**3. Results and discussion**

## **3.1. Milk yield and the chemical composition of milk**

The average milk yield and its content of fat, proteins, lactose, dry matter, non-fat dry matter, total number of microorganisms, and somatic cell count in different periods of the experiment by groups is shown in Table 2. In the preparatory period, only somatic cell count statistically significantly differed among groups. Statistically significant differences among groups in the experimental period appeared in dry matter, somatic cell count, and logarithm of the somatic cell count. In the third period of the experiment, statistically significant differences among groups appeared in the majority of observed traits.

It seems that the short time fatty acid supplementation into goat's diet does not negatively affect their milk yield. Milk yield did not vary statistically significant during the observed period (Table2). As found by Sampelayo et al. (2002), the supplemented fatty acids into the diet of Granadina goats did not affect their milk yield and the content of fat, proteins, lactose, and dry matter in milk.


a b – values which are not marked with the same letter are statistically significantly different at least P<0.05 NFDM – non-fat dry matter; DM – dry matter; SCC – somatic cell count; MO – microorganisms;

**Table 2.** Average values of the observed traits in different periods of the experiment by groups

Milk fat yield statistically significantly increased in ALFA group from 3.15 to 3.40 g/100 ml on average when goats were supplemented with linseed oil rich in α-linoleic acid (Table 2) and it slightly decreased to 3.30 g/100 ml until the third period of the experiment. In groups EPA and DHA, milk fat yield firstly decreased, while it increased slightly after the end of supplementation with fatty acids.

12 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**3.1. Milk yield and the chemical composition of milk** 

differences among groups appeared in the majority of observed traits.

**3. Results and discussion** 

lactose, and dry matter in milk.

Proteins (g/100 ml)

Lactose (g/100 ml)

log10\_MO (n\*103/ml)

log10\_SCC (n\*103/ml)

among groups were estimated by the linear contrasts, while connections between the properties were calculated by the Pearson correlation coefficient. The limit of statistical significance was taken at P <0.05 and highly statistically significance was taken at P <0.001.

The average milk yield and its content of fat, proteins, lactose, dry matter, non-fat dry matter, total number of microorganisms, and somatic cell count in different periods of the experiment by groups is shown in Table 2. In the preparatory period, only somatic cell count statistically significantly differed among groups. Statistically significant differences among groups in the experimental period appeared in dry matter, somatic cell count, and logarithm of the somatic cell count. In the third period of the experiment, statistically significant

It seems that the short time fatty acid supplementation into goat's diet does not negatively affect their milk yield. Milk yield did not vary statistically significant during the observed period (Table2). As found by Sampelayo et al. (2002), the supplemented fatty acids into the diet of Granadina goats did not affect their milk yield and the content of fat, proteins,

Group EPA ALFA DHA KONT EPA ALFA DHA KONT EPA ALFA DHA KONT Trait/Period 1 1 1 1 2 2 2 2 3 3 3 3 Milk (ml) 780a 748a 869a 766a 790a 708a 888a 824a 765a 719a 884a 789a Fat (g/100 ml) 3.05a 3.15a 3.00a 2.99a 2.65a 3.40b 2.52a 2.91a 2.84a 3.30b 2.77a 3.06a

NFDM (g/100 ml) 8.32a 8.48a 8.26a 8.33a 8.39a 8.55a 8.29a 8.30a 8.45a 8.62b 8.53a 8.33a DM (g/100 ml) 11.37a 11.62a 11.26a 11.33a 11.04a 11.76b 10.81a 11.21a 11.29a 11.92b 11.30a 11.39a MO (n\*103/ml) 653a 609a 498a 505a 315a 334a 350a 494a 266a 267a 267a 347a SCC (n\*103/ml) 1316a 1095a 585b 526b 1631a 975b 1166a 1258a 1531a 915b 1884a 1364a

a b – values which are not marked with the same letter are statistically significantly different at least P<0.05 NFDM – non-fat dry matter; DM – dry matter; SCC – somatic cell count; MO – microorganisms;

**Table 2.** Average values of the observed traits in different periods of the experiment by groups

2.93a 3.12a 2.98a 3.01a 3.01a 3.21b 3.01a 3.06a 3.15a 3.28a 3.29a 3.07b

4.59a 4.55a 4.49a 4.53a 4.58a 4.54a 4.48a 4.44a 4.50a 4.54a 4.44a 4.46a

2.64a 2.62a 2.47a 2.58a 2.44a 2.44a 2.48a 2.62b 2.35a 2.34a 2.34a 2.48a

2.69a 2.68a 2.62a 2.54a 2.82a 2.72a 2.80a 2.99a 2.83a 2.77b 2.88a 2.99a

There were no statistical significant differences among the groups of goats in milk protein yield before the supplementation with fatty acids (Table 2). During the supplementation of goats with fatty acids, milk protein yield increased and it was increasing also after the end of supplementation. Group ALFA had the highest protein yield in milk in the whole time of the experiment.

In general, lactose in milk varies little, what was confirmed also in our research. There were no statistical significant differences in lactose yield among the observed groups, neither during the supplementation with fatty acids nor after that (Table 2).

Non-fat dry matter increased during the experiment in all observed groups which were supplemented with fatty acids, but not in the control group KONT (Table 2). Differences among groups were not statistically significant. Total dry matter decreased after supplementing with fatty acids in groups EPA, DHA, and KONT, while it increased in ALFA group. After the end of supplementing with fatty acids, total dry matter increased in all groups. Group ALFA statistically significantly differed in milk dry matter from other observed groups in the second and third period of the experiment.

The number of microorganisms in milk mostly depends on milking hygiene, which includes staff, animals, facilities, equipment, hygiene maintenance, and cleaning of the equipment. It also depends on the health of the udder and the presence of mastitis. Soon after the beginning of the experiment, the hygiene and cleaning improved, and the number of microorganisms in milk decreased (Table 2). There was no mastitis detected in the whole time of the experiment. No statistically significant differences were noticed among groups in the number of microorganisms in milk.

Somatic cell count was one of the most variable traits in our experiment, since we found that values ranged from 13.000 to 24,312.000 of somatic cells in ml of milk. Despite the great variability, transformation of somatic cell count to the logarithmic value enabled to find the possible impacts of supplementation with fatty acids on somatic cell count (Figure 1). Preliminary report by Košmelj et al. (2001) showed the impact of supplementing alphalinolenic fatty acid to goats, which was reflected in a reduction of the number of somatic cells during the supplementation and four weeks after.

The average values for medians during the supplementation with fatty acids (Me1) and for medians five days after the supplementation with fatty acids (Me2) are shown in Table 3. Results showed statistical significant differences among groups of goats for medians during the supplementation with fatty acids and also for medians five days after the supplementation with fatty acids. The average of medians (Me1 and Me2) in group ALFA is negative, so it could be affirmed, that the supplementation of linseed oil rich in α-linoleic acid decreases the number of somatic cell count in milk.

Medians for Log10 somatic cell count

**Figure 1.** Standardization and log10 value median for number of somatic cells by groups


a, b - Groups with different letter are statistically significantly different (P<0.05)

**Table 3.** Average value of Me1 in Me2 in different group

On average, somatic cells in goat milk are present in a greater number than in cow milk. Zeng et al. (1997) reported that 17% of goat milk samples recorded on goat farms which are members of the Association of goat farmers in the U.S. exceeded the standard 1.0x106 of somatic cells ml-l when the experiment of daily monitoring of somatic cells in milk was carried out.

Das & Singh (2000) studied somatic cells in goat milk and electrical conductivity of milk. In the blood samples total leucocytes and differential leucocytes (lymphocytes, monocytes, neutrophils, eosinophil, and basophils) were also determined. Somatic cell count in goat milk was high during early lactation and decreased subsequently as the lactation advanced. There were found individual variations (P<0.01) in somatic cell counts between different lactation periods as weel as among and within animals. For example, one goat had very high somatic cell count in comparison to other goats from the beginning to the end of the experiment. The goat was then tested for mastitis using California mastitis test and it was found to have normal milk. Similar results were found in our experiment. Total leucocyte count in blood also decreased as the lactation progressed and remained fluctuated during late lactation in the study by Das & Singh (2000). Lymphocytes and neutrophils were low during early lactation and with establishment of lactation stabilized to normal levels. Protein content of milk did not vary during different periods of lactation. However, lactose decreased and fat percent increased with advanced lactation. It is interesting that the connection between somatic cell count and milk yield and between somatic cell count and milk composition was not found in any stage of lactation.

Mastitis is typically associated with a large number of somatic cells in small ruminants. In our experiment, the number of somatic cells significantly reduced only in the ALFA group and lasted statistically significant 39 days after the supplementation with fatty acids. For αlinolenic fatty acid is known, that it could incorporate into phospholipids five hours after ingestion (Adam et al., 1986). The other two, eicosapentaenoic acid and docosahexaenoic acid can incorporate into phospholipids only after a few days supplementation. The statistically significant effect of the α-linolenic fatty acid only on somatic cell count could be explained by the rapidness of incorporation into membrane phospholipids of this fatty acid.

The fluctuations of the somatic cell count in goat milk are subjected to many influences. Researchers have not explored other reasons for the number of somatic cells in goat milk except the hygiene measures. Ruminants are in the last 20 years fed adding n-3 fatty acids to improve the fatty acid composition of milk and meat, but the impact on the number of somatic cells have not been monitored. Our experiment clearly shows that the supplementation of the α-linolenic fatty acid had a relatively long time impact on reducing somatic cell count or to a low level of somatic cells in milk. The interpretation may be possible, that we achieved a more appropriate relationship between n-3 and n-6 long chain fatty acids with the supplementation of α-linolenic fatty acid which was not provided by the diet.

## **3.2. Composition of fatty acids in goat milk**

14 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

Medians for Log10 somatic cell count

**Figure 1.** Standardization and log10 value median for number of somatic cells by groups

12

13

14

15

Day

EPA ALFA DHA KONT

16

17

18

19

24

29

34

39

44

49

54

59

64

a, b - Groups with different letter are statistically significantly different (P<0.05)

**Table 3.** Average value of Me1 in Me2 in different group

carried out.

Standard values for medians

1

2

3

4

5

6

7

8

9

10

11

Period / Group EPA ALFA DHA KONT Average for Me1 1.01b -3.11 a 1.68 b 3.52 b Average for Me2 1.75 b -2.47 a 1.78 b 3.20 b

On average, somatic cells in goat milk are present in a greater number than in cow milk. Zeng et al. (1997) reported that 17% of goat milk samples recorded on goat farms which are members of the Association of goat farmers in the U.S. exceeded the standard 1.0x106 of somatic cells ml-l when the experiment of daily monitoring of somatic cells in milk was

Das & Singh (2000) studied somatic cells in goat milk and electrical conductivity of milk. In the blood samples total leucocytes and differential leucocytes (lymphocytes, monocytes, neutrophils, eosinophil, and basophils) were also determined. Somatic cell count in goat milk was high during early lactation and decreased subsequently as the lactation advanced. There were found individual variations (P<0.01) in somatic cell counts between different lactation periods as weel as among and within animals. For example, one goat had very high somatic cell count in comparison to other goats from the beginning to the end of the experiment. The goat was then tested for mastitis using California mastitis test and it was found to have normal milk. Similar results were found in our experiment. Total leucocyte count in blood also decreased as the lactation progressed and remained fluctuated during late lactation in the study by Das & Singh (2000). Lymphocytes and neutrophils were low during early lactation and with establishment of lactation stabilized to normal levels. Protein content of milk did not vary during different periods of lactation. However, lactose Chemical analysis of goat milk fat was done for fatty acids from 10:00 to 24:6, n-9. The fat composition of goat milk was studied by each milking during the experiment time. Therefore, values listed below (Table 3) represent the percentage of the all analyzed fatty acids rather than total fat in goat milk.

During our experiment, there was from 9.0 to 14.0 wt % of the **capric** acid (10:0) in the goat milk fat. Some authors (Hurley, 2009; Jandal, 1996; Sanz Sampelayo et al., 2002) indicated values from 8.4 to 11.1%. EPA group had the lowest level of capric acid before supplementing with fatty acids, while its level exceeded groups ALFA and KONT during the supplementation and declined to the lowest level among groups in the last period of the experiment. DHA group had the highest level of the capric acid during the supplementation with fatty acids as well as all the time after the supplementation. It is known that goat milk has more short-chain fatty acids (C4:0 to C10:0) than cow's milk, which are easier to digest than long-chain fatty acids.

We found that the **lauric** acid (12:0) in goat milk fat presented between 3.8 and 7.7 wt %. During the supplementation with fatty acids, the lauric acid increased for 2% in DHA group and for 1% in EPA group. The increase in EPA group lasted two days after the end of the supplementation, and four days in DHA group. Hurley (2009) found that there is 3.3% of the lauric acid in goat milk fat, Jandal (1996) reported about 6.0%, while Sanz Sampelayo et al. (2002) found from 4.69 to 5.11% of the lauric acid in goat milk fat.


16 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

a b – values which are not marked with the same letter, are statistically different at least P<0.05

FA – fatty acid; CLA – conjugated linoleic acid; LC PUFA – long chain polyunsaturated fatty acid

**Table 4.** Average values of fatty acids, secreted in milk in different periods of the experiment by groups (wt %)

**Myristic** acid (14:0) in goat milk fat represented from 10.0 to 13.5 wt % of fatty acids. The content was similar than in Sanz Sampelayo's et al. (2002) research. Throughout supplementing the fatty acids, a statistically significant reduction of myristic acid level in milk fat was noticed only in the ALFA group (p<0.05). Other variations were not statistically significant and the level of myristic acid was similar among groups. Myristic content in goat milk fat was very stable during the experiment.

**Miristoleic** acid (14:1) in goat milk fat was detected in the content from 0.12 to 0.40 wt %, while Sanz Sampelayo et al. (2002) listed the values between 0.41 and 0.64%. We have not observed differences among groups and even daily fluctuations of miristoleic acid in goat milk fat were very small. Miristoleic acid values were fluctuating at least in DHA group. Differences among groups were not found in any period of the experiment.

16 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

1 1 1 1 2 2 2 2 3 3 3 3

26.19a 23.08a 22.29a 23.68a 21.89a 22.80a 19.94b 22.62a 24.25a 24.58a 22.50a 26.00a

1.06 a 0.94 a 0.90 a 0.90 a 6.33 b 1.68 a 5.32 b 1.42 a 3.26 b 1.64 a 3.43 b 1.17 a

LC n-3 : n-6 (1:X) 3.11 a 2.93 a 2.91 a 3.00 a 12.17 b 5.41 a 8.44 b 4.58 a 8.35 b 4.96 a 7.80 b 3.24 a

**Table 4.** Average values of fatty acids, secreted in milk in different periods of the experiment by groups

**Myristic** acid (14:0) in goat milk fat represented from 10.0 to 13.5 wt % of fatty acids. The content was similar than in Sanz Sampelayo's et al. (2002) research. Throughout supplementing the fatty acids, a statistically significant reduction of myristic acid level in milk fat was noticed only in the ALFA group (p<0.05). Other variations were not statistically significant and the level of myristic acid was similar among groups. Myristic content in goat

**Miristoleic** acid (14:1) in goat milk fat was detected in the content from 0.12 to 0.40 wt %, while Sanz Sampelayo et al. (2002) listed the values between 0.41 and 0.64%. We have not observed differences among groups and even daily fluctuations of miristoleic acid in goat

a b – values which are not marked with the same letter, are statistically different at least P<0.05 FA – fatty acid; CLA – conjugated linoleic acid; LC PUFA – long chain polyunsaturated fatty acid

milk fat was very stable during the experiment.

FA / GROUP EPA ALFA DHA KONT EPA ALFA DHA KONT EPA ALFA DHA KONT 10:0 9.55a 11.63a 10.84a 11.16a 11.64a 11.20a 13.13b 11.60a 9.84a 10.64a 12.09b 10.08a 12:0 4.43a 6.06a 4.97a 5.38a 5.41a 5.65a 6.56a 6.06a 4.93a 5.56a 6.21a 4.96a 14:0 10.79a 12.31a 11.44a 11.88a 11.88a 10.90b 11.62a 12.49a 11.25a 11.25a 11.39a 11.14a 16:0 25.62a 24.92a 27.61a 26.01a 23.98a 22.79a 25.24a 25.04a 24.75a 23.29a 24.12a 23.81a 16:1, n-7 1.25a 1.19a 1.32a 1.18a 1.24a 1.18a 1.51b 1.16a 1.22a 1.25a 1.57b 1.25a 18:0 11.09a 9.98a 10.65a 10.24a 7.26b 10.32a 4.23b 9.54a 11.14a 11.08a 7.31b 11.04a

CLA (1) 0.82a 0.81a 0.74a 0.79a 1.73b 1.35b 2.89b 0.92a 1.19b 1.06a 2.50b 0.93a 18:2, n-6c 2.31a 2.14a 2.22a 2.17a 2.81b 3.10b 2.40a 2.19a 2.22a 2.48a 2.39a 2.55a 18:3, n-3 0.70a 0.57a 0.53a 0.52a 0.97a 2.98b 0.60a 0.80a 0.87a 0.95b 0.66a 0.78a 18:3, n-6 0.05a 0.04a 0.03a 0.00a 0.14a 0.29b 0.14a 0.17a 0.08a 0.12a 0.09a 0.04a 20:3, n-3 0.03a 0.02a 0.02a 0.02a 0.32b a 0.03a 0.04a 0.03a 0.12b 0.04a 0.02a 0.03a 20:3, n-6 0.02a 0.03a 0.02a 0.02a 0.06b 0.03a 0.04a 0.02a 0.03a 0.02a 0.04a 0.03a 20:4, n-6 0.25a 0.21a 0.22a 0.21a 0.39b 0.21a 0.47b 0.20a 0.32b 0.22a 0.30b 0.23a 20:5, n-3 0.08a 0.07a 0.07a 0.06a 2.41b 0.14a 0.48a 0.13a 0.50b 0.15a 0.30a 0.09a 22:3, n3 0.00a 0.00a 0.00a 0.00a 0.04b 0.00a 0.07b 0.00a 0.00a 0.00a 0.07b 0.02a 22:4, n6 0.07a 0.08a 0.07a 0.07a 0.10a 0.07a 0.10a 0.09a 0.10a 0.09a 0.10a 0.10a 22:5, n-3 0.18a 0.15a 0.14a 0.13a 0.64b 0.19a 0.49b 0.15a 0.60b 0.24a 0.33b 0.18a 22:6,n-3 0.07a 0.06a 0.05a 0.06a 0.13a 0.16a 2.27b 0.13a 0.15a 0.11a 0.79b 0.10a n-3 0.99 a 0.81 a 0.76 a 0.73 a 4.13 b 3.34 b 1.68 a 1.11 a 1.99 b 1.38 a 1.38 a 1.10 a n-6 2.70 a 2.50 a 2.56 a 2.47 a 3.47 b 3.70 b 3.17 b 2.67 a 2.69 b 2.93 a 2.92 a 2.95 a n-3/n-6 0.37 a 0.32 a 0.30 a 0.30 a 1.19 a 0.90 b 0.53 b 0.42 a 0.74 b 0.47 a 0.47 a 0.37 a n-3 : n-6 (1:X) 2.73 a 3.09 a 3.37 a 3.38 a 0.84 b 1.11 b 1.89 a 2.41 a 1.35 b 2.12 a 2.12 a 2.68 a LC PUFA n-3 0.36 a 0.30 a 0.28 a 0.27 a 3.29 b 0.52 a 3.35 b 0.44 a 1.27 b 0.54 a 1.51 b 0.42 a LC PUFA n-6 0.34 a 0.32 a 0.31 a 0.30 a 0.52 b 0.31 a 0.63 b 0.31 a 0.39 a 0.33 a 0.44 a 0.36 a

Experimental period

18:1, n-9c, 18:1, n-9t, 18:1, n-12t, 18:1, n-7c

LC PUFA n-3/ LC PUFA n-6

(wt %)

There was between 20 and 29 wt % of the **palmitic** acid (16:0) in the goat milk fat. Sanz Sampelayo et al. (2002) indicated values of the palmitic acid between 24.6 and 27.7%. There were no statistically significant differences observed among groups before the supplementation of the fatty acids to the goat diet. There was a trend of decreasing values during and immediately after the supplementation of fatty acids, especially in groups DHA and ALFA as well as in the EPA group.

In goat milk fat, between 1.06 and 1.73 wt % of the **palmitoleic** acid (16:1, n-7) was determined. There were no differences in the level of this fatty acid among groups before the supplementation with fatty acids. Among groups EPA, ALFA, and KONT, no statistically significant differences in the content of the palmitoleic acid in milk fat were observed neither during the supplementation with fatty acids nor after that. The content of palmitoleic acid in DHA group increased statistically significant (from 1.30% to 1.70%) from the second day of the supplementation with fatty acids. The high level of this fatty acid lasted till the ninth day after the supplementation (p<0.001). The supplementation with unprotected n-3 fatty acids in cows reduced the content of palmitoleic acid in milk fat (Chilliard et al., 2001), what is contrary to our results.

**Stearic** fatty acid (18:0) in the goat milk fat was presented in the level from 2 to 14 wt %. There were no differences in the stearic acid content among groups before supplementation with fatty acids. Differences appeared during the supplementation with fatty acids, which were expressed the most in DHA group, where the percentage of stearic fatty acid fell from about 10 to less than 3% (p<0.001). The fall of stearic acid during the supplementation with fatty acids was detected also in EPA group (p<0.05), which was somewhat less pronounced, and the level of stearic acid re-established to the previous level within two days after the end of the supplementation with fatty acids. The previous level of stearic acid in DHA group was re-established five days after the end of supplementation with fatty acids. In ALFA and KONT group, there were no statistically significant differences in the levels of stearic fatty acid throughout the experiment. This information is a further indication, that the biodegradation of long-chain fatty acids (DHA) does not expire until the stearic acid, but there are several isomers of conjugated cis- and trans- C 18:2 fatty acids (Gulati et al., 1997; Gulati et al., 2000).

The content of **oleic** fatty acid (18:1, n-9) was in our experiment determined in the concentration from 19.0 to 28.0 wt %. During the supplementation with fatty acids, the content of oleic acid statistically significantly declined in EPA and DHA groups. An increase of the content of oleic acid in milk was observed in groups KONT and ALFA, as during as well as after the supplementation with fatty acids, but differences in these two groups before and after the supplementation were not statistically significant. Sanz Sampelayo et al. (2002) noted the content of oleic acid in goat milk around 22 to 24% and stated that despite the addition of various concentrations of protected polyunsaturated fatty acids the content of oleic fatty acid in goat milk remained fairly constant.

**Conjugated linoleic** acids (CLA) are a family of at least 28 isomers of linoleic acid found mainly in the meat and dairy products derived from ruminants. Several names could be

found for conjugated linoleic acid, most often conjugated linoleic acid, then rumenic or ruminal acid or cis-9, trans-11 octadecadienoic acid. It is one of those found only in ruminants and is a product of incomplete hydrogenation of fatty acids in the rumen (Clegg et al., 2001; Chouinard et al., 1999). In goats fed with fish oil (Gulati et al., 2000) mainly vaccenic fatty acid is formed due to the altered pattern of the biohydrogenation. In our experiment (Figure 2), goat milk of all observed groups contained less than 1.0% of the conjugated linoleic acid before the supplementation with fatty acids. During the second period, groups EPA, ALFA, and DHA statistically significantly differed (p<0.05) from KONT group. The largest increase of the conjugated linoleic acid content during the supplementation appeared in DHA group, to over 3.0%. The content of conjugated linoleic acid in EPA group increased to 2.0%, and in ALFA group to 1.5%. The effect of the conjugated linoleic acid in DHA group was detected ten days after the supplementation with fatty acids. In nature, the most of the conjugated linoleic acids have their origin in alpha linolenic acid (Gulati et al., 2000), while in our experiment, the conjugated linoleic acid increased the most after feeding goats with docosahexaenoic acid (group DHA). Chilliard et al. (2001) fed cows with 200 to 300 g of the fish oil daily where the content of the conjugated linoleic acid increased from 0.2 to 0.6% to 1.5 to 2.7%. Authors mentioned that mainly rumenic acid increased which is presented also in our results, whereas the vaccenic acid occurred only in trace amounts and only a short time so that the findings published by Gulati et al. (2000 ) we could not confirm.

**Figure 2.** Average value of rumenic acid in goat milk

Conjugated linoleic acid is an intermediate product of the biohydrogenation, therefore its high concentration in DHA group was logical, since the degradation of the docosahexaenoic acid in the rumen is the slowest. The concentration of the conjugated linoleic acid in goat milk fat was relatively high also in ALFA group, knowing that the biohydrogenation of the α-linoleic acid is the fastest (Gulati et al., 1999), what we also observed in an increased concentration of C 18:1 in ALFA group. The conjugated linoleic acid is synthesised in the mammary gland of lactating animals and in the muscles of young animals. In our experiment, the conjugated linoleic acid probably did not originate only from the supplemented fatty acids, what was found also by Griinari et al. (2000).

18 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

Gulati et al. (2000 ) we could not confirm.

**Figure 2.** Average value of rumenic acid in goat milk

0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00

wt %

found for conjugated linoleic acid, most often conjugated linoleic acid, then rumenic or ruminal acid or cis-9, trans-11 octadecadienoic acid. It is one of those found only in ruminants and is a product of incomplete hydrogenation of fatty acids in the rumen (Clegg et al., 2001; Chouinard et al., 1999). In goats fed with fish oil (Gulati et al., 2000) mainly vaccenic fatty acid is formed due to the altered pattern of the biohydrogenation. In our experiment (Figure 2), goat milk of all observed groups contained less than 1.0% of the conjugated linoleic acid before the supplementation with fatty acids. During the second period, groups EPA, ALFA, and DHA statistically significantly differed (p<0.05) from KONT group. The largest increase of the conjugated linoleic acid content during the supplementation appeared in DHA group, to over 3.0%. The content of conjugated linoleic acid in EPA group increased to 2.0%, and in ALFA group to 1.5%. The effect of the conjugated linoleic acid in DHA group was detected ten days after the supplementation with fatty acids. In nature, the most of the conjugated linoleic acids have their origin in alpha linolenic acid (Gulati et al., 2000), while in our experiment, the conjugated linoleic acid increased the most after feeding goats with docosahexaenoic acid (group DHA). Chilliard et al. (2001) fed cows with 200 to 300 g of the fish oil daily where the content of the conjugated linoleic acid increased from 0.2 to 0.6% to 1.5 to 2.7%. Authors mentioned that mainly rumenic acid increased which is presented also in our results, whereas the vaccenic acid occurred only in trace amounts and only a short time so that the findings published by

Conjugated linoleic acid is an intermediate product of the biohydrogenation, therefore its high concentration in DHA group was logical, since the degradation of the docosahexaenoic acid in the rumen is the slowest. The concentration of the conjugated linoleic acid in goat milk fat was relatively high also in ALFA group, knowing that the biohydrogenation of the α-linoleic acid is the fastest (Gulati et al., 1999), what we also observed in an increased concentration of C 18:1 in ALFA group. The conjugated linoleic acid is synthesised in the

5,5 6,5 7,5 8,5 9,5 10,5 11,5 12,5 13,5 14,5 15,5 16,5 17,5 18,5 19,5 24,5 29,5 34,5 Day

EPA ALFA DHA KONT

18:2, n-6 cis-9,trans-11

Before the supplementation with fatty acids, there was from 2.00 to 2.66 wt % of the **linoleic** acid (18:2, n-6) determined in goat milk fat in all groups. During the supplementation, the percentage increased in EPA group to 2.92% and in ALFA group to 3.4% (p<0.001). Three days after the end of supplementation, the percentage dropped back to the previous value. There were no changes in the content of linoleic acid during the whole experiment in DHA and KONT groups.

**α-linolenic** (18:3, n-3 or octadecatrienoic) acid in goat milk was found in 0.50 to 1.00 wt %. During the supplementation with fatty acids, the percentage of α-linolenic acid increased only in the ALFA group to 3.20% and it dropped back to the previous level 0.50% (p<0.001) four days after ending the supplementation. Thus, goats can successfully build linolenic fatty acid into milk fat when they are supplemented with this fatty acid.

There was less than 0.06 wt % of the **γ-linolenic** or cis-6,9,12-octadecatrienoic acid (18:3, n-6) in goat milk fat in all observed groups at the beginning of the experiment. After the addition of fatty acids into the goat diet, the content of the γ-linolenic acid increased in EPA group to 0.18%, in DHA group to 0.20% (p<0.05), while the maximum increase to 0.33% appeared in ALFA group (p<0.001). The increased content reflected three days after the end of supplementation with fatty acids and then decreased to the started value. Thus, γ-linolenic fatty acid is also successfully transferred into the milk fat, the fastest from α-linolenic fatty acid.

The content of **cis-11,14,17-eicosatrienoic** acid (20:3, n-3) in goat milk fat at the beginning of the experiment was 0.02 to 0.04 wt %. During the supplemementation with fatty acids, the content increased only in the EPA group to 0.43% (p<0.001). The content did not statistically significantly change in the other three groups. It is obviously, that eicosapentaenoic fatty acid was formed as a product of biohydrogenation, which occurried as an intermediate product only in milk fat of the EPA group.

At the beginning of the experiment, the content of **cis-8,11,14-eicosatrienoic** acid (20:3, n-6) was 0.02 to 0.03 wt %. During the supplementation with fatty acids, a slight increase of the content of cis-8,11,14-eicosatrienoic acid in DHA group to 0.04 to 0.05% and in EPA group to 0.08% was detected. Statistically significant increase of the cis-8,11,14-eicosatrienoic acid in goat milk fat occurred only in EPA group, from the third to the fifth day of the supplementation (p<0.05). Immediately after ending the supplementation, the percentage of the cis-8,11,14-eicosatrienoic acid decreased in all observed groups to the value before the supplementation.

**Arachidonic** acid (20:4, n-6) was found in goat milk fat at the beginning of the experiment on average 0.20 wt %. During the supplementation with fatty acids, the percentage increased to 0.40% in EPA group and even to 0.60% in DHA group. Three days after ending the supplementation, the content of arachidonic acid in EPA group decreased to its starting level, while in DHA group, the content of arachidonic acid decreased after five days after the end of the supplementation. The statistically significant increase in arachidonic acid content during the supplementation with fatty acids occurred in EPA and DHA groups (p <0.05).

**Eicosapentaenoic** acid (20:5, n-3 or EPA) was determined in the goat milk fat at the beginning of the experiment in the content from 0.10 to 0.25 wt %. During the supplementation with fatty acids, the percentage changed in DHA group to 0.50 to 0.69%, while in EPA group the percentage rose to 2.00 to over 3.23%, as shown in Figure 3. Results showed that the level of eicosapentaenoic acid increased more than 30-times in milk, when animals consumed the eicosapentaenoic acid in the diet (p 0.001). Statistically significantly higher content of eicosapentaenoic acid was observed in goat milk fat also five days after the end of supplementation, but only in EPA group.

**Figure 3.** Average value of cis-5,8,11,14,17-eicosapentaenoic acid in goat milk

The maximum concentration of **eicosapentaenoic** acid was found on the fourth day of the supplementation with fatty acids, while Kitessa et al. (2001) noted the maximum on the sixth day, but they added only 160 mg of eicosapentaenoic acid per day as unprotected supplement, which was 125-times lower than in our case. Chilliard et al. (2001) stated the efficiency of transfer of the unsaturated fatty acids into cow's milk. The transfer was 2.6% for the eicosapentaenoic acid into cow's milk. In goats fed unprotected fatty acids, the transfer was 3.5% and 7.6% in goats fed protected fatty acids (Kitessa et al., 2001). The transfer of the eicosapentaenoic acid in our experiment was 7.1%, what had probably several reasons. The first reason can be relatively large dose of the supplememented eicosapentaenoic acid, the second short-term administration, whereas the ruminal microflora could not adapt for biohydrogenation of the eicosapentaenoic acid in this short time, and third, that according to the method of administering fatty acids the eicosapentaenoic acid partially passed through the rumen over esophageal gutter directly into the stomach.

According to the fact that the transfer of eicosapentaenoic acid through diet into the milk can be so effective, it is important how to produce milk enriched with n-3 and n-6 fatty acids. Consumers are increasingly use milk with lower fat content. Thus, milk enriched with n-3 and n-6 fatty acids would significantly help to more correct and balanced diet, especially in children and elderly people.

20 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**Figure 3.** Average value of cis-5,8,11,14,17-eicosapentaenoic acid in goat milk

rumen over esophageal gutter directly into the stomach.

The maximum concentration of **eicosapentaenoic** acid was found on the fourth day of the supplementation with fatty acids, while Kitessa et al. (2001) noted the maximum on the sixth day, but they added only 160 mg of eicosapentaenoic acid per day as unprotected supplement, which was 125-times lower than in our case. Chilliard et al. (2001) stated the efficiency of transfer of the unsaturated fatty acids into cow's milk. The transfer was 2.6% for the eicosapentaenoic acid into cow's milk. In goats fed unprotected fatty acids, the transfer was 3.5% and 7.6% in goats fed protected fatty acids (Kitessa et al., 2001). The transfer of the eicosapentaenoic acid in our experiment was 7.1%, what had probably several reasons. The first reason can be relatively large dose of the supplememented eicosapentaenoic acid, the second short-term administration, whereas the ruminal microflora could not adapt for biohydrogenation of the eicosapentaenoic acid in this short time, and third, that according to the method of administering fatty acids the eicosapentaenoic acid partially passed through the

6 7 8 9 10 11 12 13 14 15 16 17 18 19 24 29 34 39,5 Day

EPA ALFA DHA KONT

According to the fact that the transfer of eicosapentaenoic acid through diet into the milk can be so effective, it is important how to produce milk enriched with n-3 and n-6 fatty

end of supplementation, but only in EPA group.

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

wt %

end of the supplementation. The statistically significant increase in arachidonic acid content during the supplementation with fatty acids occurred in EPA and DHA groups (p <0.05).

**Eicosapentaenoic** acid (20:5, n-3 or EPA) was determined in the goat milk fat at the beginning of the experiment in the content from 0.10 to 0.25 wt %. During the supplementation with fatty acids, the percentage changed in DHA group to 0.50 to 0.69%, while in EPA group the percentage rose to 2.00 to over 3.23%, as shown in Figure 3. Results showed that the level of eicosapentaenoic acid increased more than 30-times in milk, when animals consumed the eicosapentaenoic acid in the diet (p 0.001). Statistically significantly higher content of eicosapentaenoic acid was observed in goat milk fat also five days after the

20:5, n-3

Before supplementation with fatty acids, the content of **docosatrienoic** fatty acid in goat milk fat (22:3, n-3) was in all groups below the detection limit. During the supplementation, the increased content of docosatrienoic fatty acid was detected in EPA group, 0.03 to 0.06 wt %, and in DHA group, 0.6 to 0.11 wt % (p<0.001). The increased value of the docosatrienoic fatty acid lasted until the 18th day of the experiment, and then it fell again below the detection limit. The value of the KONT group and ALFA group was below the detection limit through the whole time of experiment.

There was from 0.046 to 0.136 wt % of the **docosatetraenoic** fatty acid (22:4, n-6) in goat milk fat. During the supplementation, a slight increase of the docosatetraenoic fatty acid in EPA and DHA groups was noticed, but differences between groups in different periods of the experiment were not statistically significant.

**Docosapentaenoic** fatty acid (22:5, n-3) in goat milk fat was found in the concentration from 0.15 to 0.22 wt %. During the supplementation with fatty acids, the percentage of docosapentaenoic fatty acid increased in DHA group to 0.59% and in EPA group to 0.85% (p <0.001). In both groups, an increased concentration of docosapentaenoic fatty acid reflected still 15 to 20 days after the end of the supplementation. The concentration was statistically highly significantly greater than the ALFA and KONT groups. It looks like docosapentaenoic fatty acid passes into the udder directly by blood, as it is not produced in the mammary gland de novo.

Only 0.05 to 0.1 wt % was the concentration of **docosahexaenoic** (22:6, n-3 or DHA) fatty acid in goat milk fat at the beginning of the experiment. During the supplementation with fatty acids, the percentage increased only in DHA group to 2.80%, and after the end of supplementation, it gradually declined. Even nine days after the end of supplementation with fatty acids, milk fat contained more than 0.50% of docosahexaenoic fatty acid (Figure 4). There was 3 to 4-times higher content of docosahexaenoic fatty acid in DHA group than in other groups (p <0.001) even 20 days after the supplementation. The maximum concentration of docosahexaenoic fatty acid in goat milk fat in our experiment was found on the fifth day, while Kitessa et al. (2001) found the maximum concentration on the sixth day, but they added only 580 mg of docosahexaenoic fatty acid per day as an unprotected supplement, which is 34.5 times less than in our experiment.

The effectiveness of transfer the docosahexaenoic fatty acid into milk was observed in cows by Chilliard et al. (2001), which amounted 4.1%. In goats, it amounted 3.5% for unprotected fatty acids and 7.6% for protected fatty acids (Kitessa et al., 2001). The estimated transfer of docosahexaenoic fatty acid in our experiment was 7.84.

There was 53 to 57 wt % of the **medium chain** fatty acids in goat milk fat before the supplementation with fatty acids. After the supplementation, a decrease of the medium chain fatty acids was noticed in EPA, DHA, and ALFA group to 46 to 50%. The level of

medium chain fatty acids re-established to the starting level in three days after ending the supplementation in EPA and ALFA groups and in ten days in DHA group (p<0.05).

**Figure 4.** Average value of cis-4,7,10,13,16,19- docosahexaenoic fatty acid in goat milk

As reported Kitessa et al. (2001), a significant decrease appeared in C10 to C16 fatty acids after adding fish oil into the diet for goats, but when Chilliard et al. (2001) fed cows with fish oil only, they noticed a slight decrease in C4 to C14 fatty acids, or even 1.3% increase of these fatty acids when adding fish oil in the duodenum. In the experiment by Kitessa et al. (2001), a group of animals were supplemented a protected fish oil from 19th to 26th day and then unprotected fish oil from the 37th to 42nd day. Due to the significantly reduced feed intake and milk production in sheep the unprotected fish oil was administered a short time. Between one and another type of feeding was only eight days, which is questionable. It is possible that there was an influence of the previous supplementation, because our data showed that the effect of supplementation with some types of fatty acids can take more than 10 days on changes in the fermentation of medium chain fatty acids. Even Sanz Sampelayo et al. (2002) in goats found that the percentage of total unsaturated fatty acids reduced after the supplementation with protected polyunsaturated fatty acids.

The content of **monounsaturated** fatty acids in goat milk fat in our experiment ranged from 23 to 28 wt %, which reduced during the supplementation with fatty acids to 22% in EPA group and to 21% in DHA group. The decrease was statistically significant (p<0.05) during the supplementation in EPA and DHA groups, while the reduction of monounsaturated fatty acids did not occur in ALFA and KONT groups. As reported Sanz Sampelayo et al. (2002), the supplementation of 9% polyunsaturated fatty acids only slightly increased the content of monounsaturated fatty acids, while the supplementation of 12% polyunsaturated fatty acids significantly increased the content of monounsaturated fatty acids.

Before the supplementation with fatty acids, **polyunsaturated** fatty acids were found in goat milk fat in the concentration from 4 to 6 wt %. The same level of polyunsaturated fatty acids stayed in ALFA group throughout the whole time of experiment. A statistically significant (p=0.001) increase of the polyunsaturated fatty acids concentration appeared during the supplementation with fatty acids in EPA group (to 11%), ALFA group (9 to 10%), and in DHA group (11 to 11.9%). The peak in concentration of polyunsaturated fatty acids was achieved in EPA and DHA group on the forth and fifth day of the supplementation and in ALFA group on the third day of the supplementation. The increased percentage of polyunsaturated fatty acids in goat milk fat persisted from 10 to 14 days in EPA, ALFA, and DHA groups.

22 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

medium chain fatty acids re-established to the starting level in three days after ending the

22:6, n-3

supplementation in EPA and ALFA groups and in ten days in DHA group (p<0.05).

**Figure 4.** Average value of cis-4,7,10,13,16,19- docosahexaenoic fatty acid in goat milk

0,00

0,50

1,00

1,50

wt %

2,00

2,50

3,00

the supplementation with protected polyunsaturated fatty acids.

fatty acids significantly increased the content of monounsaturated fatty acids.

As reported Kitessa et al. (2001), a significant decrease appeared in C10 to C16 fatty acids after adding fish oil into the diet for goats, but when Chilliard et al. (2001) fed cows with fish oil only, they noticed a slight decrease in C4 to C14 fatty acids, or even 1.3% increase of these fatty acids when adding fish oil in the duodenum. In the experiment by Kitessa et al. (2001), a group of animals were supplemented a protected fish oil from 19th to 26th day and then unprotected fish oil from the 37th to 42nd day. Due to the significantly reduced feed intake and milk production in sheep the unprotected fish oil was administered a short time. Between one and another type of feeding was only eight days, which is questionable. It is possible that there was an influence of the previous supplementation, because our data showed that the effect of supplementation with some types of fatty acids can take more than 10 days on changes in the fermentation of medium chain fatty acids. Even Sanz Sampelayo et al. (2002) in goats found that the percentage of total unsaturated fatty acids reduced after

6 7 8 9 10 11 12 13 14 15 16 17 18 19 24 29 34 39,5 Day

EPA ALFA DHA KONT

The content of **monounsaturated** fatty acids in goat milk fat in our experiment ranged from 23 to 28 wt %, which reduced during the supplementation with fatty acids to 22% in EPA group and to 21% in DHA group. The decrease was statistically significant (p<0.05) during the supplementation in EPA and DHA groups, while the reduction of monounsaturated fatty acids did not occur in ALFA and KONT groups. As reported Sanz Sampelayo et al. (2002), the supplementation of 9% polyunsaturated fatty acids only slightly increased the content of monounsaturated fatty acids, while the supplementation of 12% polyunsaturated

Before the supplementation with fatty acids, **polyunsaturated** fatty acids were found in goat milk fat in the concentration from 4 to 6 wt %. The same level of polyunsaturated fatty acids The passage of the supplemented polyunsaturated fatty acids from the gastrointestinal tract into milk was estimated on the basis of the differences between the content of fatty acids before supplementation and the difference between KONT group and other groups during the supplementation and thereafter, taking into account the amount of milked milk during the supplementation and 14 days thereafter. The results are shown in Table 3, where it is clear that the passage of the conjugated linoleic acid into milk was 12.79%, 14.03% of the eicosapentaenoic acid, and 21.13% of the docosahexaenoic acid. The differences were statistically significant (p <0.05).


EPA – eicosapentaenoic acid; CLA – conjugated linoleic acid; DHA - docosahexaenoic acid; PUFA –polyunsaturated fatty acids

**Table 5.** Estimated passage of the supplemented polyunsaturated fatty acids from food into milk

The **ratio between n-3:n-6** fatty acids before the supplementation with fatty acids was the same in all groups (1:3.50) and remained unchanged throughout the experiment only in KONT group. In all other groups, the ratio reduced during the supplementation with fatty acids to 1:1 and even to 1:0.67. It was gradually establishing back more than 20 days after the end of the supplementation. The differences before and after supplementation were statistically highly significant (p<0.001).

### **3.3. Correlations between somatic cell count and some fatty acids**

Correlations between somatic cell count and some fatty acids during the experiment were calculated by the Pearson correlation coefficient. The same correlations were calculated also for the second and third period of the experiment (from the 11th to the 65th day) and for the period from the 21st to the 65th day of the experiment. Statistically significant correlations between somatic cell count and C10 throughout the whole experiment were found in EPA group (r=0.24), ALFA (r=-0.18), and (r=-0.17) KONT group. The correlations between somatic cell count and C12 and between somatic cell count and C14 were statistically significant

throughout the whole experiment only in EPA group (r=0.25 and r=0.24, respectively; p<0.01). From the 11th to the 65th day of the experiment, there were only correlations between somatic cell count and C10 in DHA group (r=-0.30), between somatic cell count and C12 in DHA group (r=-0.37), and between somatic cell count and C14 in ALFA (r=0.26) and DHA (r=-0.29) groups found statistically significant (p<0.05). From the 21st to the 65th day of the experiment, correlations between somatic cell count and C10 in EPA (r=-0.45) and DHA (r=- 0.46) groups, between somatic cell count and C12 in EPA (r=-0.43), DHA (r=-0.53), and KONT (r=0.39) groups, and between somatic cell count and C14 in ALFA (r=-0.59), DHA (r=- 0.57), and KONT (r=0.44) groups were statistically significant (p<0.05).

Correlation between somatic cell count and C18:1 was statistically significant only in EPA group (r=-0.24) throughout the whole experiment, in DHA group (r=0.47) from the 11th to the 65th day of the experiment, and in EPA (r=0.42), ALFA (r=-0.49), and DHA (r=0.67) groups from the 21st to the 65th day of the experiment. Between somatic cell count and C18:3, the correlation was statistically significant only in ALFA (r=-0.43) group from the 11th to the 65th day of the experiment. No correlations between somatic cell count and C20:4 throughout the whole experiment were statistically significant. There were only correlations between somatic cell count and C20:4 in EPA group from the 11th to the 65th day of the experiment (r=0.36) and from the 21st to the 65th day of the experiment (r=0.66) statistically significant.

Statistically significant correlation between somatic cell count and monounsaturated fatty acids throughout the whole experiment was found only in ALFA group (r=-0.22) and from the 11th to the 65th day of the experiment in DHA group (r=0.50). From the 21st to the 65th day of the experiment, this correlation was statistically significant in EPA (r=0.43), ALFA (r=- 0.50), and DHA (r=0.68) groups. Between somatic cell count and polyunsaturated fatty acids, only the correlation in ALFA group from the 21st to the 65th day of the experiment was found statistically significant (r=-0.49).
