PANNON UNIVERSITY SCHOOL OF ANIMAL AND ENVIRONMENTAL SCIENCES Leader of School: Dr. Angela Anda

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1 PANNON UNIVERSITY SCHOOL OF ANIMAL AND ENVIRONMENTAL SCIENCES Leader of School: Dr. Angela Anda THE ROLE OF RUMEN PROTECTED CONJUGATED LINOLEIC ACID ISMERS (cis-9, trans-11; trans-10, cis-12) IN NUTRITION OF HIGH PRODUCING DAIRY RUMINANTS PhD THESIS By GALAMB ESZTER Supervisor DR. HUSVÉTH FERENC KESZTHELY

2 1. OBJECTIVES The objective of our experiments was to investigate the effect of dietary supplementation of rumen protected conjugated linoleic acid isomers on milk yield, milk composition, liver fat content, and fatty acid composition of hepatic lipids and milk fat in high producing dairy ewes and cows. In addition, we intended to study whether CLA supplementation started prepartum or after calving, respectively is able to decrease the length of the negative energy balance period which causes notable economic losses. 2

3 2. MATERIAL AND METHODS 2.1. Experiment 1 In this experiment the ruminal stability of the CLA supplement, containing c-9, t-11 and a t-10, c-12 CLA-isomers in similar proportions and used in the further investigations with dairy ewes and cows, were determined by an in situ method developed to measure feed degradability Experiment 2 In this study the effect of the CLA supplement with high rumen stability index was examined on milk yield, milk composition liver fat content as well as on the fatty acid composition of milk- and liver lipids of high lactating dairy ewes. Fifty multiparous Awassi ewes were allotted into two treatment groups. The diet of one the groups was supplemented with 25 g CLA containing product (ewe/day) from d 2 to 42 postpartum while ewes in the Control group were fed an isocaloric-isonitrogenous diet without CLA supplementation. The individual milk production of ewes was recorded daily, however, milk fat and protein content as well as fatty acids compositions were determined weekly. Liver tissue was collected from ewes by needle biopsy, randomly from 10 animals per groups on day 2, 21 and 42 postpartum, respectively and the total lipid and 3

4 fatty acid composition were determined. First, the ruminal stability of different CLA isomers in product were determined by an in situ method developed to measure feed degradability, what was used in further investigations Experiment 3 The objective of this experiment was to determine the optimal period of CLA feeding in high-producing Holstein dairy cows. For this reason the effect of CLA applications were compared if the supplementation to the diet started in the dry period or only after calving, respectively. The influence of CLA feeding on milk production, liver lipid content, as well as some plasma metabolites indicating energy homeostasis were studied. Furthermore, the changes of fatty acid compositions in milk and liver lipids were determined. The experiment was carried out with 60 multiparous Holstein dairy cows. The experimental period started 21 days prior to the expected day of calving and ended on day 77 postpartum. Animals were allotted into 3 treatment groups as matching triplet method, in order to the efficiency of prepartal CLA supplementation and postpartal CLA supplementation can be compared. The diet of CLA1 group was supplemented with 70 g (cow/day) CLA product from d 21 prepartum until 77 DIM. The diet of CLA2 group was supplemented with 70 g CLA product from parturition until 77 DIM. The Control animals were fed an isocaloric-isonitrogenous diet without CLA supplementation. 4

5 The individual milk production was recorded daily, milk fat and protein contents were determined every second weeks. Milk fatty acid composition was analyzed on week 1, 5 and 11 postpartum. Liver samples were collected by needle biopsy, randomly from 10 animals per group on week 3 before calving and week 1, 5 in lactation. Total lipid content and fatty acid composition of liver lipids were measured. For the determination of plasma metabolites blood samples were collected 8 times from every single cow during the experimental period in the 3 rd ( 3) and 1 st ( 1) weeks pre partum and on the day of calving (2 hours after calving; 0), and subsequently in the 1 st, 2 nd, 3 rd, 5 th and 11 th weeks postpartum. 3. RESULTS 3.1. Experiment 1 The rumen stability of CLA isomers was 70% at 12.5 hours of incubation Experiment 2 As a result of CLA supplementation increased the milk yield by 9%, at the same time the milk fat decreased by 10% (P<0,05) in lactating ewes. The liver lipid content was lower in CLA supplemented group than in control on day 21 postpartum (P<0,05). The c-9, t-11 and a t-10, c-12 CLA isomers showed higher values on day 21 and 42 due to CLA supplementation 5

6 (P=0,05). The changes of stearic (C18:0) and oleic acid (C18:1n-9) correlated with DIM (days in milk) (P=0,01). The c-9, t-11 CLA increased with elevation of DIM in both groups (P=0,03). However, those was higher in milk of CLA supplemented animals, than in control (P=0,01) Experiment 3 The milk production of CLA supplemented groups was higher compared to control between week 3 and week 6 of lactation (P<0,05). The fat content of milk decreased following week 3 of lactation due to CLA supplementation (P<0,05). As a result of CLA supplementation started from dry period the BCS loss was lower in early lactation period (P<0,05). The BCS increased only to week 11 of lactation due to postpartum CLA supplementation (P<0,05). The plasma NEFA concentration was lower in early postpartum period if the CLA supplementation began before calving. The liver lipid content was lower in CLA 1 group than in control on week 5 postpartum (P<0,05). The oleic acid (C18:1n-9) content of liver lipids was lower in lactation than during dry period (P<0,001). Contrarily, the stearic acid (C18:0) values reduced in all groups after calving (P<0,001). The proportion of oleic acid (C18:1n-9) was lower in CLA groups compared to control (P<0,05). The CLA supplementation decreased the arachidon acid (C20:4n-6) in liver lipids (P<0,05). The c-9, t-11 and t-10, c-12 isomers increased due to CLA supplementation in liver and also in milk lipids (P<0,05). The incorporation of c-9, t-11 CLA isomers to milk lipids is preferred 6

7 against to liver. While ratio of the t-10, c-12 CLA isomer is higher in the liver lipids than milk fat. 4. CONCLUSION These results demonstrate that CLA supplementation suitable method to alleviate the body mass mobilization of high producing ruminants during early lactation period. This results in smaller BCS loss and reduced lipid infiltration of the liver, lower plasma NEFA levels, shortened negative energy balance period as well as reduces the prevalence of subclinical ketosis. There effects can be enhanced if the CLA supplementation started from dry period. Furthermore, the CLA supplementation can results increased milk yield of dairy ruminants, when decreases milk fat at least 10%. 7

8 NEW SCIENTIFIC RESULTS 1. The CLA supplementation enhanced milk yield of dairy ewes from the first week postpartum, and at the same time decreases milk fat. On the other hand, in dairy cow, milk fat depression appears only from the third week of lactation, irrespectively of whether CLA treatment started during the dry period or only after calving. 2. In overconditioned dry cows CLA supplementation started 3 weeks prior to calving improves energy balance during the critical, early phase of lactation. This results in smaller BCS loss and reduced lipid infiltration of the liver during the first 5 weeks of lactation. The above mechanism is supported by lower plasma NEFA levels following calving, which reduces the prevalence of subclinical ketosis with more than 50%. 3. In high producing ruminants the incorporation of cis-9, trans-11 CLA isomers to milk lipids is preferred against to liver. While ratio of the trans-10, cis-12 CLA isomer is higher in the liver lipids than milk fat. 4. The proportion of the cis-9, trans-11 CLA isomer, known to have anticarcinogenic effect, is higher in sheep milk lipids compared to cow milk. Considering the usual milk fat levels in the two species, the conclusion is that sheep milk is a more favourable CLA source for its human health effects than cow milk. 8

9 5. During early lactation, when in lactating ruminant the lipid mobilization increased, CLA supplementation reduces total liver lipid content, in conjunction with higher stearic acid and lower oleic acid proportion of hepatic lipids. CLA treatment decreases the arachidonic acid (C20:4n-6) ration of liver lipids in lactating cows. 9

10 SCIENTIFIC PUBLICATIONS RELATED TO THIS RESEARCH Publications released in peer reviwed periodicals in English Husvéth, F., Galamb, E., Gaál, T., Dublecz, K., Wágner, L., Pál, L. (2010). Milk production, milk composition, liver lipid contents and C18 fatty acid composition of milk and liver lipids in Awassi ewes fed a diet supplemented with protected cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers. Small Rum. Res. 94: (IF: 1,395) Husvéth, F., Galamb, E., Farkas, V., Wágner, L., Jolánkai, R., Pál, L. (2011). Conjugated linoleic acid and other C18 fatty acid composition of muscle and adipose tissues in lambs fed on diets containing vegetable oil supplementations or grass silage. Acta Aliment. 40(3): (IF: 0,444) Galamb, E., Faigl, V., Keresztes, M., Csillik, Z., Tröscher, A., Elek, P., Kulcsár, M., Huszenicza, Gy., Fébel, H. and Husvéth, F. (2013). Effects of lipid-encapsulated conjugated linoleic acid (CLA) isomers on milk yield and metabolic responses in lactating dairy cows. Animal, Under revision. (IF: 1,648) 10

11 Publications released in peer reviwed periodicals in Hungarian Galamb, E., Fébel, H., Kulcsár, M., Husvéth, F. (2013). A takrmányozás és a tejzsírdepresszió összefüggései tejelő tehenekben. Magy. Állatorv. Lapja, Accepted for publication. (IF: 0,146) Publications released in peer reviwed proceedings of confereces in English Galamb, E., Pál, L., Wágner, L., Husvéth F. (2010). Effects of lipid encapsulated conjugated linoleic acid (CLA) supplements on lipid metabolism of lactating ewes In: Proceedings of the 19th International Scientific Symposium on Nutrition of Farm Animals. Murska Sobota, Szlovénia. ZED10 ZED22- Galamb.doc Publications released in proceedings of confereces in English Wágner, L., Dublecz, K., Husvéth, F., Pál, L., Jolánkai, R., Farkas, R., Galamb, E. and Kovács, G. (2009). Conjugated linoleic acid determination in chicken egg yolk. WPSA, XIII. European Symposium on the Quality of Eggs and Egg Products June, Turku, Finland. 11

12 Publications released in proceedings of confereces in Hungarian Galamb, E., Pál, L., Dublecz, K., Wágner, L., Husvéth, F. (2010). Védett konjugált linolsav (CLA) kiegészítés hatásának vizsgálata intenzíven tejelő juhokkal. Agrár- és Vidékfejlesztési Szemle 5(2): 109. Galamb, E., Elek, P., Jolánkai, R., Husvéth, F. Bendővédett CLA készítmény hatása tejelő juhok tejtermelésére, tejösszetételére valamint a majlipidekre. XXVI. Ifjúsági Tudományos Fórum, Keszthely, márc. 25. In: Pannon Egyetem Georgikon Kar szerk. Galamb, E., Pál, L., Dublecz, K., Wágner, L., Husvéth, F. A konjugált linolsav (CLA) kiegészítés hatása intenzíven tejelő anyajuhok tejtermelésére, a tej összetételére, valamint a tej és májlipidek zsírsavösszetételére. XXXIII. Óvári Tudományos Nap, Mosonmagyaróvár, okt. 07. A magyar élelmiszergazdaság jövője a KAP reform tükrében. In: Kovácsné Gaál Katalin szerk. ISBN: Galamb, E., Huszenicza, Gy., Faigl, V., Fébel, H., Elek, P., Csillik, Z., Husvéth, F. Bendővédett konjugált linolsav (CLA) takarmány-kiegészítés hatásának vizsgálata intenzíven tejelő Holstein-fríz tehenek tejtermelésére. XXVII. Ifjúsági Tudományos Fórum, Keszthely, ápr. 21. In: Pannon Egyetem Georgikon Kar szerk. 12

13 Galamb, E., Faigl, V., Keresztes, M., Kulcsár, M., Huszenicza, Gy., Fébel, H., Csillik, Z., Troescher, A., Husvéth, F. Bendővédett konjugált linolsav (CLA) takarmánykiegészítő-termék hatása tejelő tehenek tejtermelésére, a máj zsírtartalmára, valamint a metabolikus státuszt jellemző vérparaméterekre. Magyar Buiatrikus Társaság XXI. Nemzetközi Kongresszusa, Sümeg, okt Galamb, E., Pál, L., Gaál, T., Wágner, L., Husvéth, F. (2010). A konjugált linolsav alkalmazásának lehetősége az intenzíven termelő anyajuhok takarmányozásában. Akadémiai Beszámolók, Takarmányozástan Szekció. 36. Galamb, E., Faigl, V., Keresztes, M., Kulcsár, M., Huszenicza, Gy., Fébel, H., Csillik, Z., Husveth, F. (2011). Bendővédett konjugált linolsav (CLA) hatása tejelő tehenek májának zsírtartalmára, a májlipidek zsírsavösszetételére és az energia egyensúlyt jelző vérparaméterekre. In: Akadémiai Beszámolók, Állathigénia, Állattenyésztés, Genetika, Takarmányozástan Szekció

14 Faigl, V., Galamb, E., Csillik, Z., Keresztes, M., Tröscher, A., Kulcsár, M., Husvéth, F., Huszenicza, Gy. (2011) Bendővédett mikrolipid burokba zárt konjugált linolsav (CLA) izomerek (Lutrel Pure-BASF) etetésének hatása nagy tejtermelésű holstein fríz tehenek kondiciójára, szárazanyagfelvételére, és tejtermelésére. In: Akadémiai Beszámolók. Állathigénia, Állattenyésztés, Genetika, Takarmányozástan. 17. Csillik, Z., Faigl, V., Keresztes, M., Galamb, E., Tröscher, A., Kulcsár, M., Husvéth, F., Huszenicza, Gy. (2011). Bendővédett mikrolipid burokba zárt konjugált linolsav (CLA) izomerek (Lutrel-Pure-BASF) etetésének hatása nagy tejtermelésű holstein fríz tehenek metabolikus státuszára, valamint egyes szaporodásbiológiai paramétereire. In: Akadémiai Beszámolók. Állathigénia, Állattenyésztés, Genetika, Takarmányozástan