Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic

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1 NEURAL REGENERATION RESEARCH Volume 7, Issue 22, August Cite this rticle s: Neurl Regen Res. 212;7(22): Neuroprotective effects of ovine colostrum on intrcererl hemorrhge-induced poptotic neuronl cell deth in rts Sung Eun Kim 1, Il Gyu Ko 1, Ml Soon Shin 1, Chng Ju Kim 1, Young Gwn Ko 2, Hnjin Cho 3 1 Deprtment of Physiology, College of Medicine, Kyung Hee University, Seoul 13-71, Repulic of Kore 2 Deprtment of Emergency Medicine, College of Medicine, Kyung Hee University, Seoul 13-71, Repulic of Kore 3 Deprtment of Emergency Medicine, Kore University Ansn Hospitl, Ansn , Repulic of Kore Astrct Brin cell deth fter intrcererl hemorrhge my e medited in prt y n poptotic mechnism. Colostrum is the first milk produced y mmmls for their young. It plys n importnt role in protection nd development y providing vrious ntiodies, growth fctors nd nutrients, nd hs een used for vrious diseses in mny countries. In the present study, we investigted the nti-poptotic effects of ovine colostrum using orgnotypic hippocmpl slice cultures nd n intrcererl hemorrhge niml model. We performed densitometric mesurements of propidium iodide uptke, step-down voidnce tsk, Nissl stining, nd cspse-3 immunohistochemistry. The present results reveled tht colostrum tretment significntly suppressed N-methyl-D-sprtic cid-induced neuronl cell deth in the rt hippocmpus. Moreover, colostrum tretment improved short-term memory y suppressing hemorrhge-induced poptotic neuronl cell deth nd decresing the volume of the lesion induced y intrcererl hemorrhge in the rt hippocmpus. These results suggest tht colostrum my hve eneficil role in recovering rin function following hemorrhgic stroke y suppressing poptotic cell deth. Key Words intrcererl hemorrhge; orgnotypic hippocmpl slice culture; ovine colostrum; poptotic cell deth; N-methyl-D-sprtic cid; cspse-3; hippocmpus; memory Reserch Highlights (1) In vitro experiment results confirmed tht ovine colostrum cn inhiit N-methyl-D-sprtic cid-induced neuronl cell deth in the rt hippocmpus. (2) In vivo experimentl results confirmed tht ovine colostrum cn inhiit intrcererl hemorrhge-induced neuronl cell deth, decrese cspse-3 expression, nd reduce the size of intrcererl hemorrhge-induced lesions in the hippocmpus, nd improve the cognitive function of rts with intrcererl hemorrhge. Arevition NMDA, N-methyl-D-sprtic cid Sung Eun Kim, Ph.D., Deprtment of Physiology, College of Medicine, Kyung Hee University, #1 Hoigi-dong, Dongdemoon-gu, Seoul 13-71, Repulic of Kore Corresponding uthor: Hnjin Cho, M.D., Ph.D., Assistnt professor, Deprtment of Emergency Medicine, Kore University Ansn Hospitl, #516 Gojn-dong, Dnwon-gu, Ansn , Kore chohj327@hotmil.com Received: Accepted: (NY /H) Kim SE, Ko IG, Shin MS, Kim CJ, Ko YG, Cho HJ. Neuroprotective effects of ovine colostrum on intrcererl hemorrhge-induced poptotic neuronl cell deth in rts. Neurl Regen Res. 212;7(22): doi:1.3969/j.issn INTRODUCTION Brin injury fter intrcererl hemorrhge occurs through multiple mechnisms including direct tissue destruction, the spce-occupying effect of hemtoms, ischemic dmge to djcent tissue, clot-derived toxic fctors, nd edem [1-2]. Brin cell deth fter intrcererl hemorrhge my e medited in prt y n poptotic mechnism [3]. Apoptosis, the process of progrmmed cell deth, plys n importnt role in norml development nd tissue homeostsis through functions in cell replcement, tissue remodeling, nd the 1715

2 removl of dmged cells [4-5]. However, inpproprite or excessive poptosis is implicted in severl types of neurodegenertive disorders, including stroke [3-6]. Inflmmtion contriutes to secondry rin injury induced y intrcererl hemorrhge. Inflmmtion is chrcterized y the ccumultion nd ctivtion of inflmmtory cells nd meditors within the hemorrhgic rin [7]. Therefore, it hs een suggested tht ctivtion nd regultion of inflmmtory responses in the hemorrhgic rin could e therpeutic trget for intrcererl hemorrhge [8]. Colostrum is the first milk produced y femle mmmls during the first few dys postprtum. It provides vrious ntiodies, growth fctors, nd nutrients such s proteins, crohydrtes, fts, vitmins nd minerls for the neonte. Moreover, colostrum contins mny iologiclly ctive constituents tht ply importnt roles in protection nd development [9]. Bovine colostrum hs een used for the tretment of vrious gstrointestinl disorders, intestinl inflmmtion, respirtory infections, rheumtoid rthritis, nd the heling of injured tissues [1-12]. Recently, Schuster et l [13] reported tht colostrinin, clss of proline-rich polypeptides derived from colostrum, hd protective effect on neurolstom cells y reducing firil formtion nd cell deth induced y et-myloid. As mentioned ove, mny studies hve reported on the eneficil effects of colostrum. However, these reports hve minly focused on its protective effects ginst vrious infectious microorgnisms, nd few studies hve investigted the nti-poptotic effect of colostrum. Therefore, in the present study, we investigted the nti-poptotic effects of ovine colostrum in orgnotypic hippocmpl slice cultures nd in n intrcererl hemorrhge niml model. neuronl cell deth in the hippocmpus 24 hours fter NMDA ppliction. When the vlue of the NMDA-treted group ws set s 1% dmge, the vlue of slices incuted with ovine colostrum t concentrtion of.1,.5, nd 1. mg/ml ws ± 9.47%, ± 3.58%, nd ± 5.55%, respectively. Pre-tretment with.5 nd 1. mg/ml ovine colostrum significntly reduced the uptke of propidium iodide (Figure 1). B D % PI uptke A C E RESULTS Quntittive nlysis of experimentl nimls In in vivo experiments, thirty rts were initilly included nd rndomly divided into three groups, with 1 rts in ech group: shm-opertion group, hemorrhge-induced group (induction of intrcererl hemorrhge plus intrgstric dministrtion of distilled wter), nd colostrum-treted group (induction of intrcererl hemorrhge plus intrgstric dministrtion of colostrum). During this study, there ws no spontneous deth of rts, nd 3 rts were included in the finl nlysis. Effect of colostrum on N-methyl-D-sprtic cid (NMDA)-induced neuronl cell deth in orgnotypic hippocmpl slice cultures As shown in Figure 1, 1 μm NMDA tretment cused A B C D E Figure 1 Effect of colostrum on N-methyl-D-sprtic cid (NMDA)-induced neuronl cell deth in orgnotypic hippocmpl slice cultures. (A) Control group; (B) 1 μm NMDA-treted group; (C) 1 μm NMDA plus.1 mg/ml colostrum-treted group; (D) 1 μm NMDA plus.5 mg/ml colostrum-treted group; (E) 1 μm NMDA plus 1. mg/ml colostrum-treted group. Upper: Fluorescence photomicrogrphs of propidium iodide (PI) uptke, showing the effects of ovine colostrum t different concentrtions. The scle r represents 5 μm. The red is Cy3 leling. Lower: Reltive percentge of propidium iodide uptke. Vlues re represent men ± SEM (n = five tches in ech group). P <.5, vs. the control group. P <.5, vs. NMDA-treted group (one-wy nlysis of vrince followed y Duncn s post hoc test). 1716

3 Body weight chnges The ody weights of rts on the 22 nd dy of the experiment were 232 ± 4.14, 225 ± 5.32, nd 238 ± 3.22 g in the shm-opertion group, hemorrhge- induced group, nd colostrum-treted group, respectively. There ws no significnt difference in ody weight mong the groups. Effect of colostrum on short-term memory of cererl hemorrhge rts The short-term memory of rts ws impired y induction of intrcererl hemorrhge (P <.5). However, tretment with colostrum significntly llevited hemorrhge-induced short-term memory impirment (P <.5; Figure 2). Ltency (second) A B C Figure 2 Effect of colostrum on ltency in the step-down voidnce tsk fter intrcererl hemorrhge induction in rts. (A) Shm-opertion group; (B) hemorrhge-induced group; (C) colostrum-treted group. Vlues represent the men ± SEM of 1 rts in ech group. P <.5, vs. shm-opertion group. P <.5, vs. hemorrhge-induced group (one-wy nlysis of vrince followed y Duncn s post hoc test). Effect of colostrum on the size of the intrcererl hemorrhge-induced lesion Photomicrogrphs of the lesioned re in the hippocmpus re presented in Figure 3. The verge neuronl lesion size in the hemorrhge-induced group ws ± 7.42% of the norml hippocmpus re. No noticele lesions were oserved in the shm- opertion group. However, the size of intrcererl hemorrhge-induced lesion ws significntly reduced to 4.1 ± 2.45% following tretment with colostrums (Figure 3). These results showed tht lesion size ws incresed following intrcererl hemorrhge (P <.5), nd tretment with colostrum significntly decresed hemorrhge-induced lesion size in the hippocmpus (P <.5). Lesion volume (%) Figure 3 Effect of colostrum on the size of the intrcererl hemorrhge-induced lesion. (A) Shm-opertion group; (B) hemorrhge-induced group; (C) colostrum-treted group. Upper: Photomicrogrphs showing Nissl stining in the hippocmpus. The scle r represents 8 μm. Lower: Men size of the lesion re compred to the norml re in ech group. Vlues represent men ± SEM (n = 2 slices in ech group). P <.5, vs. shm-opertion group; P <.5, vs. hemorrhge-induced group (one-wy nlysis of vrince followed y Duncn s post hoc test). Effect of colostrum on cspse-3 expression in the hippocmpus Photomicrogrphs of cspse-3-positive cells in the hippocmpus re presented in Figure 4. The numer of cspse-3-positive cells ws 3.35 ± 1.51/mm 2 in the shm-opertion group, ± 9.31/mm 2 in the hemorrhge-induced group, nd ± 16/mm 2 in the colostrum-treted group (Figure 4). These results showed tht intrcererl hemorrhge incresed cspse-3 expression in the hippocmpus (P <.5) nd tretment with colostrum significntly suppressed hemorrhge-induced cspse-3 expression (P <.5). DISCUSSION The excessive ctivtion of postsynptic NMDA receptors results in neurotoxicity [14]. In prticulr, ctivtion of NMDA receptors cuses the cellulr influx of clcium ions nd then intrcellulr ccumultion of these ions, which leds to edem nd cell deth [15-16]. Along with the rekdown of the lood-rin rrier, edem nd neuronl cell deth re the min pthophysiologic chnges induced y hemtoms fter intrcererl hemorrhge [17-18]. A B C 1717

4 Numer of cspse-3- positive cells (/mm 2 ) Figure 4 Effect of colostrum on cspse-3 expression in the hippocmpus of intrcererl hemorrhge rts. Upper: Photomicrogrphs showing immunostining for cspse-3. The numers of cspse-3-positive cells in fields 1, 2, 3, nd 4 were counted. Cspse-3-positive cells ppered rown in color. The scle r represents 25 μm in (A) (C) nd 1 μm in () (c). (A) nd () Shm-opertion group; (B) nd () hemorrhge-induced group; (C) nd (c) colostrum-treted group. Lower: The numer of cspse-3-positive cells in ech group. Vlues represent men ± SEM (n = 2 slices in ech group). P <.5, vs. shm-opertion group. P <.5, vs. hemorrhge-induced group (one-wy nlysis of vrince followed y Duncn s post hoc test). In ddition, Ardizzone et l [19] suggested tht intrcererl hemorrhge medites injury through ctivtion of the protein phosphoryltion of NMDA receptors. In the present study, ovine colostrum tretment prevented NMDA-induced cell deth in hippocmpl slices dose-dependently. Cognitive impirments evolve fter stroke, with motor deficits. In prticulr, it ws reported tht hemorrhgic strokes hd 6 times greter frequency of cognitive impirment thn ischemic stroke [2]. Recently, neuropsychologicl study reported tht the stritum plys key roles in some forms of lerning nd memory [21]. Lekic et l [22] reported tht collgense-induced intrcererl hemorrhge in the sl gngli cuses significnt memory deficits. Jeon et l [23] reported tht surchnoid hemorrhge significntly incresed the numer of poptotic neurons in the hippocmpus, the cererl cortex nd the cereellum, nd tht cognitive nd memory functions were impired. However, they suggested tht poptosis in the hippocmpus ws not sufficient to cuse neuroehviorl deficits, nd other fctors in the hippocmpl cell deth A B C pthwy my contriute to the impirment. The hippocmpus, including the dentte gyrus, plys pivotl role in lerning nd memory [24]. In the present study, we induced intrcererl hemorrhge y directly injecting collgense into the hippocmpl CA1 region. Consequently, intrcererl hemorrhge induction in the hippocmpus shortened the ltency in the step-down voidnce tsk, mesure of short-term memory. On the other hnd, tretment with ovine colostrum significntly incresed the ltency. This result indictes tht ovine colostrum improved short-term memory following hippocmpl hemorrhge in rts. The eneficil effects of colostrum on cognitive function hve een investigted previously. Popik et l [25] reported tht colostrinin, one of the polypeptides derived from colostrum, fcilitted the cquisition nd retrievl of sptil memory nd long-term memory in ged rts, nd Bilikiewicz nd Gus [26] reported tht colostrinin retrded the progression of Alzheimer s disese. Apoptosis ppers to ply key role in neuronl cell deth following stroke [7, 1]. Intrcererl hemorrhge injury induces the ctivtion of cspse-3, which plys n importnt role in poptotic cell deth [6, 27]. An increse in cspse-3 expression precedes DNA frgmenttion, reches its pek level t 24 hours fter intrcererl hemorrhge induction, nd then declines [28]. Our results showed tht intrcererl injection of collgense incresed the lesion volume nd the expression of cspse-3 in the hippocmpl CA1 region, indicting tht collgense-induced intrcererl hemorrhge triggers poptotic neuronl cell deth in the hippocmpus. On the other hnd, tretment with ovine colostrum significntly reduced the lesion volume nd suppressed the expression of cspse-3. Recently, Dourghi-Zdeh et l [29] suggested tht colostrinin my ply role in preventing Alzheimer s disese through the inhiition of Fs-medited poptosis. In conclusion, we demonstrted tht tretment with ovine colostrum prevents poptosis induced y NMDA, nd improves short-term memory impired y the induction of intrcererl hemorrhge y significntly reducing the lesion volume nd suppressing the expression of cspse-3 in the hippocmpl CA1 region. Although we investigted the nti-poptotic effect of ovine colostrum in the present study, we could not give detiled explntion of the mechnism y which ovine colostrum induces its nti-poptotic effect. Moreover, ovine colostrum is suggested to hve nti-inflmmtory effects [3]. In fct, inflmmtion contriutes to secondry rin injury induced y intrcererl hemorrhge [11], nd colostrum hs een shown to lock interleukin-1β- induced proinflmmtory gene expression nd Cox-2 protein expression in intestinl epithelil cells through IκB-α degrdtion nd inhiition of NF-κB signling. However, the pre- 1718

5 cise inflmmtory suppression mechnism ovine colostrum intercts with remins unknown [3]. Therefore, further studies re required to clrify the moleculr nd iologicl mechnisms y which colostrum exhiits its nti-poptotic nd nti-inflmmtory effects. However, it is importnt to note tht this study showed the therpeutic potentil of ovine colostrum in the recovery of rin function following hemorrhgic stroke nd its nti-poptotic effect. MATERIALS AND METHODS Design A rndomized, controlled niml study. Time nd setting This study ws performed in the Physiology Lortory of the College of Medicine, Kyung Hee University, Repulic of Kore, etween June nd Decemer 21. Mterils The experimentl procedures were performed in ccordnce with the niml cre guidelines of the Ntionl Institutes of Helth nd the Koren Acdemy of Medicl Sciences. Mle Sprgue-Dwley rts weighing 2 ± 1 g, ged 7 weeks, were used in the experiments. The rts were housed t 2 ± 2 C with light cycle t 7:-19:, nd hd free ccess to food nd wter efore nd fter surgery, Methods Intrcererl hemorrhge induction To induce intrcererl hemorrhge, the rts were intrperitonelly nesthetized with Zoletil 5 (1 mg/kg; Vic Lortories, Crros, Frnce) nd plced in stereotxic frme s previously descried [6]. Through hole drilled in the skull, 26-guge needle ws implnted into the hippocmpl CA1 region t the following coordintes: 2.4 mm lterl to the midline nd 4.2 mm posterior to the coronl suture t depth of 2.4 mm from the surfce of the rin. In totl, 1 μl of sline contining.2 U collgense (type 4; Sigm Chemicl Co., St. Louis, MO, USA) ws then infused over 1-minute period. The needle remined in plce for n dditionl 3 minutes following the infusion, nd fterwrds it ws slowly withdrwn. The nimls in the shm-opertion group received 1 μl of physiologicl sline y the sme method. Colostrum tretment Rts in the colostrum-treted group received ovine colostrum (.4 g/kg) vi n orogstric tue once dy for 21 consecutive dys eginning 1 dy fter surgery. The nimls in the shm-opertion nd hemorrhge-induced groups received equl mounts of distilled wter. Step-down voidnce tsk Short-term memory ws evluted y ssessing the ltency of the step-down voidnce tsk s previously descried [3]. For trining, the rts were plced on 7 cm 25 cm pltform tht ws 2.5 cm in height nd were llowed to rest on the pltform for 2 minutes. The pltform fced 42 cm 25 cm grid with prllel.1-cm clier stinless steel rs spced 1 cm prt. During the trining session, the nimls received.3 ma scrmled foot shock for 2 seconds immeditely fter stepping down. The retention time ws determined 2 dys fter the trining session on the 2 th dy fter strting the experiment. The intervl etween the time when the rts first stepped down nd the time when they plced ll four pws on the grid ws defined s the ltency of the step-down voidnce tsk. Ltencies over 3 seconds were counted s 3 seconds. Brin tissue preprtion Brin tissue preprtion ws mde s previously descried [31]. The rts were scrificed on the 22 nd dy of the experiment immeditely fter determintion of the ltency. The nimls were weighed nd then given n overdose of Zoletil 5 (1 mg/kg, i.p.; Vic Lortories). After complete lck of response ws oserved, the rts were trnscrdilly perfused with 5 mm phosphte uffered sline nd fixed with freshly prepred solution consisting of 4% (w/v) prformldehyde in 1 mm phosphte uffer (ph 7.4). The rins were dissected nd post-fixed in the sme fixtive overnight nd then trnsferred to 3% (w/v) sucrose solution for cryoprotection. Seril coronl sections (4 μm thick) were mde using freezing microtome (Leic, Nussloch, Germny). On verge, four hippocmpl tissue sections were collected from ech rt for Nissl stining nd immunohistochemistry. Determintion of lesion size y Nissl stining To determine lesion size, Nissl stining ws performed s previously descried [6]. A digitl imge of the Nissl stined cells ws otined from the field of view under light microscope (Olympus, Tokyo, Jpn). The lesion re of the collgense injection site ws determined using n Imge-Pro Plus imge nlyzer (Medi Cyernetics Inc., Silver Spring, MD, USA). The lesion size in the hippocmpl CA1 region (%) ws clculted s follows: the hemorrhge-induced lesion size (collgense injection side)/intct CA1 region size (contrlterl side) 1%. Cspse-3 immunohistochemistry For visuliztion of cspse-3 expression in the hippo- 1719

6 cmpl CA1 region, cspse-3 immunohistochemistry ws performed. Eight sections on verge were selected from ech rin region spnning from Bregm -3.8 mm to -4.5 mm. Free-floting tissue sections were incuted overnight with mouse nti-cspse-3 ntiody (1:1, sc-7272; Snt Cruz Biotechnology, Snt Cruz, CA, USA) in order to visulize cspse-3 expression. The sections were then incuted for 1 hour with iotinylted nti-mouse secondry ntiody (Vector Lortories, Burlingme, CA, USA). The sections were susequently incuted with vidin-iotin-peroxidse complex (Vector Lortories) for 1 hour t room temperture. Immunorectivity ws visulized y incuting the sections in solution consisting of.5% (w/v) 3,3-diminoenzidine nd.1% (v/v) H 2 O 2 in 5 mm Tris uffer (ph 7.6) for 3 minutes. The numer of cspse-3-positive cells ws quntittively ssessed in four fields (25 μm 25 μm in ech field) within the hippocmpl CA1 region djcent to the hemtom ccording to previously descried method [6]. The dt nlysis for cspse-3-positive cells ws performed in linded fshion. Orgnotypic hippocmpl slice cultures nd quntifiction of cell dmge Orgnotypic hippocmpl slice cultures were prepred from the hippocmpi of 7-8 dy-old Sprgue-Dwley rts (n = 3) using the method of Stoppini et l [32]. Briefly, trnsverse slices t thickness 35 μm were cut from the hippocmpi using McIlwin tissue chopper (Mickle Lortory Engineering Ltd., Surrey, UK). Five slices were plced on Millicell culture inserts (Millipore, Billeric, MA, USA). The slices were cultured for 1 dys in vitro with culture medium consisting of 5% (v/v) minimum essentil medium, 25% (v/v) Hnk s lnced slt solution, nd 25% (v/v) het-inctivted horse serum (Gico BRL, Grnd Islnd, NY, USA) supplemented with 6.5 g/l glucose. The slices were incuted t 37 C in 5% CO 2, 95% O 2 humidified incutor, nd culture medi were chnged twice week. The slices were incuted for 1 dys, nd then ovine colostrum (Alph Lortories, Aucklnd, New Zelnd) t concentrtions of.1,.5, nd 1. mg/ml were dded to independent wells 1 hour efore tretment with 1 μm NMDA. Therefter, the slices were incuted for 24 hours in humidified incutor. Control slices were treted with only 1 μm NMDA, which resulted in cell deth. Cell dmge in orgnotypic hippocmpl slice cultures ws evluted with densitometric mesurements of cellulr uptke of propidium iodide (Sigm Chemicl Co., St. Louis, MO, USA), used s mrker of ded or dying cells [33]. Propidium iodide (5 μg/ml) ws dded to the culture medium 24 hours fter NMDA tretment nd then incuted for 2 hours. All imges were cptured with digitl cmer under fluorescence microscope (AMG, Bothell, WA, USA), nd quntified using Imge-Pro R Plus imge nlyzer (Medi Cyernetics Inc., Silver Spring, MD, USA). The vlue otined from the control slices ws set s 1% dmge nd ws then compred with vlues otined from the slices treted with ovine colostrum. Sttisticl nlysis Sttisticl nlysis ws performed using IBM SPSS (version 2.; IBM Corp., Armonk, NY, USA). For the comprison etween groups, one-wy nlysis of vrince nd Duncn's post-hoc test were performed. The results were expressed s men ± SEM. P vlues <.5 were considered sttisticlly significnt. Author contriutions: Hnjin Cho nd Young Gwn Ko designed this study. Sung Eun Kim nd Il Gyu Ko performed the experiments. Sung Eun Kim wrote the mnuscript. Ml Soon Shin nlyzed the dt. Chng Ju Kim supervised lortory procedures. Conflicts of interest: None declred. Ethicl pprovl: This study ws pproved y the Kyung Hee University Institutionl Animl Cre nd Use Committee in Repulic of Kore. REFERENCES [1] Lee KR, Colon GP, Betz AL, et l. Edem from intrcererl hemorrhge: the role of thromin. J Neurosurg. 1996;84(1): [2] Lee HH, Kim H, Lee MH, et l. Tredmill exercise decreses intrstritl hemorrhge-induced neuronl cell deth vi suppression on cspse-3 expression in rts. Neurosci Lett. 23;352(1): [3] Mtsushit K, Meng W, Wng X, et l. Evidence for poptosis fter intercererl hemorrhge in rt stritum. J Cere Blood Flow Met. 2;2(2): [4] Thompson CB. Apoptosis in the pthogenesis nd tretment of disese. Science. 1995;267(523): [5] Woodle ES, Kulkrni S. Progrmmed cell deth. Trnsplnttion. 1998;66(6): [6] Johnson EM Jr, Greenlund LJ, Akins PT, et l. Neuronl poptosis: current understnding of moleculr mechnisms nd potentil role in ischemic rin injury. J Neurotrum. 1995;12(5): [7] Wng J. Preclinicl nd clinicl reserch on inflmmtion fter intrcererl hemorrhge. Prog Neuroiol. 21; 92(4): [8] Teng W, Wng L, Xue W, et l. Activtion of TLR4-medited NFkppB signling in hemorrhgic rin in rts. Meditors Inflmm. 29;29: [9] Dvis PF, Greenhill NS, Rown AM, et l. The sfety of New Zelnd ovine colostrum: nutritionl nd 172

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