DOI: 10.1111/jpn.12344
ORIGINAL ARTICLE
Quality and fatty acid profile of the milk of indigenous goats subjected to different local diets in Tunisian arid lands N. Ayeb1,2, M. Addis3, M. Fiori3, S. Khorchani1, M. Atigui1 and T. Khorchani1 1 Laboratory of livestock and Wild life, Institute of Arid Lands (IRA Medenine), Medenine, Tunisia rieur Agronomique, Sousse, Tunisie, and 2 Institut Supe 3 Department of Animal Production Research, AGRIS-Sardegna, Olmedo, Italy
Summary The study tested the hypothesis that certain pastoral forages and olive by-products, available in arid areas, may positively influence fatty acid composition and physicochemical properties of goat’s milk. Thirty indigenous goats (body weight = 25.2 kg; age = 4.1 years) were allocated to three groups. During 60 days, the goats received ad libitum either dried olive leaves + Stipa tenacissima (group OL), khortane grass hay (group Ko) or oat hay (control diet, group OH). Milk samples were collected and analysed for total solids, fat, protein, lactose and ash content and fatty acid profile. Average milk yield did not statistically differ among groups. Milk total solids from OL group were higher in comparison with Ko and C groups (15.3, 14.7 and 14.5%, respectively; p < 0.05). Fat content was also higher for the OL group as compared to the other groups (5.44 vs. 5.01 and 4.66%, respectively, for Ko and OH). No significant differences were observed for the milk content of lactose, protein and ash. The percentage of saturated fatty acids of total milk fat was higher in OL and Ko groups compared to the C group (p < 0.001); the milk whereof was characterized by the highest percentage of monounsaturated (p < 0.01) and total unsaturated fatty acids. Milk fat of Ko and C groups showed significantly higher proportions of rumenic (CLA cis-9 trans-11) and vaccenic acids (C18:1 trans-11) compared to OL milk. The feeding system based on Stipa tenacissima and dried olive leaves resulted in the milk lowest proportion of trans-fatty acids and the highest proportion of polyunsaturated x3-fatty acids (p < 0.05). Keywords arid land, local resource, goat production, milk quality, fatty acids Correspondence N. Ayeb, Livestock and Wildlife Laboratory, Arid Regions Institute, 4119, Medenine, Tunisia. Tel: +216-96-593848; Fax: +216-75-633006; E-mail: [email protected] Received: 29 December 2014; accepted: 17 April 2015
Introduction In arid regions, milk is mainly produced by goats. Goats are better adapted than cows to extensive conditions and to harsh environments, because of their better ability to consume cell wall-rich plant resources, such as shrubs and trees (Martınez, 2002). In these regions, particularly in southern Tunisian, goat farming is predominant and goats use rangelands as main resources for their diet. When these resources cannot meet the maintenance and growth requirements of goats, the breeders developed several strategies to ensure the nutritional requirements of their livestock, such as the exploitation of some droughttolerant shrubs and pastoral plants, which represents an effective strategy for livestock feeding. Several studies have demonstrated that cactus (Opuntia ficusindica) cladodes (Ben Salem et al., 1996; Misra et al.,
2006) or fruits (Ben Salem et al., 2003) and saltbush (Atriplex nummularia; Ben Salem et al., 2002) have a good nutritional value, when used as supplements in small ruminant diets. In addition, breeders profit from favourable seasons to establish reserves by harvesting rangeland species as Stipa tenaacissima and grass hay traditionally called Khortane which is a mixture of grass species (Ayeb et al., 2011). In summer and in drought period, khortane is a very important forage resource for small ruminants as well as equines and camels. Khortane is a mixture of annual and perennial species, which are always collected in the spring and stored in a closed room. Four species are the most searched to make the khortane: Launeae residifolia, Chrysanthemum coronarium, Lolium multiflorum and Erodium glaucophyllum (Ayeb, 2009). Other resources are commonly used such agro-industrial by-products that, in animal nutrition, have been successfully
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
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adopted as a strategy to reduce feeding costs. This is the case, for instance, for by-products deriving from olive oil production industries. In several studies, citrus pulp (Bueno et al., 2002), olive cake (Ben Salem et al., 2003), dried olive leaves (Abbeddou et al., 2011a,b,c; Ayeb et al., 2013) sugar beet pulp (Olfaz et al., 2005) and tomato pomace (Denek and Can, 2006; Abbeddou et al., 2011a,b,c, 2015) have been successfully used as supplements for small ruminant diets. However, little information is available on the effects of some of these by-products on quality of animal products. Whatever the source of lipids, linseed (Chilliard et al., 2009; Ferlay et al., 2010), sunflower seed oil (Bernard et al., 2009), olive cake (Abbeddou et al., 2011b,c) or pasture, feeding has a significant impact on milk quality and milk fatty acid (FA) profile (Nudda et al., 2006). Milk is an important source of protein and fat in human diet. This is also true for unsaturated fatty acids, especially conjugated linoleic acid (CLA) isomers, principally cis-9, trans-11 and trans-10, cis-12 that have functional properties affecting positively consumers health (Nudda et al., 2006). The CLA cis-9–trans-11 is considered to have anticarcinogenic properties (Piperova et al., 2004), whereas CLA trans-10 cis-12 actively participates in lipid metabolism decreasing the fat content of cow milk (Baumgard et al., 2000). Important local feeds available in southern Tunisia (dried olive leaves, Stipa tenacissima and khortane) were rarely valorized in ruminant diets. As far as we know, no study had reported the use and the effect of these resources, except olive leaves, on milk quality and FA composition of small ruminants. Furthermore, the use of local resources could partially replace green forages or cereals obtained by irrigation in a context of very high water constraint. Thus, the aim of this study was to assess the effect of local feed resources on milk production, physicochemical composition and fatty acid profile of the milk of Tunisian indigenous goats. Materials and methods Animals and diets
This study was carried out at the experimental station of the Arid Regions Institute (Medenine, Tunisia) situated at N33°300 E10°380 and characterized by an arid bioclimatic stage with an annual average rainfall of 180 mm. Thirty indigenous goats in late lactation stage (mean weight 25.20 3.36 kg and mean age 4.10 0.70 years) were kept indoors, treated against internal and external parasites and used. Goats were separated in three homogeneous groups of 10 goats each, according to their milk production and live 102
weight. Each group received individually and ad libitum one of the three dietary treatments: the control group (OH) received oat hay; the Ko group received mixed khortane; and the OL group of received dried olive leaves in the morning and Stipa tenacissima in the evening. In all groups, each animal received 500 g/ day of the same commercial concentrate (g/kg dry matter (DM): crude protein (CP), 146; ash, 74; neutral detergent fibre (NDF), 286). Khortane, a traditional form of mixed plant species, was composed of dried annual and perennial species (Table 1), which were harvested during the spring season, from the steppe in Ben-Guerdane (Tunisia), and immediately sun-dried for 3–4 days. Stipa tenacissima or Sparta is a perennial herbaceous plant of the Poaceae family, native to arid regions of the western Mediterranean basin; it is used in the manufacturing of paper and also for animal feeding. In this study, it was hand-harvested on native rangelands from the neighbouring Matmata Mountains during the late growing stage (April), air-dried and stocked in a dry area. Dried olive leaves were supplied from the neighbouring private farmers. The composition of the experimental forages is reported in Table 2. For all groups, the refusals of all feeds were weighed daily. The total duration of the experiment was 60 days (15 days of adaptation), starting at the 15 May 2012 until 15 July 2012. Table 1 Floristic composition and specific contribution of the mixed grass hay (Khortane) used in the experiment Species
SC (%)
Anacyclus cyrtolepidioides Chamaemelum nobile Chenopodium murale Chrysanthemum coronarium Cymicio caliens Cynodon dactylon Dactylis glomerata Diplotaxis harra Erodium triangulaire Hordeum murinum Launaea glomerata Launaea resedifolia Lolium multiflorum Malva aegyptiaca Medicago sativa Paronychia arabica Polygonum equisetiforme Polypogon monspeliensis Cutandia dichotoma Schismus barbatus
1.38 2.77 6.94 20.83 2.08 2.77 1.38 4.86 3.47 2.77 2.77 4.86 5.55 18.05 2.08 4.16 2.08 2.77 4.16 4.16
SC: specific contribution (Csi) = 100 9 (Fsi/Σ Fsi) = 100 9 (specific frequency of individual species/the sum of the specific frequencies of all species recorded on 100 sample points).
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
Effect of local forage resources on goat milk
N. Ayeb et al.
Table 2 Chemical composition and fatty acid profile of the feeds used in the experiment Diets
OH
Feeds
Oat hay
Chemical composition (% DM) DM 89.35 Ash 3.09 CP 6.34 NDF 60.06 ADF 42.72 Fatty acids (% of total fatty acid) C16:0 30.35 C18:0 5.47 C18:1 15.68 C18:2 21.00 C18:3 12.51 SFA 45.99 MUFA 17.5 PUFA 36.52 Daily intake 0.66 C18:2 (g/d) Daily intake 0.34 C18:3 (g/d) Intake 0.88 DM (kg DM/d)
OL Stipa tenacissima
Olive leaves
Ko Grass hay
92.87 4.91 6.33 85.00 55.51
92.33 8.74 10.16 34.59 30.35
86.63 7.99 9.44 42.18 29.32
20.48 6.08 5.42 11.2 10.25 67.09 6.96 25.95 0.09
32.98 6.43 16.01 9.84 12.49 57.18 18.1 24.72 0.20
26.68 4.09 5.38 20.5 24.33 44.65 7.64 47.71 1.77
0.09
0.26
1.71
0.79
1.07
DM, dry matter; CP, crude protein; NDFom, neutral detergent fibre (exclusive of residual ash); ADFom, acid detergent fibre (exclusive of residual ash); SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; OH, oat hay; OL, dried olive leaves; Ko, khortane.
Sampling and analysis
The feed samples were dried at 105°C to determine dry matter (DM). Forage samples were dried at 65 °C for 48 h to determine the chemical composition. Ash was determined by incinerating samples in a furnace at 550 °C for 6 h, and crude protein (CP) was determined by Kjeldahl method (AOAC, 1991). Analysis of neutral and acid detergent fibre (NDF, ADF) was performed according to the method described by Van Soest et al. (1991). Milk yield was recorded daily once a day, and samples were collected at the morning milking once every week for the determination of gross composition. Total solids (TS), protein (CP), fat and lactose contents were measured by an automatic milk analyser device (Lactoscan MCC milk analyser, Stara Zagora, Bulgaria) calibrated for goat milk. The analyses were achieved on aliquots of fresh milk just after milking. The pH value was determined at room temperature (20 °C) using a pH meter (inoLab, Weilheim, Germany). Milk samples were stored at 20 °C before fat extraction.
Fatty acids analysis
Milk fat was extracted according to the method proposed by Murphy et al. (1990), and fatty acid methyl esters (FAMEs) were obtained from milk fat with basic transmethylation according to ISO 15884/FIL 182 methods and analysed through GC-FID. Separation and quantification of the methyl esters were carried out using a gas chromatograph (Varian 3600; Varian, Harbor City, CA, USA), equipped with a split/splitless injector and a flame ionization detector. The methyl ester separation was carried out on capillary column SP2560 (100 m 9 0.25 mm i.d., 0.25 lm of phase; Supelco, Bellefonte, PA, USA) using helium as the carrier gas (331 kPa). The injector and detector temperature was set at 290 °C. The injection was carried out in split mode with 1:100 split ratio. The temperature of the column was initially at 75 °C for 1.5 min, then increased to 190 °C at 8 °C/min, held at this temperature for 25 min, then increased again to 230 °Cat 15 °C/min and held for a further 4.5 min at 230 °C. The results were analysed by the software STAR SYSTEM 4.5 (Varian 3900; Varian). Individual FAMEs (Sigma-Aldrich, St. Louis, MO, USA) were identified on the basis of the retention time and the comparison with a standard mixture of 37 pure components (Supelco 37 Component FAME Mix; Sigma-Aldrich). In addition, the identification of the isomers of the conjugated linoleic acid (CLA) has been accomplished by the comparison between the retention time of each chromatographic peak and those of a mixture of chromatographic standards (C18:2 cis-9–trans-11; C18:2 trans-10–cis-12; C18:2 cis-9–cis-11; C18:2 trans-9– trans-11). Furthermore, a comparison of the chromatographic profile obtained with those described in literature (Kramer et al., 2004) has been taken into account for confirmatory purposes. Each fatty acid was quantified with respect to the following internal standards (Sigma-Aldrich): Me-C5:0 (C4:0-C7:0), Me-C9:0 (C8:0-C10:0), Me-C13:0 (C11:0-C17:0) and Me-C19:0 (C18:0-C26:0). The concentration of each internal standard added to the sample was 100 mg/g of fat. Calculations and statistical analyses
Before statistical analyses, the data on individual fatty acids were also summarized in groups, namely all saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), total unsaturated fatty acids (UFA), total x-6 fatty acids, total x-3 fatty acids and total CLA isomers. Moreover, the following ratios were calculated: x-6/x-3, vaccenic acid (VA)/rumenic acid (CLA) (VA/CLA). The
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activity of Δ9-desaturase enzyme in mammary gland was estimated as the ratio of C14:1cis-9/C14:0. Finally, the atherogenicity index was calculated according to Chilliard et al. (2003), as follows: (C12:0 + 4 9 C14:0 + C16:0)/(MUFA + x-6 + x-3). Statistical analysis of the data was performed using the statistical package (SPSS 11.5 software, Chicago, IL, USA) by subjecting data to analysis of variance. Diet (OH, OL, Ko) was the explanatory factor and was considered significant for p < 0.05 or as a trend if p < 0.10. Residual plots were examined for homogeneity, and no transformations were required for any of the variables. Duncan’s multiple range test was used to test differences between means. Results and discussion Animal performance and milk quality
Feed intake was higher (p < 0.001) in the Ko group than in OL and OH groups (Table 3). This indicates the lower palatability of oat hay and S. tenacissima + dried olive leaves in comparison with khortane. Le Houerou and Ionesco (1987) reported a very high palatability index (IP = 5) for Chrysanthemum coronarium and Malva aegyptiacae which are the major components of khortane (21% and 18%, respectively, for the two species; Table 1). Sulas et al. (1999) found a high herbage intake of Chrysanthemum coronarium grazed by Sarda sheep during flowering stage, and Avondo et al. (2002) reported that this forage is in the list of the most palatable species for dairy sheep grazing native Mediterranean pastures. Milk yield did not significantly differ among the groups although it tended to be higher in the Ko group, which could be related to the higher feed intake and, possibly, a better ruminal utilization of
Table 3 Effect of the experimental diets on intake, milk yield and composition and properties of the milk
Yield (ml/day) pH Density Acidity (D°) Total solids (%) Protein (%) Fat (%) Lactose (%)
OH
OL
Ko
SEM
p
216 6.30 29 20.9 14.7b 3.68 5.01b 4.83
199 6.39 29 20.9 15.3a 3.71 5.44a 4.93
243 6.38 29 20.5 14.5b 3.76 4.66b 4.90
93 0.01 0.2 0.2 0.1 0.02 0.09 0.03
0.58 0.87 0.19 0.57 0.023 0.21 0.002 0.40
OH, oat hay control group; OL, Stipa tenancissima + dried olive leaves group; Ko, khortane group. a,b Means within a row with different superscripts differ significantly, SEM, standard error means; p, probability.
104
diet components (Maltz et al., 1991). Couvreur et al. (2006) showed that the milk production increases linearly with the increase in the proportion of grass in the diet (+0.21 kg/day with the addition 10% of grass). It seems that adverse factors, such as the high acid detergent fibre content in Stipa tenacissima compared to khortane (Table 2) and the relatively high content of phenols in olive leaves (Abbeddou et al., 2011b), were unfavourable in lactating sheep. The use of olive leaves to feed small ruminants, as tested in other studies, resulted in an unchanged milk yield in other studies (ammonia-treated olive leaves, Fegeros et al., 1995; fresh olive leaves, Abbeddou et al., 2011b). There were no significant diet effects on the physical properties of the milk (pH, density and acidity). Milk from group OL had a higher (p < 0.01) fat content than groups OH and Ko. Also, total solids content was higher for OL group than the other groups. Sanz Sampelayo et al. (2007) indicated that fat contained in goats’ feed affected the quantity and composition of milk fat more than the other feed components. Morand-Fehr et al. (2007) suggested that the fat content in goat milk is not only linked to the fibre content of the diets, but also to energy intake. No significant differences were observed in the other chemical parameters of milk. In general, in our study, the low milk production level is explained by the late lactation stage (May and June), which also explains the high total solids and fat contents. Strzałkowska et al. (2010) reported an increased milk fat content at the final stage of goat lactation. Fatty acids in the milk
The effect of diet type was significant for the majority of milk fat proportions of the individual and groups of milk fatty acids (Table 4). The percentage of saturated fatty acids was higher (p < 0.001) in OL and Ko groups compared to OH group where MUFA proportion was highest. The saturated short- and mediumchain fatty acids in milk are mainly de novo-synthesized (almost all C4:0 to C14:0 and more than 50% of C16:0) at the mammary gland level (Chilliard et al., 2001). Long-chain FA (from diet or body fat mobilization) and trans-FA (especially 18:1 trans-10 and 18:2 trans-10–cis-12 originating from hydrogenated dietary fat or from ruminal biohydrogenation) can, partly, inhibit the de novo synthesis process (Bauman and Griinari, 2003; Chilliard et al., 2007). As expected, the oat hay diet, containing high proportions of longchain FA, particularly C18:2 (Table 2), effectively decreased both SCFA (C4:0, C10:0) and MCFA
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
Effect of local forage resources on goat milk
N. Ayeb et al.
Table 4 Effect of the experimental diets on the fatty acid composition of milk fat g/100 g esters
fatty
acid
methyl
C4:0 C6:0 C7:0 C8:0 C10:0 C11:0 C12:0 C14:0 C14:1 cis-9 C16:0 C18:0 C20:0 C18:1 trans-6, trans-8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 trans-12 C18:1 cis-9 C18:1 cis-11 C18:1 cis-12 C18:1 cis-16 C18:2 cis-9, trans-13 C18:2 cis-9, trans-12 C18:2 trans-9, cis-12 C18:2 trans-11, cis-15 C18:2 cis-9, cis-15 C18:2 cis-9, cis-15 C18:2 cis-9, trans-11 (CLA) C18:2 trans-10, cis-12 (CLA) C18:3 cis-9, cis-12, cis-15 C20:3 cis-5, cis-8, cis-11 C20:4 cis-5, cis-8, cis-11, cis-14 C20:5 cis-5, cis-8, cis-11, cis-14, cis17 C22:5 cis-7, cis-10, cis-13, cis-16, cis-19 C22:6 cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 Total saturated fatty acids Total monounsaturated fatty acids Total polyunsaturated fatty acids Total unsaturated fatty acids Total x-3 Total x-6 x6/x3 Atherogenicity indexc D9-desaturase indexd
OH
OL
Ko
SEM
p
2.70b 2.81 0.030 3.38 12.2 0.51b 4.7b 10.0b 0.19b 25.9b 6.6a 0.41a 0.089a 0.202a 0.24 0.71a 0.198a 20.2a 0.38 0.14b 0.067a 0.219a 0.095a 0.08 0.014b 1.74b 0.008b 0.58a 0.045a 0.27 0.04 0.122
2.89a 2.87 0.036 3.57 13.5 0.67a 4.9b 10.8ab 0.27a 32.3a 3.8b 0.16b 0.051b 0.120b 0.48 0.34b 0.100b 15.4b 0.39 0.12b 0.047b 0.130b 0.056b 0.02 0.040a 1.76b 0.028a 0.33b 0.033b 0.30 0.04 0.145
2.69a 2.84 0.034 3.59 13.6 0.62a 5.6a 11.5a 0.26a 27.7b 4.5b 0.21b 0.093a 0.179a 0.31 0.81a 0.179b 16.1b 0.36 0.24a 0.065a 0.206a 0.079a 0.01 0.013b 2.11a 0.007b 0.71a 0.037ab 0.26 0.04 0.144
0.04 0.04 0.002 0.08 0.3 0.02 0.1 0.2 0.01 0.5 0.2 0.02 0.006 0.007 0.07 0.05 0.009 0.4 0.01 0.01 0.004 0.008 0.004 0.02 0.005 0.06 0.002 0.03 0.002 0.01 0.002 0.005
0.037 0.83 0.38 0.49 0.085 0.009 0.011 0.019 0.016
ORIGINAL ARTICLE
Quality and fatty acid profile of the milk of indigenous goats subjected to different local diets in Tunisian arid lands N. Ayeb1,2, M. Addis3, M. Fiori3, S. Khorchani1, M. Atigui1 and T. Khorchani1 1 Laboratory of livestock and Wild life, Institute of Arid Lands (IRA Medenine), Medenine, Tunisia rieur Agronomique, Sousse, Tunisie, and 2 Institut Supe 3 Department of Animal Production Research, AGRIS-Sardegna, Olmedo, Italy
Summary The study tested the hypothesis that certain pastoral forages and olive by-products, available in arid areas, may positively influence fatty acid composition and physicochemical properties of goat’s milk. Thirty indigenous goats (body weight = 25.2 kg; age = 4.1 years) were allocated to three groups. During 60 days, the goats received ad libitum either dried olive leaves + Stipa tenacissima (group OL), khortane grass hay (group Ko) or oat hay (control diet, group OH). Milk samples were collected and analysed for total solids, fat, protein, lactose and ash content and fatty acid profile. Average milk yield did not statistically differ among groups. Milk total solids from OL group were higher in comparison with Ko and C groups (15.3, 14.7 and 14.5%, respectively; p < 0.05). Fat content was also higher for the OL group as compared to the other groups (5.44 vs. 5.01 and 4.66%, respectively, for Ko and OH). No significant differences were observed for the milk content of lactose, protein and ash. The percentage of saturated fatty acids of total milk fat was higher in OL and Ko groups compared to the C group (p < 0.001); the milk whereof was characterized by the highest percentage of monounsaturated (p < 0.01) and total unsaturated fatty acids. Milk fat of Ko and C groups showed significantly higher proportions of rumenic (CLA cis-9 trans-11) and vaccenic acids (C18:1 trans-11) compared to OL milk. The feeding system based on Stipa tenacissima and dried olive leaves resulted in the milk lowest proportion of trans-fatty acids and the highest proportion of polyunsaturated x3-fatty acids (p < 0.05). Keywords arid land, local resource, goat production, milk quality, fatty acids Correspondence N. Ayeb, Livestock and Wildlife Laboratory, Arid Regions Institute, 4119, Medenine, Tunisia. Tel: +216-96-593848; Fax: +216-75-633006; E-mail: [email protected] Received: 29 December 2014; accepted: 17 April 2015
Introduction In arid regions, milk is mainly produced by goats. Goats are better adapted than cows to extensive conditions and to harsh environments, because of their better ability to consume cell wall-rich plant resources, such as shrubs and trees (Martınez, 2002). In these regions, particularly in southern Tunisian, goat farming is predominant and goats use rangelands as main resources for their diet. When these resources cannot meet the maintenance and growth requirements of goats, the breeders developed several strategies to ensure the nutritional requirements of their livestock, such as the exploitation of some droughttolerant shrubs and pastoral plants, which represents an effective strategy for livestock feeding. Several studies have demonstrated that cactus (Opuntia ficusindica) cladodes (Ben Salem et al., 1996; Misra et al.,
2006) or fruits (Ben Salem et al., 2003) and saltbush (Atriplex nummularia; Ben Salem et al., 2002) have a good nutritional value, when used as supplements in small ruminant diets. In addition, breeders profit from favourable seasons to establish reserves by harvesting rangeland species as Stipa tenaacissima and grass hay traditionally called Khortane which is a mixture of grass species (Ayeb et al., 2011). In summer and in drought period, khortane is a very important forage resource for small ruminants as well as equines and camels. Khortane is a mixture of annual and perennial species, which are always collected in the spring and stored in a closed room. Four species are the most searched to make the khortane: Launeae residifolia, Chrysanthemum coronarium, Lolium multiflorum and Erodium glaucophyllum (Ayeb, 2009). Other resources are commonly used such agro-industrial by-products that, in animal nutrition, have been successfully
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
101
Effect of local forage resources on goat milk
N. Ayeb et al.
adopted as a strategy to reduce feeding costs. This is the case, for instance, for by-products deriving from olive oil production industries. In several studies, citrus pulp (Bueno et al., 2002), olive cake (Ben Salem et al., 2003), dried olive leaves (Abbeddou et al., 2011a,b,c; Ayeb et al., 2013) sugar beet pulp (Olfaz et al., 2005) and tomato pomace (Denek and Can, 2006; Abbeddou et al., 2011a,b,c, 2015) have been successfully used as supplements for small ruminant diets. However, little information is available on the effects of some of these by-products on quality of animal products. Whatever the source of lipids, linseed (Chilliard et al., 2009; Ferlay et al., 2010), sunflower seed oil (Bernard et al., 2009), olive cake (Abbeddou et al., 2011b,c) or pasture, feeding has a significant impact on milk quality and milk fatty acid (FA) profile (Nudda et al., 2006). Milk is an important source of protein and fat in human diet. This is also true for unsaturated fatty acids, especially conjugated linoleic acid (CLA) isomers, principally cis-9, trans-11 and trans-10, cis-12 that have functional properties affecting positively consumers health (Nudda et al., 2006). The CLA cis-9–trans-11 is considered to have anticarcinogenic properties (Piperova et al., 2004), whereas CLA trans-10 cis-12 actively participates in lipid metabolism decreasing the fat content of cow milk (Baumgard et al., 2000). Important local feeds available in southern Tunisia (dried olive leaves, Stipa tenacissima and khortane) were rarely valorized in ruminant diets. As far as we know, no study had reported the use and the effect of these resources, except olive leaves, on milk quality and FA composition of small ruminants. Furthermore, the use of local resources could partially replace green forages or cereals obtained by irrigation in a context of very high water constraint. Thus, the aim of this study was to assess the effect of local feed resources on milk production, physicochemical composition and fatty acid profile of the milk of Tunisian indigenous goats. Materials and methods Animals and diets
This study was carried out at the experimental station of the Arid Regions Institute (Medenine, Tunisia) situated at N33°300 E10°380 and characterized by an arid bioclimatic stage with an annual average rainfall of 180 mm. Thirty indigenous goats in late lactation stage (mean weight 25.20 3.36 kg and mean age 4.10 0.70 years) were kept indoors, treated against internal and external parasites and used. Goats were separated in three homogeneous groups of 10 goats each, according to their milk production and live 102
weight. Each group received individually and ad libitum one of the three dietary treatments: the control group (OH) received oat hay; the Ko group received mixed khortane; and the OL group of received dried olive leaves in the morning and Stipa tenacissima in the evening. In all groups, each animal received 500 g/ day of the same commercial concentrate (g/kg dry matter (DM): crude protein (CP), 146; ash, 74; neutral detergent fibre (NDF), 286). Khortane, a traditional form of mixed plant species, was composed of dried annual and perennial species (Table 1), which were harvested during the spring season, from the steppe in Ben-Guerdane (Tunisia), and immediately sun-dried for 3–4 days. Stipa tenacissima or Sparta is a perennial herbaceous plant of the Poaceae family, native to arid regions of the western Mediterranean basin; it is used in the manufacturing of paper and also for animal feeding. In this study, it was hand-harvested on native rangelands from the neighbouring Matmata Mountains during the late growing stage (April), air-dried and stocked in a dry area. Dried olive leaves were supplied from the neighbouring private farmers. The composition of the experimental forages is reported in Table 2. For all groups, the refusals of all feeds were weighed daily. The total duration of the experiment was 60 days (15 days of adaptation), starting at the 15 May 2012 until 15 July 2012. Table 1 Floristic composition and specific contribution of the mixed grass hay (Khortane) used in the experiment Species
SC (%)
Anacyclus cyrtolepidioides Chamaemelum nobile Chenopodium murale Chrysanthemum coronarium Cymicio caliens Cynodon dactylon Dactylis glomerata Diplotaxis harra Erodium triangulaire Hordeum murinum Launaea glomerata Launaea resedifolia Lolium multiflorum Malva aegyptiaca Medicago sativa Paronychia arabica Polygonum equisetiforme Polypogon monspeliensis Cutandia dichotoma Schismus barbatus
1.38 2.77 6.94 20.83 2.08 2.77 1.38 4.86 3.47 2.77 2.77 4.86 5.55 18.05 2.08 4.16 2.08 2.77 4.16 4.16
SC: specific contribution (Csi) = 100 9 (Fsi/Σ Fsi) = 100 9 (specific frequency of individual species/the sum of the specific frequencies of all species recorded on 100 sample points).
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
Effect of local forage resources on goat milk
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Table 2 Chemical composition and fatty acid profile of the feeds used in the experiment Diets
OH
Feeds
Oat hay
Chemical composition (% DM) DM 89.35 Ash 3.09 CP 6.34 NDF 60.06 ADF 42.72 Fatty acids (% of total fatty acid) C16:0 30.35 C18:0 5.47 C18:1 15.68 C18:2 21.00 C18:3 12.51 SFA 45.99 MUFA 17.5 PUFA 36.52 Daily intake 0.66 C18:2 (g/d) Daily intake 0.34 C18:3 (g/d) Intake 0.88 DM (kg DM/d)
OL Stipa tenacissima
Olive leaves
Ko Grass hay
92.87 4.91 6.33 85.00 55.51
92.33 8.74 10.16 34.59 30.35
86.63 7.99 9.44 42.18 29.32
20.48 6.08 5.42 11.2 10.25 67.09 6.96 25.95 0.09
32.98 6.43 16.01 9.84 12.49 57.18 18.1 24.72 0.20
26.68 4.09 5.38 20.5 24.33 44.65 7.64 47.71 1.77
0.09
0.26
1.71
0.79
1.07
DM, dry matter; CP, crude protein; NDFom, neutral detergent fibre (exclusive of residual ash); ADFom, acid detergent fibre (exclusive of residual ash); SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; OH, oat hay; OL, dried olive leaves; Ko, khortane.
Sampling and analysis
The feed samples were dried at 105°C to determine dry matter (DM). Forage samples were dried at 65 °C for 48 h to determine the chemical composition. Ash was determined by incinerating samples in a furnace at 550 °C for 6 h, and crude protein (CP) was determined by Kjeldahl method (AOAC, 1991). Analysis of neutral and acid detergent fibre (NDF, ADF) was performed according to the method described by Van Soest et al. (1991). Milk yield was recorded daily once a day, and samples were collected at the morning milking once every week for the determination of gross composition. Total solids (TS), protein (CP), fat and lactose contents were measured by an automatic milk analyser device (Lactoscan MCC milk analyser, Stara Zagora, Bulgaria) calibrated for goat milk. The analyses were achieved on aliquots of fresh milk just after milking. The pH value was determined at room temperature (20 °C) using a pH meter (inoLab, Weilheim, Germany). Milk samples were stored at 20 °C before fat extraction.
Fatty acids analysis
Milk fat was extracted according to the method proposed by Murphy et al. (1990), and fatty acid methyl esters (FAMEs) were obtained from milk fat with basic transmethylation according to ISO 15884/FIL 182 methods and analysed through GC-FID. Separation and quantification of the methyl esters were carried out using a gas chromatograph (Varian 3600; Varian, Harbor City, CA, USA), equipped with a split/splitless injector and a flame ionization detector. The methyl ester separation was carried out on capillary column SP2560 (100 m 9 0.25 mm i.d., 0.25 lm of phase; Supelco, Bellefonte, PA, USA) using helium as the carrier gas (331 kPa). The injector and detector temperature was set at 290 °C. The injection was carried out in split mode with 1:100 split ratio. The temperature of the column was initially at 75 °C for 1.5 min, then increased to 190 °C at 8 °C/min, held at this temperature for 25 min, then increased again to 230 °Cat 15 °C/min and held for a further 4.5 min at 230 °C. The results were analysed by the software STAR SYSTEM 4.5 (Varian 3900; Varian). Individual FAMEs (Sigma-Aldrich, St. Louis, MO, USA) were identified on the basis of the retention time and the comparison with a standard mixture of 37 pure components (Supelco 37 Component FAME Mix; Sigma-Aldrich). In addition, the identification of the isomers of the conjugated linoleic acid (CLA) has been accomplished by the comparison between the retention time of each chromatographic peak and those of a mixture of chromatographic standards (C18:2 cis-9–trans-11; C18:2 trans-10–cis-12; C18:2 cis-9–cis-11; C18:2 trans-9– trans-11). Furthermore, a comparison of the chromatographic profile obtained with those described in literature (Kramer et al., 2004) has been taken into account for confirmatory purposes. Each fatty acid was quantified with respect to the following internal standards (Sigma-Aldrich): Me-C5:0 (C4:0-C7:0), Me-C9:0 (C8:0-C10:0), Me-C13:0 (C11:0-C17:0) and Me-C19:0 (C18:0-C26:0). The concentration of each internal standard added to the sample was 100 mg/g of fat. Calculations and statistical analyses
Before statistical analyses, the data on individual fatty acids were also summarized in groups, namely all saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), total unsaturated fatty acids (UFA), total x-6 fatty acids, total x-3 fatty acids and total CLA isomers. Moreover, the following ratios were calculated: x-6/x-3, vaccenic acid (VA)/rumenic acid (CLA) (VA/CLA). The
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
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Effect of local forage resources on goat milk
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activity of Δ9-desaturase enzyme in mammary gland was estimated as the ratio of C14:1cis-9/C14:0. Finally, the atherogenicity index was calculated according to Chilliard et al. (2003), as follows: (C12:0 + 4 9 C14:0 + C16:0)/(MUFA + x-6 + x-3). Statistical analysis of the data was performed using the statistical package (SPSS 11.5 software, Chicago, IL, USA) by subjecting data to analysis of variance. Diet (OH, OL, Ko) was the explanatory factor and was considered significant for p < 0.05 or as a trend if p < 0.10. Residual plots were examined for homogeneity, and no transformations were required for any of the variables. Duncan’s multiple range test was used to test differences between means. Results and discussion Animal performance and milk quality
Feed intake was higher (p < 0.001) in the Ko group than in OL and OH groups (Table 3). This indicates the lower palatability of oat hay and S. tenacissima + dried olive leaves in comparison with khortane. Le Houerou and Ionesco (1987) reported a very high palatability index (IP = 5) for Chrysanthemum coronarium and Malva aegyptiacae which are the major components of khortane (21% and 18%, respectively, for the two species; Table 1). Sulas et al. (1999) found a high herbage intake of Chrysanthemum coronarium grazed by Sarda sheep during flowering stage, and Avondo et al. (2002) reported that this forage is in the list of the most palatable species for dairy sheep grazing native Mediterranean pastures. Milk yield did not significantly differ among the groups although it tended to be higher in the Ko group, which could be related to the higher feed intake and, possibly, a better ruminal utilization of
Table 3 Effect of the experimental diets on intake, milk yield and composition and properties of the milk
Yield (ml/day) pH Density Acidity (D°) Total solids (%) Protein (%) Fat (%) Lactose (%)
OH
OL
Ko
SEM
p
216 6.30 29 20.9 14.7b 3.68 5.01b 4.83
199 6.39 29 20.9 15.3a 3.71 5.44a 4.93
243 6.38 29 20.5 14.5b 3.76 4.66b 4.90
93 0.01 0.2 0.2 0.1 0.02 0.09 0.03
0.58 0.87 0.19 0.57 0.023 0.21 0.002 0.40
OH, oat hay control group; OL, Stipa tenancissima + dried olive leaves group; Ko, khortane group. a,b Means within a row with different superscripts differ significantly, SEM, standard error means; p, probability.
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diet components (Maltz et al., 1991). Couvreur et al. (2006) showed that the milk production increases linearly with the increase in the proportion of grass in the diet (+0.21 kg/day with the addition 10% of grass). It seems that adverse factors, such as the high acid detergent fibre content in Stipa tenacissima compared to khortane (Table 2) and the relatively high content of phenols in olive leaves (Abbeddou et al., 2011b), were unfavourable in lactating sheep. The use of olive leaves to feed small ruminants, as tested in other studies, resulted in an unchanged milk yield in other studies (ammonia-treated olive leaves, Fegeros et al., 1995; fresh olive leaves, Abbeddou et al., 2011b). There were no significant diet effects on the physical properties of the milk (pH, density and acidity). Milk from group OL had a higher (p < 0.01) fat content than groups OH and Ko. Also, total solids content was higher for OL group than the other groups. Sanz Sampelayo et al. (2007) indicated that fat contained in goats’ feed affected the quantity and composition of milk fat more than the other feed components. Morand-Fehr et al. (2007) suggested that the fat content in goat milk is not only linked to the fibre content of the diets, but also to energy intake. No significant differences were observed in the other chemical parameters of milk. In general, in our study, the low milk production level is explained by the late lactation stage (May and June), which also explains the high total solids and fat contents. Strzałkowska et al. (2010) reported an increased milk fat content at the final stage of goat lactation. Fatty acids in the milk
The effect of diet type was significant for the majority of milk fat proportions of the individual and groups of milk fatty acids (Table 4). The percentage of saturated fatty acids was higher (p < 0.001) in OL and Ko groups compared to OH group where MUFA proportion was highest. The saturated short- and mediumchain fatty acids in milk are mainly de novo-synthesized (almost all C4:0 to C14:0 and more than 50% of C16:0) at the mammary gland level (Chilliard et al., 2001). Long-chain FA (from diet or body fat mobilization) and trans-FA (especially 18:1 trans-10 and 18:2 trans-10–cis-12 originating from hydrogenated dietary fat or from ruminal biohydrogenation) can, partly, inhibit the de novo synthesis process (Bauman and Griinari, 2003; Chilliard et al., 2007). As expected, the oat hay diet, containing high proportions of longchain FA, particularly C18:2 (Table 2), effectively decreased both SCFA (C4:0, C10:0) and MCFA
Journal of Animal Physiology and Animal Nutrition 100 (2016) 101–108 © 2015 Blackwell Verlag GmbH
Effect of local forage resources on goat milk
N. Ayeb et al.
Table 4 Effect of the experimental diets on the fatty acid composition of milk fat g/100 g esters
fatty
acid
methyl
C4:0 C6:0 C7:0 C8:0 C10:0 C11:0 C12:0 C14:0 C14:1 cis-9 C16:0 C18:0 C20:0 C18:1 trans-6, trans-8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 trans-12 C18:1 cis-9 C18:1 cis-11 C18:1 cis-12 C18:1 cis-16 C18:2 cis-9, trans-13 C18:2 cis-9, trans-12 C18:2 trans-9, cis-12 C18:2 trans-11, cis-15 C18:2 cis-9, cis-15 C18:2 cis-9, cis-15 C18:2 cis-9, trans-11 (CLA) C18:2 trans-10, cis-12 (CLA) C18:3 cis-9, cis-12, cis-15 C20:3 cis-5, cis-8, cis-11 C20:4 cis-5, cis-8, cis-11, cis-14 C20:5 cis-5, cis-8, cis-11, cis-14, cis17 C22:5 cis-7, cis-10, cis-13, cis-16, cis-19 C22:6 cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 Total saturated fatty acids Total monounsaturated fatty acids Total polyunsaturated fatty acids Total unsaturated fatty acids Total x-3 Total x-6 x6/x3 Atherogenicity indexc D9-desaturase indexd
OH
OL
Ko
SEM
p
2.70b 2.81 0.030 3.38 12.2 0.51b 4.7b 10.0b 0.19b 25.9b 6.6a 0.41a 0.089a 0.202a 0.24 0.71a 0.198a 20.2a 0.38 0.14b 0.067a 0.219a 0.095a 0.08 0.014b 1.74b 0.008b 0.58a 0.045a 0.27 0.04 0.122
2.89a 2.87 0.036 3.57 13.5 0.67a 4.9b 10.8ab 0.27a 32.3a 3.8b 0.16b 0.051b 0.120b 0.48 0.34b 0.100b 15.4b 0.39 0.12b 0.047b 0.130b 0.056b 0.02 0.040a 1.76b 0.028a 0.33b 0.033b 0.30 0.04 0.145
2.69a 2.84 0.034 3.59 13.6 0.62a 5.6a 11.5a 0.26a 27.7b 4.5b 0.21b 0.093a 0.179a 0.31 0.81a 0.179b 16.1b 0.36 0.24a 0.065a 0.206a 0.079a 0.01 0.013b 2.11a 0.007b 0.71a 0.037ab 0.26 0.04 0.144
0.04 0.04 0.002 0.08 0.3 0.02 0.1 0.2 0.01 0.5 0.2 0.02 0.006 0.007 0.07 0.05 0.009 0.4 0.01 0.01 0.004 0.008 0.004 0.02 0.005 0.06 0.002 0.03 0.002 0.01 0.002 0.005
0.037 0.83 0.38 0.49 0.085 0.009 0.011 0.019 0.016
