Int.J.Vet.Res. 3,2:113-128,29 113 2 Effets of Fish Oil, Sfflower Oil nd Monensin Supplementtion on Performne, Rumen Fermenttion Prmeters nd Plsm Metolites in Chll Sheep 1* 2 1 1 3 Mirzei, F., Rezein, M., Towhidi, A., Nik-khh, A., Sereshti, H. 1 Deprtment of Animl Siene, Fulty of Agronomy nd Animl Siene, University of Tehrn, Krj-Irn. Deprtment of Animl Helth nd Nutrition, Fulty of Veterinry Mediine, University of Tehrn, Tehrn-Irn. 3 Deprtment of Chemistry, Fulty of Siene, University of Tehrn, Tehrn-Irn. (Reeived 1 July 28, Aepted 15 June 29) Astrt: Thirty-two ltting Chll ewes were ssigned to eight groups nd reeived one of eight dietry tretments for ten weeks s follows: ontrol diet (CON); fish oil-supplemented diet (2 g/kg dry mtter intke (DMI); FO); sfflower oil-supplemented diet (2 g/kg DMI; SO); monensin-supplemented diet (15 mg/kg DMI; MO); diet supplemented with fish oil (2 g/kg DMI) nd sfflower oil (2 g/kg DMI; FS); diet supplemented with fish oil (2 g/kg DMI) nd monensin (15 mg/kg DM;I FM); diet tht ontined sfflower oil (2 g/kg DMI) nd monensin (15 mg/kg DMI; SM); nd diet tht ontined fish oil (2 g/kg DMI), sfflower oil (2 g/kg DMI) nd monensin (15 mg/kg DMI; FSM). During the experimentl period, ewes were kept in individul pens nd the mounts of feed tht were offered to individul ewes were reorded dily. Milk smples were olleted weekly nd nlyzed for their omposition. Rumen fluid nd plsm smples were otined from eh ewe on the finl dy of the tril nd stored t -2 C efore nlysis. Results showed tht the SO diet hd no signifint effet (p<.5) on DMI, However, other diets redued the DMI signifintly. Milk yield ws signifintly lower for ewes tht were fed diets supplemented with monensin ompred to the other groups (p<.5). The milk ft perentge ws reltively low for ll tretments, lthough only FS, FM nd FSM tretments were signifintly different from the ontrol group. The yield of milk ft ws signifintly redued in the MO, FS, FM, SM nd FSM groups. The perentge of protein in milk ws signifintly redued in the FS nd FSM diets. However, the milk protein yield ws redued signifintly s diret result of the deresed milk yield in ewes tht were fed the MO, FS, FM, SM nd FSM diets. The MO diet inresed signifintly the onentrtion of ure nitrogen in milk. Fish oil nd sfflower oil-supplemented diets with or without monensin resulted in deresed numers of infetive protozo, nd deresed ette-to-propionte rtio in rumen fluid when ompred to the CON group. The onentrtions of plsm gluose nd ure were not ffeted y ny of the tretments, ut the plsm onentrtions of triglyerides, totl holesterol, nd HDL-holesterol were higher in ewes tht onsumed the oil-ontining diets thn the other groups. It ws onluded tht dietry supplementtion with omintion of fish oil, sfflower oil nd monensin ould lter the omposition of milk from ewes nd, in prtiulr, use redution in the perentge of ft in the milk. This my mke this milk more suitle for humn onsumption. Keywords: Milk, ewe, fish oil, sfflower oil, monensin. *Corresponding uthor: Mirzei@um..ir Tel:+98(261)224882 Fx: +98(261)2246752
114 Introdution Reltive to ovine nd prine milk, ovine milk is hrterized y its higher ontent of totl solids nd ft, whih mkes it idel for heese prodution (Henlein, 21). However, the high ft ontent in the milk of sheep might limit the demnd for milk nd milk produts y helth onsious onsumers (Zhng et l., 26). In reent yers, onsiderle ttempts hve een mde to lter the omposition of milk to mke it more suitle for humn onsumption. Modifition of the diet of ewes shows the most promise s mngement strtegy to hnge the omposition of milk in the short-term (Cnt et l., 1997). Dietry fts, whih re rih in polyunsturted ftty ids, hve een used to mnipulte the ftty id omposition of milk from ewes y n improvement in the unsturted: sturted ftty id rtio (Zhng et l., 26). Most studies regrding the effets of supplementtion with fts in ruminnts hve involved diry ows. However, dt is limited on giving feed supplementtion with dietry fts, suh s fish oil nd sfflower oil, to ltting ewes. Fish oil nd sfflower oil would e good hoie for feed supplementtion from the point of view of onsumers s oth re rih in polyunsturted ftty ids. Fish oil is soure of C2:5 nd C22:6 ftty ids, whih re n-3 ftty ids, wheres sfflower oil hve plentiful mount of linolei id (LA), whih is n-6 ftty id. Previous studies hve shown tht n-3 ftty ids my help to prevent oronry hert disese (Donovn et l., 2; Shingfield et l., 23).The n-3 ftty ids suh s eiospentenoi id (EPA [(C2:5 (n-3)]) nd dooshexenoi id (DHA [(C22:6 (n-3)]) re present in sustntil quntities in fish oil. Therefore, the inlusion of fish oil in the diet of ruminnts hs inresed the onentrtion of these long-hin polyunsturted ftty ids in milk ft tht my e enefiil for humn helth (Shingfield et l., 23; Lee et l., 25). The supplementtion of feeds with high mounts of long-hin ftty ids hs een shown to inrese milk ft nd inhiit de novo synthesis of short- nd medium-hin ftty ids in ovine Mirzei, F. mmmry glnds (Plmquis et l., 1993). The evidene is limited with regrds to the metolism of fish oil in the rumen, ut less thn 5% of the lipids in fish oils re likely to e hydrolyzed in the rumen, ompred to more thn 9% of plnt oils (Donovn et l., 2). Sfflower oil ws hosen in this study euse of its high level of is-9, is-12 18:2, whih my represent fesile dietry soure of LA for livestok. Lms tht were fed with sfflower seeds hd inresed unsturted ftty ids nd onentrtions of is-9, trns-11 onjugted linolei id (CLA) in their musle tissue (Bolte et l., 22; Kott et l., 23; Boles et l., 25). Monensin, whih is one of monoroxyli id ionophore ntiiotis, hs een known to inhiit the growth of Grm-positive teri tht produe hydrogen nd lter lipid metolism in the rumen (Wng et l., 25). Previous studies hve shown tht ionophores interfere with the iohydrogention of is-9, is-12 18:2 nd used redution in the extent of is-9, is-12 18:2 iohydrogention with n umultion of intermedite produts, inluding CLA (Fellner et l., 1997; Bell et l., 26). Monensin my inrese the protein ontent in the milk of diry nimls (Brown nd Hogue, 1985; Lynh et l., 199). Monensin n lso use redution in the perentge of milk ft (Cnt et l., 1997; Suer et l., 1998). Ionophores disrupt ruminl iohydrogention in similr mnner to unsturted ft supplements, nd monensin supplementtion of feeds enhned the levels of LA nd trns ftty ids in the milk of ltting ows (Jenkins et l., 23). The ojetive of this study ws to evlute the effets of supplementing the feeds of ltting ewes with fish oil nd sfflower oil with or without monensin. Dt ws olleted with regrds to their milk yield nd omposition, their plsm metolites nd ruminl prmeters. Mterils nd Methods Animls nd diets Thirty-two ltting Chll ewes (2, 3 or 4 yrs old nd 5 ± 1.5 kg ody weight [BW]) were ssigned to one of eight groups (n =4ineh group), nd were
Effets of Fish Oil, Sfflower Oil... 115 lned for their milk prodution t 1 nd 14 d fter lming. Eh group ws rndomized to e fed one of eight different dietry tretments (Tle 1). The diets with 52-55: 48-45, forge:onentrte rtio (dry mtter intke (DMI) sis) were formulted to meet the nutritionl requirements of 5 kg ewe in the first six to eight weeks of lttion with twin sukling lms (CNCPS, 21). The experimentl diets onsisted of: 1) ontrol diet, without supplementtion (CON); 2) diet supplemented with fish oil (2 g/kg of DMI; FO); 3) diet supplemented with sfflower oil (2 g/kg of DMI; SO); 4) diet supplemented with monensin (15 mg/kg of DMI; MO); 5) diet supplemented with fish oil (2 g/kg of DMI) nd sfflower oil (2 g/kg of DMI; FS); 6) diet supplemented with fish oil (2 Tle 1: Ingredient nd hemil omposition of experimentl diets. 1 Tretment diets (on DM sis) Items CON FO SO MO FS FM SM FSM Diet Ingredients (g/kg) Alflf hy 339.9 Corn silge 15.1 Brley strw 76.5 Brley grin 25.5 Cottonseed mel 94.5 Beet pulp 85.4 Soyen mel 71.4 Fish oil Sfflower oil Monensin Dilium phosphte 9.1 Limestone 3.7 Minerl-vitmin mix 2 4.5 Slt 4.6 Chemil ompositions (%) CP (g/kg) 16.2 EE 2.2 NDF 39.31 ADF 24.11 Ash 8.4 C.89 P.42 ME, (Ml/kg DM) 2.271 Dietry forge (%) 52.2 373. 83.4 86.8 146.8 122.8 82.4 62.2 2. 9.2 4.8 4.7 3.9 16.25 4.21 4.53 25.38 8.7.9.43 2.313 54.3 373. 83.4 86.8 146.8 122.8 82.4 62.2 2. 9.2 4.8 4.7 3.9 16.28 4.17 4.15 25.35 8.5.88.43 2.313 54.3 339.9 15. 76.5 25.4 94.5 85. 71.4.15 9.1 3.7 4.7 4.7 16.2 2.23 38.94 23.9 8.5.91.42 2.271 52.2 321.3 11.3 118.2 86.3 135.3 89.3 75.1 2. 2. 9.6 4.9 4.9 4.8 16.19 6.25 4.8 26.33 8.8.93.44 2.385 55. 372.9 83.4 86.7 146.8 122.7 82.4 62.2 2..15 9.3 4.8 4.7 4. 16.23 4.2 4.62 25.46 8.9.9.43 2.312 54.3 372.9 83.4 86.7 146.8 122.7 82.4 62.2 2..15 9.3 4.8 4.7 4. 16.26 4.19 4.2 25.2 8.8.87.43 2.311 54.3 321.3 11.3 118.2 86.3 135.3 89.3 75.1 2. 2..15 9.5 4.9 4.9 4.8 16.18 6.23 4.83 26.35 8.8.93.45 2.385 55. 1 CON: ontrol; FO: ontrol supplemented with fish oil; SO: sfflower oil; MO: monensin; FS: fish oil nd sfflower oil; FM: fish oil nd monensin; SM: sfflower oil nd monensin; FSMN: fish oil, sfflower oil nd monensin. 2 Contined Vitmin A(4 IU/kg), Vitmin D3 (1 IU/kg), Vitmin E (2 IU/kg), Clium (18 g/kg); Phosphte (7 g/kg), Mg (3 g/kg), N (5 g/kg), Fe (4 g/kg), Cu (.3 g/kg), Zn (3 g/kg), Mn (5 g/kg), I (.1 g/kg), Co (.1 g/kg), Se (.2 g/kg).
116 Mirzei, F. g/kg of DMI) nd monensin (15 mg/kg of DMI; FM); 7) diet supplemented with sfflower oil (2 g/kg of DMI) nd monensin (15 mg/kg of DMI; SM); nd 8) diet supplemented with fish oil (2 g/kg of DMI), sfflower oil (2 g/kg of DMI) nd monensin (15 mg/kg of DMI; FSM). During the experimentl period, ewes were kept in individul pens, nd wter ws ville freely t ll times. The diets were fed with three mels of equl size every dy of the tril t 8: h, 12: h, nd 16: h s Totl Mixed Rtion (TMR) for 12 weeks, whih llowed two weeks for dietry dpttion nd ten weeks for dt olletion. The mounts of the feeds tht were offered nd the mount of dily remined feed (orts) for eh ewe were reorded dily for eh individul ewe, nd the feed orts were djusted to mintin etween 5% to 1% of intke on s fed sis. Smpling nd hemil nlysis The TMR offered nd orts were mesured on dily sis. Feed nd orts were tken one weekly during the experimentl period nd nlyzed for the levels of DMI, CP, EE, Ash (AOAC 2) NDF, ADF (Vn Soest et l. 1991). The volume of milk prodution ws reorded weekly for ten weeks. It ws mesured over six hour period in dy y the use of omined hnd-milking nd the use of oxytoin (2 IU of oxytoin, Snphrm Denmrk Compny) s desried y Perez Al et l. (1997), nd the dily milk yield ws then lulted. Milk smples were then tken from eh ewe nd omined with preservtive (dihromte potssium) efore eing stored t 4 C until nlysis. The smples were then nlyzed for the levels of ft, protein, ltose, totl solids (TS), nd solids tht were not fts (SNF) using Milko-Sn 133 B (Foss Eletri, Hillerod, Denmrk) nd somti ell ount (SCC) using Fossomti 9. Approximtely 3 ml of ruminl fluid ws olleted from eh ewe t 7: h on the lst dy of week ten prior to the morning feed vi stomh tue (Jones et l., 2). The ph of the smples ws mesured immeditely fter the smpling, nd then 1 ml of rumen fluid from eh niml ws strined through four lyers of heeseloth. Three milliliters of metphosphori id (25 g/l) were dded to 15 ml from eh of the ruminl fluid smples, nd then these were stored t 2 C efore nlysis of the totl voltile ftty ids (VFA). Additionlly, 2 ml of hydrohlori id (HCl;.2 N) ws dded to 2 ml from eh of the ruminl fluid smples nd stored t - 2 C for free mmoni (NH3 N) nlysis (Heldt et l., 1999). The smples were thwed nd the onentrtion of NH3-N in ruminl fluid ws determined with the use of titrtion method, s desried y Crooke nd Simpson (1971). The onentrtion of VFAs ws determined s desried y Ottenstein nd Btler (1971) with gs hromtogrph (Philips PU 441). Four-Methyl Vleri Aid (Sigm) ws used s the internl stndrd. Briefly, 1 ml of the ruminl fluid smple nd 6 μl of the internl stndrd were trnsferred into 15 ml glss test tue, nd these were entrifuged t 3 g for 2 min t 4 C. The upper lyer ws then trnsferred into len 15 ml glss tue with Psteur pipette, nd 8 μl ws injeted into the gs hromtogrph. The initil olumn temperture ws 8 C, whih ws then inresed t rte of 1 C/min to 18 C. The injetor nd detetor tempertures were mintined t 2 C. Hydrogen ws the rrier gs. Ruminl fluid smples from ewes in eh of the tretment groups were lso used to ount the numer of protozo; 1 ml of strined ruminl fluid were mixed with 1 ml of 5% formlin (18.5% formldehyde, V/V) nd stored t 1 C efore estimting the protozol popultion (Dehority, 23). One milliliter of the preserved smples ws the pipetted into 16 15 mm test tue, two drops of Brillint green dye were dded, the tues were mixed nd then llowed to stnd overnight. Eh smple ws then ounted twie to determine the men verge of protozo in eh smple. Blood smples (5 ml) were olleted in heprin-ontining tues from the jugulr vein efore the morning feed on the lst dy of week ten in order to estimte the plsm onentrtion of
Effets of Fish Oil, Sfflower Oil... 117 metolites (Chillird nd Ottou, 1995) Blood ws entrifuged t 3 gt4 Cfor15minndplsm ws hrvested nd stored t -2 C until they were nlyzed for metolites (Loor et l., 25). Plsm onentrtion of gluose, triglyeride, totl holesterol, high density lipoprotein holesterol (HDL-holesterol), nd low-density lipoprotein holesterol (LDL-holesterol) were determined with the enzymti, olorimetri-endpoint method nd the enzymti, fixed-time method using ommeril kits (Prs Azmun Irn Compny) with spetrophotometer (Shimdzu 21, VIS spetrophotometer Shimdzu, Kyoto, Jpn). Sttistil nlysis The dt of milk yield nd omposition were nlyzed ording to ompletely rndomized ftoril design with time (weeks) s repeted mesure using the MIXED proedure of the Sttistil Anlyzer Softwre (SAS, 1996). The model for sttistil nlyses of dry mtter intke, milk yield, milk omponent perentges nd yields inluded: ovrite djustment, supplement (FO, SO, MO), week, supplement week intertion, nd residul error. Oservtions during the first two weeks prior to initition of the 1-wk lttion tril were used s ovrites for the respetive nd lest squre mens (LSM) of the ovrites. Dt were reported s LSM with pooled stndrd error (SE). The LSM were seprted y lest squres differene. Overll differenes etween tretment mens were onsidered signifint when the p-vlue ws less thn.5. Ruminl fluid mesurements nd plsm metolites were nlyzed using the generl liner model (GLM) proedure of the SAS progrm (SAS, 1996). Results were reported s LSM with pooled SE. The differenes etween oservtions were designted s signifint when the p-vlue ws less thn.5. Results Dry mtter intke (DMI), milk yield nd omposition The effet of diets on the DMI is presented in Tle 2. The supplementtion of the ontrol diet with fish oil signifintly redued the DMI (p<.5). Supplementtion with sfflower oil deresed DMI, lthough this differene ws not signifint etween SO nd CON tretments, nd only slight redution in DMI ws oserved for the SO diet when ompred with the CON group. The redution in DMI ws higher with the FO diet ompred to the SO diet. Addition of fish oil to sfflower oil (FS diet) produed greter derese in DMI, nd this rehed signifine for FS ompred with oth the CON nd SO diets (1.82 kg/d vs. 2.36 kg/d nd 2.7 kg/d, respetively). The drop in DMI ppered to e more pronouned for the diets tht ontined monensin thn for the ontrol diet, nd this rehed signifine for the MO, FM, SM, nd FSM diets when ompred with the CON (1.88 kg/d, 1.62 kg/d, 1.87 kg/d nd 1.58 kg/d vs. 2.36 kg/d, respetively). The highest derese in DMI ws oserved with the FSM nd FM diets when ompred with the CON diet. Milk yield nd omposition dt re shown in Tle 2. Milk yield delined for ll groups when ompred with the CON diet, lthough it ws signifintly lower (p<.5) in ewes tht were fed with MO, FM, SM, nd FSM diets thn those fed with the CON diet (17.9 g/d, 926.9 g/d, 1123.2 g/d nd 172.3 g/d vs. 144.4 g/d, respetively). However, no signifint differenes were found etween the milk yield nd omposition for the FO, SO, FS diets ompred with the CON diet. The perentge of milk ft ws reltively low for ll tretments (Tle 2), ut only the FS, FM, nd FSM diets were signifintly different when ompred with the CON diet (8.42%, 7.72% nd 8.9% vs. 9.55%, respetively). Due to the derese in the ontent of milk ft, the yields of ft were lso signifintly redued for ewes tht were fed with the MO, FS, FM, SM, nd FSM diets when ompred with the CON diet group (92.73 g/d, 12.17 g/d, 71.85 g/d, 14.9 g/d nd 85.6 g/d vs. 134.55 g/d, respetively). The ft ontent of milk ws lower in ewes tht were fed with diets ontining monensin
118 Mirzei, F. Tle 2: Dry mtter intke, milk yield nd omposition. Items Tretment diets 1 CON FO SO MO FS FM SM FSM SE DMI, (kg/d) Milk yield, (g/d) Ft, (%) Ft yield, (g/d) Protein, (%) Protein yield, (g/d) Ltose, (%) Ltose yield, (g/d) Totl solids, (%) Solids (not fts), (%) Milk ure N, (mg/dl) SCC, ( 1³/ml),,, d 1 2.36 144.4 9.55 134.55 5.31 74.13 5.36 74.45 2.42 11.29 16.97 16.75 2.7 1248.6 9.41 118.57 5.45 67.35 5.27 64.97 2.87 11.23 18.22 125.75 2.22 1271.4 9.48 12.91 5.18 65.22 5.38 68.73 2.83 11.18 17.8 133.25 1.88 d 17.9 8.74 d 92.73 5.17 d 54.2 5.28 56.75 19.92 11.6 19.97 14.13 1.82 d 1178.7 8.42 12.17 4.76 d 55.97 5.32 62.95 19.35 1.8 17.78 154.88 1.62 d 926.9 7.72 d 71.85 5.2 d 47.47 5.45 5.66 19.21 11.26 18.5 1.25 1.87 d 1123.2 9.28 14.9 5.23 58.85 5.25 59.35 2.65 11.9 17.98 142. 1.58 d 172.3 8.9 d 85.6 4.78 d 5.68 5.51 59.68 19.22 1.99 18.36 152.88.7 71.5.65 4.97.15 2.41.6 3.5.52.14.79 28.48 Vlues within row with different supersripts re signifintly different (p<.5) CON: ontrol; FO: ontrol diet supplemented with fish oil; SO: sfflower oil; MO: monensin; FS: fish oil nd sfflower oil; FM: fish oil nd monensin; SM: sfflower oil nd monensin; FSMN: fish oil, sfflower oil nd monensin. thn in ewes tht hd no monensin in their diets, nd the omintion of monensin with fish oil used signifint derese in the perentge of milk ft nd the milk yield. However, ddition of monensin to the sfflower diet did not hve signifint effet on these prmeters. The perentge of milk protein ws signifintly redued (Tle 2) for ewes fed with the FS nd FSM diets ompred with the FO diet (4.76% nd 4.78% vs. 5.45%, respetively). However, no signifint differenes were oserved etween the experimentl diets ompred to the CON diet (p<.5). Our dt showed tht fish oil nd sfflower oil together led to the signifint derese in the perentge of milk protein nd yield. The perentge of milk protein ws not ffeted y the MO diet. Among the experimentl diets used in the present study, the milk protein yield ws signifintly deresed (p<.5) y the MO, FS, FM, SM nd FSM diets when ompred with the CON diet (54.2, 55.97, 47.47, 58.85 nd 5.68 vs. 74.13 grm/dy, respetively). The perentge of ltose, TS nd SNF in milk were not signifintly different etween the tretments in this study. However, the milk ltose yield ws signifintly deresed (p<.5) y diets tht ontined monensin (MO, FM, SM nd FSM) ompred with the CON diet euse of the deresed milk yield of ewes tht were fed with these diets (56.75, 5.66, 59.35 nd 59.68 vs. 74.45 grm/dy, respetively). No differene ourred etween the other diets. The milk ure nitrogen (N) onentrtion ws inresed y monensin supplementtion (Tle 2), lthough only MO diet inresed signifintly the onentrtion of milk ure N ompred to the CON diet (19.97 vs. 16.97 mg/dl). The supplementtion of oils with or without monensin lso resulted in inresed milk ure N onentrtions, ut these effets were not sttistilly signifint. No signifint differenes (p>.5) were oserved in the somti ell ount (SCC) nlysis etween tretments.
Effets of Fish Oil, Sfflower Oil... 119 Rumen prmeters Protozo numers, ph nd NH3-N onentrtion Feeds tht were supplemented with oils with or without monensin signifintly deresed (p<.5) the totl numer of protozo in the rumen fluid 6 smples from pproximtely 2.5 1 /ml in the CON 5 diet to slightly less thn 6 1 /ml in thefm nd FSM diets (Tle 3). The effets on this prmeter in ll of the experimentl diet groups were signifintly different when ompred to the ontrols. In the present study, supplementtion of diets with oils nd monensin did not signifintly ffet the ph of the rumen (Tle 3). The onentrtion of ruminl NH3- N ws deresed y the tretment diets, nd this tended to e lower in ewes tht were fed with diets tht ontined oil nd monensin together (Tle 3). VFAonentrtion The totl onentrtion of VFAs (mmol/l) ws not signifintly different etween the groups with different diets. However, the supplementtion of diets with oils or monensin resulted in redution in the onentrtion of VFA when ompred with the ewes in the ontrol diet (Tle 3). The omintion of the different oils with monensin used greter redution in the onentrtion of VFA reltive to their use lone. Dietry supplementtion with sfflower oil in oth the SO nd FS diets signifintly redued (p<.5) the onentrtion of eti id in rumen fluid. The ddition of fish oil nd monensin lso redued the eti id onentrtion. The eti id onentrtions were lower numerilly in diets with sfflower oil thn other tretments. The signifint inrese in the onentrtion of propionte (p<.5) when oils or monensin ws dded. Our dt showed tht dding fish oil nd sfflower oil together my result in higher inrese in the onentrtion of propionte ompred the ontrols (23.34% vs. 9.73%)(p<.5). We oserved tht the onentrtion of propionte ws higher in diets with sfflower oil ompred to diets without it. The onentrtion of utyrte deresed when fish oil, sfflower oil or monensin ws dded to the diets, ut this redution ws not signifint. Dietry supplementtion hd no signifint effet on the proportion of vleri id, ut the level of isovlerte ws signifintly higher (p<.5) in ruminl fluid from ewes fed with the FO nd MO diets ompred with ontrols (2.7% nd 1.5% vs. 4.65%, respetively) (p<.5). The SO diet did not ffet the onentrtion of isovleri id, ut the umultive intertion of monensin nd oils were oserved in this reserh. The onentrtion of isovleri id deresed (p<.5) when diet ws supplemented with fish oil or sfflower oil withdietry supplementtions with oils or monensin resulted in signifint redution (p<.5) in the ette-to-propionte rtio (A:P) ompred with the ontrols. The lowest rtio ws oserved signifintly in the FSM diet (3.7 vs. 7.49) (p<.5). Plsm metolites The effets of the different diets on the levels of plsm metolites re presented in Tle 4. Plsm onentrtions of gluose nd ure were not signifintly ltered. The ddition of oil inresed the plsm onentrtions of totl holesterol nd triglyeride (p<.5). Plsm onentrtions of totl holesterol nd triglyeride were the highest (p<.5) fter ewes were fed with the FSM diet ompred to the CON diet (82.68 mg/dl nd 46.12 mg/dl vs. 56.39 mg/dl nd 39.11 mg/dl, respetively). No signifint differene ws found etween fish oil nd sfflower oil on the plsm onentrtions of totl holesterol nd triglyeride. The ddition of monensin hd no signifint effet on the plsm onentrtions of totl holesterol, HDL-holesterol, LDLholesterol, nd triglyerides. The onentrtions of HDL-holesterol nd LDL-holesterol were greter (p<.5) in ewes fed with diets supplemented y oils thn the diets without oils. Disussion Dry mtter intke (DMI) The derese in DMI hs een ttriuted to derese in the degrdtion of fier nd its
12 Mirzei, F. Tle 3: ph, protozo numers nd men onentrtion (Mm/l) of VFA in the ruminl fluid of ewes fed with experimentl diets. Tretment diets 1 Constituents CON FO SO MO FS FM SM FSM SE Ruminl fluid, (ph) Protozo numer, ( 1 ) NH3-N, (mg/1 ml) Totl VFA, ( mm/l) Aeti id, (%) Propioni id, (%) Butyri id, (%) Iso-vleri id, (%) Vleri id, (%) A: P 6.81 2.532 18.53 57.48 74.33 9.73 13.47 4.65.68 7.49 6.91 1.151 13.47 5.95 69.32 18.4 1.18 2.7.63 4.2 6.98 1.525 14.8 56.33 64.2 19.4 12.68 3.97.54 3.47 7.4 1.22 13.95 56.7 67.64 19.83 1.47 1.5.4 3.37 7.13 1.16 12.59 51.3 69.93 19.43 7.65 1.72.47 3.9 7.17 d.53 12.4 44.83 7.96 18.29 8.4 1.55.71 3.98 7.23 1.34 13.86 4.6 67.42 21.53 1.14 1.42.65 3.27 7.9 d.547 12.81 42.43 67.43 23.34 7.75 1.36.6 3.7.2.16.8 7.12 2.31 1.69 1.43.69.9.45,,, d Vlues within row with different supersripts re signifintly different (p<.5) 1 CON, ontrol; FO, ontrol supplemented with fish oil; SO, sfflower oil; MO, monensin; FS, fish oil plus sfflower oil; FM, fish oil plus monensin; SM, sfflower oil plus monensin; FSMN, fish oil plus sfflower oil plus monensin. Tle 4: Men onentrtion (mg/dl) of plsm metolites in ewes fed with experimentl diets. Tretment diets 1 Items CON FO SO MO FS FM SM FSM SE Gluose Triglyeride Totl holesterol HDL-holesterol LDL-holesterol Ure 63.93 39.11 d 56.39 38.23 18.16 32.2 64.14 42.96 7.15 46.35 24.8 28.6 64.94 42.84 69.29 43.46 25.73 29.45 66.79 39.4 55.86 37.63 18.29 31.65 67.36 44.6 83.5 51.6 31.9 34.9 67.75 42.75 67.34 46.18 22.16 29.52 64.92 42.86 68.68 46.58 23.25 32.22 67.35 46.12 82.68 51.49 31.32 34.22 3.44.9 3.23 3.52 3.11 2.58,,, d Vlues within row with different supersripts re signifintly different (p<.5) 1 CON: ontrol; FO: ontrol diet supplemented with fish oil; SO: sfflower oil; MO: monensin; FS: fish oil nd sfflower oil; FM: fish oil nd monensin; SM: sfflower oil nd monensin; FSMN: fish oil, sfflower oil nd monensin. digestiility in the rumen, whih lters the rtio of ruminl ette to propionte. In turn, is due to the diret dverse effet of fts, espeilly from high in unsturted ftty ids on ruminl miroes nd fermenttion(nrc, 21). Previous reserhers hve indited tht plnt oil or fish oil supplementtion of feeds uses signifint inhiition of the digestion of fier (Jenkins, 1993; 25). The high onentrtion of unsturted ftty Cnt et l., 1997; Donovn et l., 2; He et l., 25). The high onentrtion of unsturted ftty ids in fish oil is likely to e effetive inhiitors of fier digestion in the rumen (Jenkins nd Jenny, 1989). Unsturted ftty ids inhiit ellulr respirtion nd use lysis of teril ells (Glrith nd Miller, 1973; Donovn et l., 2). The negtive effet of dietry oil hs een ttriuted to four ftors (He et l., 25): 1) the physil
Effets of Fish Oil, Sfflower Oil... 121 oting of the feed with oil, whih restrits the ess of miroes to feed prtiles; 2) the formtion of insolule sops, whih dereses the vilility of tions suh s lium; 3) the inhiition of miroil tivity; nd (4) the toxiity to ertin miroes, espeilly protozo. The effet of ft supplementtion on the DMI ws relted to vrious ftors inluding the volume nd degree of the unsturtion of ftty ids, nd the type of ft tht supplements the diet (NRC, 21). Fish oil is omposed of highly polyunsturted ftty ids ompred with sfflower oil, whih is soure of polyunsturted ftty ids (Thoms et l., 1997). Therefore, it ould e expeted tht the DMI deresed to greter extent for fish oil ompred to sfflower oil. Consistent with this, the ddition of fish oil to the sfflower oil (FS diet) produed further derese in the DMI in our study. A similr effet of sfflower oil on DMI hs een oserved y Bell et l. (26) when diet supplemented with 6% DMI sfflower oil. The effet of monensin on DMI hs een demonstrted efore (Cnt et l., 1997; Suer et l., 1998; Jenkins et l., 23; Bell et l., 26). Cnt et l., (1997) reported derese in DMI when monensin ws used, nd this ould e relted to the ft tht monensin is toxi to speifi miroorgnisms in the rumen. In generl, this inludes those without n outer memrne suh s some ellulolyti speies. Johnson et l. (1988) lso found tht the omintion of fish oil with monensin produed greter degree in the derese of DMI in diry ows reltive to their use lone. Cnt et l., (1997) reported tht monensin my hve inresed the suseptiility of the rumen miroil popultion to the memrne disrupting effets of fish oil. Milk yield nd omposition Our results re onsistent with the report of Bell et l., (26) who showed tht the milk yield deresed in nimls fed with diet tht ontined sfflower oil nd monensin during the tretment period. However, Cnt et l. (1997) oserved tht there ws no effet on the milk yield when fish oil nd monensin were dded together to the diry ow diet. The redution of milk yield in ewes tht were fed with diets tht ontined oils, nd fish oil in prtiulr, my e relted to redution of DMI nd derese in ruminl funtion (Gonthier et l., 25; Zhng et l., 26) tht were exerted y supplementtion with monensin (Cnt et l., 1997). Generlly, the response of different speies of diry nimls to supplementl ft were different nd my e ffeted y the soure of ft, the volume of supplementl ft in the field nd the stge of lttion (NRC, 21). Milk ft depression is oserved ommonly when unproteted oil is fed (Cnt et l., 1997; Donovn et l., 2; Bell et l., 26; Fthni et l., 28). Fish oil nd other rumen-tive fts indued milk ft depression in ltting ows (Ber et l., 21). It is epted generlly tht diets with high level of polyunsturted ftty ids inhiits the de novo synthesis of milk ft, whih leds to low milk ft proportion (Zhng et l., 26). The ontent of milk ft ws lower in ewes tht were fed diets ontining monensin in our study, whih ws in greement with the results of Benhr (26) who reported tht the ddition of 35 mg monensin to the diet resulted in the redution of milk ft. This redution ws ssoited with higher level of trns-1 18:1, whih is potent inhiitor of the synthesis of milk ft. The supplementtion of diry ow diets with 14.5 mg/kg monensin hd no signifint effet on the perentge of milk ft, lthough monensin only used derese of 7.5%. However, omintion of 2% fish oil nd 14.5 mg/kg monensin used signifint deline in the perentge of milk ft (Cnt et l., 1997). Bell et l. (26) found no signifint differene in the perentge of milk ft nd yield when 24 ppm monensin ws fed to Holstein ows. However, omintion of 6% sfflower oil nd 24 ppm monensin used signifint deline in the oth the perentge of milk ft nd the milk yield. Our dt showed tht supplementtion with monensin lone used slight redution in the perentge of milk ft ompred with the ontrol group (8.74% vs. 9.55%). Bell et l. (26) suggested tht dietry supplementtion with monensin did not pper to hve independent effet
122 Mirzei, F. on the perentge of ft in milk. Our dt showed tht omintion of monensin with fish oil used signifint derese in the perentge of milk ft nd the milk yield, whih onurred with previous reserh (Cnt et l., 1997). The results of this ltter study suggested tht the redution in milk ft ould e result of the modultion of rumen fermenttion towrds inresed levels of propionte nd redued ette prodution in the rumen. However, in the present study, the ddition of monensin to sfflower diet did not hve signifint effet. Our results showed tht the omintion of fish oil with sfflower oil nd monensin (FSM) resulted in signifint deline in the perentge of milk ft nd yield. This my e relted to the ddition fish oil, whih my hve ltered the environment in the rumen suffiiently for diets tht ontined sfflower oil nd monensin to indue depressnt effet on milk ft. The perentge of milk protein redued signifintly in ewes tht were fed FS nd FSM diets. Severl studies showed tht feeding ft to ltting ewes (Kitess et l., 23 nd Zhng et l., 26) nd gots (Mir et l., 1999) hd either no effet or deresed the protein ontent of milk of ewes (Rotunno et l., 1998; Csls et l., 1999; Chioflo et l., 24). Cnt et l. (1993, 1997) reported tht lood flow to the udder glnd ws deresed fter feeding ruminnts with 4% dded dietry ft. They hypothesized tht this ws due to n inresed energeti effiieny of milk synthesis, whih mnifested s drop in the mmmry lood flow to milk volume rtio, resulting in deresed ontent of milk protein. Their proposl ws supported y the results of this experiment, in whih the level of dded dietry ft ws 4% (2% fish oil nd 2% sfflower oil). Additionlly, the milk yield ws not inresed in response to the lipids in feed ut the protein ontent ws deresed. The FO nd SO diets hd no signifint effet on the perentge of protein in milk, whih ws in greement with others (Mir et l., 1999; Donovn et l., 2; Zheng et l., 25; Bell et l., 26; Zhng et l., 26). However, our results were different from tht of Cnt et l. (1997), in whih the milk protein onentrtion of ows ws signifintly redued y dietry fish oil supplementtion. Also, Rotunno et l., (1998), nd Csls et l., (1999) found negtive effet of ft supplementtion on the perentge of the milk protein of ewes. Wu nd Huer (1994) onluded tht redution in milk protein perentge during ft supplementtion might e ttriuted to n inrese in the milk yield without n inrese in the numer of mino ids ville to the mmmry glnd for protein synthesis. The perentge of milk protein ws not ffeted y the MO diet. This onurs with previous reserh studies (Suer et l., 1989; Dhimn et l., 1999; Bell et l., 26). However, Cnt et l. (1997) oserved redution in the milk protein ontent when monensin lone or monensin with fish oil ws dded to the diet. The perentge of ltose, TS nd SNF in milk were not signifintly different etween the tretment groups, whih ws onsistent with previous findings (Cnt et l., 1997; Mir et l., 1999; Zhng et l., 26; Ber et l., 21; Bell et l., 26). In the present study, the onentrtion of ure N in milk ws inresed y monensin supplementtion. A higher milk ure N onentrtion hs een reported when diets were supplemented with monensin (Duffield et l., 1998). Aording to tht study, monensin supplementtion inresed the mount of protein tht rehed the smll intestine nd therefore inresed the use of Amino Aids (AA) for gluoneogenesis. This inresed the mount of demintion nd the onentrtion of lood ure N. Cnt et l. (1997) reported tht monensin inresed the espe of dietry undegrded protein from the rumen, nd, therefore, it ws expeted tht the levels of ruminl NH3-N nd susequent ure N of milk nd plsm would inrese. The numeril inrese of milk ure N onentrtion of ewes tht were fed diets supplemented with oils my hve een due redution in the mount of non-firous rohydrtes (NFC) in their diets tht redued the energy ville for miroil protein synthesis nd resulted in the susequent inrese in the onentrtion of ruminl
Effets of Fish Oil, Sfflower Oil... 123 NH3-N (Chouinrd et l., 1998). In the present study, SCC ws not ffeted y experimentl tretments. Similr results were reported y Ber et l. (21) fter the inlusion of 2% fish oil in the diet of diry ows. However, Aughzleh et l., (23) oserved signifint inrese in the SCC when the diet of diry ows ws supplemented with 1% fish oil plus 2% fts from high LAsunflower seeds. Protozo numers, ph nd NH3-N onentrtion Deresed numers of protozo in the ruminl fluid of sheep due to dietry supplementtion with fts nd oils hve een reported in severl studies (Ikwuegu nd Sutton., 1982; Sutton et l., 1983; Broudisou et l., 1994; Ivn et l., 21). Protozo hve limited ility to tke up, ssimilte nd trnsform dietry lipid, nd high dietry lipid onentrtion is toxi to protozo (Ivn et l., 21). Ikwuegu nd Sutton (1982), reported tht dietry linseed oil supplementtion in sheep resulted in redued fun nd no signifint effets on the ph of the rumen. However, Ivn et l. (21) found tht the quntity of fun ws redued fter supplementing feeds with sunflower seed oil, ut ph ws inresed signifintly nd ws more stle in sheep fed with the supplemented diet ompred to those on the ontrol diet. Supplementtion with monensin tended to inrese the ruminl ph in our study, lthough this ws not signifint. Other studies hve reported similr inrese in ruminl ph (Plizer et l., 2; Benhr et l., 26) when diry ows were fed with diet supplemented with monensin. The lower NH3-N onentrtion of ewes tht were fed with supplementl oils or monensin ws most proly due to the redution of the protozol popultion (Tle 3). Therefore, this resulted in the redution of miroil protein proteolysis nd the derese in miroil nitrogen reyling (Broudisou et l., 1994; Ivn et l., 21). VFAonentrtion The results otined regrding to totl VFA onentrtion (mmol/l) in this study ws onsistene with previous findings (Jenkins et l., 23; Wng et l., 25; Benhr et l., 26). However, the supplementtion of diets with oils or monensin resulted in numeril redution in the onentrtion of VFA ompred with ontrol diet. The ddition of ft prtilly reples the nonstruturl rohydrtes in the feed nd so redues the fermentle rohydrte ville for VFA prodution, whih results in derese in the totl VFA onentrtion in the rumen (Chihlowski et l., 25). In the present study, the higher redution in VFA onentrtion in the omintion of oil with monensin reltive to their use lone my e relted to lower DMI in ewes fed from diets tht ontin oils nd monensin together. Dietry supplementtion of oils nd monensin tended to redue the onentrtion of eti id, whih ould e relted to the negtive effets of unsturted ftty ids (Jenkins et l., 1993) nd monensin (Cnt et l., 1997) on ellulolyti teri nd redution in the digestility of fier. Ftty id esters in polyunsturted fts (suh s sfflower oil), prtiulrly olei (C18:1) nd linolei (C18:2), re hydrolyzed nd iohydrogented with very high effiieny (>9%) in the rumen. These tend to modify fermenttion ptterns strongly in fvor of propionte, ut ftty id esters from highly polyunsturted fts (suh s fish oil), re hydrolyzed less effiiently (<35%), prtiulrly due to the presene of 2-ron polyunsturted ftty ids. The ltter tend to pss through the rumen unmodified (Jenkins, 1993; Donovn et l., 2). Monensin normlly provokes derese in C2 nd n inrese in C3 (Cnt et l., 1997; Wng et l., 25). The results of the present study re lso ordne with their findings (Tle 3). The signifint inrese in the onentrtion of propionte (p<.5) when oils or monensin ws dded is lso omprle with the report of Chillird et l. (2). Hrfoot nd Hzlewood (1997) hypothesized tht the inrese in propionte ould e relted to the redution of methne prodution nd the onversion of glyerol of triylglyerols to
124 Mirzei, F. propionte. The propionte onentrtion ws higher in diets with sfflower oil ompred to diets without. This ould e relted to LA, whih is mjor onstituent of sfflower oil tht is known to enhne the prodution of propionte in the rumen prodution (Toms et l., 1997). Our results relted to utyrte were onsistent with previous findings (Ivn et l., 21; Jenkins et l., 23). The derese of isovleri ould e relted to the effets of unsturted ftty ids on the degrdtion of mino ids y rumen teri (Hrfoot et l., 1997). The effet of the omintion of monensin with oils is in greement with other findings (Jenkins et l., 23). The effets of oils or monensin on the A:P rtio ould e relted the ft tht unsturted oils typilly interfere with ruminl fermenttion, whih uses drop in A:P rtio, nd methne prodution. This is similr to the effets of the ionophores (Jenkins et l., 23). The effiy of monensin in modifying the A:P rtio hs lso een shown to vry with diet omposition, prtiulrly with referene to the dietry proportion of the onentrte (Benhr et l., 26). In the urrent study, the FSM diet ontined 45% onentrte ompred with 48% in the CON diet (Tle 1), whih my explin the signifint effets of monensin on thea:p rtio. Briefly, our dt showed tht the omintion of oil nd monensin in the diet of ewes uses greter redution in the totl VFA onentrtion, A:P rtio, isovleri id onentrtion, nd inrese in the onentrtion of propionte reltive to their use lone. The effet of sfflower oil ws gret thn tht of fish oil. Plsm metolites Supplementtion of feed with oils does not inrese lood gluose (Grummer nd Crrol., 1991), lthough there ws numeril inrese in the gluose onentrtion in experimentl diets in our study, whih ws likely to e due to the inrese of the onentrtion of propionte (Tle 3). Propionte is n importnt sustrte for gluose synthesis in the gluoneogenesis pthwy in ruminnts (MDonld et l., 1997). Other studies reported similr effet to tht seen in our study (Chillird et l., 1993; Kumr et l., 24), or redution (Chillird nd Ottou, 1995; Sims et l., 1995) of plsm gluose, when diry ows were fed with diets tht were supplemented with ft. Aording to Duffield et l. (1998), monensin supplementtion inresed the mount of protein tht rehes the smll intestine nd the use of AA for gluoneogenesis. The ddition of ft tended to inrese plsm onentrtions of totl holesterol nd triglyerides (p<.5). These results re in greement with others (Thoms et l., 1997) who reported higher holesterol nd triglyeride onentrtions fter feeding ft to diry ows. The inrese of the plsm onentrtion of triglyerides n e relted to the higher digestiility of unsturted fts thn sturted fts (Nik-Khh et l., 23). This finding ould lso e relted to inrese to the synthesis of holesterol nd triglyerides in the epithelium of the smll intestine nd liver ells, nd the inrese of the sorption of these fts from the smll intestine fter dietry supplementtion of ft (Demeyer et l., 1999; Chihlowski et l., 25). Rumen teri nd protozo hydrolyze omplex lipids into their onstituent long-hin ftty ids, sugrs, orgni ses, nd glyerol. Thus, the rumen is the primry site of omplex lipid hydrolysis rther thn the smll intestine. The higher serum levels of triglyerides indited tht the epithelium of the smll intestine is ple of soring the dietry ftty ids postruminlly nd inorporting them into triglyeride lipoproteins (Chihlowski et l., 25). Our results re in greement with others (Grummer et l., 1991; Thoms et l., 1997) who reported tht oils omposed of predominntly polyunsturted ftty ids hve lso een shown to inrese the serum onentrtions of lipoprotein holesterol. Briefly, our dt showed the ddition of oil tended to inrese plsm onentrtions of totl holesterol nd triglyerides, ut no signifint differene etween the effet of fish oil nd sfflower
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