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Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function, composition and morphology

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Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function, composition and morphology
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  J. Dairy Sci. 85:1093–1102 ©  American Dairy Science Association, 2002. Bovine Somatotropin Administration to Dairy Goats in Late Lactation:Effects on Mammary Gland Function, Composition and Morphology 1 A. Baldi,* S. Modina,† F. Cheli,* F. Gandolfi,† L. Pinotti,* L. Baraldi Scesi,† F. Fantuz‡ and V. Dell’Orto* *Department of Veterinary Sciences and Technology for Food Safety, and†Institute of Domestic Animals, Faculty of Veterinary Medicine,Via Trentacoste 2, 20134 Milan, Italy‡Department of Veterinary Sciences, Faculty of Veterinary Medicine,University of Camerino,Via Circonvallazione 93/95, 62024 Matelica (MC), Italy ABSTRACT We investigated the effects of bovine somatotropin(bST) on mammary gland function and composition inthe declining phase of lactation in goats. SixteenSaanengoats,180 ± 11daysinmilk(DIM),weredividedequally into control and treated groups. The treatedgroup received 120 mg/2 wk of slow-release bST forthree cycles.Milk yield, milk composition,milk clotting measures, and plasmin-plasminogen activator activi-ties were recorded weekly. Milk Na and K were deter-mined in individual milk samples collected weekly dur-ingthethirdcycle.Bloodsampleswerecollectedweeklyduring the second cycle and the plasma analyzed fornonesterified fatty acids (NEFA), glucose, and urea. Atthe end of the 6 wk, three goats from each group wereslaughtered, and the udders were removed. Mammaryglandweight, composition,andtotal DNAcontentweredetermined. The histological effects of bST on mam-mary tissue were investigated. The analyzed parame-ters included numbers of alveoli, corpora amylacea,apoptoticcells,andlamininfibronectindistributionandlocalization. An extensive morphological analysis onthe epithelial and stromal components was performed.Milkyieldwassignificantlyhigherinthetreatedgroup,fatcontentwasnotaffected,butproteinandnonproteinnitrogen were lower in treated goats milk. Treatmentwith bST did not influence milk pH but reduced coagu-lation time. Plasmin and plasminogen activator activi-ties were not affected. Milk K levels were higher andthe Na/K ratio was lower in treated animals. Plasmaglucose, NEFA, and urea were unaffected. Mammarygland weight and total DNA were higher in treatedthan control animals, suggesting that with advancing  Received May 29, 2001. Accepted November 27, 2001.Correspondingauthor:ProfessorA.Baldi;e-mail:antonella.baldi@ unimi.it. 1 Studysupportedby60%granttoProf.A.BaldifromItalianMinis-try of University. 1093 lactation bST treatment maintains cells. Fat, protein,and collagen content of the mammary tissue did notdiffer between the groups. Treatment with bST signifi-cantly increased the number of lactating alveoli (LA)and significantly reduced the number of regressing al- veoli (RA) and corpora amylacea, both within and out-side the alveolar lumen. Laminin and fibronectin local-ization were not affected, and very few apoptotic cellswere found in both treated and control samples. OurfindingssuggestthatbSTadministrationtodairygoatsin late lactation can modulate mammary gland activityand improve lactation persistency; this is associatedwith maintained total mammary parenchyma weightand lactating alveoli.( Keywords: dairygoat,mammarygland,bovinesoma-totropin)  Abbreviationkey:a 30 =curdfirmness, aCA  = alveolarcorpora amylacea, CA  =  corpora amylacea, iCA  =  inter-stitial corpora amylacea,  K  20  = firming rate of curd,  LA  =  lactating alveoli,  PA   =  plasminogen activator,  PL  = plasmin,  r  =  coagulation time,  RA   =  regressing alveoli, TA  =  total alveoli, tCA  =  total corpora amylacea, TdT = terminal deoxynucleotidyl transferase,  TUNEL =  TdT-mediated dUTP nick-end labeling technique. INTRODUCTION The progress of lactation is characterized by majorchanges in the population of mammary gland cells(Knight, 1997; Capuco and Akers, 1999). In goats, afterlactationpeak,themammaryglandgraduallyinvolutesso that at drying off, after 36 to 40 wk of lactation,secretory cell numbers have decreased by 40 to 50%(Wilde et al., 1999). The secretory epithelium regressesto the nonproductive state (Quarrie et al., 1996; Li etal., 1999; Wilde et al., 1999), accompanied by gradualtissue remodeling (Turner and Huynh, 1991). Involu-tionischaracterizedbydecreasingalveolarlumenarea,increasing interalveolar connective tissue, regressing alveoli ( RA  ), and increasing corpora amylacea ( CA  )  BALDI ET AL. 1094 (Sordillo and Nickerson, 1988; Anderson and Wahab,1990; Gonzalez-Romanoet al., 2000),although alveolarstructures are maintained (Li et al., 1999). Reductionin cell number and the associated decline in milk pro-ductionandtissueDNAafterthelactationpeakintheseanimals has been correlated with cell death byapoptosis (Wilde et al., 1997).It is established that administration of growth hor-mone enhances milk production in dairy ruminants,including goats (Bauman, 1999). Administration of aslow-release formulation of bST to dairy ruminants im-proves lactation persistency by slowing down the post-peak rate of decline (Gallo et al., 1997; Baldi, 1999;Chiofalo et al., 1999). Administration of growth hor-mone in midlactation seems to cause an increase inthe quantity of mammary parenchyma (Sejrsen et al.,1999). A number of studies (Politis et al., 1990; Baldiet al., 1997; Baldi, 1999; Tonner et al., 2000) indicatethat, via an effect on IGF-I, bST can delay involutionof the mammary gland by reducing the activity of theplasmin-plasminogen system, an important initiator of tissue remodeling during late lactation in dairy rumi-nants (Baldi et al., 1996a). Effect of bST on plasmin-plasminogen system is one of the possible mechanismsthatcandelayregressionofthemammarytissueduring the transition from late lactation to dry period. Theseremodeling phenomena are represented mainly by pro-teolysis,forwhichlamininandfibronectinarepotentialtargets (Turner and Huynh, 1991).Milk composition and quality change with stage of lactation, with quality deteriorating markedly in theadvanced stages. Therefore, bST administration maybe of considerable benefit in modulating the rate of mammary gland involution in small dairy ruminants,particularly goats, for which stage of lactation is syn-chronized as a result of the seasonal breeding cycle(Chiofalo et al., 1999).To better understand the mechanisms by which bSTacts on tissue remodeling during the declining phaseof lactation, we studied the effects of bST administra-tion on milk production, milk quality, and mammarygland composition and morphology in dairy goats inlate lactation. MATERIALS AND METHODSAnimals and Diet Sixteen nonpregnant Saanen goats in late lactationfrom the Gian Paolo Guidobono Cavalchini experimen-tal farm of the University of Milan were used. On May20, 1999 (pre-experimental period) the goats were di- videdintocontrol(treatedwithsaline)andbST-treatedgroups homogeneous for milk production (2.10  ±  0.27kg/d)anddays oflactation(180 ± 11 DIM).Theanimals Journal of Dairy Science Vol. 85, No. 5, 2002 were fed a TMR, given once daily, containing alfalfahay (45%), beet pulp (20%), wheat straw (8%), and con-centrate(27%),whichprovided1.5McalofNE L  /kg(DM)and 17% of CP (DM). TMR was balanced to provide (ona DM basis) 1.3 Mcal of NE L  /kg and 12.2% of CP. TMRsamples were analyzed according to AOAC procedures(1980).Feedintakeforeachgroupwasrecordedweekly.Theanimalshadfreeaccesstowater.Theyweremilkedautomatically twice a day with a rotating milking ma-chine. The animals used in this experiment were caredforinaccordancewiththeguidelinesestablishedbytheEuropean Union and approved by the Italian Ministryof Health. Treatment Starting June 2, goats assigned to the treatmentgroupreceived120mgofslow-release-formulationbST(Monsanto Agricultural Co., St. Louis, MO) every 2 wkfor 3 cycles (42 d). Injections were subcutaneous in thetailregion.Doseswerecalculatedasinpreviousstudies(Chiofalo et al., 1999), considering also the late stageof lactation.On d 42 three goats of the bST group received anadditional injection of bST. Seven days later, corres-ponding to the expected maximum response to bST,the three treated animals and three control goats weremilked out and slaughtered; the udders were excisedand processed for compositional and histological analy-sis. The six slaughtered goats had very similar BW,lactation stages, and milk production at the beginning of the experiment. They were also similar in terms of udder morphology, udder volume, and teat length. Milk Data and Sampling Individual milk yields from the two daily milkings(7:30 a.m. and 5:30 p.m.) were recorded. Milk samplesfor analysis were taken from the milkings of each goatonce a week. In these samples, fat, protein, and lactoseweredeterminedbyinfraredanalyzerwithaMilkoscan(Foss Electric, Hillerød, Denmark); SCC was deter-mined by a Fossomatic (Foss Electric); milk casein andNPN were determined by the FIL-IDF method (IDF,1964); milk pH was determined within 2 h of milking by a pH meter. Na and K were determined by atomicabsorption spectrophotometry (Perkin-Elmer Corp.,Norwalk, CT) in milk samples collected on d 28, 35,and 42 (during the third cycle of treatment). Milk Clotting Measures The Formagraph method (Foss Electric) was used todetermine milk-clotting measures (by the movement  bST EFFECTS ON GOAT MAMMARY GLAND FUNCTION  1095 Figure 1 . Representative examples of alveolar structures in goat mammary glands. A,B, Lactating alveoli showing well-differentiatedepithelial cells with abundant apical secretory vesicles (arrow, A), rounded basally displaced nuclei (arrow, B) and a typical abundantcytoplasmic-to-nuclear ratio. C,D, Regressing alveoli showing irregularly shaped epithelial cells (arrow, C), large lipid droplets (arrow, D),and reduced cytoplasmic-to-nuclear ratio compared to epithelial cells of lactating alveoli (srcinal magnification 250  × ). of small pendulums immersed in linearly oscillating samples of coagulating milk). Coagulation time ( r ),firming rate of curd ( K  20 ), and curd firmness ( a 30 ), weredetermined at 35 ° C using the method of Ambrosoli etal. (1988). Determination of Plasmin andPlasminogen Activator Activity in Milk Plasmin ( PL ) and plasminogen activator ( PA  ) activi-ties were determined in individual milk samples by anenzymatic colorimetric method (Baldi et al., 1996b).Briefly, for PA analysis, milk samples (5 ml) were cen-trifuged at 2000  ×  g  for 15 min at 4 ° C to produce cream,skim milk, and somatic cell pellet. The cream fractionwas discarded, and the skim milk was centrifuged at100,000  ×  g  for 1 h at 4 ° C to obtain milk serum andcaseinpellet.Thislatterwasredispersedto5mlin0.05  M   Tris-HCL buffer (pH 8.0). Assays were performed on250 µ lof0.1  M  Trisbuffer(pH8.0)containingplasmino-gen (50  µ g/ml; P5661; Sigma Chemical Co., St. Louis,MO), 0.6 m  M   Val-Leu-Lys-p-nitroanilide (V7127; Journal of Dairy Science Vol. 85, No. 5, 2002 Sigma Chemical Co.), and 2  µ l of casein suspension.ThePL activitywas determinedin milksamples(3 ml),which were mixed with 1 ml of 0.4  M   sodium citrateand centrifuged at 27,000  ×  g  for 20 min. The superna-tant was recovered, and assays were performed in 250 µ l of 0.1  M   Tris buffer (pH 7.4), containing 0.6 m  M   Val-Leu-Lys-  p -nitroanilide (V7127; Sigma Chemical Co.),and 30  µ l of sample. The reaction mixture was incu-batedat37 ° C,andabsorbanceat405nmwasmeasuredafter2and3h.ThePLandPAactivitieswereexpressedas units, one unit being the amount of enzyme thatproduced a change in absorbance of 0.1 at 405 nm in60 min. Blood Sampling and Analysis Bloodsamples,collectedintoheparinizedtubes(Ven-oject; Terumo Europe, Leuven, Belgium), were takenweekly from the jugular vein during the second experi-mental cycle before TMR administration. Blood wascentrifuged at 14,000  ×  g  for 15 min at 10 ° C to obtainplasma. Plasma was stored at  − 20 ° C pending analysis  BALDI ET AL. 1096 for glucose (Sigma Chemical Co.), NEFA (Enzycolor;Boehringer-Mannheim,Monza,Italy),andurea(Boher-inger-Mannheim). Mammary Gland Analysis Uddersfromthethreecontrolandthreetreatedgoatsslaughtered were removed, weighed, and kept on iceduring transport to the laboratory. The milk was al-lowed to drain and the mammary tissue was dissectedaway from surrounding structures (skin, teat, subcuta-neousfat)andweighed.Eachglandwasthenseparatedand weighed separately. After samples for morphological analysis were col-lected, the glands of each animal were homogenizedand samples taken, frozen in liquid nitrogen, and keptat  − 80 ° C pending analysis for protein (Kjeldahl proce-dure), fat (Soxhlet ether extraction), total DNA (La-barcaandPaigen,1980)andhydroxyproline(Woessner,1961). Collagen content was calculated from hydroxy-proline, according to Anderson and Wahab (1990). Tissue Sampling and Fixation Several blocks of mammary tissue were dissectedfrom two zones of each gland of treated and controlanimals (Akers et al., 1990): deep tissue was collectedfrom the dorso-lateral portion of the gland at a depthof 2 to 3 cm from the abdominal wall, and cisternaltissuewascollectedfromtheareaimmediatelyadjacentto the cistern. For morphological studies, tissue blockswere fixed for 48 h in 10% formalin buffered to pH 7with 0.025  M   phosphate buffer, and blocks of approxi-mately 1 cm 3 were embedded in paraffin and sectioned. Alveoli and Corpora Amylacea Five- µ m-thick sections were mounted on poly-L-ly-sine(SigmaChemicalCo.)coatedslidesforquantitativeanalyses of histological structures. The total numberof alveoli ( TA  ) were counted on hematoxylin and eosin-stained sections. Alveoli were classified as lactating al- veoli ( LA  ) or RA based on the following criteria. TheLA epithelium was columnar, with well-differentiatedcellscontainingroundedbasallydisplacednuclei,abun-dant apical secretory vacuoles, and evidence of secre-tion;thecellswerewellpolarizedwithlargecytoplasm-to-nucleus ratio. The RA epithelium was no longer co-lumnar; the cytoplasm-to-nucleus ratio was lower; se-cretory vesicles were absent; but large lipid dropletswere present, and frequently nuclei were located api-cally. Some alveoli were collapsed. The alveolar classi-fication is illustrated in Figure 1.The numbers and positions of CA were estimated onsections stained with Congo red (Highman technique) Journal of Dairy Science Vol. 85, No. 5, 2002 Figure2 .Representativeexamplesofcorporaamylacea(A)withinthe alveolar lumen and (B) in the interstitial tissue (srcinal magnifi-cation 250  × ). and Mayer’s hematoxylin, using a kit (Bio-Optica, Mi-lano, Italy). The CA within the lumen of the alveoluswere classified as alveolar ( aCA, ) and those in interal- veolarconnectivetissuewereclassifiedasinteralveolar( iCA  ). Examples of the two types of CA are shown inFigure 2. Alveoli and CA were quantified with the aidof a grid placed in the eyepiece. For each animal, 16independent tissue blocks were examined: four blocksfrom four different zones in each gland (left and right).Five random fields from eight sections of each blockwere evaluated at  ×  250. Therefore, for each treatment,alveoli and CA were counted in 640 fields of 384sections.ThenumbersofTA,LA,andRAwerecountedineachfield,selectingalveolarsectionsinwhichmostsecretorycells presented sagittal section. The CA were countedin the same fields; for each field the number of tCA,aCA, and iCA were counted. Counts were performed bytwo independent observers.  bST EFFECTS ON GOAT MAMMARY GLAND FUNCTION  1097 Immunohistochemistry and Apoptosis For immunohistochemical studies and in situ detec-tion of cell death, 1-cm 3 mammary gland samples, col-lected as described above from treated and controlgoats, were embedded in embedding medium (Bio-Op-tica) and rapidly frozen in isopentane cooled in liquidnitrogen ( − 150 ° C). For each zone, 30 10- µ m sectionswerecut, thaw-mountedonto slidescoated withpoly-L-lysine(Sigma ChemicalCo.) andfixed incooled acetone( − 20 ° C) for 10 min.Laminin was detected with a polyclonal antibody(Sigma Chemical Co.) and fibronectin with a mono-clonal antibody (clone IST3; Sigma Chemical Co.). Theimmuno-reactionswererevealedwiththestreptavidin-biotin horseradish peroxidase complex technique (ABCkit;VectorLabs,Burlingame,CA)and3 ′ ,3-diaminoben-zidine dehydrate (DAB; Sigma Chemical Co. [Gandolfiet al., 1995]). Nuclei were counterstained with 0.1 %(wt/vol) nuclear red (Sigma Chemical Co.). Apoptotic cells were localized using the terminaldeoxynucleotidyl transferase ( TdT )-mediated dUTPnick-end labeling technique (TUNEL). Frozen sectionswere permeabilized with 0.1% aqueous sodium citrate(wt/vol) supplementedwith 0.1% TritonX-100 (vol/vol);DNA strand breaks generated during apoptosis weredirectly identified using fluorescein-dUTP incubatedwithaTdTthatcatalyzespolymerizationofnucleotidestoDNAends(insitucelldeathdetectionkit,fluorescein,Boehringer Mannheim, East Sussex, UK). All sections were observed and photographed with alight microscope equipped for epifluorescence analysis(Nikon Diaphot TMD; Nikon Instruments, GardenCity, NY) Statistical Analysis Data on milk yield and composition were analyzedby ANOVA, using the general linear model procedureof SAS (1988), considering treatment, sampling time,and the interaction between treatment and time. Val-ues at the beginning of treatment were included in themodel as covariates. Treatment  ×  sampling time inter-actions were nonsignificant for all variables analyzed,and therefore only the main effects are considered. Todetect differences in time trends, the data were ana-lyzed by repeated measures of ANOVA (SAS, 1988) ina model that included the effects of bST treatment,sampling time, and residual error. SCC was linearizedbylogSCC/1000.Plasmaconstituentsofindividualani-mals collected at different times did not differ and,therefore, were pooled for each group and analyzed byone-wayANOVA.TheeffectsofbSTonmammaryglandcomposition and morphological measures were ana-lyzed by one-way ANOVA, followed by Fisher’s LSD Journal of Dairy Science Vol. 85, No. 5, 2002 Table 1 . Least-squares means of milk production, milk composition,and milk clotting parameters, plasmin (PL) activity, plasminogenactivator (PA) activity, sodium Na, and K concentrations and Na/K ratio, in control and bST-treated goats.Control bST-treated SEM  P Goats 7 7Milk yield, kg/d 1.82 2.19 0.06 0.02Fat, % 2.88 2.96 0.08 0.55Protein, % 3.19 3.03 0.03 0.01Casein, % 2.37 2.32 0.04 0.40Whey protein, % 0.59 0.56 0.02 0.40NPN, % 0.21 0.18 0.007 0.03Lactose, % 4.69 4.66 0.02 0.42SCC, Log/1000  2.81 2.47 0.07 0.03pH 6.77 6.72 0.03 0.29 r , 1 min 14.6 13.3 0.20 0.05k 20 , 2 min 5.61 4.87 0.40 0.24a 30 , 3 mm 21.1 20.9 0.70 0.93PL activity, U/ml 19.81 17.91 0.91 0.15PA activity, U/ml 1110.68 1035.71 59.39 0.37Na, mM 16.22 15.20 0.58 0.22K, mM 42.65 48.03 1.57 0.02Na/K. ratio 0.39 0.31 0.02 0.02 1 Coagulation time. 2 Firming rate of curd. 3 Curd firmness. test. Effects were considered significantly different at  P  ≤  0.05. RESULTSMilk Yield, Composition, and Coagulating Properties Two of the 16 recruited goats were excluded from theexperiment: one (control) dried before the end of theexperiment, and another (treated) developed clinicalmastitis.The othergoats remainedhealthy throughoutthe study, and began and ended the study at similarBW (63.75  ±  2.81 vs. 65.56  ±  2.92 kg, for control andtreatedgoats, respectively).DMI didnot differbetweencontrol and treated goats (2.11  ±  0.09 kg/d vs. 2.13  ± 0.09 kg/d, for control and treated goats, respectively).The overall milk yield and composition are shown inTable 1. Milk production in the treated group was 20%higher (  P  =  0.02) than in the control group throughouttheexperimentalperiod.Milkyieldinthetreatedgoatspeaked 7 d postinjection in a cyclic pattern (Figure 3),and the response to the third injection was greater ( + 60% vs. control;  P  <  0.01) than to the second ( +  46%;  P <  0.01) and the first ( +  31%;  P  <  0.05) injections. Milklactose and fat content were not affected by the treat-ment (Table 1). Milk protein was reduced by the treat-ment; however, casein and whey protein were unaf-fected (  P  =  0.40), whereas NPN content was signifi-cantly lower (  P  <  0.05) in the treated group. Treatedgoats had a lower  (  P  <  0.05) SCC than controls.
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