APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper

CHANGES IN COMPOSITION OF DOMESTIC BALKAN DONKEYS’ BREED DURING PERIODS

Jasmina M. Lazarević*, Tatjana A. Tasić, Sanja J. Popović, Vojislav V. Banjac, Olivera M. Đuragić, Bojana M. Kokić, Ivana S. Čabarkapa

Univeristy of Novi Sad, Institute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

Changes in the milk composition during early, middle and late stage of lactation period were monitored in 10 individual autochthonous donkeys of the Domestic Balkan breed. Animals were grazing ad libitum during early and middle lactation, while supple- ments (meadow hay, corn and corn stalks) were added during the late lactation period. The aim of this study was to evaluate the main compounds of donkeys’ milk during different periods of lactation. Based on the obtained results it was noticed that nutritional effects on milk compo- sition were the greatest in the early and middle lactation. Chemical analysis indicated that donkeys’ milk was quite poor in dry matter (8.82-9.68%), (0.54-0.67%) and protein (1.44-1.79%), and conversely was high in content (6.06-7.12%). Concen- tration of vitamin C varied significantly (P<0.05) during the lactation period and was very high (12.84-26.89 μg/mL) in comparison to milk from other animal species. Like- wise, the content of lysozyme (1.95-3.29 g/L) and lactoferrin (1.56-3.14 g/L) during the early and middle lactation period showed an increase. Donkeys' milk did not show any significant differences in MUFA, n6-PUFA and UFA contents, while n3-PUFA, n6/n3 and UFA/SFA ratio showed a significant variability (P<0.05) during the lactation period.

KEY WORDS: Balkan donkeys’ milk, fractions, protein fractions, fatty acids

INTRODUCTION

The research interest in donkeys’ milk has increased in Europe in recent years, since its composition is similar to human milk (1, 2), and that has consequently increased the price of donkeys’ milk. In the available literature, there is a lack of data regarding accurate characterization and changes in chemical composition of milk during lactation for the Balkan donkeys’ breed. In the Serbian farm "Zasavica breeding of Domestic Bal- kan donkeys" they started a project of the revitalization and preservation of this low reproductive breed, based on different diet systems consisted of feeding on pasture. Pas- ture is the major dietary source for donkeys’, but there is little information regarding its

* Corresponding author: Jasmina M. Lazarević, University of Novi Sad, Institute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected] 187 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper nutritional composition. It is widely recognized that pasture, during efficient grazing, is the cheapest feedstuff (3), with highly variable chemical composition. The sources of variation in pasture composition are plant species, growth stage, temperature, and light intensity. Fresh grass contains a high proportion of total fatty acids (50-75%) in the form of n3 linolenic acid (2, 4). In donkeys’ milk, total protein content is very low, in particular it shows a low level of casein (CN), and on the other hand, high level of lactose, linoleic acid (n6-PUFA), linolenic acid (n3-PUFA) and lysozyme (5, 6). The content of CN ranges from 37.56 to 39.98% of total protein content (1). Guo et al. (6) reported that the content of whey pro- teins (WP) in donkeys’ milk is in the range of 53.03 to 57.06% of total protein content. The main components of WP are -lactalbumin (-LA) and -lactoglobulin (-LG), which account from 35% to 50% of the total proteins, compared to 20% present in cows’ milk (1). Also, donkeys’ milk is a very rich source of lysozyme (LYS) (1.0 g/L) (4, 5, 7), and has significantly higher content than bovine milk (0.13 mg/L), while the level of LYS is quite similar to that in (0.4-1 g/L) (8, 9). The lipid content of donkeys’ milk is quite poor, according to Guo et al. (6), who showed significant variability (0.20-1.82%), indicating that the lipid content could be af- fected by breed, breeding area and forage, technique, interval between milking, and mainly by the stage and year of lactation. Similar results were reported by Fox (10). Saturated fatty acids (SFA) are the most representative fatty acids in donkeys’ milk, which was indicated by different studies (10, 20). However, it has been noticed that SFA content is lower in donkeys’ milk than in ruminant milk (11, 12). During the lactation phase, content of SFA decreases while the content of unsaturated fatty acids (UFA) shows an increase (2). In view of the above, the aim of the present study was to determine the influence of lactation period on the chemical composition of the milk from Balkan donkeys’ when diet of animals was based on pasture feeding.

EXPERIMENTAL

Animals feeding

During the early (40-100 days) and middle (130-190 days) (from spring to autumn) periods of lactation. the donkeys’ were grazing ad libitum. During the winter feeding (late period of lactation; 220-280 days) meadow hay, corn and corn stalks were used as supplements (two times a day). Some of the species found in pasture vegetation included Poaceae, Fabaceae, Asteraceae and Plantaginaceae families. Water was available ad libitum.

Milk sample collection

Individual samples were collected during the lactation period (between 40 and 280 day) from ten healthy female donkeys’. Sample collection was performed four

188 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper times per each lactation period. Milking was performed two times a day, in the morning (08:00 am) and afternoon (14:00 am), after which samples were merged for every indivi- dual animal. The samples were stored in sterile plastic bottles at 4 °C until further ana- lysis. The samples that were used for the ascorbic acid determination were stored in plastic bottles wrapped in aluminum foil. The experiment was repeated during two years.

Chemical analysis

Basic chemical composition of the milk, i.e. dry matter, ash, protein, and fat were analyzed by methods of the IDF (13). Lactose content was determined by GEA Niro ana- lytical method A18b (14). The vitamin C (L-ascorbic acid) content was determined by enzymatic method (Megazyme International Ireland 2011), and the absorbance was mo- nitored using GBC CINTRA 303UV/VIS spectrophotometer.

Determination of protein profile

Protein profiles (CN and whey fractions) of milk were analyzed using two-dimen- sional difference gel electrophoresis. Sample preparation was carried out according to Tidona et al. (15) with some modifications. A 1:1.5 (v/v) dilution of each milk sample was made with buffer (0.125m Tris-HCL, 4% SDS, 2% glycerol, 2% -mercaptoethanol, pH 6.8) and heated at 100 °C for 5min. The chip-based separation was performed on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) in combination with Protein 80 Plus Lab Chip kit.

Fatty acid analysis

Fat was extracted from the milk samples using Folch method (16). The preparation of fatty acid methyl esters was done by transmethylation with 14% wt. boron trifluoride/ methanol solution, which is recommended for this type of substrates. Fatty acid analysis was done on an Agilent 7890A gas chromatographer (Agilent Technologies, CA, USA) with flame ionization detector, and fused silicia capillary column (DB-WAX 30 m, 0.25 mm, 0.50 μm). The carrier gas was helium (purity  99.9997 vol%, flow rate = 1.5 ml/min).

Statistical analysis

To analyze variations of the results, one-way ANOVA and Post-hoc Duncan test were used. Differences between the means with probability P<0.05 were accepted as statisti- cally significant. The analysis was performed using the software package Statistics 12.0 for Windows (Stat Soft, Tulsa, Oklahoma, USA). All measurements were made in tripli- cates, and the results were expressed as mean value ± standard deviation.

189 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper

RESULTS AND DISCUSSION

The chemical composition of Balkan donkeys’ milk samples are summarized in Table 1. Depending on the lactation period, dry matter, fat and lactose showed higher content in the early and late lactation in comparison to the middle lactation. The milk fat level did not vary significantly (P>0.05) within the period of lactation. The fat content was slightly higher in the early lactation due to the quality and availability of fresh grass in the pasture, and in the late lactation due to addition of supplements to the diet, which is consistent with the results of Ivanković et al. (17). A similar trend in fat content decrease in donkeys’ milk after 100 days of lactation was reported by Salimei et al. (1) and Guo et al. (7).

Table 1. Chemical composition of Domestic Balkan donkeys’ milk during various lactation periods

Lactation period Composition Early Middle Late (40-100 days) (130-190 days) (220-280 days) Dry matter, % 9.27ab ± 0.36 8.82a ± 0.23 9.68b ± 0.47 Ash, % 0.43a ± 0.04 0.64b ± 0.10 0.47a ± 0.02 Protein,% 1.79b ± 0.10 1.57a ± 0.08 1.44a ± 0.06 Fat,% 0.66a ± 0.27 0.54a ± 0.10 0.67a ± 0.15 Lactose,% 6.38a ± 1.88 6.06b ± 0.83 7.12c ± 0.86 Vitamin C, μg/ml 26.89a ± 0.19 24.15a ± 0.18 12.84b ± 0.20 a−b Means with different letter indexes in the same row are statistically significantly different (P<0.05).

The highest concentrations of milk protein and vitamin C were determined during the early lactation period, and gradually decreased from the beginning to the end of the lactation period. Similar trends were observed in mares’ (18), cows’ (11) and donkeys’ milk (19). The protein content did not vary significantly (P>0.05) during the middle and late lactation. In the middle period of lactation, statistically higher (P<0.05) ash content was observed compared to other stages of lactation. The obtained results of donkeys’ milk chemical composition do not appear to differ significantly from those in the litera- ture based on the other donkeys breeds, with the exception of the average content of ash, which was slightly higher than the reported values (1, 7, 19). In the present study, che- mical composition of milk was improved when donkeys’ gained access to pasture in the early and middle lactation period. The changes observed in CN and its fractions in the various lactation periods are shown in Table 2. The average content of CN showed a significant decrease after the middle period of lactation. The obtained results for CN content are similar to those reported for Amiata donkeys' breed (20, 21), but lower than for ruminant's milk (22). The

CN fractions present in the analyzed samples were -CN, S1-CN and S2-CN. The chan-

190 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper ges in the concentration of CN fractions during the early and middle period were not statistically significant (P>0.05), while the observed decrease of all CN fractions was significant in the late lactation. The highest concentration of CN, S1-CN and -CN was observed during the middle lactation (4.82%, 1.72%, 2.88%, respectively), and for S2- CN (0.24%) during the early lactation.

Table 2. Mean concentration of CN and casein fractions in Domestic Balkan donkeys’ milk

CN fractions Lactation Casein (CN)  -CN  -CN -CN period (g/L) S1 S2 (g/L) (g/L) (g/L) Early 3.84ab ± 0.58 1.37a ± 0.36 0.24a ± 0.07 2.23a ± 0.37 Middle 4.82b ± 1.19 1.72a ± 0.71 0.22a ± 0.03 2.88a ± 0.62 Late 2.63a ± 0.50 1.10a ± 0.10 0.10b ± 0.03 1.45b ± 0.20 a−b Means with different letter indexes in the same column are statistically significantly different (P<0.05).

Table 3. Mean concentration of WP and WP fractions in Domestic Balkan donkeys’ milk

WP fractions Lactation -LA -LG LYZ LF period (WP) (g/L) (g/L) (g/L) (g/L) (g/L) Early 11.05a ± 0.68 2.66ab ± 0.10 2.10a ± 0.20 3.29a ± 0.28 2.06a ± 0.62 Middle 11.60a ±1.62 3.03b ± 0.65 1.97a ± 0.24 2.71a ± 0.49 3.14b ± 0.44 Late 7.49b ± 1.71 2.11a ± 0.30 1.50b ± 0.34 1.95b ± 0.39 1.56a ± 0.74 a−b Means with different letter indexes in the same column are statistically significantly different (P<0.05).

Table 3 summarizes the data of total WP and its fractions determined in donkeys’ milk during various lactation periods. The WP content was the highest during the early and middle period (11g/L), and then rapidly decreased during the late period of lactation, which was not in accordance with the study of Malacarne et al. (23), where the content of WP remained constant throughout the lactation period. The content of WP fractions -LG and LYZ slightly decreased in the middle in comparison to early lactation period, but the decrease was not statistically significant (P>0.05). In the middle of the lactation period, the content of -LA and LF was significantly higher (P<0.05) compared to the early and late periods. All WP fractions significantly decreased (P<0.05) during the late period of lactation. Table 4 shows the level of fatty acids. The fatty acids were affected by the lactation period and showed a statistically significant decrease (P < 0.05) in the SFA content, and a significant increase (P<0.05) in the PUFA content. Overall, the average SFA content was higher than the MUFA and PUFA contents. The most of UFA present in milk, whose content increased from the beginning to the end of the lactation period, were MUFA. The period of lactation did not affect (P>0.05) the MUFA content that averaged around 32%. 191 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper

The UFA/SFA ratio increased from the early to late lactation and averaged around 1.0, which was similar to that in mare’s milk (22), and higher than the ratio found in the Ragusana donkeys’ milk (UFA/SFA ratio = 0.48) (11).

Table 4. Fatty acids composition of Balkan donkeys’ milk (% of total fatty acid)

Lactation period Fatty acid composition Early Middle Late (40-100 days) (130-190 days) (220-280 days) SFA 59.75a ± 4.88 57.61a ± 6.41 42.47b ± 7.49 MUFA 31.74a ± 6.87 33.12a ± 3.56 30.71a ± 3.56 PUFA 15.91a ± 3.30 21.53ab ± 6.53 25.80b ± 4.36 n3-PUFA 8.86b ± 0.53 6.80a ± 0.81 7.21a ± 1.20 n6-PUFA 9.16a ± 1.88 11.29a ± 0.83 11.18a ± 0.86 n6/n3 ratio 1.03a ± 0.17 1.67b ± 0.03 1.61b ± 0.14 UFA 47.66a ± 7.20 54.64a ± 9.71 56.51a ± 6.51 UFA/SFA ratio 0.81a ± 0.19 0.97a ± 0.25 1.37b ± 0.37 a−b Means with different letter indexes in the same row are statistically significantly different (P<0.05). SFA - saturated fatty acids MUFA - monounsaturated fatty acids PUFA - polyunsaturated fatty acids UFA - unsaturated fatty acids

The changes in the PUFA and n6-PUFA contents in the analyzed samples showed a tendency to increase until the end of the lactation. The maximum content of n3-PUFA was in the early lactation period. The lowest levels of n6-PUFA and n-6/n-3 ratio were observed in the early lactation period, and did not change during the middle and late lactation. The higher n6/n3 ratio may be explained by the increase in pasture feeding of donkeys on richer grass during the middle and late lactation period (2, 11).

CONCLUSION

On the basis of these results it could be concluded that Balkan donkeys’ milk represents a rich source of WP, LF and LYS, as well as essential fatty acids. During feeding of donkeys’, the fresh forage in the pasture and the addition of meadow hay, as well as of corn during the winter, increased dry matter, fat and lactose content in the milk in the early and late period of lactation. The higher n3-PUFA content and lower n6/n3 ratio is of great interest in adopting donkeys’ milk.

192 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper

Acknowledgement

This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project No. III46012. Authors would like to thank Messrs. Slobodan Simić and Nikola Nilić (Special Nature Reserve “Zasavica”, Serbia) for their great cooperation in this research.

REFERENCES

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ПРОМЕНЕ САСТАВА МЛЕКА МАГАРИЦЕ БАЛКАНСКЕ РАСЕ ТОКОМ ПЕРИОДА ЛАКТАЦИЈЕ

Јасмина М. Лазаревић, Татјана Т. Тасић, Сања Ј. Поповић, Војислав В. Бањац, Оливера М. Ђурагић, Бојана М. Кокић, Ивана С. Чабаркапа

Универзитет у Новом Саду, Научни институт за прехрамбене технологије, Булевар Цара Лазара 1, 21000 Нови Сад, Србија

Промене састава млека су испитиване на 10 магарица аутохтоне балканске расе током раног, средњег и касног периода лактације. Животиње су се храниле на паш- њаку током ране и средње лактације, док су додаци исхрани (ливадско сено, куку- 194 APTEFF, 48, 1-323 (2017) UDC: 637.1+636.18]:612.664 https://doi.org/10.2298/APT1748187L BIBLID: 1450-7188 (2017) 48, 187-195 Original scientific paper

руз и кукурузна стабљика) уведени током касног периода лактације. Циљ овог ис- траживања је било испитивање састава млека магарица током различитих периода лактације. На основу добијених резултата примећено је да су нутритивне промене млека биле највеће током раног и средњег периода лактације. Хемијском анализом је утврђено да је млеко магарице имaло низак садржај суве материје (8,82-9,68%), млечне масти (0,54-0,67%) и протеина (1,44-1,79%), док је садржај лактозе био ви- сок (6,06-7,12%). Концентрација витамина C је значајно варирала (P <0,05) током периода лактације и била је веома висока (12,84-26,89 μg/mL) у поређењу са мле- ком других врста животиња. Такође, садржај лизозима (1,95-3,29 g /L) и лактофе- рина (1,56-3,14 g/L) је био повећан током раног и средњег периода лактације. Мле- ко магарице није показало статистички значајне разлике у садржају MUFA, n6- PUFA и UFA, док је садржај n3- PUFA, n6/n3 и UFA/SFA значајно варирао (P <0.05) током периода лактације.

Кључне речи: млеко магарице балканске расе, фракције казеина, фракције проте- ина сурутке, масне киселине

Received: 15 August 2017. Accepted: 11 October 2017.

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