Using dried orange pulp in the diet of dairy goats: effects on milk yield and composition and blood parameters of dams and growth performance and carcass quality of kids

Using dried orange pulp in the diet of dairy goats: effects on milk yield and composition and blood parameters of dams and growth performance and carcass quality of kids J. L. Guzmán1, A. Perez-Ecija2, L. A. Zarazaga1, A. I. Martín-García3, A. Horcada4and M. Delgado-Pertí˜nez4† 1Departamento de Ciencias Agroforestales, Escuela Técnica Superior de Ingeniería, Universidad de Huelva, “Campus de Excelencia Internacional Agroalimentario, ceiA3”, Campus de la Rábida, 21819 Palos de la Frontera, Huelva, Spain; 2Departamento de Medicina y Cirugía Animal, Universidad de C ´ordoba, Campus Rabanales, 14104 C ´ordoba, Spain; 3Estaci´on Experimental del Zaidín (CSIC), Profesor Albareda 1, 18008 Granada, Spain; 4Departamento de Ciencias Agroforestales, Escuela Técnica Superior de Ingeniería Agron´omica, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain (Received 10 January 2020; Accepted 6 April 2020; First published online 5 May 2020) Although dried orange pulp ( DOP ) may conveniently replace cereals in ruminant diets, few studies have considered similar diet substitution for goats. We hypothesised that DOP could replace cereal-based concentrate in goat diets without detrimental effects on growth performance and carcass quality of suckling kids and milk performance and blood biochemical parameters of dams in early lactation. We also hypothesised that DOP substitution may increase the levels of antioxidants, such as phenolic compounds and vitamin E, in milk and improve its total antioxidant capacity ( TAC) . Therefore, 44 primiparous Payoya dairy goats were allocated to three experimental groups, each fed a different diet: control (CD, n=14) based on a commercial concentrate with alfalfa hay as forage; and DOP40 (n=16) in which 40% and DOP80 (n=14) in which 80% of the cereal in the concentrate were replaced by DOP. The experiment lasted from the final month of pregnancy to 55 days postpartum. The DOP diets did not affect suckling kids’carcass quality, but at 28 days, led to improvement in live weight ( LW ) and average daily gain ( ADG ) from birth, although no differences were found between DOP40 and DOP80 (for CD, DOP40 and DOP80, LW at 28 days was 8.00, 8.58 and 8.34 kg and ADG was 184, 199 and 195 g/day, respectively). Diet had no significant effect on milk yield (average daily milk yield and total yield at 55 days were 1.66 l/day and 90.6 l, respectively) and commercial and fatty acid composition. Nevertheless, α-tocopherol, total phenolic compound ( TPC ) and TAC concentration in milk increased with substitution of cereals by DOP (for CD, DOP40 and DOP80, concentration of α-tocopherol was 21.7, 32.8 and 42.3 μg/100 g, TPCs was 63.5, 84.1 and 102 mg gallic acid equivalents/l, and TAC was 6.63, 11.1 and 12.8 μmol Trolox equivalents/ml, respectively). Every plasma biochemistry parameter considered was within reference values for healthy goats; therefore, no pathological effect was detected for these variables due to dietary treatment. However, DOP diets caused a reduction in plasmatic creatine kinase and aspartate aminotransferase, implying reduced oxidative damage to muscles. In conclusion, DOP may be an interesting alternative to cereals in early lactation goat diets for increasing farmers’income and the healthy antioxidant capacity of milk. Keywords: Payoya breed, citrus by-product, fatty acids, phenolic compounds, fat-soluble vitamins Implications Although dried orange pulp (DOP) may conveniently replace conventional cereals in ruminant diets, few studies have considered similar diet substitution for dairy goats. Our results show that DOP could replace cereals in goat diets without detrimental effects on suckling kid growth and dam milk performance and blood parameters. DOP increases phenolic and vitamin E content in milk. These findings may be used to improve goat production with the aim of increasing farmers’ income and the healthy antioxidant capacity of milk. Introduction In recent decades, goat farms have specialised in milk production for the cheese industry, particularly in economically developed countries in Mediterranean Europe (Castel et al., 2011). In the European Union, Spain has the second highest number of goats (3.1 million head), with Andalusia (in southern Spain) being the region with the largest herd †E-mail: [email protected] Animal (2020), 14:10, pp 2212–2220 © The Author(s), 2020. Published by Cambridge University Press on behalf of The Animal Consortium doi:10.1017/S1751731120000932 animal 2212 (FAO, 2017). Although most of the goats in Spain are dairy breeds, such as Payoya, goat farms produce suckling kids (the preferred goat carcass) which are exclusively fed with milk from their mothers for a month until they are slaughtered at live weight (LW) of 8 to 10 kg (Guzmán et al.,2019). To achieve greater milk yields, farmers have increased the supply of concentrates fed to goats, thus reducing or even eliminating pasture as a feed source. The demand for cereals and other grains for animal feeding increases the price of concentrate (Castel et al.,2011). This justifies the need to decrease the alimentation cost of livestock by developing strategies, such as greater dependence on local feed resources, which would increase the sustainability of livestock production systems (L´opez et al.,2014). In 2017, Spain was the leading producer of oranges in Europe (3.4 million tonnes; FAO, 2017). The orange juice industry produces organic waste such as dried orange pellets that can be used for animal feeding as a source of dietary energy (Bampidis and Robinson, 2006). Furthermore, citrus pulp can be a source of residual bioactive molecules with antioxidant properties, including phenolic compounds (Santos et al.,2014; Luciano et al.,2017) and vitamin E (Luciano et al.,2017). Replacing cereal grain (Bueno et al., 2002; Oni et al., 2008) with dehydrated citrus pulp (DCP) improved the post-weaning growth performance of kids. Nevertheless, research on the effects of citrus by-products in the diet on the growth and carcass quality of suckling kids is lacking. Further, studies involving the dietary replacement of starch sources by DCP and its effect on milk yield and composition for dairy goats are scarce. When cereal grain (L´opez et al., 2014; Ibá˜nez et al., 2016) was completely replaced by DCP in goat feed, the intake, digestibility of most nutrients, milk yield and commercial composition were not affected. With respect to the milk fatty acid (FA) profile of goat-fed orange pulp, only the study of Ibá˜nez et al.(2016) is known and their results showed that the FA profile was barely altered by the treatment. Furthermore, the effects of DCP on milk antioxidants and blood metabolites are limited to dairy cows, while no reports are available for dairy goats. In this respect, the addition of DCP to cows’diets increased the total polyphenol and flavonoid concentration in milk (Santos et al., 2014) and no significant differences were observed in most blood parameters (Belibasakis and Tsirgogianni, 1996). We hypothesised that the partial replacement of cereals by DOP in the concentrated fraction of the dairy goat diet would not have detrimental effects on milk production or composition, or the performance of suckling kids, and could even improve the antioxidant status of milk by increasing compounds, such as phenolics and vitamin E. Therefore, this study evaluated the effects of replacement of cereal by DOP (as a natural antioxidant source) in the diet of dairy goats in early lactation on suckling kid growth performance and carcass quality and dam milk yield, milk quality and blood metabolites. Material and methods Experimental diets, goats and chemical analyses of feed This study was carried out at the experimental farm of the University of Huelva (Huelva, Spain). All goats included in the study were of the Payoya breed. All procedures were performed by trained personnel in strict accordance with Spanish guidelines for the protection of experimental animals (RD 53/2013). Forty-four primiparous goats were assigned to three experimental groups, each with a different diet; the groups were balanced according to the goats’LW and body condition scores (BCSs). The animals were housed in a communal pen and each experimental group was separated by metal barriers with free access to a yard. The three experimental diets were as follows: control (CD, n=14), with a commercial concentrate plus alfalfa hay as forage; DOP40 (n=16) based on CD, but with 40% of the cereals in the concentrate replaced by DOP; and DOP80 (n=14), based on CD, with 80% of the cereals in the concentrate replaced by DOP. From the beginning of the third month of gestation, DOP was gradually introduced into the diet of the animals in the DOP40 and DOP80 groups. During the last month of pregnancy, each group of animals received its corresponding experimental prepartum-restricted diet (Supplementary Table S1), with a 60:40 concentrate to forage ratio. After parturition, the animals were offered the experimental diets adapted to early lactation (Table 1), which consisted of a concentrate to forage ratio of 80:20. The formulation of the rations and the chemical composition of the isoenergetic and isoproteic diets are shown in Supplementary Table S1 and Table 1. The nannies followed these nutrition regimens, which were designed using the Feed Ration Balancer (Format Solutions) software, version 2.0 (2017; Cargill, Inc., USA; www.formatsolutions.com). Goats were allowed ad libitum access to water and fed once daily in the mornings. After delivery, all goats received 0.4 kg of alfalfa hay daily and the amount of concentrate per animal increased every day, adjusting the amount supplied based on the real capability of consumption (from 0.70 kg/animal/day, after delivery, to 1.88 kg/animal/day at 55 days postpartum). Food intake for each group was calculated daily by subtracting the orts (uneaten food) from the amount of food offered every day. On average, goats were fed 0.4 kg of alfalfa hay and 1.67 kg (during the suckling period) or 1.88 kg (after weaning the kids) of concentrate per animal on a daily basis. After the weaning, the total average intake per goat in the diet groups was determined (DM was 2.04, 2.00 and 1.98 kg/day, CP was 0.40, 0.37 and 0.40 kg/day, and gross energy (GE) was 9.25, 8.91 and 8.71 Mcal/day in the CD, DOP40 and DOP80 groups, respectively). After weaning, and coinciding with the test-day milk yield recordings (at 45 ± 5 days and at 55 ± 5 days postpartum), LW and BCS were recorded for all animals. The BCS was always tested by the same handler. Using orange pulp in early lactation diet of goats 2213 A sample of hay and concentrates were prepared for analysis by mixing equal amounts of five subsamples collected during the trial and storing at −4°C. The samples were dried and ground using a Wiley mill with a 1-mm screen before analysis. AOAC (2005) methods were used to determine DM (method 934.01), ash (method 942.05), ether extract (method 920.39) and N (method 984.13) content in the samples of hay and concentrates. Total N was determined by the Kjeldahl procedure and converted to CP by multiplying by a factor of 6.25. The analyses of NDF and ADF were carried out according to Van Soest et al. (1991). Amylase was not used in the analysis of NDF, and both NDF and ADF were expressed as exclusive of residual ash. Acid detergent lignin was determined through solubilisation of cellulose with 72% sulphuric acid. All fibre fractions were analysed with a Fibertec 1030 Hot Extractor (Foss Tecator AB, Höganäs, Sweden). Fat content was measured by extraction with petroleum ether (boiling point, 40°C to 60°C) on a Soxtec System 1040 Extraction Unit (Foss Tecator AB). Ca and P content was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES Jobin Yvon Ultima 2, Longjumeau, France), according to the manufacturer’s instructions. The GE content of diets was determined using an adiabatic calorimeter (model AC600, LECO Corporation, St. Joseph, MI, USA), according to the manufacturer’s instructions. Sugar, starch, the forage unit for lactation (UFL) and protein digestible in the small intestine (PDI) were calculated using Feed Ration Balancer software. Total antioxidant capacity (TAC) was analysed by the DPPH (2,2-diphenyl-l- picrylhydrazyl) assay as described by Shin et al. (2018). The water-soluble vitamin E analogue Trolox was used as a standard for antioxidant capacity, and TAC was expressed as mmol Trolox equivalents/kg of sample. Total phenolic compound (TPC) content was estimated in methanolacetone extracts from feedstuffs and a subsequent analysis with Folin–Ciocalteu reagent, according to the procedure described by Seiquer et al. (2015), with some modifications. Standard solutions of gallic acid (GA) were used to express the phenolic compounds as g of GA equivalents/kg of DM. The procedures described by Delgado-Pertí˜nez et al. (2013) and Gutiérrez-Pe˜na et al. (2018) were used for the measurement of FA composition (absolute values expressed as mg/g DM) and vitamin E (α-tocopherol) content in lactation diets (expressed as mg/kg DM). The details for TAC, TPC, FA and vitamin E analyses are given in Supplementary Material S1,S2,S3 and S4, respectively. Growth performance of kids After parturition, 63 kids of both sexes and goat prolificacy (single or double birth) were individually analysed in the study (n=20 each for CD and DOP80 groups; n=23 for DOP40 group). Kids remained with their dams from birth to weaning (at 28 days), with free constant access to mothers’milk. The LW of kids was recorded at 0, 14 and Table 1 Ration ingredients and proximate and fatty acid (FA) composition of the experimental diets used to feed goats during early lactation Items Early lactation experimental diets1 Control DOP40 DOP80 Ration ingredients, % DM basis Alfalfa hay 17.4 17.5 17.6 Concentrate Dried orange pulp (pellets) 0 20.0 40.0 Grain oats 22.1 13.2 4.38 Grain barley 8.53 5.11 1.70 Grain corn 19.3 11.6 3.89 Soy flour, 44% 7.31 10.2 13.0 Sunflower pellets, 28% 12.8 12.5 13.8 Grain peas 10.3 8.12 4.05 Salt 0.41 0.41 0.41 Stabilised lard 0.41 0.00 0.00 Vitamins and minerals21.31 1.31 1.32 Proximate composition and nutritive value, % DM DM, % 90.5 89.8 90.0 CP 17.8 16.6 18.4 NDF 24.3 22.6 25.4 ADF 12.3 15.9 19.0 ADL 4.14 4.19 5.45 Sugar and starch 39.3 31.4 22.4 Ether extract 3.42 2.31 1.71 Ash 4.82 6.44 7.50 Ca 0.42 0.80 1.02 P 0.39 0.34 0.37 Gross energy, kcal/g DM 4.56 4.44 4.42 Forage unit for lactation, UFL/kg 0.98 0.98 0.97 Protein digestible in the intestine (PDI) 10.5 11.0 11.5 Total phenolic compounds, g/kg DM 5.29 6.74 8.57 TAC3, mmol Trolox®equivalents/kg DM 14.9 20.4 26.3 α-tocopherol, mg/kg DM 3.18 26.5 70.1 FA composition, mg/g DM C8:0–C14 1.54 3.12 4.54 C16:0 31.4 23.6 26.3 C16:1 1.04 0.63 0.62 C18:0 11.8 9.70 10.7 C18:1 n-9 cis 34.1 23.3 19.3 C18:2 n-6 cis 48.3 34.8 28.2 C18:3 n-6 0.18 0.18 0.44 C18:3 n-3 2.92 3.81 4.12 ∑SFA 44.7 36.4 41.5 ∑MUFA 35.1 24.0 19.9 ∑PUFA 51.4 38.8 32.8 ∑n-6 48.5 35.0 28.7 ∑n-3 2.92 3.81 4.12 n-6/n-3 16.6 9.17 6.97 SFA =saturated FA; MUFA =monounsaturated FA; PUFA =polyunsaturated FA. 1Control, diet based on commercial concentrates plus alfalfa hay; DOP40, diet based on concentrate with 40% of cereals replaced by dried orange pulp (DOP) plus alfalfa hay; DOP80, diet based on concentrate with 80% of cereals replaced by DOP plus alfalfa hay. 2Nutral cabras LD granulado, Cargill®, Spain. 3TAC, total antioxidant capacity by DPPH (2,2-diphenyl-l-picrylhydrazyl) assay. Guzmán, Perez-Ecija, Zarazaga, Martín-García, Horcada and Delgado-Pertí˜nez 2214 28 days after birth for all groups, and the average daily gain (ADG) was calculated. Slaughter and carcass quality Thirty male suckling kids were used for the carcass study. Ten male suckling kids from each experimental group were selected and slaughtered at an average weight of 8.90 (±0.17) kg and 32 (±1.5) days old. All kids were only fed with maternal milk until slaughter. The chemical composition and energy content of mother’s milk are shown in Table 2. After the kids reached their target commercial LW, the farm LW was recorded on the day prior to slaughter. Suckling kids were sacrificed at the slaughterhouse, after 13.5 ±0.02 h of fasting with free access to water, using standard commercial procedures in accordance with the guidelines of EU Directive 2010/63/EU for the protection of animals used for experimental and other scientific purposes. Slaughter LW was recorded immediately prior to slaughter. The tail, kidneys, pelvic fat and testes were retained in the carcass, and the hot carcass weight was recorded immediately after slaughter. The carcasses were kept in a chilling room at 4°C for 24 h. After chilling, the cold carcass weight was recorded. The carcass dressing percentage, chilling losses, carcass linear measurements and the indices from these carcass conformation measurements were calculated as described in Guzmán et al. (2019). After chilling, the carcasses were split down the dorsal midline, and then the left half of each carcass was removed, weighed and transported under refrigeration to the University of Huelva. The carcass was then physically dissected into five prime cuts (shoulder, flank, neck, ribs and long leg). The prime cuts were weighed and grouped into three categories: extra (long leg and ribs), first (shoulder) and second (neck and flank). Shoulder tissue composition was evaluated according to Guzmán et al. (2019). The muscle/bone and muscle/fat indices were calculated. The details of carcass measurements are given in Supplementary Material S5. Milk sample collection and chemical analyses After weaning the kids, the dams were milked once a day (at 0900 h) in a 12-stall Casse system milking parlour; individual recorder jars were used to measure the volume of milk produced. A repeated measures design was used for recording milk yield from each animal; test-day yields were recorded at 45 ±5 days and 55 ±5 days postpartum, and the total production in early lactation (from birth to 55 days postpartum) was calculated according to the A4 method of the International Committee for Animal Recording. The following formula was used to compute milk yield (MY) for the lactation record: MY ¼I0M1 þI1 M1 þM2ðÞ½=2; where M1 and M2 are the volumes (in litres) of milk yielded in the 24 h of the recording day, I0 is the interval, in days, between the lactation period start date and the first recording date, and I1 is the interval, in days, between recording dates. During the suckling period, representative samples (aliquots from each animal for milk traits and TAC analyses) were taken between the third and fourth week postpartum. After 45 days postpartum, representative samples (aliquots from each animal for milk traits, TAC, TPC, FA and fatsoluble vitamin analyses) were taken from the volumetric flask of the parlour during machine milking carried out at the control. The aliquots were placed in 50 ml plastic bottles and wrapped with aluminium foil to protect them from light. Azidiol was used as a preservative in one of the samples used to analyse commercial traits. Samples were deposited in iceboxes, sent to the laboratory and frozen at −20 ºC until analysis, except for the samples for vitamin analysis which were frozen at −80 ºC. Milk traits (DM, protein, fat and lactose content) were estimated by near-IR (NIR) spectroscopy according to Delgado-Pertí˜ nez et al. (2013). The procedures described by Delgado-Pertí˜nez et al. (2013) and Gutiérrez-Pe˜na et al. (2018) were used to measure FA composition (absolute values Table 2 Effects of experimental diets1and goat prolificacy on the chemical composition of milk during the natural suckling period Item Diet (D)1Prolificacy (PR) SEM P2 Control DOP40 DOP80 Single Twins D PR No. of goats 12 12 12 17 19 DM, % 12.4 12.2 12.2 12.1 12.3 0.32 ns ns CP, % 4.02b4.84a3.94b4.58 3.99 0.14 ** * Fat, % 4.92 5.09 4.29 4.77 4.76 0.21 ns ns Lactose, % 5.63 5.54 5.35 5.50 5.51 0.16 ns ns TAC3,μmol Trolox®equivalents/ml 7.25c9.38b13.1a9.25 10.5 0.49 *** ns ME4, MJ/kg 3.45 3.52 3.20 3.39 3.39 0.08 ns ns a,b,cMeans with different letters within each row differ significantly (P≤0.05). 1Control , diet based on commercial concentrates plus alfalfa hay; DOP40, diet based on concentrate with 40% of cereals replaced by dried orange pulp (DOP) plus alfalfa hay; DOP80, diet based on concentrate with 80% of cereals replaced by DOP plus alfalfa hay. 2Statistical probability for comparisons: ns, not significant (P>0.05); *P≤0.05; **P<0.01; ***P<0.001. No significant interactions between these factors were noted (P>0.05). 3Total antioxidant capacity (TAC) by ABTS (2,20-azinobis[3-ethylenbenzothiazoline-6-sulphonic acid]) assay. 4The metabolisable energy (ME) concentration was estimated according to Nsahlai et al. (2004). Using orange pulp in early lactation diet of goats 2215 expressed as mg/g DM); the intra- and inter-assay CVs for this analysis were less than 7% and 10%, respectively. Fat extraction and direct methylation of FAs were performed in a single-step procedure based on the method by Sukhija and Palmquist (1988)andrevisedbyJuárezet al. (2008). Fat-soluble vitamin (A and E) analyses (expressed as μg/100 g) were based on the methods by Herrero-Barbudo et al. (2005) and Chauveau-Duriot et al. (2010) and modified by Gutiérrez- Pe˜na et al. (2018); the intra- and inter-assay CVs were less than 3% and 6%, respectively. Total antioxidant capacity was analysed by the ABTS method (2,20-azino- bis[3-ethylbenzothiazoline-6-sulphonic acid]) of Pellegrini et al. (1999) and modified by Delgado-Pertí˜nez et al. (2013). Total antioxidant capacity was expressed as mmol vitamin E analogue Trolox equivalents/ml of sample; the intra- and inter-assay CVs were less than 4% and 6%, respectively. Total phenolic compounds were quantified according to the procedure described by Vázquez et al (2015)usingthe Folin–Ciocalteu method, with some modifications. The values were reported in mg of GA equivalents/l; the intra- and inter-assay CVs were less than 4% and 7%, respectively. The details for the analyses of NIR, FA, fat-soluble vitamins, TAC and TPC are given in Supplementary Material S6,S3, S4,S7 and S8, respectively. Blood analyses Biochemical analysis was performed during the last month of pregnancy and at 45 ±5 days postpartum. Blood was collected aseptically from the left jugular vein in fasting animals in the morning and poured into tubes containing lithium heparin. After centrifugation at 3500 r.p.m. for 30 min, the plasma was kept frozen at −20 ºC until analysis. Every parameter was measured in an automated spectrophotometer (Biosystems A25, Biosystems, Barcelona, Spain) using specific kits previously validated for small ruminants (Biosystems, Barcelona, Spain), according to the manufacturer’s instructions. The intra- and inter-assay CVs for all blood parameters were less than 3.5% and 6%, respectively. Data treatment and statistical analysis The data for the kids’growth were analysed using factorial ANOVA and a GLM, with birth weight as a covariate and dietary treatment (CD, DOP40 or DOP80), sex and the interactions between these factors as fixed effects. The carcass parameters were analysed using a one-way ANOVA model to test the effect of dietary treatment. The data for milk composition during the suckling period were analysed using a factorial model, with dietary treatment and goat prolificacy (single or double birth) and the interactions between these factors as fixed effects. The goats’weight, BCSs, commercial milk traits and daily milk yield recorded at 45 ±5 days and at 55 ±5 days postpartum were analysed with the repeated measures procedure. The model included the fixed betweensubjects factors dietary treatment and prolificacy, the fixed within-subjects factor week of lactation (repeated measures), and the interactions between these factors. For simplification, the results for the factor week of lactation in both repeated measures analyses have not been presented in this paper. The data for total milk yield from birth to 55 days postpartum and composition parameters of milk samples taken at 55 ±5 days postpartum (FA, vitamins, TAC and TPC) were analysed using a factorial model which included dietary treatment and prolificacy and the interactions between these factors as fixed effects. Plasma biochemical parameters at pregnancy and at 45 ±5 days postpartum were analysed using a factorial model which included dietary treatment and prolificacy and the interactions between these factors as fixed effects. In all the statistical analyses, the individual kids or goats were considered experimental units and the significance level was set at P≤0.05. Tukey’s honestly significant difference test was used where appropriate for pairwise comparisons of means. The statistical analyses were performed using IBM SPSS Statistics for Windows (version 25.0; IBM Corp., Armonk, NY, USA). Results Growth performance and carcass quality of kids No significant effects of diet treatment on milk components and metabolisable energy during the suckling period were observed, except for CP (P<0.01) and TAC (P<0.001; Table 2). Crude protein content was significantly higher in the milk of the DOP40 group (4.84%) than in the CD (4.02%) and DOP80 (3.94%) groups. Furthermore, TAC in the milk increased as the percentage substitution of cereals by DOP increased (7.25, 9.38 and 13.1 ±0.49 μmol Trolox® equivalents/ml in CD, DOP40 and DOP80 treatments, respectively). Diet treatment had a significant effect on the LW of kids at 28 days and on the postnatal growth rate from birth to 28 days (Table 3). The CD treatment showed lower values for both LW at 28 days and ADG from birth to 28 days (P<0.05), but no differences were found between DOP40 and DOP80 treatments (LW at 28 days was 8.00, 8.58 and 8.34 ±0.20 kg and ADG from birth to 28 days was 184, 199 and 195 ±4.3 g in CD, DOP40 and DOP80, respectively). Finally, replacement of cereals by DOP did not influence carcass weight and quality (P>0.05; Supplementary Tables S2 and S3). Yield and composition of milk in early lactation The milk production and composition and the LW and BCS of goats are presented in Table 4. Live weight and BCS were similar for all three diets (38.9 kg and 2.67 on average, respectively). Although milk yield was numerically higher in the DOP diet groups than in CD, this difference was not statistically significant (average daily milk yield and total yield at 55 days were 1.66 l/day and 90.6 l, respectively). Similar results were obtained for commercial composition, with the exception of DM (P<0.05). Milk DM percentage differed between DOP40 (12.4%) and DOP80 (11.9%), while it was an intermediate value for CD (12.2%) which did not Guzmán, Perez-Ecija, Zarazaga, Martín-García, Horcada and Delgado-Pertí˜nez 2216 differ significantly from the values for the DOP diets. The level of α-tocopherol (P<0.01), TPC (P<0.001) and TAC (P<0.001) in the milk increased as the rate of substitution of cereals by DOP increased (the concentration of α-tocopherol was 21.7, 32.8 and 42.3 ± 2.99 μg/100 g, TPC was 63.5, 84.1 and 102.4 ±2.94 mg GA equivalents/l, and TAC was 6.63, 11.1 and 12.8 ±0.53 μmol Trolox®equivalents/ml in CD, DOP40 and DOP80 treatments, respectively). The individual FA concentrations and the sum of FAs (mg/g DM) are shown in Supplementary Table S4. The FA profile was not changed by different diets for any of the variables examined (P>0.05). Clinical blood parameters Biochemistry results showed no significant differences in most blood parameters between the diet groups (Table 5). However, both DOP40 and DOP80 showed significantly lower (P<0.05) plasma glucose compared to CD (a 21% and 19% reduction, respectively, at early lactation, and a 16% reduction for both groups at the end of gestation). Creatine kinase (CK) and aspartate aminotransferase (AST) values were significantly lower (P<0.05) in the DOP40 and DOP80 groups compared to those in CD at the end of pregnancy (a 50% and 31% reduction for CK and a 31% and 24% reduction for AST, respectively). The total Ca Table 3 Effects of the experimental diet1of goats and kids’sex on the growth performance of naturally suckling kids Item Diet (D)1Sex (S) SEM P2 Control DOP40 DOP80 Female Male D S No. of kids 20 23 20 33 30 Live weight, kg Birth 2.84 3.06 2.95 2.92 2.99 0.11 ns ns 14 day 5.56 5.80 5.72 5.45 5.97 0.14 ns *** 28 day 8.00b8.58a8.34a7.93 8.76 0.20 * *** Average daily gain, g/day Birth to14 day 194 196 198 181 213 4.8 ns *** 14 to 28 day 174b206a194ab 177 209 5.5 ** *** Birth to–28 day 184b199a195a179 209 4.3 * *** a,bMeans with different letters within each row differ significantly (P≤0.05). 1Control, diet based on commercial concentrates plus alfalfa hay; DOP40, diet based on concentrate with 40% of cereals replaced by dried orange pulp (DOP) plus alfalfa hay; DOP80, diet based on concentrate with 80% of cereals replaced by DOP plus alfalfa hay. 2Statistical probability for comparisons: ns, not significant (P>0.05); *P≤0.05; **P<0.01; ***P<0.001. No significant interactions between these factors were noted (P>0.05). Table 4 Effects of experimental diets1and prolificacy on the early (0 to 55 days) lactation performance of goats Item2 Diet (D)1Prolificacy (PR) SEM P3 Control DOP40 DOP80 Single Twins D PR D ×PR No. of goats214 16 14 23 21 Live weight, kg 38.0 39.3 39.3 38.9 38.9 0.65 ns ns ns Body condition score 2.74 2.63 2.64 2.73 2.60 0.03 ns ns ns Daily milk yield, l/day 1.62 1.68 1.67 1.47 1.85 0.06 ns ** ns Milk yield (55 day), l 84.7 92.7 93.7 80.9 100.7 4.1 ns * ns DM, % 12.2ab 12.4a11.9b12.2 12.2 0.09 * ns ns CP, % 2.94 3.07 2.94 2.96 3.01 0.03 ns ns ns CP yield, g/day 50.0 53.1 53.7 49.5 57.5 3.10 ns * ns Fat, % 3.78 3.84 3.53 3.66 3.77 0.06 ns ns ns Fat yield, g/day 64.1 66.5 65.0 59.5 72.0 4.26 ns * ns Lactose, % 4.64 4.72 4.64 4.71 4.63 0.03 ns ns ns Lactose yield, g/day 78.4 82.0 81.5 75.5 88.3 4.74 ns * ns Retinol, μg/100 g 2.46 1.92 2.56 2.43 2.15 0.28 ns ns ns α-Tocopherol, μg/100 g 21.7b32.8ab 42.3a35.4 29.7 2.92 ** ns ns Total phenolic compounds, mg GA equivalents/l 63.5c84.0b102.3a80.6 86.2 2.94 *** ns ns TAC4,μmol Trolox®equivalents/ml 6.63c11.1b12.8a9.62 10.7 0.53 *** ns * GA, gallic acid. a,b,cMeans with different letters within each row differ significantly (P≤0.05). 1Control, diet based on commercial concentrates plus alfalfa hay; DOP40, diet based on concentrate with 40% of cereals replaced by dried orange pulp (DOP) plus alfalfa hay; DOP80, diet based on concentrate with 80% of cereals replaced by DOP plus alfalfa hay. 2For vitamins, total antioxidant capacity (TAC) and total phenolic compound analyses, n=12 in each group. 3Statistical probability for comparisons: ns, not significant (P>0.05); *P≤0.05; **P<0.01; ***P<0.001. 4TAC by ABTS (2,20-azinobis[3-ethylenbenzothiazoline-6-sulphonic acid]) assay. Using orange pulp in early lactation diet of goats 2217 concentration during early lactation also showed significant differences (P<0.05) between groups, with higher values in DOP80 compared to CD and DOP40. Mg levels were significantly different (P<0.05) between groups at the end of pregnancy, with the highest level in CD and the lowest in DOP80. Discussion Growth performance and carcass traits of kids Dried orange pulp can replace cereals in the diet of nannies, without depressing the growth performance and carcass quality of their suckling kids, as well as improving ADG and LW at 28 days of life. Growth essentially depends on ingested energy and nevertheless was not evaluated in this study. Although no significant effects of diet treatment on milk components and metabolisable energy were observed, the incorporation of DOP into the diet could be expected to influence energy intake. Indeed, milk production in DOP diets is about 10% higher (although not significant; see Table 4) than in CD goats, which is the same range as the difference in kids’ADG, and could explain our results, at least partially. Furthermore, and in light of the results for the antioxidant status parameters measured in milk during the suckling period (Table 2) and after weaning (Table 4), the kids’growth might also be attributable to antioxidants and redox homeostasis (McGrath et al.,2018). Antioxidant supplementation may help to counter negative effects of oxidative stress associated with growth in young ruminants, reducing the incidence of morbidity and mortality, and ultimately improving their performance (McGrath et al., 2018). In this study, partial replacement of cereals by DOP in the goats’feed increased TAC, TPC and α-tocopherol in the diet (see Table 1) and, consequently, in milk (see Table 4). To our knowledge, no previous studies have reported the effect of feeding DOP to small ruminants on the concentration of these antioxidants in their milk. However, the addition of 9% and 18% of pelleted citrus pulp to normal diets increases the total polyphenol and flavonoid concentration, and ferric reducing antioxidant power in cows’milk (Santos et al.,2014). Vitamin E has been extensively demonstrated to play a major role in protecting animal tissue against lipid peroxidation. Citrus phenolic compounds, mainly flavonoids, possess antioxidant activity, and their intake with the diet has been suggested to increase the antioxidant capacity of animal tissue. However, the possible mechanisms for the antioxidant effects are not clear (Luciano et al.,2017). Indeed, Luciano et al. (2017) reported that despite the fact that flavonoids are generally considered the main antioxidants in citrus fruits, α-tocopherol plays the major role in improving antioxidant capacity of muscle of lambs fed diets supplemented with DCP. Further research should undoubtedly clarify the antioxidant effect of dietary orange pulp in ruminants. Table 5 Effects of experimental diets1on blood biochemical parameters of goats in late (120 to 150 days) gestation and early (0 to 55 days) lactation Parameter (units)2 Early lactation1,3 SEM Late gestation1,3 SEMControl DOP40 DOP80 Control DOP40 DOP80 Total proteins, g/l 76.4 78.3 75.6 1.2 69.1 68.1 67.6 1.4 Albumin, g/l 30.1 32.1 31.4 1.0 30.4 31.2 30.3 1.0 Triglycerides, mmol/l 0.26 0.32 0.29 0.02 0.42 0.34 0.33 0.02 Cholesterol, mmol/l 2.03 2.07 1.76 0.09 1.90 1.75 1.83 0.10 Glucose, mmol/l 3.95a3.15b3.23b0.12 3.54a3.00b3.00b0.15 Urea nitrogen, mmol/l 6.07 5.14 4.53 0.28 4.57 4.58 4.67 0.22 Creatinine, μmol/l 57.4 54.8 54.8 2.5 58.3 60.1 61.8 2.4 Total bilirubin, μmol/l 3.77 4.95 4.44 0.35 4.19 5.39 5.30 0.39 Alanine aminotransferase, IU/l 12.9 12.1 14.6 1.5 13.1 9.6 13.6 1.2 Aspartate aminotransferase, IU/l 99.3 93.3 87.8 4.1 98.6a68.5b75.2b3.0 Alkaline phosphatase, IU/l 80.4 231.5 78.6 30.1 172.8 216.4 127.2 33.3 Creatine kinase, IU/l 178.9 212.2 149.2 21.0 273.4a137.0c191.3b18.7 Amylase, IU/l 53.6 62.9 35.6 11.1 40.4 32.5 36.0 3.0 P, mmol/l 2.70 2.49 2.03 0.19 2.22 2.48 2.23 0.13 Total Ca, mmol/l 2.17b1.82b2.75a0.09 2.59 2.24 2.42 0.11 Mg, mmol/l 1.22 1.18 1.44 0.09 1.43a1.04c1.26b0.06 Na, mmol/l 147.3 149.1 150.9 1.5 149.7 144.9 145.7 0.6 K, mmol/l 4.59 4.59 4.96 0.10 5.40 5.52 5.30 0.15 Cl, mmol/l 108.6 107.4 109.6 0.9 111.1 109.0 108.9 0.6 Fe, μmol/l 23.1 23.2 24.5 1.6 22.9 25.2 22.15 2.0 a,b,cMeans with different letters () within each row differ significantly (P≤0.05). 1Control, diet based on commercial concentrates plus alfalfa hay; DOP40, diet based on concentrate with 40% of cereals replaced by dried orange pulp (DOP) plus alfalfa hay; DOP80, diet based on concentrate with 80% of cereals replaced by DOP plus alfalfa hay. 2For every blood parameter analysis, n=10 in each group. 3No effect of prolificacy or diet ×prolificacy interaction was observed for any variable examined (P>0.05). Guzmán, Perez-Ecija, Zarazaga, Martín-García, Horcada and Delgado-Pertí˜nez 2218 Milk yield and composition In this study, individual intake was not measured, but the average intake per animal obtained indirectly through the consumption of the group shows similar values in the different dietary treatments. Similarly, in L´opez et al. (2014), when total cereal (corn) was replaced by DCP for lactating Murciano–Granadina goats, the intake and apparent total tract digestibility of most nutrients were not affected. Incorporation of DCP (about 20% DM) in diets of dairy cows often depresses DM intake (DMI; Solomon et al., 2000; Santos et al.,2014), probably due to the increased rumen fill by hydration of DCP and the slightly lower rumen degradation rate of pectin than that of starch (Santos et al., 2014). Studies on inclusion of DCP to replace cereals in lactating dairy ewe diets did not report a reduction in DMI (Fegeros et al.,1995; Santos-Silva et al.,2016), suggesting that the response of ewes and goats may be different from that of dairy cows. The lack of an effect of diet on LW and BCS and the similar intake values observed in the dietary treatments may be consistent with the lack of change observed in milk yield and commercial traits in the present study, as previouslyreported for DCP (L´opez et al.,2014; Ibá˜nez et al.,2016). With respect to the FA profile, Ibá˜nez et al. (2016) found that when barley was fully replaced by DCP for goats in mid-lactation, there was no effect on FAs with 4 to 15 C atoms, but there were significant effects on other FAs. Ibá˜nez et al. (2016) specifically highlighted the lower C15:0 and higher C17:0 FA content in the milk of goats fed DCP, suggesting a negative impact on rumen bacterial fermentation activity and fat mobilisation. However, in the present study, the FA composition of milk was not affected by replacement of cereals by DOP in the diet; these resultsare similar to those obtained by Fegeros et al. (1995) for lactating sheep fed concentrate rations with 30% DCP. As discussed above, DOP antioxidants in the present study were transferred from the diet to the milk, as previously reported by Santos et al. (2014) for cows fed citrus pulp. The result for α-tocopherol is rather unexpected, as there are no data currently for the effects of feeding DOP to ruminants on the concentration of α-tocopherol in milk and other dairy products. From a global perspective, our results are very promising. In recent years, foods containing natural antioxidants have become popular all over the world as antioxidants can neutralise and scavenge free radicals and reduce their harmful effects, which are continuously produced in the body (Khan et al.,2019). Clinical blood parameters All the values determined for plasma biochemistry were within reported reference values for healthy goats (Tschuor et al.,2008). Contrary to previous reports on dairy cows (Belibasakis and Tsirgogianni, 1996), dietary citrus pulp supplementation did not cause changes in the plasma levels of cholesterol in goats. Both DOP diets caused a significant decrease in plasma glucose compared to the CD group, as previously observed for calves fed with citrus by-products (Alnaimy et al.,2017). Although improved insulin sensitivity has been reported as a general benefit of diets containing Citrus spp. (Flores-Fernández et al.,2017), our lack of data on insulin concentrations makes further studies necessary to elucidate the importance of this finding. Interestingly, both CK and AST were significantly lower in supplemented groups at the end of pregnancy. Both enzymes are related to muscular damage and oxidative muscular stress (Brancaccio et al.,2010), which implies a beneficial impact of DOP supplementation under those conditions. The higher total Ca found during early lactation in the DOP80 group may be related to the effect of calcium oxide addition during citrus pellet preparation. Finally, Mg levels also differed between groups at the end of pregnancy, although a clear relationship between DOP content in the diet and the plasma levels of Mg ions does not seem straightforward. Conclusions Our data suggest that DOP can be an interesting alternative to cereals in the concentrate fraction of diets for dairy goats in early lactation. Replacement of 40% and 80% of cereals by DOP in the diet of Payoya dairy goats modifies growth performance of suckling kids, leading to an improvement in LW at 28 days and ADG from birth to 28 days, although no differences were found between the DOP40 and DOP80 treatments. The levels of α-tocopherol, TPC and TAC in milk increased as the percentage of pelleted orange pulp replacing cereals increased. DOP also caused lower plasmatic levels of CK and AST, which implies reduced muscular oxidative damage. Furthermore, DOP can be used as a cereal grain substitute without negatively affecting the carcass quality of suckling kids and the milk performance and health status of dairy goats. Acknowledgements The authors express special thanks to the Excma. Diputaci´on Provincial de Huelva for financial support through the Integrated Project ‘Aprovechamiento de Subproductos de las Empresas Agroalimentarias para Alimentaci´on del Ganado’. The authors also thank Cítricos del Andévalo, S.A. (J. García Carri´on) for supplying the orange pellets, OVIPOR, Soc. Coop. And. for their contribution to the preparation of diets, and the Servicio General de Investigaci´on Agraria (Universidad de Sevilla) and the Laboratorio Agroalimentario de Sevilla (Junta de Andalucía) for technical assistance with laboratory analysis. M. Delgado-Pertí˜nez 0000-0003-3589-5011 Declaration of interest The authors declare no conflict of interest. Ethics statement All experiments performed followed Spanish standards for the protection of animals used for scientific purposes. The Using orange pulp in early lactation diet of goats 2219 procedures applied were supervised by the Bioethics Committee of the University of Huelva (Spain). Software and data repository resources None of the data were deposited in an official repository. Supplementary material To view supplementary material for this article, please visit https://doi.org/10.1017/S1751731120000932 References AOAC 2005. Official methods of analysis of AOAC International, 18th edition. AOAC International Gaithersburg, MD, USA, Official Method 2005.08. Alnaimy A, Gad AE, Mustafa MM, Atta MAA and Basuony HAM 2017. Using of citrus by-products in farm animals feeding. Open Access Journal of Science 1, 58–67. Bampidis VA and Robinson PH 2006. Citrus by-products as ruminant feeds: a review. Animal Feed Science and Technology 128, 175–217. Belibasakis NG and Tsirgogianni D 1996. Effects of dried citrus pulp on milk yield, milk composition and blood components of dairy cows. Animal Feed Science and Technology 60, 87–92. Brancaccio P, Lippi G and Maffulli N 2010. Biochemical markers of muscular damage. Clinical Chemistry and Laboratory Medicine 48, 757–67. Bueno MS, Ferrari E, Bianchini D, Leinz FF and Rodrigues CFC 2002. Effect of replacing corn with dehydrated citrus pulp in diets of growing kids. Small Ruminant Research 46, 179–185. Castel JM, Mena Y, Ruiz FA, Camu˜nez-Ruiz J and Sánchez-Rodríguez M 2011. Changes occurring in dairy goat production systems in less favoured areas of Spain. Small Ruminant Research 96, 83–92. Chauveau-Duriot B, Doreau M, Nozière P and Graulet B 2010. Simultaneous quantification of carotenoids, retinol, and tocopherols in forages, bovine plasma, and milk: validation of a novel UPLC method. Analytical and Bioanalytical Chemistry 397, 777–790. Delgado-Pertí˜nez M, Gutiérrez-Pe˜na R, Mena Y, Fernández-Cabanás VM and Laberye D 2013. Milk production, fatty acid composition and vitamin E content of Payoya goats according to grazing level in summer on Mediterranean shrublands. Small Ruminant Research 114, 167–175. FAO 2017. FAOSTAT data. Retrieved on 4 September 2019 from http://www.fao. org/faostat/en/#data Fegeros K, Zervas G, Stamouli S and Apostolaki E 1995. Nutritive value of dried citrus pulp and its effect on milk yield and milk composition of lactating ewes. Journal of Dairy Science 78, 1116–1121. Flores-Fernández JM, Barragán-Álvarez CP, Díaz-Martínez NE, Villanueva- Rodríguez S and Padilla-Camberos E 2017. In vitro and in vivo postprandial glycemic activity of Citrus limetta peel flour. Pharmacognosy Magazine 13, 613–616. Gutiérrez-Pe˜na R, Fernández-Cabanás VM, Mena Y and Delgado-Pertí˜nez M 2018. Fatty acid profile and vitamins A and E contents of milk in goat farms under Mediterranean wood pastures as affected by grazing conditions and seasons. Journal of Food Composition and Analysis 72, 122–131. Guzmán JL, de la Vega F, Zarazaga LA, Argüello A and Delgado-Pertí˜nez M 2019. Carcass characteristics and meat quality of conventionally and organically reared suckling dairy goat kids of the Payoya breed. Annals of Animal Science 19, 1143–1159. Herrero-Barbudo MC, Granado-Lorencio F, Blanco-Navarro I and Olmedilla- Alonso B 2005. Retinol, α-and γ-tocopherol and carotenoids in natural and vitamin A- and E-fortified dairy products commercialized in Spain. International Dairy Journal 15, 521–526. Ibá˜nez C, Criscioni P, Arriaga H, Merino P, Espin´os FJ, Fernández C 2016. Murciano-Granadina goat performance and methane emission after replacing barley grain with fibrous by-products. PLoS ONE 11, 1–21. Juárez M, Polvillo O, Contò M, Ficco A, Ballico S and Failla S 2008. Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat. Journal of Chromatography A, 1190, 327–332. Khan IT, Nadeem M, Imran M, Ullah R, Ajmal M and Jaspal MH 2019. Antioxidant properties of Milk and dairy products: a comprehensive review of the current knowledge. Lipids in Health and Disease 18, 41–53. L´opez MC, Estellés F, Moya VJ and Fernández C 2014. Use of dry citrus pulp or soybean hulls as a replacement for corn grain in energy and nitrogen partitioning, methane emissions, and milk performance in lactating Murciano-Granadina goats. Journal of Dairy Science 97, 7821–7832. Luciano G, Roscini V, Mattioli S, Ruggeri S, Gravador RS, Natalello A, Lanza M, De Angelis A and Priolo A 2017. Vitamin E is the major contributor to the antioxidant capacity in lambs fed whole dried citrus pulp. Animal 11, 411–417. McGrath J, Duval SM, Tamassia LFM, Kindermann M, Stemmler RT, de Gouvea VN, Acedo TS, Immig I, Williams SN and Celi P 2018. Nutritional strategies in ruminants: a lifetime approach. Research in Veterinary Science 116, 28–39. Nsahlai IV, Goetsch AL, Luo J, Johnson ZB, Moore JE, Sahlu T, Ferrell CL, Galyean ML and Owens FN 2004. Metabolizable energy requirements of lactating goats. Small Ruminant Research 53, 253–273. Oni AO, Onwuka CFI, Oduguwa OO, Onifade OS and Arigbede OM 2008. Utilization of citrus pulp based diets and Enterolobium cyclocarpum (JACQ. GRISEB) foliage by West African dwarf goats. Livestock Science 117, 184–191. Pellegrini N, Ke R, Yang M and Rice-Evans C 1999. Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,20-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid) radical cation decolourisation assay. Methods in Enzymolology 299, 379–389. Santos GT, Lima LS, Schogor ALB, Romero JV, De Marchi FE, Grande PA, Santos NW, Santos FS and Kazama R 2014. Citrus pulp as a dietary source of antioxidants for lactating Holstein cows fed highly polyunsaturated fatty acid diets. Asian-Australasian Journal of Animal Sciences 27, 1104–1113. Santos-Silva J, Dentinho MT, Francisco A, Portugal AP, Belo AT, Martins APL, Alves SP and Bessa RJB 2016. Replacing cereals with dehydrated citrus pulp in a soybean oil supplemented diet increases vaccenic and rumenic acids in ewe milk. Journal of Dairy Science 99, 1173–1182. Seiquer I, Rueda A, Olalla M and Cabrera-Vique C 2015. Assessing the bioavailability of polyphenols and antioxidant properties of extra virgin argan oil by simulated digestion and Caco-2 cell assays. Comparative study with extra virgin olive oil. Food Chemistry 188, 496–503. Shin EC, Shurson GC and Gallaher DD 2018. Antioxidant capacity and phytochemical content of 16 sources of corn distillers dried grains with solubles (DDGS). Animal Nutrition 4, 435–441. Solomon R, Chase LE, Ben-Ghedalia D and Bauman DE 2000. The effect of nonstructural carbohydrate and addition of full fat extruded soybeans on the concentration of conjugated linoleic acid in the milk fat of dairy cows. Journal of Dairy Science 83, 1322–1329. Sukhija PS and Palmquist DL 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. Journal of Agricultural and Food Chemistry 36, 1202–1206. Tschuor AC, Riond B, Braun U and Lutz H 2008. Hämatologische und klinischchemische Referenzwerte für adulte Ziegen und Schafe. Schweizer Archiv für Tierheilkunde, 150, 287–295. Van Soest PJ, Robertson JB and LewisBA 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 3583–3597. Vázquez CV, Rojas MG, Ramirez CA, Chávez-Servin JL, Garcia-Gasca T, Ferriz- Martinez RA, Garcia OP, Rosado JL, Lopez-Sabater CM, Castellote AI, Montemayor HM and de la Torre CK 2015. Total phenolic compounds in milk from different species. Design of an extraction technique for quantification using the Folin-Ciocalteu method. Food Chemistry 176, 480–486. Guzmán, Perez-Ecija, Zarazaga, Martín-García, Horcada and Delgado-Pertí˜nez 2220