TAC was expressed as mmol Trolox equivalents. Finally, TPC was analyzed according to the method described by Vázquez et al. [ 34 ] as modified by Guzmán et al. [ 21 ]. The phenolic compounds in the liquid extract were quantified at an absorbance of 750 nm using the Folin–Ciocalteu method, adapted to test tubes. Standard solutions of gallic acid (GA) were used to express the phenolic compounds as g of GA equivalents. 2.3. Data Treatment and Statistical Analysis The data for the milk composition of the goats was recorded at each lactation stage and analyzed according to the repeated measures procedure using IBM SPSS Statistics for Windows (version 26.0; IBM Corp., Armonk, NY, USA). The model included the fixed between-subjects factors of dietary treatment (CD, DOP40, or DOP80) and prolificacy (single or double birth) and the fixed within-subjects factor of lactation stage (repeated measures), as well as the interactions between these factors. For simplification, the results for the factor prolificacy have not been presented in this paper. Tukey’s Honest Significant Difference (HSD) test was used for pairwise means comparisons. Finally, Pearson correlation coefficients were calculated for some of the variables used. 3. Results 3.1. Fat-Soluble Vitamins, Phenolic Compounds, and Antioxidant Capacity Average milk fat yield and percentage were only affected by the lactation phase (Table 2). The percentage increase in fat between the early and late stages produced the highest values for the mid and late stages and the lowest values for the early stage; the Animals 2021,11, 2421 6 of 17 opposite pattern was observed for fat yield, with the highest value obtained at the early stage and the lowest values being detected at the mid and late stages. Table 2. Effects of experimental diets 1 and lactation phase on resultant milk’s fat-soluble vitamins, phenolic compounds, and antioxidant capacity. Item Diet (D) 1Lactation Phase (LP)) SEM p2 Control DOP40 DOP80 Early Mid Late D LP D ×LP Fat, % 4.11 4.19 3.85 3.72c 3.96b 4.52a 0.07 ns *** ns Fat yield, g/day 60.7 57.4 56.6 66.5a 54.7b 52.6b 2.16 ns ** ns Retinol, µg/100 g 4.84 6.64 6.07 3.09c 4.59b 9.11a 0.94 ns ** ns α -Tocopherol, µ g/100 g 27.1b 28.2b 45.2a 35.6a 25.1b 38.8a 2.07 ** ** ns Total phenolic compounds, mg gallic acid equivalents/L 46.3c 69.1b 97.0a 83.3a 75.7b 56.1c 2.73 *** *** *** Total antioxidant capacity, µmol trolox equivalents/mL 6..44c 9.39b 12.0a 10.1a 9.06b 8.61c 0.27 *** *** * Means with different letters (a, b, c) within each row differ significantly (p≤0.05); 1See Table 1;2ns, not significant (p> 0.05); *, p≤0.05; **, p< 0.01; ***, p< 0.001. The average content of all antioxidant parameters changed significantly between diets and lactation phases (Table 2), with the exception of retinol content, which was not affected by diet, although it did increase from the early to the late lactation phase (p< 0.01). The milk fat of goats fed the DOP80 diet presented higher values of α -tocopherol (45.2 µ g/100 g) than the other diets (21.1 and 28.2 µ g/100 for the CD and DOP40 diets) (p< 0.01). This vitamin was also affected by the lactation phase (p< 0.01), decreasing between the early and mid-phases and increasing thereafter. Significant effects of the main factors and interaction between them were observed in the content of TPC and TAC. The TPC and TAC content were higher for the DOP80 diet (97.0 mg GA equivalents/l; 12.0 µ mol Trolox equivalents/mL) than for the CD diet (46.3 mg GA equivalents/l; 6.44 µ mol Trolox equivalents/mL), while the DOP40 diet (69.1 mg GA equivalents/l; 9.39 µ mol Trolox equivalents/mL) presented an intermediate value between the other two (p< 0.001). Regarding the interaction between diet and lactation stage, the content of both parameters decreased (p< 0.001) from the early to the late lactation phase, with the exception of TPC for the DOP80 diet, which did not decrease during the late phase, and the TAC in the DC diet, which did not present any between-phase differences (p< 0.05). Finally, Figure 1 demonstrates the positive correlation between TAC and TPC (r = 0.78, p< 0.001) for all three diets and throughout lactation. 3.2. Fatty Acid Composition Significant effects of the main factors and interactions between them were observed for most individual FAs, FA groups, and FA indices, with differences between diets especially notable in the late stage (Table 3). Effects of the main factors were observed in the C10:0, C11:0, C12:0, and C20:0 content. Effects for only the lactation phase were observed in the content of C4:0, C13:0, C22:0, C22:2, C24:0, and total n-3, as well as impacting the AI index. Finally, no effects were observed in C14:1, C15:1, C17:0, C20:1 n9, C20:2, C20:3 n3, C20:5 n-3 (EPA), C21, C22:1 n9, C22:6 n-3 (DHA), C23:0 or C24:1 content. Animals 2021,11, 2421 7 of 17 Figure 1. Correlation between total antioxidant capacity (TAC) and total phenolic compounds (TPC) in milk derived from goats fed different diets and at different lactation stages. Table 3. Effects of experimental diets 1and lactation phase on fatty acid (FA) composition of milk. Item 3(g/100 g FA) Diet (D) 1Lactation Phase (LP)) SEM p2 Control DOP40 DOP80 Early Mid Late D LP D ×LP C4:0 3.65 3.48 3.57 3.60a 3.74a 3.34b 0.03 ns *** ns C6:0 4.85a 4.52b 4.67ab 4.78a 4.84a 4.39b 0.04 ** *** ** C8:0 4.17a 3.87b 4.00b 4.10a 4.15a 3.76b 0.03 *** *** * C10:0 9.86a,b 9.53b 10.13a 9.69b 10.62a 9.14c 0.08 ** *** ns C11:0 0.07a 0.06b 0.06b 0.07a 0.07a 0.06b 0.00 *** *** ns C12:0 5.58a 5.40b 5.39b 5.46a 5.59a 5.29b 0.04 * ** ns C13:0 0.06 0.06 0.06 0.06a 0.06a 0.05b 0.00 ns *** ns C14:0 7.30b 7.46a,b 7.54a 7.09b 6.99b 8.22a 0.06 * *** *** C14:1 0.19 0.19 0.19 0.19 0.19 0.19 0.00 ns ns ns C15:0 0.52 0.51 0.51 0.52 0.52 0.49 0.01 ns ns *** C15:1 0.03 0.03 0.03 0.03 0.03 0.03 0.00 ns ns ns C16:0 24.4b 25.1a 24.4b 24.6b 24.2c 25.3a 0.10 ** *** * C16:1 1.01 1.01 1.00 1.01 1.01 1.00 0.01 ns ns ** C17:0 0.33 0.35 0.32 0.32 0.32 0.35 0.00 ns ns ns C17:1 0.09b 0.11a 0.10a,b 0.09b 0.09b 0.12a 0.00 ** *** *** C18:0 11.4 11.3 10.9 11.5a 10.8b 11.2a,b 0.10 ns ** *** C18:1 n-9 trans 0.97a 0.91b 0.89b 0.94a 0.96a 0.85b 0.01 *** *** ** C18:1 n-11 trans (VA) 0.68 0.70 0.67 0.67 0.68 0.71 0.01 ns ns ** C18:1 n-9 cis 19.6b 20.0a,b 20.3a 19.9a,b 19.7b 20.3a 0.09 * * * C18:2 n-6 trans 0.15b 0.19a 0.17a 0.14b 0.15b 0.23a 0.01 ** *** *** C18:2 n-6 cis 3.20 3.30 3.23 3.31a 3.35a 3.08b 0.03 ns *** *** γ-C18:3 n-6 0.11a 0.10b 0.09b 0.09b 0.09b 0.12a 0.00 *** *** *** α-C18:3 n-3 0.21 0.22 0.22 0.21b 0.21b 0.23a 0.00 ns * ** CLA cis-9, trans-11 (RA) 0.69a 0.67a,b 0.65b 0.68a,b 0.69a 0.64b 0.01 * * ** CLA trans-10, cis-12 0.01 0.02 0.02 0.01b 0.01b 0.02a 0.00 ns *** *** C20:0 0.22a 0.20b 0.20b 0.21a 0.21a 0.19b 0.00 * * ns C20:1 n-9 0.04 0.04 0.04 0.04 0.04 0.04 0.00 ns ns ns C20:2 0.06 0.06 0.06 0.06 0.06 0.06 0.00 ns ns ns Animals 2021,11, 2421 8 of 17 Table 3. Cont. Item 3(g/100 g FA) Diet (D) 1Lactation Phase (LP)) SEM p2 Control DOP40 DOP80 Early Mid Late D LP D ×LP C20:3 n-3 0.02 0.03 0.02 0.03 0.03 0.03 0.00 ns ns ns C20:3 n-6 0.03 0.03 0.03 0.03 0.03 0.03 0.00 ns ns *** C20:4 n-6 0.20 0.20 0.20 0.19b 0.20b 0.21a 0.00 ns ** *** C20:5 n-3 0.03 0.03 0.03 0.03 0.03 0.03 0.00 ns ns ns C21:0 0.02 0.02 0.02 0.02 0.02 0.02 0.00 ns ns ns C22:0 0.11 0.11 0.10 0.12a 0.12a 0.09b 0.00 ns *** ns C22:1 n-9 0.02 0.02 0.02 0.02 0.02 0.02 0.00 ns ns ns C22:2 0.01 0.01 0.01 0.01b 0.01b 0.02a 0.00 ns *** ns C22:5 n-3 0.05 0.05 0.05 0.05 0.06 0.05 0.00 ns ns *** C22:6 n-3 0.02 0.02 0.02 0.02 0.02 0.02 0.00 ns ns ns C23:0 0.02 0.02 0.02 0.02 0.02 0.02 0.00 ns ns ns C24:0 0.02 0.02 0.02 0.02a 0.02a 0.01b 0.00 ns * ns C24:1 0.01 0.01 0.01 0.01 0.01 0.01 0.00 ns ns ns SFA 72.5a 72.0a,b 72.0b 72.2 72.3 72.0 0.09 * ns *** MUFA 22.6b 23.0a,b 23.2a 23.0a,b 22.7b 23.3a 0.08 * * * PUFA 4.82 4.94 4.81 4.87a,b 4.94a 4.77b 0.03 ns * *** SCFA 22.5a 21.4b 22.4a 22.2b 23.3a 20.6c 0.14 *** *** ** MCFA 39.6b 40.3a 39.6b 39.4b 39.1c 41.1a 0.13 ** *** ** LCFA 37.9 38.3 38.0 38.4a 37.6b 38.3a 0.11 ns ** *** n-3 0.34 0.34 0.34 0.33b 0.35a,b 0.36a 0.00 ns *** ns n-6 3.69 3.83 3.72 3.77a,b 3.82a 3.67b 0.03 ns * *** CLA total 0.71a 0.69a,b 0.67b 0.69a,b 0.70a 0.66b 0.01 * * *** n-6:n-3 10.8 11.2 10.9 11.5a 11.0b 10.4c 0.11 ns *** ** MUFA/SFA 0.31b 0.32a,b 0.33a 0.32 0.31 0.32 0.00 * ns ** PUFA/SFA 0.07 0.07 0.07 0.07 0.07 0.07 0.00 ns ns *** DI C16:0 0.04 0.04 0.04 0.04 0.04 0.04 0.00 ns ns ** DI C18:0 0.65b 0.66a,b 0.67a 0.65 0.66 0.66 0.00 ** ns *** CLA index 0.50 0.49 0.49 0.50a 0.50a 0.48b 0.00 ns * *** AI 2.16 2.16 2.14 2.10b 2.10b 2.27a 0.01 ns *** ns TI 3.02a 3.02a 2.94b 3.00b 2.92c 3.06a 0.02 * *** ns For each main effect type, means with different letters (a, b, c) within each row differ significantly (p ≤ 0.05); 1 See Table 1; 2 ns, not significant (p> 0.05); *, p ≤ 0.05; **, p< 0.01; ***, p< 0.001; 3 SCFA, short-chain FAs (C4:0-C10:0); MCFA, medium-chain FAs (C11:0-C17:1); LCFA, long-chain FAs (C18:0-C24:1); DI C16:0, desaturation index of C16:0 [C16:1 FAs/(C16:0 + C16:1 FAs)]; DI C18:0, desaturation index of C18:0 [C18:1 FAs/(C18:0 + C18:1 FAs)]; CLA desaturase index [rumenic acid, RA/(vaccenic acid, VA + RA)]; AI, atherogenic index [(C12:0 + 4 x C14:0 + C16:0)/(MUFA + PUFA)]; TI, thrombogenic index [(C14:0 + C16:0 + C18:0)/(0.5 x MUFA + 0.5 x n-6 PUFA + 3 x n-3 PUFA) + (n-3 PUFA/n-6 PUFA)]. With the exception of C4:0 content, which was not affected by diet, total and individual short-chain FAs (SCFA) in milk fat were significantly affected by this factor (p< 0.001; although the milk fat of goats fed the CD diet presented higher values than the DOP diets, the DOP80 diet did not differ significantly from the CD diet) and by the lactation phase (p< 0.001; content was generally lower in late lactation compared to early and midlactation) (Table 3). Additionally, C6:0 (p< 0.01), C8:0 (p< 0.05) and total SCFA (p< 0.01) demonstrated interaction between the two main factors (Tables 3and 4and Figure 2); thus, no significant differences were found between the diets during mid-lactation. Animals 2021,11, 2421 9 of 17 Table 4. Effect of interaction between dietary treatment 1 and lactation phase (early, mid, late) on the fatty acid (FA) composition of milk. Item (g/100 g FA) Early Mid Late Control DOP40 DOP80 Control DOP40 DOP80 Control DOP40 DOP80 C6:0 4.97a 4.57b,c 4.84a,b 4.90a,b 4.88a,b 4.72a,b,c 4.70a,b,c 4.10d 4.45c,d C8:0 4.28a 3.91b,c 4.15a,b 4.22a,b 4.19a,b 4.04a,b,c 4.02a,b,c 3.52d 3.82c,d C14:0 7.15c 7.10c 7.01c 7.01c 7.06c 6.89c 7.72b 8.18b 8.71a C15:0 0.49a.b 0.56a 0.50a,b 0.52a,b 0.51a,b 0.54a 0.55a 0.45b 0.49a,b C16:0 24.3b,c 25.2a,b 24.0c 24.3b,c 24.6a,b,c 23.6c 24.5a,b,c 25.5a 25.6a C16:1 0.95b 1.09a 0.98b 1.00a,b 0.99a.b 1.04a.b 1.08a 0.96b 0.97b C17:1 0.09c 0.08c 0.09c 0.09c 0.09c 0.09c 0.09c 0.15a 0.12b C18:0 11.0b,c,d 11.9a,b 11.6a,b,c 10.7c,d 10.6c,d 11.2b,c 12.4a 11.4a,b,c 10.0d C18:1 n-9 trans 0.97a 0.90a,b 0.96a 0.98a 0.97a 0.93a 0.95a 0.84b,c 0.78c C18:1 n-11 trans 0.70a,b 0.64b 0.66b 0.69a,b 0.69a,b 0.66b 0.66b 0.76a 0.69a,b C18:1 n-9 cis 19.8a,b,c 20.0a,b,c 20.0a,b,c 19.4b,c 19.3c 20.3a,b,c 19.5b,c 20.6a 20.5a,b C18:2 n-6 trans 0.15b 0.14b 0.15b 0.15b 0.15b 0.14b 0.15b 0.28a 0.23a C18:2 n-6 cis 3.45a 3.16a,b 3.35a,b 3.41a,b 3.38a,b 3.26a,b 2.73c 3.36a,b 3.07b,c γ-C18:3 n-6 0.08b,c 0.09b,c 0.08b,c 0.09b,c 0.09b,c 0.09b,c 0.16a 0.10b 0.11b,c α-C18:3 n-3 0.22a,b,c 0.20c 0.21b,c 0.22a,b,c 0.21b,c 0.21b,c 0.21b,c 0.24a 0.23a,b CLA cis-9, trans-11 0.65b,c 0.71a,b 0.68a,b 0.68a,b 0.68a,b 0.71a,b 0.74a 0.63b,c 0.57c CLA trans-10, cis-12 0.01b 0.01b 0.01b 0.01b 0.01b 0.01b 0.01b 0.02a 0.02a C20:3 n-6 0.03b 0.04a 0.03b 0.03b 0.03b 0.04a 0.04a 0.03b 0.03b C20:4 n-6 (ARA) 0.20b 0.18b 0.20b 0.20b 0.20b 0.19b 0.19b 0.22a 0.20b C22:5 n-3 (DPA) 0.04b 0.05a,b 0.04b 0.06a 0.06a 0.06a 0.06a 0.04b 0.05a,b n-6 3.92a,b 3.61b,c 3.81a,b 3.88a,b 3.86a,b 3.72a,b 3.28c 4.01a 3.64a,b,c CLA total 0.66b,c 0.72a,b 0.69a,b 0.70a,b 0.69a,b 0.72a,b 0.76a 0.65b,c 0.59c n-6:n-3 11.8a 11.2a,b 11.6a 11.1a,b 11.1a,b 10.8a,b 9.41c 11.3a,b 10.2b,c MUFA/SFA 0.32a,b 0.32a,b 0.32a,b 0.31b 0.31b 0.32a,b 0.31b 0.33a 0.33a PUFA/SFA 0.07a 0.07a 0.07a 0.07a 0.07a 0.07a 0.06b 0.07a 0.07a DI C16:0 0.04b 0.04b 0.04b 0.04b 0.04b 0.04b 0.05a 0.04b 0.04b DI C18:0 0.66a,b 0.64b,c 0.65b,c 0.66a,b 0.66a,b 0.66a,b 0.63c 0.66a,b 0.69a CLA index 0.48b,c 0.52a,b 0.50a,b 0.50a,b 0.50a,b 0.52a,b 0.53a 0.46c 0.45c Means with different letters (a, b, c, d) within each row differ significantly (p≤0.05). Significant effects of the main factors and interaction between them were observed in the C14:0, C16:0, and total medium-chain FAs (MCFA) of milk fat (Tables 3and 4and Figure 2). These effects were generally noted in early and mid rather than late lactation; in particular, the C16:0 content presented the most substantial difference between the diet treatments (generally, the milk fat from goats fed the DOP80 diet produced lower values than the other two groups). Opposite patterns were observed for the other MCFA, which was generally lower in the DOP diets compared to the CD diet and lower in the late stage compared to the early and mid-stages (Table 3). 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