**1.2. Fatty acid composition in goat milk fat**

Average goat milk fat differs in contents of its fatty acids significantly from average cow milk fat, being much higher in butyric (C4:0), caproic (C6:0), caprylic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), linoleic (C18:2), but lower in stearic (C18:0), and oleic acid (C18:1) (Table 1). Three of the medium chain fatty acids (caproic, caprilyc, and capric) have actually been named after goats, due to their predominance in goat milk. They contribute to 15% of the total fatty acid content in goat milk in comparison to 5% in cow milk (Haenlein, 1993). The presence of relatively high levels of medium chain fatty acids (C6:0 to C10:0) in goat milk fat could be responsible for its inferior flavour (Skjevdal, 1979).


**Table 1.** Fatty acid composition (mg FA 100 g−1 milk) in goat milk fat in comparison to cow milk (1Posati & Orr, 1976; 2Žan et al., 2005)

#### **1.3. The effect of nutrition on goat milk fat and fatty acids composition**

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

while in monogastric animals, the precursor is glucose (Clegg et al., 2001).

**1.2. Fatty acid composition in goat milk fat** 

Fatty acid Goat milk1 Goat milk (from

(1Posati & Orr, 1976; 2Žan et al., 2005)

(Hurley, 2009).

they are eliminated from the cell as fat globules of the milk. The synthesis is endogenous in a large extent, where the presence of the conjugated linoleic acid plays an important role

Fatty acids in goat milk are synthesized in epithelial cells of the mammary gland de novo or they are passing over from the blood (Chilliard et al., 2003). Two coenzymes have a major role in the synthesis of fatty acids in goat milk: acetyl-coenzyme A-carboxylase, which participates in the synthesis of fatty acids de novo and fatty acid synthase, which is a complex of enzymatic active substances and is responsible for the extension (elongation) of the fatty acid chain (Hurley, 2009). Fatty acids of exogenous origin are presented via the circulation to mammary epithelial cells either in the form of non-esterified fatty acids or esterified as the acyl groups of the triacylglycerol component of lipoprotein particles. In the mammary gland of ruminant animals, short and medium chain saturated fatty acids are the major products of de novo lipogenesis whereas plasma lipids contribute longer chain and mono unsaturated species. The acetate is the precursor of fatty acids synthesis in ruminants,

Average goat milk fat differs in contents of its fatty acids significantly from average cow milk fat, being much higher in butyric (C4:0), caproic (C6:0), caprylic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), linoleic (C18:2), but lower in stearic (C18:0), and oleic acid (C18:1) (Table 1). Three of the medium chain fatty acids (caproic, caprilyc, and capric) have actually been named after goats, due to their predominance in goat milk. They contribute to 15% of the total fatty acid content in goat milk in comparison to 5% in cow milk (Haenlein, 1993). The presence of relatively high levels of medium chain fatty acids (C6:0 to C10:0) in goat milk fat could be responsible for its inferior flavour (Skjevdal, 1979).

highland flock)2

C4:0 butyric 130 - - 110 C6:0 caproic 90 - - 60 C8:0 caprylic 100 106 85 40 C10:0 capric 260 433 321 80 C12:0 lauric 120 228 149 90 C14:0 myristic 320 441 392 340 C16:0 palmitic 910 984 990 880 C16:1 palmitoleic 80 - - 80 C18:0 stearic 440 333 300 400 C18:1 oleic 980 - - 840 C18:2 linoleic 110 103 76 80 C18:3 linolenic 40 32 26 50

**Table 1.** Fatty acid composition (mg FA 100 g−1 milk) in goat milk fat in comparison to cow milk

Goat milk (from mountain flock)2 Cow milk1

Nutrition (forage-to-concentrate ratio, type of forages, etc.) is the main environmental factor regulating milk fat synthesis and fatty acid composition in ruminants (Nudda et al., 2003; Bernard et al., 2009). Forage in the diet is known to affect milk fat composition responses to plant oils, including trans-18:1 and conjugated linoleic acid isomer concentrations. Inclusion of fat in the diet enhances milk fat secretion in the goat in the absence of systematic changes in milk yield and protein content (Bernard et al., 2009; Chilliard et al., 2003, 2007). Bernard et al. (2009) found out that, changes in goat milk fatty acid composition were dependent on forage type and plant oil composition, with evidence of an interaction between these nutritional factors. Responses to lipid supplements were characterised as a reduction in fatty acids synthesised de novo (C10:0–C16:0) and an increase in C18:0, cis-C18:1, conjugated linoleic acid and polyunsaturated fatty acid concentrations, indicating that plant oils can be used to effect potentially beneficial changes in milk fat composition without inducing detrimental effects on animal performance. Moreover, goats fed a high level of pasture forage had higher milk fat contents of C4:0, C6:0, C18:0, C18:l, C18:3, C20:0, iso-, ante-iso-, and odd fatty acids, but lower values of C10:0, C12:0, C14:0, C16:0, and C18:2, than those fed the low levels of forage. However, high levels of alfalfa forage also produced the lowest contents of the less desirable trans-C18:1 fatty acids (LeDoux et al., 2002). The conclusion was that decreasing the fibre content and increasing the grain part in the goat daily ration would lead to higher contents of the undesirable trans-C18:1 fatty acids in milk. The composition of goat milk fatty acids differed also in goats grazing one flock on highland (615-630 m altitude) and one flock on mountain (1060-1075 m altitude) pasture by Žan et al. (2005). The most abundant fatty acids in milk of both flocks were C16:0, C18:1, n−9, C14:0 and C10:0 (Table 1). The average content of saturated fatty acids was 74.52 and 73.05% in milk from the highland and mountain flocks, respectively. Three saturated fatty acids (caprylic (C8:0), capric (C10:0) and lauric acid (C12:0)), were present at significantly higher amounts in milk from the highland flock than in milk from the mountain flock. Monounsaturated fatty acids represented 20.49 and 22.32% and polyunsaturated fatty acids 3.73 and 3.24% of the milk from the highland and mountain flocks, respectively. Among the monounsaturated fatty acids, palmitoleic + palmitelaidic acid (C16:1, n−7) showed a significantly higher concentration in milk from mountain flock than in milk from the highland flock. The content of linolelaidic acid (C18:2, n−6) was signicantly higher in comparison to milk from the highland flock. The average quantity (32 mg 100 g−1 milk) of essential α-linolenic acid (C18:3, n−3) was slightly higher in milk of the highland ock than in milk from the mountain flock (26 mg 100 g−1 milk). Hou et al. (2011) stated that the supplementation of fish oil can significantly increase the production of cis-9, trans-11 conjugated linoleic acid, and trans-11 C18:1, while lowering the amount of trans-10 C18:1 and trans-10, cis-12 conjugated linoleic acid in the ruminal fluid of goats. Increased cis9, trans-11 conjugated linoleic acid, and trans-11 C18:1 can lead to a higher output of cis-9, trans-11 conjugated linoleic acid in milk product, and the decrease in trans-10 C18:1 and trans-10, cis-12 conjugated linoleic acid supports the role of fish oil in the alleviation of milk fat depression.

## **1.4. Conjugated linoleic acid**

Conjugated linoleic acid consists of a series of positional and geometric dienoic isomers of linoleic acid that occurs naturally in foods. It is a product of biohydrogenation in the rumen of ruminants and has a great influence on synthesis of fatty acids in milk in low concentrations (Bessa et al. 2000; Chouinard et al. 1999; Griinari & Bauman, 1999; Griinari et al. 2000; Khanal & Dhiman, 2004). Actually, the conjugated linoleic acid found in goat milk fat originate from two sources (Griinari & Bauman, 1999). One source is conjugated linoleic acid formed during ruminal biohydrogenation of linoleic acid (C18:2 n-6) that leads first to vaccenic (trans-11 C18:1) and finally to stearic acid (C18:0) (Nudda et al., 2003). The second source is conjugated linoleic acid synthesized by the animal's tissues from trans-11 C18:1, another intermediate in the rumen biohydrogenation of unsaturated FA. Thus, the uniqueness of conjugated linoleic acid in food products derived from ruminants relates to the incomplete biohydrogenation of dietary unsaturated fatty acids in the rumen. Ruminal biohydrogenation combined with mammary lipogenic and ∆-9 desaturation pathways considerably modifies the profile of dietary fatty acids and thus milk composition (Chilliard et al., 2007).

Dietary sources from ruminants such as milk, cheese and meats contain more conjugated linoleic acid than foods of non-ruminant origin (Bessa et al. 2000; Khanal & Dhiman, 2004). The increase of linoleic acid intake is one of the feeding strategies for conjugated linoleic acid enrichment in ruminant fat since linoleic acid is the main precursor of conjugated linoleic acid (Bessa et al., 2000). The main available sources of linoleic acid in animal feeds are cereal and oilseed grains or oils obtained from these. Goat milk conjugated linoleic acid content increases sharply after either vegetable oil supplementation (Bernard et al., 2009) or fresh grass feeding containing unsaturated fatty acids, but does not change markedly when goats receive whole untreated oilseeds (Chilliard et al., 2003). Mir et al. (1999) found that it is possible to increase conjugated linoleic acid content of goat milk by manipulation of dietary regimen such as supplementation with canola oil. The pasture has major effects by decreasing saturated fatty acids and increasing fatty acids considered as favourable for human health (C9-18:1, C18:3n-3 and C9t11-CLA), compared to winter diets, especially those based on maize silage and concentrates (Chilliard et al., 2007). Investigations have shown that milk fat conjugated linoleic acid content can be also enhanced by manipulation of the rumen fermentation (Bessa et al., 2000; Griinari et al., 1999) or by direct addition of a dietary supplement of conjugated linoleic acid (Lock et al., 2008).

## **1.5. Effect of fatty acids on health**

Milk, apart from its nutritional traits, contains substances which have beneficial effects on human health and is, therefore, considered essential to a correct nutrition. In particular, in milk are present vitamin A, vitamin E, β-carotene, sphingomyelins, butyric acid, and conjugated linoleic acid, all with a strong antitumor effect (Parodi, 1999). Different FA (short and medium chain, saturated, branched, mono and polyunsaturated, *cis* and *trans*, conjugated) in the lipid fraction of milk are potentially involved as positive or negative predisposing factors for human health (Parodi, 1999; Williams, 2000). In this respect, conjugated linoleic acid is the most characteristic one. One of the goat milk significance in human nutrition is treating people afflicted with cow milk allergies and gastro-intestinal disorders, which is a significant segment in many populations of developed countries. Fat in goat milk is more digestible than bovine milk fat which may be related to the lower mean milk fat globule size, higher C8:0–C10:0 concentrations and a larger proportion of short- and medium-chain fatty acids (Chilliard et al., 2006 as cited in Bernard et al., 2009). Because of predominance of smaller fat globules in goat milk, it is easier to digest than cow milk and this may be attributed to faster lipase activity on smaller fat globules due to a greater surface area (Chandan et al., 1992). Goat milk is therefore recommended for infants, old, and convalescent people.

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

Conjugated linoleic acid consists of a series of positional and geometric dienoic isomers of linoleic acid that occurs naturally in foods. It is a product of biohydrogenation in the rumen of ruminants and has a great influence on synthesis of fatty acids in milk in low concentrations (Bessa et al. 2000; Chouinard et al. 1999; Griinari & Bauman, 1999; Griinari et al. 2000; Khanal & Dhiman, 2004). Actually, the conjugated linoleic acid found in goat milk fat originate from two sources (Griinari & Bauman, 1999). One source is conjugated linoleic acid formed during ruminal biohydrogenation of linoleic acid (C18:2 n-6) that leads first to vaccenic (trans-11 C18:1) and finally to stearic acid (C18:0) (Nudda et al., 2003). The second source is conjugated linoleic acid synthesized by the animal's tissues from trans-11 C18:1, another intermediate in the rumen biohydrogenation of unsaturated FA. Thus, the uniqueness of conjugated linoleic acid in food products derived from ruminants relates to the incomplete biohydrogenation of dietary unsaturated fatty acids in the rumen. Ruminal biohydrogenation combined with mammary lipogenic and ∆-9 desaturation pathways considerably modifies the profile of dietary fatty acids and thus milk composition (Chilliard

Dietary sources from ruminants such as milk, cheese and meats contain more conjugated linoleic acid than foods of non-ruminant origin (Bessa et al. 2000; Khanal & Dhiman, 2004). The increase of linoleic acid intake is one of the feeding strategies for conjugated linoleic acid enrichment in ruminant fat since linoleic acid is the main precursor of conjugated linoleic acid (Bessa et al., 2000). The main available sources of linoleic acid in animal feeds are cereal and oilseed grains or oils obtained from these. Goat milk conjugated linoleic acid content increases sharply after either vegetable oil supplementation (Bernard et al., 2009) or fresh grass feeding containing unsaturated fatty acids, but does not change markedly when goats receive whole untreated oilseeds (Chilliard et al., 2003). Mir et al. (1999) found that it is possible to increase conjugated linoleic acid content of goat milk by manipulation of dietary regimen such as supplementation with canola oil. The pasture has major effects by decreasing saturated fatty acids and increasing fatty acids considered as favourable for human health (C9-18:1, C18:3n-3 and C9t11-CLA), compared to winter diets, especially those based on maize silage and concentrates (Chilliard et al., 2007). Investigations have shown that milk fat conjugated linoleic acid content can be also enhanced by manipulation of the rumen fermentation (Bessa et al., 2000; Griinari et al., 1999) or by direct addition of a

Milk, apart from its nutritional traits, contains substances which have beneficial effects on human health and is, therefore, considered essential to a correct nutrition. In particular, in milk are present vitamin A, vitamin E, β-carotene, sphingomyelins, butyric acid, and conjugated linoleic acid, all with a strong antitumor effect (Parodi, 1999). Different FA (short and medium chain, saturated, branched, mono and polyunsaturated, *cis* and *trans*, conjugated) in the lipid fraction of milk are potentially involved as positive or negative

dietary supplement of conjugated linoleic acid (Lock et al., 2008).

**1.5. Effect of fatty acids on health** 

**1.4. Conjugated linoleic acid** 

et al., 2007).

The physiological and biochemical facts of the unique qualities of goat milk are just barely known and little exploited, especially not the high levels in goat milk of short and medium chain fatty acids, which have recognized medical values for many disorders and diseases of people (Haenlein, 2004). Goat milk exceeds cow and sheep milk in monounsaturated, polyunsaturated fatty acids, and medium chain triglycerides, which all are known to be beneficial for human health, especially for cardiovascular conditions. Capric, caprylic acids and medium chain triglycerides have become established medical treatments for an array of clinical disorders, including malabsorption syndromes, chyluria, steatorrhea, hyperlipoproteinemia, intestinal resection, premature infant feeding, non-thriftiness of children, infant malnutrition, epilepsy, cystic fibrosis, coronary by-pass, and gallstones, because of their unique metabolic ability to provide direct energy instead of being deposited in adipose tissues, and because of their actions of lowering serum cholesterol, inhibiting and limiting cholesterol deposition (Alferez et al., 2001; Greenberger & Skillman, 1969; Kalser, 1971; Schwabe et al., 1964; Tantibhedhyanangkul & Hashim, 1978).

Conjugated linoleic acid was recognized as having antioxidative and anticarcinogenic properties in animal model studies (Ip et al., 1991; Jiang et al., 1996; Parodi, 1997). Several *in vitro* and *in vivo* studies showed also antiatherogenic, anti-obesity, anti-diabetes and immune-stimulating properties of conjugated linoleic acid (McGuire & McGuire, 1999). By Parodi (1997), conjugated linoleic acid inhibited proliferation of human malignant melanoma, colorectal, breast and lung cancer cell lines. Anticarcinogenic effects of conjugated linoleic acid appear to be dose dependent, from 0.1 to 1% in the diet (Ip et al., 1991). Conjugated linoleic acid reduced the incidence of chemically induced mouse epidermal tumors, mouse forestomach neoplasia and aberrant crypt foci in the rat colon. They have been also shown to stimulate immune response and protect against arteriosclerosis (Cook et al., 1993; Lee et al., 1994). When rabbits were fed conjugated linoleic acid, LDL cholesterol to HDL cholesterol ratio and total cholesterol to HDL cholesterol ratio were significantly reduced. Examination of the aortas of conjugated linoleic acid fed rabbits showed less atherosclerosis (Lee et al., 1994).

Somatic cells in milk are the total sum of white blood cells present in milk and udder epithelial cells, which may be an indicator of the udder health status (Das & Singh, 2000; Manlongat et al., 1998; Zeng & Escobar, 1996; Wilson et al., 1995). They are present in milk

all the time. In cows, a somatic cell count above the regulatory standard is generally considered as an indication of mastitis. An increased number of somatic cell count is either the consequence of an inflammatory process due to the presence of an intramammary infection or under non-pathological conditions due to physiological processes such as oestrus or advanced stage of lactation. For this reason, the somatic cell count of milk represents a sensitive marker of the health of the udder and is considered a useful parameter to evaluate the relationship between intramammary infection and changes in milk characteristics. The standard for the permissible number of somatic cell count for cow milk exists, while it is still under study for goat milk due to considerable fluctuations. When the udder is tired during late lactation, the number of somatic cells in normal conditions can considerably enlarge, and approximately 80% of the cells may be polymorphonuclear leukocytes (Manlongat et al., 1998). The same authors found that normal nonmastitic latelactation-stage goat milk is significantly higher in polymorphonuclear leukocytes chemotactic activity than early-lactation-stage goat milk. The chemotactic factor(s) present in the milk of normal late-lactation-stage goats is nonpathological and may play a physiologic regulatory role in mammary gland involution. On the other hand, the increase of leucocytes is a response to the inflammatory process in the mammary gland or somewhere in the body. The number of leucocytes increases due to bacterial infections, but it could also be increased due to the stage of lactation, age of the animal, stress, season of the year, nutrition and udder injuries. The variability of somatic cell count in goat milk is very high, which exists among the animals and within the time span of individual animals (Das & Singh, 2000). Therefore, it is important to determine how nutrition can influence the reduction of somatic cell count in goat milk. Gantner & Kompan (2009) found that a five-day supplementation of α-linoleic acid in Alpine goat diet had a significant effect on lower somatic cell count in milk. Based on this experiment, it was concluded that α-linoleic acid supplementation had no effect on milk yield; it had low effect on milk components and significant effect on somatic cell count. A decrease in somatic cell count was determined in the 1st day of the treatment period and continued until 30th day after the treatment period. The supplementation of the goat diet with α-linoleic acid could be used as a method of choice for reduction of somatic cell count in goat milk.

The aim of our study was therefore to ascertain the changes in goat milk yield and its contents of fat, protein, lactose, dry matter, somatic cell count, and total number of microorganisms when goats are supplemented with the following fatty acids: α-linoleic acid, eicosapentanoic acid, and docosahexanoic acid and how these three fatty acids influence on the content of particular fatty acids during and after the supplementation.

## **2. Material and methods**

## **2.1. Material**

The research was performed on the farm with 90 Slovenian Alpine and Slovenian Saanen goats. Goats were machine milked. During the experiment, goats were in different stages of lactation. The average body weight of the goats was 51 ± 6 kg. All kids were weaned. Goats were arranged into three pens according to their stage of lactation, namely, after kidding from the forth to the tenth week of lactation (pen A), from the 11th to the 20th week of lactation (pen B), and after the 20th week of lactation (pen C). Goats were milked twice a day, at 6 a.m. (± 30 min) and at 6 p.m. (± 30 min). Diet was composed from hay (2 kg/animal/day) which was given to goats twice a day. Goats were supplemented with feed mixture at milking parlor during the milking time. Supplemental feed mixture contained 50% of grounded maize grains, 30% of dried beet pulp, and 20% of wheat bran. Goats from pen A were supplemented with 500 g, goats from pen B with 350 g, and goats from pen C with 250 g of feed mixture. Vitamin-mineral supplement and water were offered to goats *ad libitum*. After the tenth day preparing period, 62 goats from pens A and B were selected and randomly arranged into four experimental groups. At the beginning of the experiment (September 17th, 2000), goats were 28 to 105 days after kidding. The experiment lasted 63 days. During this time, experimental goats were added fats or oils.

## **2.2. Methods**

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

choice for reduction of somatic cell count in goat milk.

**2. Material and methods** 

**2.1. Material** 

all the time. In cows, a somatic cell count above the regulatory standard is generally considered as an indication of mastitis. An increased number of somatic cell count is either the consequence of an inflammatory process due to the presence of an intramammary infection or under non-pathological conditions due to physiological processes such as oestrus or advanced stage of lactation. For this reason, the somatic cell count of milk represents a sensitive marker of the health of the udder and is considered a useful parameter to evaluate the relationship between intramammary infection and changes in milk characteristics. The standard for the permissible number of somatic cell count for cow milk exists, while it is still under study for goat milk due to considerable fluctuations. When the udder is tired during late lactation, the number of somatic cells in normal conditions can considerably enlarge, and approximately 80% of the cells may be polymorphonuclear leukocytes (Manlongat et al., 1998). The same authors found that normal nonmastitic latelactation-stage goat milk is significantly higher in polymorphonuclear leukocytes chemotactic activity than early-lactation-stage goat milk. The chemotactic factor(s) present in the milk of normal late-lactation-stage goats is nonpathological and may play a physiologic regulatory role in mammary gland involution. On the other hand, the increase of leucocytes is a response to the inflammatory process in the mammary gland or somewhere in the body. The number of leucocytes increases due to bacterial infections, but it could also be increased due to the stage of lactation, age of the animal, stress, season of the year, nutrition and udder injuries. The variability of somatic cell count in goat milk is very high, which exists among the animals and within the time span of individual animals (Das & Singh, 2000). Therefore, it is important to determine how nutrition can influence the reduction of somatic cell count in goat milk. Gantner & Kompan (2009) found that a five-day supplementation of α-linoleic acid in Alpine goat diet had a significant effect on lower somatic cell count in milk. Based on this experiment, it was concluded that α-linoleic acid supplementation had no effect on milk yield; it had low effect on milk components and significant effect on somatic cell count. A decrease in somatic cell count was determined in the 1st day of the treatment period and continued until 30th day after the treatment period. The supplementation of the goat diet with α-linoleic acid could be used as a method of

The aim of our study was therefore to ascertain the changes in goat milk yield and its contents of fat, protein, lactose, dry matter, somatic cell count, and total number of microorganisms when goats are supplemented with the following fatty acids: α-linoleic acid, eicosapentanoic acid, and docosahexanoic acid and how these three fatty acids influence on the content of particular fatty acids during and after the supplementation.

The research was performed on the farm with 90 Slovenian Alpine and Slovenian Saanen goats. Goats were machine milked. During the experiment, goats were in different stages of lactation. The average body weight of the goats was 51 ± 6 kg. All kids were weaned. Goats

#### *2.2.1. Measuring performance and milk sampling*

The whole experiment was performed in three periods:

**1st period**: Preparatory period - measuring before adding fats or oils. The preparatory period lasted 10 days. During this period, milk yield in goats was measured, milk samples were collected, and animals were adapting to the working group. Goats were adapted to the work and people after a week, so they were not under the stress any more. Milk yield was measured every day at morning and evening milking, when 70 ml of milk sample was taken for the analysis of milk content, somatic cells, and bacteriological analysis, and 2 ml for fatty acid content analysis.

**2nd period**: Experimental period – adding fatty acids. After the tenth day preparing period, 62 goats from pens A and B were randomly selected into four experimental groups, named EPA, ALFA, DHA, and KONT. There were 15 goats in groups EPA, ALFA, and DHA and 17 goats in the group KONT. Supplementation of the fats was performed 5 days (from the 11th to the 15th day), after morning milking in groups EPA, ALFA, and DHA. Each goat was cached and individually administered the appropriate quantity of fatty acids into its mouth with a special sound. Group EPA was receiving a preparation rich in eicosapentaenoic acid (EPA; 20 g/day), group ALFA was receiving a linseed oil rich in α-linoleic acid (ALA; 20 g/day), and group DHA was receiving a preparation rich in docosahexaenoic acid (DHA; 20 g/day). Group KONT was a control group, which was receiving no preparation. Measuring of the milk yield and collecting milk samples was followed the same procedure as in the first period.

**3rd period**: This period lasted from the 16th day, after the end of administering fatty acids to goats. Milk yield measuring and milk samples collection was continuing until the 20th day. From the 21st day of the experiment, milk yield measuring and milk samples collection was performing every five days, at the morning and evening milking, until the end of the

experiment (63rd day). All together, 30 morning and 30 evening records were collected by each goat.

## *2.2.2. Milk yield measuring*

There were 90 goats all together in the flock, which were milked on the milking parlor with 24 places for milking goats connected to milk pipeline. Goats were milked every morning between 5:40 and 7:20 a.m. and every evening between 6:20 and 8:00 p.m. A measuring gauged flask was connected to milking unit to measure milk yield. Milk yield was written down for every goat. A milk sample was also taken for the analysis. During the experiment, 30 daily records were collected for every goat, which means 60 records for each goat and 60 milk samples by 70 ml for milk analysis (sample A) and 60 samples by 2 ml (sample B) for fatty acid analysis. The preservative azidiol on the basis of NaN3 in concentration 0.02% with the addition of chloramphenicol for the stabilization of microorganisms was added to the sample A. For every 50 to 70 ml of the milk sample, 0.2 ml of the preservative was added. Milk samples A were then delivered to the Laboratory for dairying, while milk samples B were delivered to the Chemical laboratory at Biotechnical Faculty in Ljubljana.

### *2.2.3. Analyses of milk samples*

**Chemical composition, somatic cell count, and total number of microorganisms**: Fat, protein, lactose, and dry matter content, somatic cell count and total number of microorganisms were determined in the collected milk samples A in the Laboratory of dairying at Biotechnical Faculty in Ljubljana. Furthermore, fatty acid composition of milk lipids was determined. Chemical composition of goat milk was determined by the instrument MilkoScan 133 B, which operates on the principle of infrared spectrometry. Somatic cell count was determined using apparatus Fossomatic 5000, which operates on the basis of automatic epifluorescent technique, by the principle of flow cytometry. The total number of microorganisms was determined using the apparatus Bactoscan 8000, type 27000.

**Fatty acid composition of milk lipids**: Milk samples B were stored in liquid nitrogen immediately after milk recording. They were stored then in freezer chamber at -70ºC until the analysis. Before the analysis, milk samples were warmed to 38-40ºC in water bath and mixed up. After that, 500 mg of the milk sample were weighed out into tubes, where 300 μl of methylenchloride and 3 ml of fresh prepared 0.5M of sodium hydroxide in methanol were added. To determine the fatty acid composition of milk lipids, the analysis of methyl esters of fatty acids was done. This analysis was performed on gas chromatograph Hewlett Packard HP AGILENT 6890 SERIES GC SYSTEM, USA. Processing of chromatographic data was conducted using ChemStation Plus software. Furthermore, factor of the responsiveness of the flame ionization detector was determined. Total lipids in the sample are composed of both fatty acids and glycerol from triglycerids, phosphate from phospholipids, and sterol. For the calculation of the fatty acid value in the sample in mg, special factors are used, which express the proportion of acids in total fat.

#### *2.2.4. Statistical analysis of the data*

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

each goat.

*2.2.2. Milk yield measuring* 

*2.2.3. Analyses of milk samples* 

which express the proportion of acids in total fat.

type 27000.

experiment (63rd day). All together, 30 morning and 30 evening records were collected by

There were 90 goats all together in the flock, which were milked on the milking parlor with 24 places for milking goats connected to milk pipeline. Goats were milked every morning between 5:40 and 7:20 a.m. and every evening between 6:20 and 8:00 p.m. A measuring gauged flask was connected to milking unit to measure milk yield. Milk yield was written down for every goat. A milk sample was also taken for the analysis. During the experiment, 30 daily records were collected for every goat, which means 60 records for each goat and 60 milk samples by 70 ml for milk analysis (sample A) and 60 samples by 2 ml (sample B) for fatty acid analysis. The preservative azidiol on the basis of NaN3 in concentration 0.02% with the addition of chloramphenicol for the stabilization of microorganisms was added to the sample A. For every 50 to 70 ml of the milk sample, 0.2 ml of the preservative was added. Milk samples A were then delivered to the Laboratory for dairying, while milk samples B were delivered to the Chemical laboratory at Biotechnical Faculty in Ljubljana.

**Chemical composition, somatic cell count, and total number of microorganisms**: Fat, protein, lactose, and dry matter content, somatic cell count and total number of microorganisms were determined in the collected milk samples A in the Laboratory of dairying at Biotechnical Faculty in Ljubljana. Furthermore, fatty acid composition of milk lipids was determined. Chemical composition of goat milk was determined by the instrument MilkoScan 133 B, which operates on the principle of infrared spectrometry. Somatic cell count was determined using apparatus Fossomatic 5000, which operates on the basis of automatic epifluorescent technique, by the principle of flow cytometry. The total number of microorganisms was determined using the apparatus Bactoscan 8000,

**Fatty acid composition of milk lipids**: Milk samples B were stored in liquid nitrogen immediately after milk recording. They were stored then in freezer chamber at -70ºC until the analysis. Before the analysis, milk samples were warmed to 38-40ºC in water bath and mixed up. After that, 500 mg of the milk sample were weighed out into tubes, where 300 μl of methylenchloride and 3 ml of fresh prepared 0.5M of sodium hydroxide in methanol were added. To determine the fatty acid composition of milk lipids, the analysis of methyl esters of fatty acids was done. This analysis was performed on gas chromatograph Hewlett Packard HP AGILENT 6890 SERIES GC SYSTEM, USA. Processing of chromatographic data was conducted using ChemStation Plus software. Furthermore, factor of the responsiveness of the flame ionization detector was determined. Total lipids in the sample are composed of both fatty acids and glycerol from triglycerids, phosphate from phospholipids, and sterol. For the calculation of the fatty acid value in the sample in mg, special factors are used, The statistical package SAS (SAS/STAT, 2000) and partly the statistical package S-PLUS (1966) were used to analyse the data. The statistical analysis did not include records collected during the first six days of the preparation period. In the meantime, the situation in the stable was stabilizing and the team who participated in the experiment was introducing in the everyday milk measuring and collecting samples.

Due to the large fluctuations in individual values of the somatic cell count and number of microorganisms among animals and among observations within animals, we analyzed each animal individually as its time series, and for the most variable ones the logarithm of the values was found (X = log10Y).

The time series were first standardized (S) in the way that last four days (from the7th to 10th day) of the preparatory period (before supplementing with fatty acids) were took as a starting point. Mean value of this period was calculated by the median (Me), the measure of variability was the average absolute deviation (AD). In this way we reduced the impact of outliers. Although, it is usual to standardize by the average and standard deviation, we decided for median and absolute deviation. In this way, the standardized time series for the animal was calculated using the following equation:

$$\mathbf{S} \Leftarrow (\text{(X-Me)/AD}) \dots \tag{1}$$

In this way, the standardized time series (S) are comparable for animals with different values. Then, we calculated the median for the three periods on the standardized time series:


For each animal, the corresponding median has become an input data for the statistical analysis. In this way, we analyzed milk yield (ml), the content of milk proteins (g/100 ml), milk fat (g/100 ml), milk lactose (g/100 ml), dry matter (g/100 ml), non-fat dry matter (g/100 ml), total number of microorganisms (n\*103/ml), and somatic cell count (n\*103/ml) in milk.

In this way, a comparison of groups with a simple analysis of variance was made where the zero assumption was checked for that the averages by groups were the same. If a statistically significant difference test was found (5% level of significance was considered), then the groups were compared also by the Duncan test or by the contrast analysis, where each group was compared with the control group.

All other traits were analyzed by the GLM procedure (General Linear Model) with statistical package SAS, which included the impacts of the group (4) and period (3). Differences

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.
