**3. The role of the breed on oligosaccharides: a special focus on the Mediterranean goats**

*2.2.5. Case study 5: degree of antioxidant protection*

milk and cheese.

250 Goat Science

(modified from Ref. [13]). a, b and c = *P* < 0.05.

In this case study, Ref. [13], in order to trace and identify milk and cheese from different feeding systems, proposed an interesting tool. Milk and cheese samples from ten feeding systems as grazing, grazing plus different types of supplement and indoor and zero grazing were studied to identify a tracing parameter correlated to the feeding system. In particular, α-tocopherol and cholesterol were measured in milk and cheese and were combined to calculate the degree of antioxidant protection (DAP). This tracing parameter was calculated as molar ratio between antioxidant compounds and a selected oxidation target. In dairy products from goats, only α-tocopherol was selected as the antioxidant because β-carotene is absent in goat's milk, and cholesterol was selected as oxidation target. All samples were analysed for α-tocopherol and cholesterol content. Briefly, all samples were hydrolysed in alkaline solution, and the extracted residue was dissolved in 2-propanol (1%) in n-hexane and analysed by the normal phase chromatographic method described in Ref. [13]. This index allows an evaluation of milk and cheese resistance to oxidative reactions, the main determinants of food quality and functionality for human nutrition. The DAP values (**Figure 11**) greater than 7.0 × 10−3 were found in grazing feeding systems, and values lower than 7.0 × 10−3 were found in indoor and zero grazing feeding systems, for

**Figure 11.** (a) Degree of antioxidant protection (DAP) of milk from goats fed with G = grazing, GBCM = grazing plus 0.6 kg/d mixed barley and chickpeas grain, GMBM = grazing plus 0.6 kg/d mixed corn and broad beans grain and HS = pasture hay *ad libitum* plus 0.6/kg/d of commercial concentrate. (b) DAP of *Caciotta* cheese from goats fed with G = grazing, GUC = grazing plus unlimited concentrate and HUC = hay plus unlimited concentrate. (c) DAP of *Caciotta* cheese from goats fed with GVC = grazing on valley pasture, GMC = grazing on mountain pasture and ZG = zero grazing Besides the feeding system, the breed plays a fundamental role in affecting the nutritional profile of goat milk and cheese. The breed may be considered the result of the adaptation of a species to a given environment, basically in order to go over the climate and feeding and water resource limits that might affect the reproduction and kidding. The goats are present in high mountains as far as in the internal lands and coastal regions; they are reared in technological farms but also in extensive, grazing systems in the Mediterranean area, an environment characterised by high variability, that was able to select very different breeds [62].

The so-called native breed has become able to optimise the resources in terms of water and feedstuff [63]. The breed's answer is expressed as phenotype, quantity and, overall, quality of production. The differences are both in micro and macronutrients, and they are affected by the environment directly or indirectly. In the first case, we can say that different breed means different feeding behaviour and thus milk yield and quality, since it is well known that feeding largely affects the milk composition [64]. Moreover, the genetic polymorphism may affect the milk features.

Within the same breed, in the same environment and diet, it is expectable to have very similar performances. Contrarily, especially for goat, significant differences have been found for quality but also quantity parameters. This variability has been explained, in part, by the genetic polymorphism of caseins that are αs2-casein, β-casein and k-casein but in particular at the locus αs1-casein, first discovered by Boulanger et al. [65]. It was found that goats carrying strong alleles (AA) for high α-s<sup>1</sup> casein present higher percentage of milk casein, fat, calcium, phosphorus and smaller micelles than the milk from goats with weak alleles (FF) [66, 67]. Several goat breeds have been characterised for this variability: the Vallesana, Roccaverano, Jonica, Garganica and Maltese breeds [68] and Alpine breed [69]. Spanish Malagueña goats with a high (HG) and low (LG) genetic capability for αS1-casein synthesis were used to determine whether the different genotypes were related to differences in feed utilisation (13.6 vs. 17.7% crude protein content for diets 1 and 2, respectively). The findings have let to explain the differences in milk composition between the two genotype groups by the greater nitrogen and energy utilisation of HG vs. LG goats [70]. Moreover, the interaction genotype x feeding system was studied (e.g., see Ref. [71] on Malagueña dairy goat breed).

The breed effect on milk oligosaccharide (OS) composition, and in particular sialyloligosaccharide (SOS) content, is scarcely studied. The milk from the Spanish Murciana-Granadina goat breed was found characterised by 25 OS [72], later [73] isolated 15 new oligosaccharide structures from fresh milk of Spanish goats, obtaining a virtually lactose and salt-free product, containing more than 80% of the original oligosaccharide content. Evenly, the effect of interaction of breed x feeding is scarcely studied.

Within the management's strategies, the choice of the breed is a key element to weigh up towards the type of livestock, the available resources and the business plan, in terms of destination of the milk, namely, if destined to the market of drinking milk or to dairy production. Some speculations can be made whether optimizing production, rheological properties, and bioactive profile and content may be feasible with feeding management modulated in terms of energy and protein supply depending upon genotype. The following three case studies are presented, in order to partially cover this gap on Mediterranean goats.

#### **3.1. Case studies 1 and 2: oligosaccharides in colostrum and milk**

A study on the content of three SOSs, namely, 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL) and di-sialyllactose (DSL) in colostrum and milk, was carried out [22] with two Italian goat breeds, Garganica goat (a native breed from Gargano Mountain in Apulia region, Southern Italy) and the Maltese goat breed, native from Malta isle in the Mediterranean area. The animals were fed indoor, receiving hay (from polyphytic cultivated meadows) *ad libitum* and concentrate supplementation (400 and 600 g/h/d, respectively) at 14% crude protein, according to their milk yield (800 and 1200 g/day milk, respectively). The SOSs were isolated from individual colostrum and milk samples obtained in five periods (at kidding, 24 h, 7 days, 30 days and 90 days after delivery). Briefly, after centrifugation (2000 × g, 4°C, 10 min) the supernatant lipid layer was removed, and the proteins were precipitated by addition of 0.5 volumes of 1.8 g 100 mL−1 Ba(OH)<sup>2</sup> ·8H<sup>2</sup> O and 0.5 volumes of 2 g 100 mL−1 ZnSO<sup>4</sup> ·7H<sup>2</sup> O. The blend was vortexed and centrifuged (12,000 × g, 10 min, 4°C). The supernatant was removed and centrifuged again. The second supernatant was filtered with a 0.45 μm nylon filter prior to analysis by high-performance anion-exchange chromatography (HPAEC) on a Dionex PA100 column (Dionex, Sunnyvale, California, USA). Elution was monitored by pulsed amperometric detection (Dionex ED40) and the gradient controlled by a Varian ProStar pump system. Data were collected and analysed by Star Chromatography Workstation 6.41 (Varian, Inc. Walnut Creek, California, USA), and 6′-SL, 3′-SL and DSL external standards were used to generate standard curves for comparison.

The results showed a significant effect of breed and sampling time on SOS content. Garganica breed showed the highest values of 3′-SL and 6′-SL while Maltese breed the lowest content of DSL (**Figure 12**).

Also the interaction breed x sampling time affected the SOS content in milk and colostrum; in particular, 3′-SL content was significantly higher in Garganica colostrum at 24 h after kidding and in milk at the 7th and 30th day. DSL was affected by interaction, showing higher values in Garganica's colostrum at parturition and Maltese's colostrum 24 h and milk. The content of the

three SOSs was higher than values found by Ref. [73] on Spanish goats, confirming the breed effect and, consequently, the importance of the choice of the breed among the management options aimed at improving the nutraceutical quality of milk, namely, the oligosaccharide content.

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**Figure 12.** Effect of breed and sampling time on SOS concentration in milk [22].

Bioactive Compounds in Goat Milk and Cheese: The Role of Feeding System and Breed http://dx.doi.org/10.5772/intechopen.70083 253

**Figure 12.** Effect of breed and sampling time on SOS concentration in milk [22].

The breed effect on milk oligosaccharide (OS) composition, and in particular sialyloligosaccharide (SOS) content, is scarcely studied. The milk from the Spanish Murciana-Granadina goat breed was found characterised by 25 OS [72], later [73] isolated 15 new oligosaccharide structures from fresh milk of Spanish goats, obtaining a virtually lactose and salt-free product, containing more than 80% of the original oligosaccharide content. Evenly, the effect of

Within the management's strategies, the choice of the breed is a key element to weigh up towards the type of livestock, the available resources and the business plan, in terms of destination of the milk, namely, if destined to the market of drinking milk or to dairy production. Some speculations can be made whether optimizing production, rheological properties, and bioactive profile and content may be feasible with feeding management modulated in terms of energy and protein supply depending upon genotype. The following three case studies are

A study on the content of three SOSs, namely, 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL) and di-sialyllactose (DSL) in colostrum and milk, was carried out [22] with two Italian goat breeds, Garganica goat (a native breed from Gargano Mountain in Apulia region, Southern Italy) and the Maltese goat breed, native from Malta isle in the Mediterranean area. The animals were fed indoor, receiving hay (from polyphytic cultivated meadows) *ad libitum* and concentrate supplementation (400 and 600 g/h/d, respectively) at 14% crude protein, according to their milk yield (800 and 1200 g/day milk, respectively). The SOSs were isolated from individual colostrum and milk samples obtained in five periods (at kidding, 24 h, 7 days, 30 days and 90 days after delivery). Briefly, after centrifugation (2000 × g, 4°C, 10 min) the supernatant lipid layer was removed, and the proteins were precipitated by addition of 0.5 volumes of

O and 0.5 volumes of 2 g 100 mL−1 ZnSO<sup>4</sup>

vortexed and centrifuged (12,000 × g, 10 min, 4°C). The supernatant was removed and centrifuged again. The second supernatant was filtered with a 0.45 μm nylon filter prior to analysis by high-performance anion-exchange chromatography (HPAEC) on a Dionex PA100 column (Dionex, Sunnyvale, California, USA). Elution was monitored by pulsed amperometric detection (Dionex ED40) and the gradient controlled by a Varian ProStar pump system. Data were collected and analysed by Star Chromatography Workstation 6.41 (Varian, Inc. Walnut Creek, California, USA), and 6′-SL, 3′-SL and DSL external standards were used to generate standard

The results showed a significant effect of breed and sampling time on SOS content. Garganica breed showed the highest values of 3′-SL and 6′-SL while Maltese breed the lowest content of

Also the interaction breed x sampling time affected the SOS content in milk and colostrum; in particular, 3′-SL content was significantly higher in Garganica colostrum at 24 h after kidding and in milk at the 7th and 30th day. DSL was affected by interaction, showing higher values in Garganica's colostrum at parturition and Maltese's colostrum 24 h and milk. The content of the

·7H<sup>2</sup>

O. The blend was

presented, in order to partially cover this gap on Mediterranean goats.

**3.1. Case studies 1 and 2: oligosaccharides in colostrum and milk**

interaction of breed x feeding is scarcely studied.

252 Goat Science

1.8 g 100 mL−1 Ba(OH)<sup>2</sup>

curves for comparison.

DSL (**Figure 12**).

·8H<sup>2</sup>

three SOSs was higher than values found by Ref. [73] on Spanish goats, confirming the breed effect and, consequently, the importance of the choice of the breed among the management options aimed at improving the nutraceutical quality of milk, namely, the oligosaccharide content.

In a further study [74], Garganica goat milk SOSs were compared with Saanen goats' from colostrum time to the 90th day after parturition, in their 3rd parity. In the experiment, the Saanen and Garganica goats were fed indoor, receiving hay *ad libitum* (50% polyphytic meadow and 50% alfalfa hay) and 700 g/h/day DM and 450 g/day/h DM, respectively, of concentrate supplementation at 18.3% crude protein (wheat by-products, corn grain, soybean meal, molasses, supplemented with mineral mixtures). The results confirmed a breed effect on SOS contents in colostrum and milk also between Garganica and Saanen breed, where Garganica goats showed mean higher values for the three SOSs (see **Table 2**).

considering the genotype, the diet and their interaction. The results revealed that genotype and diet affected the 3′-SL content in milk (*P* < 0.05) (**Figure 13**), while their interaction expressed only a trend of variation (*P* = 0.10). The goats fed with undernourishing diet (D)

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The 6′-SL and DSL showed only a decreasing trend. This result might be related to the reduction of the expression of genes involved in the milk synthesis after a prolonged fasting [78]. Similarly, in human milk a decrease of OS was found in milk from undernourished

**Figure 13.** Effect of genotype (a) and diet (b) on three sialyloligosaccharides in Mediterranean Red goat milk (adapted

women [11].

from Ref. [77]).

showed a 3′-SL content 58.5% lower than M goats.

Moreover, a significant interaction breed x sampling time was recorded for 3′-SL (*P* < 0.001) and 6′-SL (*P* < 0.01), while no significant interaction was found for DSL. The results may be considered under a genetic point of view. In fact, under equal feeding condition, the breeds expressed their OS synthesis potential, probably influenced by the genetic polymorphism in the locus CSN1S1 (αs1-casein). On this matter, the Saanen goats were characterised by a high frequency of defective alleles (F and E) and low frequency of strong alleles (A and B) at αs1-casein locus; contrarily Garganica goats had high frequency at strong alleles and low frequency at weak alleles (F) [75].

In a previous study, Ref. [76] have found in Alpine goats that the genotype (A/A or 0/0) affected the OS profile, even though not the total OS production. So, Claps et al. [74] speculated that an indirect link between goat breeds, about the allelic frequencies at the locus αs1-casein in Saanen and Garganica goat breeds, might have affected the SOS content in milk and colostrum, where the Saanen breed, characterised by high frequencies of defective alleles [66], could have adversely affected the production of SOSs.

#### **3.2. Case study 3: interaction of genotype with feeding regimen**

The Mediterranean Red goat was characterised for the content of three SOSs considering the polymorphism at locus CSN1S1 and its interaction with feeding regimen [77]. Six goats, with genotype A/A (strong, αs1-casein producers), and six goats with genotype F/F (weak) were fed with two diets in pellet, respectively, at 100% of energetic and 105% of protein requirements (M) and 70% of energetic and 75% of protein requirements (L). Milk samples at the 69th ± 3 day of milking were analysed for the content of three sialyllactoses (see Section 3.1),


Means within a row with different letters (a, b) differ at *P* ≤ 0.05. SE = standard error.

\* *P* < 0.05.

\*\*\**P* < 0.001.

**Table 2.** Mean content in milk of 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL) and disialyllactose (DSL) from Garganica goat breed compared with Saanen, a cosmopolitan breed, at 30 days in milk (from Ref. [74]).

considering the genotype, the diet and their interaction. The results revealed that genotype and diet affected the 3′-SL content in milk (*P* < 0.05) (**Figure 13**), while their interaction expressed only a trend of variation (*P* = 0.10). The goats fed with undernourishing diet (D) showed a 3′-SL content 58.5% lower than M goats.

**Figure 13.** Effect of genotype (a) and diet (b) on three sialyloligosaccharides in Mediterranean Red goat milk (adapted from Ref. [77]).

**SOS Garganica Saanen SE Significance**

**Table 2.** Mean content in milk of 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL) and disialyllactose (DSL) from Garganica

In a further study [74], Garganica goat milk SOSs were compared with Saanen goats' from colostrum time to the 90th day after parturition, in their 3rd parity. In the experiment, the Saanen and Garganica goats were fed indoor, receiving hay *ad libitum* (50% polyphytic meadow and 50% alfalfa hay) and 700 g/h/day DM and 450 g/day/h DM, respectively, of concentrate supplementation at 18.3% crude protein (wheat by-products, corn grain, soybean meal, molasses, supplemented with mineral mixtures). The results confirmed a breed effect on SOS contents in colostrum and milk also between Garganica and Saanen breed, where

Moreover, a significant interaction breed x sampling time was recorded for 3′-SL (*P* < 0.001) and 6′-SL (*P* < 0.01), while no significant interaction was found for DSL. The results may be considered under a genetic point of view. In fact, under equal feeding condition, the breeds expressed their OS synthesis potential, probably influenced by the genetic polymorphism in the locus CSN1S1 (αs1-casein). On this matter, the Saanen goats were characterised by a high frequency of defective alleles (F and E) and low frequency of strong alleles (A and B) at αs1-casein locus; contrarily Garganica goats had high frequency at strong alleles and low

In a previous study, Ref. [76] have found in Alpine goats that the genotype (A/A or 0/0) affected the OS profile, even though not the total OS production. So, Claps et al. [74] speculated that an indirect link between goat breeds, about the allelic frequencies at the locus αs1-casein in Saanen and Garganica goat breeds, might have affected the SOS content in milk and colostrum, where the Saanen breed, characterised by high frequencies of defective alleles

The Mediterranean Red goat was characterised for the content of three SOSs considering the polymorphism at locus CSN1S1 and its interaction with feeding regimen [77]. Six goats, with genotype A/A (strong, αs1-casein producers), and six goats with genotype F/F (weak) were fed with two diets in pellet, respectively, at 100% of energetic and 105% of protein requirements (M) and 70% of energetic and 75% of protein requirements (L). Milk samples at the 69th ± 3 day of milking were analysed for the content of three sialyllactoses (see Section 3.1),

Garganica goats showed mean higher values for the three SOSs (see **Table 2**).

frequency at weak alleles (F) [75].

254 Goat Science

3′-SL (mg/L) 195.5 a 124.8 b 12.7 \*\*\* 6′-SL (mg/L) 129.7 a 15.4 b 5.5 \*\*\* DSL (mg/L) 104.0 a 79.9 b 7.3 \*

Means within a row with different letters (a, b) differ at *P* ≤ 0.05. SE = standard error.

[66], could have adversely affected the production of SOSs.

**3.2. Case study 3: interaction of genotype with feeding regimen**

goat breed compared with Saanen, a cosmopolitan breed, at 30 days in milk (from Ref. [74]).

\* *P* < 0.05. \*\*\**P* < 0.001. The 6′-SL and DSL showed only a decreasing trend. This result might be related to the reduction of the expression of genes involved in the milk synthesis after a prolonged fasting [78]. Similarly, in human milk a decrease of OS was found in milk from undernourished women [11].

These results demonstrated that there is a different efficiency in diet utilisation and response in synthesis of metabolites such as oligosaccharides, depending on the genotype. Consequently, in systems that use selected animals, the diet may be formulated taking into account the genotype, in order to achieve certain qualitative profile of goat milk and increase the efficiency in feeding management.

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