Insulin-like growth factor (IGF)-I and -II and IGF binding proteins in serum and mammary secretions during the dry period and early lactation in dairy cows.

INSULIN-LIKE GROWTH FACTOR (IGF)-I AND -11 AND IGF BINDING PROTEINS IN SERUM AND MAMMARY SECRETIONS DURING THE DRY PERIOD AND EARLY LACTATION IN DAIRY COWS'

The Pennsylvania State University3, University Park 16802 ABSTRACT

Concentrations of IGF-I and IGF-11, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 f 7 to 24 f 3 ns/ml, P < .Ol), there was no significant change in serum IGF-II concentration during this period (150 f 17 vs 173 f 13 ns/ml, P > .05). Furthermore, a 57% increase in serum IGF-I was obsewed from the last week of lactation to the second week of drying off (100 f 5 to 157 f 8 ndml, P < .05). Changes in serum IGF-11 were not observed (126 f 11 vs 150 f 10 ng/ml, respectively; P > .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 f 1.1, 1.83 f .6, and 7.27 f .76 pg/ml, respectively), total output/quarter was highest in colostrum (394 f 119, 295 f 132, and 2,680 f 1,967 pdquarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P> .05) IGF-I or IGF-11 concentration (ns/ml) or total output (pdquarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-11, and IGFBP by the gland may be related to each other. Key Words: IGF-I, IGF-11, Binding Proteins, Mammary Secretions. Bovine, Dry Period J. Anim. Sci. 1991. 692538-2547

lntroductlon

Insulin-like growth factor I (IGF-I), insulin-lie growth factor 11 (IGF-10, and IGF binding proteins (IGFBP) have been observed in bovine prepartum mammary secretions (Malven et al., 1987; Pyke and Baumrucker, 1988), colostrum (Francis et al., 1988), and milk (Campbell and Baumrucker, 1989). Elevated concentrations of serum IGF-I (Davis et al., 1987; Kerr, 1989), IGF-11 (Collier et al.,

'This research was supported by tbe Pennsylvania Agric. Exp. Station and USDA Compctitive Grant 85-CRa-1-1881.

*Address reprint requests to C. R. Baumrucker. kept. of Dairy and Anim. Sci. Received June 15, 1990. Accepted December 6, 1990.

1989), and mammary tissue IGF-I (Glimm et al., 1988) have been reported in dairy cows treated with bovine growth hormone (bGH). Short-term infusion of IGF-I (8.25 pg/min) into the arterial supply of one mammary gland of a goat resulted in a significant increase in milk production and IGF-I secretion by the infused gland compared with the contralateral uninfused gland (Prosser et al., 1990). McGrath and Collier (1988) infused a combination of IGF-I (12.5 pg) and epidermal growth factor (12.5 pg) directly into one side of the udder of pregnant ewes every other day. Glands treated with IGF-I and epidermal growth factor contained 33% more DNA than control glands. In vitro studies have shown that IGF-I stimulated DNA synthesis in explants of bovine mammary tissue (Shamay et al., 1988; Baumrucker and Stemberger, 1989).

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J. R. Vega, C. A. Gibson, T. C. Skaar, D. L. Hadsell and C. R. BaumruckeG

IGF-I, IGP-II, AND IGFBP IN DAIRY COWS

Materials and Methods

Sample Collection. Blood was collected weekly from the tail vein of 30 Holstein cows to evaluate changes in serum IGF-I and IGF-II associated with cessation and initiation of lactation. Group 1 consisted of 21 cows sampled from 7 wk before to 1 wk after parturition. Group 2 included nine cows sampled from the last week of lactation to the first 3 wk of the dry period. Serum and mammary secretions were obtained from five Holstein cows (Group 3) starting 4 wk prepartum to evaluate changes in IGF-I and IGF-11 concentrations, IGFBP binding activity in mammary secretions, and milk yield due to weekly milking. Samples (serum and mammary secretions) were collected at weekly intervals during the dry period, immediately after parturition (colostrum), and at 2 wk and 7 wk postpartum. One front and one rear quarter

4vnless otherwise indicated a11 the reagents were Sigma Chemical Co., St Louis, MO. 'Gift from NIDDK, BaItimm, MD; Underwood and Van Wyk antibody. 6Gift from B. D. Burleigh, International Minerals and Chemical Co., Northbrook, E. 7~~ International ~nzymes, T ~ Y , VA. 8Bachem, Inc., Torrence, CA.

were milked and the other two were used as controls. During the dry period, cows were milked by hand and a milking machine was used after parturition. Milk from each quaxter was collected separately. IGF-I Radioimmunwssay.Serum and mammary secretions were assayed for IGF-I (Etherton et al., 1987). Interference from IGFJ3P was removed using the acid/ethanol extraction method (Campbell and Baumrucker, 1989) with some modifications. Two hundred microliters of serum, milk, or assay buffer (30 mM sodium phosphate4, 200 pg of protamine sulfatehl, 10 mM EDTA, .05% (voWol) Tween 20, .02% ( W o l ) sodium azide) was extracted with 800 pl of acid/ethanol (2 N HCl:ethanol, 12387.5) for 30 min at room temperature and centrifuged at 2,800 x g for 30 min. The Supernatant (500 pl) was neutralized with 200 pl of .855 M Tris base. Neutralized aliquots were used to determine IGF-I. A polyclonal antibody UBK487' was used at a final dilution of 1: 180,OOO.Recombinant human IGF-I (rhIGF@ was used as tracer and standard Iodination was by the chloramine T method to a specific activity of approximately 250 pCi/pg (Etherton et al., 1987). The sensitivity of the RIA was approximately 25 pg/tube, with an EDSO (median effective dose) of 75 pg/tube. Intra-assay CV was 7.8%, and interassay CV was 12%. Recovery studies using serum and prepartum and postpartum mammary secretions to which known concentrations of rhIGF-I had been added resulted in a recovery of 85%. Concentration of IGF-I in prepartum and postpartum mammary secretions determined by the acid/ ethanol method were validated (n = 10) by gel filtration under acid conditions (Daughaday, 1987). There was 24 f 6.6% more immunoreactive IGF-I in acid chromatographed samples than in those using the acid/ethanol extraction method. IGF-II Radioimmunoassay. Serum and mammary secretions were extracted with acid/ ethanol and assayed for IGF-II as described by Malven et al. (1987)with some modifications. An aci4ethanol extraction method similar to that used in the IGF-I RIA was used. A monoclonal antibody against hIGF-117 was used at a final dilution of 1:8,000.Recombinant human IGF-11 (rhIGF-II)8 was iodinated using the chloramine T method to a specific activity of approximately 150 pCi/pg. The sensitivity of the assay was approximately 35


Specific IGF-I and IGF-11 receptors have been characterized in bovine mammary tissue. An increase in the concentration of both receptors, especially type I (Dehoff et al., 1988; Hadsell et al., 199Oa), was associated with parturition. These changes in receptor population could play a role in modulating the activity or transport of IGF in bovine mammary tissue. Although concentrations of IGF-I and IGF-11 in blood and milk from dahy cows have been reported from within 1 wk prepartum throughout the entire lactation (Malven et al., 1987;Ken, 1989), there has been a lack of information on IGF-I, IGF-11, and IGFBP in dairy cows during the dry period. The first objective of this study was to determine whether changes occur in circulating IGF-I and IGF-II concentrations with the cessation and initiation of lactation in dahy cows. The second objective was to evaluate the effect of weekly removal of mammary secretions during the late dry period on the concentration of IGF-I and IGF-11 in CO~OS~IUII and postpamUn milk and to determine its effects on milk yield.

2539

2540

VEGA ET AL.

9pharmacia ~nc.,Piscaraway, NJ.

I, IGF-11, and milk yield (morning milking) per quarter postpartum. Results

Immunoreactive IGF-I and IGF-II in Acid Chromatographed Samples. The comparison of RIA for IGF-I and IGF-11 from acid extraction vs acid chromatography showed that acid/ ethanol extraction resulted in an underestimation of true IGF values. Both IGF-I and IGF-II exhibited a 24% increase when acid chromatographed in place of acidethanol treatment. Thus, the results presented here are an underestimation of actual IGF values. The results presented are acidethanol extractions and the changes reported are significant without correction for underestimation. Serum IGF-I and IGF-II. The profile of circulating IGF-I during the dry period and early lactation in 21 Holstein cows from Group 1 is illustrated in Figure 1A. All cows used were bled weekly until 1 wk postpartum. Concentrations of IGF-I in serum varied considerably between 7 to 1 wk before parturition; values ranged from 70 to 317 ngl ml. There was a threefold decrease in serum IGF-I within 1 wk after parturition. Circulating IGF-I in 11 cows sampled within 5 d before parturition was 81 f 7 ng/ml, whereas serum IGF-I in eight cows sampled at parturition was 24 f 3 ng/ml. Data for IGF-I were fitted to a polynomial function (serum IGF-I (nglml) = 51.5 - 6.6d - .098d2 where d = day postpartum; P < .001; R2 = S3). Based on this equation, predicted peak serum IGF-I concentration was 163 ng/ml at 34 d prepartum. Figure 1B shows serum IGF-I and IGF-II from four Holstein cows (randomly selected from Group I) during the end of the dry period. Two cows had a dry period of 57 d and the other two had a dry period of 80 d. Although there was a constant decline in circulating IGF-I as parturition approached, IGF-11 remained constant. The greatest change in serum IGF-I occurred between 4 d before and 3 d after Parturition with a 70% reduction in IGF-I (80 f 15 vs 24 f 7 ng/ml, respectively). Circulating IGF-I concentrations in nine Holstein cows (Group 2) during the early dry period are shown in Figure 2A. A significant increase in serum IGF-I was observed 9 d after cessation of milking. The IGF-I concentrations increased from 100 f 5 to 157 f 8 ndml for cows bled within 5 d before and 9 d after


pg/tube, with an ED50 of 140 pg/tube. Intraassay CV was 3.6%, and interassay CV was 7.8%. Recovery studies using serum and prepartum and postpartum mammary secretions to which known concentrations of rhIGF11 had been added resulted in an average recovery of 88%. The crossreactivity for rhIGF-I of the monoclonal antibody used was .14%. Concentrations of IGF-11 in mammary secretions determined by the acid/ethanol method were validated (n = 10) by gel filtration under acid conditions (Daughaday, 1987). Recovery of IGF-11 in acid chromatographed fractions showed increases identical to IGF-I discussed previously. Gel Filtration. Prepartum mammary secretions (wk 4 to 1 before parturition) and colostrum (n = 10) from two Holstein cows were defatted by centrifugation, filtered through glass wool, and fractioned under acid chromatography. Samples containing 1 ml of mammary secretions were preincubated for 2 h at room temperature with an equal volume of 2 M acetic acid, pH 3.5. Samples were centrifuged at 2,800 x g for 30 min to remove any precipitate. Supernatants were loaded onto a 1.6-cm x l00cm Sephadex G-75 column9 equilibrated with 1 M acetic acid, pH 3.5. Fractions containing 2 ml were collected and analyzed for immunoreactive IGF-I, IGF-II, and IGFBP activity. IGFBP Assay. Fractions from the gel filtration column were assayed for IGFBP as described by Campbell and Baumcker (1989). Labeled rhIGF-I and rhIGF-11 were used as specific ligands. An aliquot of each fraction was neutralized with .855 A4 Tris base and 5 to 15 pl of the neutral fraction was analyzed for IGFBP activity. Fractions exhibiting binding of the ligands were pooled and reanalyzed for IGFBP. Activity of IGFBP was expressed as micrograms of either [1251]rhIGFI or [lZ5IlrhIGF-II bound per milliliter of sample. Statistical Methocis. Regression analysis was performed using the GLM procedure (SAS, 1985) on immunoreactive IGF-I and IGF-II present in serum. Paired t-tests were performed on colostrum and l l d and 49-d milk values to evaluate weekly milking during the dry period on immunoreactive IGF-

2541

IGF-I, IGF-II, AND IGFBP IN DAIRY COWS

respectively, whereas two cows with dry periods of 46 and 49 d had serum IGF-I concentrations of 88 n g / d (2 and 5 d prepartum or 44 d after drying off), respective ly. Serum IGF-I and IGF-11 in four cows (randomly selected from Group 2) from the day of drying off to d 16 of the dry period are shown in Figure 2B. Whereas IGF-I concentrations showed a linear and quadratic effect with the cessation of milking ( P e .Ol), circulating IGF-11 remained unchanged (P > .lo). There was a 73% increase in circulating IGF-I from the day of drying off to d 9 of the dry period, with no significant change in IGF-II.

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Day postpor;um Figure 1. Insulin-like growth factor (IGF) concentrations in blood during the dry period and early lactation. panel A is sewn IGF-I in 21 Holstein cows from 7 wk before to 1 wk after p ~ t i o Each e data point represents an individual cow bled on the specified day. The line represents a polynomial function of the data IGF-I = 51.5 - 6.6d- .osSd2, where d = day postpamnn; P < .001; R2 = S3). Panel B is serum immunoreactive IGF-I (0) and IGFII (0)concentrations in four Holstein cows during the last 3 wk of the dry period and the 1st wk after parturition. Each point represents the mean f SE of four cows bled at the specified day.

Figure 2. Insulin-likc growth factor (IGV concentrations in blood during the late lactation and early dry period. P a l A is s e n ~ nIGF-I M I X X I I ~ ~ ~ ~ ~ OinD Snine Holstein cows from the last week of lactation to the fmt 3 wk of the dry period. Each point represents an iudividd cow bled on the specified day. The linc represents a polynomial function of the data IGF-I = 114.6 + 4.6d .17d2, where d = day aftm drying off; P < .05; R2 = 2 2 ) . Panel B is serum immunoreactive IGF-I (0) and IGF-II (0)concentrations in four Holstein cows from the last week of lactation to the 3rd wk of the dry period. Each point represents the mean f SE of four cows bled at the specified day.


drying off,respectively. The data were fitted to a polynomial function (serum IGF-I (ng/ml) = 114.6 + 4.6d - .17d2, where d = day after drying off; P < .05; R2 = .22). Based on this equation, predicted peak serum IGF-I concentration was 146 n g / d at 14 d after cessation of milking. There was a large variation due to cow in circulating IGF-I, with values ranging from 82 to 176 ndml 23 d after drying off, That variation may have been due to differences in stage of gestation among cows on the same day of a dry period. The average dry period was 68 f 5 d for all cows. Two cows with dry periods of 85 and 93 d had IGF-I concentrations of 162 and 176 ng/ml (62 and 71 d prepartum or 23 d after drying off),

2542

VEGA ET AL.

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which IGF-I concentrations were about 50% greater than IGF-II concentrations for 3 to 1 wk prepartum. The mammary secretion to serum ratios for IGF-I and IGF-TI are illustrated in Figure 3C. The average increase in IGF-I and IGF-II in mammary Secretions during the late dry period resulted in a 3.1-fold and 2.4-fold increase in the ratio of IGF-I and IGF-It in mammary m e t i o n s to serum, respectively, between 4 and 1 wk postpartum. However, at maximum IGF-I showed a concentration ratio of 23 and IGF-11 a ratio of 11. Total outputs per quarter of immunoreactive IGF-I and IGF-11 are shown in Figure 4. The total mass of IGF-I and IGF-11 during the late dry period did not change (153 f 21 and 106 f 15 figquarter, respectively). There was a 2.6- and 2.8-fold increase in total IGF-I and IGF-11 in colostrum compared with prepartum secretions (394 It 119 and 295 f 132 pgl quarter for IGF-I and IGF-II, respectively). Immunoreactive IGF-I and IGF-II in Acid Chromatographed Samples. Mammary seers tions were run under acid conditions to remove interference from IGFBP. Individual fractions were neutralized and analyzed for immunoreactive IGF-I and IGF-II. Fractions containing IGF were pooled and reanalyzed for immunoreactive IGF-I and IGF-II. There was 24.4 f 6.6% more immunoreactive IGF-I and IGF-11 (data not shown) in acid chromatographed samples than in those using the acid/ ethanol extraction method.

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Week p o s t p a r t J m Figure 3. Comparison of insulin-like growth factor (IGF)in serum and mammary secretions. IGF-Iand IGF-II concentrations in serum and mammary secretions of five Holstein cows from 4 wk prepto 7 wk postpartum. AU panels are IGF-I ( 0 ) and IGF-II(0).Panel (A) is concentrations in serum. Panel (B) is concentrations in mammasy secretions. Panel (C) is the mamplary secretion to serum ratios. Sampling was at weekly mtervals. Each point represents the mean f SE of five cows.

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Figure 4. Total insulin-like growth factor (IGF)mass and volume of mammary secretions. Mass of IGF-I (0) and IGF-II(0)and volume (A) per quarter from 4 wk prepartum to 7 wk postpartum. Each point represents the mean f SE of two quarters per cow (n = 4 cows) collected at morning millnngs.


IGF-I and IGF-I. in Mammary Secretions. Concentrations of IGF-I and IGF-11 in serum and mammary secretions of five Holstein cows (Group 3) are shown in Figure 3. Concentrations of IGF-I in serum declined between 2 wk before and 11 d after parturition (152 f 56 vs 31 f 4 ng/ml) followed by an increase by 49 d after parturition (76 f 11 ng/ml) (Figure 3A). Serum IGF-11 remained constant during the entire sampling period, with an average concentration of 182 f 7 ng/ml. Concentrations of IGF-I and IGF-11 in mammary secretions were highest 2 wk prepartum (2,949 f 1,158 and 1,825 f 608 ng/ml) and lowest in milk 49 d postpartum (5.0 f 2.0 and 1.0 f .1 nglml, respectively; Figure 3B). Serum IGF-11 concentrations were higher than IGF-I concentrations for the entire sampling period, but the opposite occurred in mammary secretions, in


2543

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Week 9 o s t o c r t u m Figure 5. Insulin-like growth factor binding pro I (IGFBP) activity in acid chromatographed ~llammary secretions. Data are from two Holstein cows daring the f d 4 wk of the dry period and colostrum. Each point represents Ihc mean at the specified week. Activity of IGFBP is expressed as ' ograms per milliliter or milligrams per quarter of FI]rhIGF-l bound (0)and [12511rbIGF-II bound (0).Panel (A) is concentration and panel (B) is total mass.

Discussion

In the present study, we have reported concentrations of immunoreactive IGF-I and IGF-11 in serum as well as IGF-I, IGF-11, and IGFBP in mammary secretions during the dry period and early lactation. The elevation of serum IGF-I during the dry period, subsequent decline around parturition, and increase after parturition have been described in other reports (Ronge and Blum, 1988; Ronge et al., 1988; Kerr, 1989). Serum IGF-II did not change throughout the dry period and early lactation. The changes in serum IGF-I concentrations during the penparturient period are likely the result of at least two processes: 1) decrease and increase in metabolic demand due to the cessation and onset of lactation, respectively, and 2) changes in transport of IGF-I by the mammary gland. During late pregnancy and early lactation the metabolism in the dairy cow


IGF Binding Activity in Mammary Secre- tum mammary secretions and colostnun (62 f tions. The profile of IGF binding activity in 29 vs 1,040 f 420 ml/quarter, respectively). A comparison between IGFBP and total two Holstein cows is shown in Figure 5. There was a parallel increase in IGF binding activity immunoreactive IGF concentration (IGF-I plus similar to the profiles of immunoreactive IGF-I IGF-II) in mammary secretion is shown in and IGF-11 concentration during the same Table 1. Binding activity of IGF determined sampling period. The IGF binding activity by [l2SI]rhIGF-I bound was 36 f 2.7% of the estimated using [lUI]hIGF-II as a tracer total immunoreactive IGF (IGF-I plus IGF-II) resulted in 2.6- to 3.6-fold higher binding detected by RIA during the prepartum period, activity compared with using [~~Q~IIGF-Iwhereas IGF binding activity estimated by (Figure 5A). The greatest concentration of [1251]IfiIGF-II bound was 106 f 7.9%. The IGFJ3P present in mammary secretions was ratio of IGF binding activity to total imobserved between 3 and 2 wk before p 6 - munoreactive IGF in colostnun changed dration (2.79 f .1 and 7.58 f 2.8 pg/ml) for matically at parturition. This ratio reflects a [1251]rhIGF-Ibound and [125TJrhIGF-IIbound, change in the affinity of the IGFBP for the two respectively. The total mass of IGFBP is ligands caused by an alteration in the relative shown in Figure 5B. A large increase in total concentrations of the various binding protein IGFBP was observed in colostrum, mainly due species at parturition. Binding activity of IGF to an increase in volume between the preparin colostrum determined by [1251'JrhIGF-I bound and [l25I]rhIGF-II bound were 121 and 336% of the total immunoreactive IGF, respectively. Effect of Weekly Milking During the Dry I Period. Milk yield, IGF-I, and IGF-11 in 0 colo~trum, and 11-d and 49-d milking of quarters milked during the dry period vs E \ ipsilateral unmilked quarters are shown in Table 2. Weekly removal of mammary secretions during the last 4 wk of the dry period did not affect ( P > .05) milk yield, IGF-I, or IGF0 11 concentration of the milked quarter compared with the unmilked, ipsilateral quarter at c the three sampling times (colostrum, 2 wk,and 0 7 wk).

2544

VEGA ET AL. TABLE 1. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP) ACTIVITY

AND TOTAL IGF LEVELS IN AClD CHROMATOGRAPHED MAMMARY SECRETIONS OF COWSa DURING THE LAST 4 WEEKS BEFORE PARTURITION AND IN COLOSTRUMb IGFBP activity

IGF-nd, Mm

IGF-IC,

w

.561 f 2.538 f 2.794 f 1.901 f .782 f

.06

.81 .10 .12 .38

1.769 f 7583 f 7267 f 5.950 f 2.166 f

Total IGP,

Mm .19 2.82 .76 .001 1.01

1.829 5.866 7.644 5.801 .564

f f f f f

.392 2.09 1.29 2.60 .314

aN = 2. bSamples were chromatographed under acid conditions as d d b e d in Materials and Methods. Volumes reflect total secretion volume. c['251]rhIGF-I bound. d['23]rhIGF-Jl bound. qGF-I plus IGF-II determined by RIA.

undergoes some changes involving a decrease in nitrogen and energy balance. This decrease has been associated with a decrease in plasma IGF-I without any alteration in plasma IGF-II. Steers fed at a maintenance level of nutrition averaged plasma IGF-I levels approximately one-half those of steers fed at a high level of nutrition, but plasma levels of IGF-11 were not affected (Breier et al., 1988). In rodents, fasting (Bomfeldt et al., 1989) and protein restriction (Moats-Staats et al., 1989; Thissen et al., 1990) caused a reduction in IGF-I. The increase in serum IGF-I and IGF-11 observed after cessation of milking agrees with a recent report (Kerr, 1989). This increase in

serum IGF-I may be due to the abrupt termhation of millcing and be caused by either an improved nutritional status or the cessation of IGF removal from circulation via the mammary gland. Serum IGF-I was positively correlated with energy and protein balances in dairy cows when such balances were positive due to low yield (Ronge et al., 1988). On the other hand, infusion of IGF-I into the arterial supply of a lactating goat mammary gland resulted in an 85% increase in IGF-I concentration in milk (Prosser et al., 1989), supporting the idea that IGF-I may be sequestered Itom the blood by the lactating gland.

TABLE 2. EFFECT OF WEEKLY REMOVAL OF MAMMARY SECRETIONS DURING THE DRY PERIOD' ON MILK YlELD AND INSULIN-LIKEGROWTH FACTOR (IGP) CONCENTRATION OF COLOSTRUM AND 2 W K AND 7-WK MORNING MILKINGS sampling period Item Milk yield, litersb Milked Unmilked IGF-I, ng/mlb Milked Unmilked IGF-Jl, ng/mlb Milked Unmilked

Milk2 wk

Colostrum 2.08 f 1.80 f

.44 .44

5.74 f .43 527 f 25

hWk7wk 7.40 f .38 7.18 f .48

190.4 f 11.0 233.8 f 26.9

10.4 f 2.5 8.8 f 1.5

4.7 f 1.0 4.4 f .9

206.6 f 26.4 216.5 f 19.7

6.4 f 1.8 6.2 f 1.2

2.1 f .8 2.9 f 1.0

'Last 4 wk. %lk yield, IGF-I, and IGF-II of the milked qunrters were not different (P > .05) from the ipsilateral, umuilked quarters.


Week Prep4 3 2 1 Colostrum

IGF-I, IGF-E, AND IGF'BP IN DAIRY COWS

Baumrucker and Blum, unpublished data). Comparing these results to the marked increase of IGF-I in serum and prepartum secretions of pregnant cows in this study may indicate the importance of the newly regenerated epithelial cell in the hypothesized transport of circulating IGF-I to the prepmammary secretions. Weekly removal of mammary secretions fiom two quarters of the udder during the last 4 wk of the dry period did not affect IGF-I and IGF-II concentrations or milk yield in colostrum, 2 wk, and 7 wk milking. Although concentrations of IGF-I and IGF-II were lower in colostrum than in prepartum mammary secretions, total mass of IGF and IGFBP were highest in colostrum, in agreement with other reports (Baxter et al., 1984; Ronge and Blum, 1988; Simmen et al., 1988; Campbell and Baumrucker, 1989). The highest concentration of IGF-I during the late dry period raises the question of IGF-I as a local mediator of mammary epithelial growth and development. Insulin-like growth factor I has been shown to stimulate bovine mammary epithelial cell growth (Shamay et al., 1988; Baumrucker and Stemberger, 1989). Furthermore, the type I IGF receptor increased at parturition in bovine mammary tissue (Dehoff et al., 1988; Hadsell et al., 199Oa) and is present both in the apical and basolateral membranes in a cloned bovine mammary epithelial cell line (Hadsell et aL, 199Ob). In vivo studies in which IGF-I was infused directly into the mammary gland have shown a lactogenic (Prosser et al., 1989) and mammogenic response to IGF-I (McGrath and Collier, 1988). The high concentration of IGFBP present in prepartum mammary secretions could have a role in sequestering IGF-I and reducing the IGF-I receptor interaction. Insulin-like growth factor binding proteins have been shown to compete with the receptor for IGF binding (Binoux et al., 1986; Ritvos et al., 1988; Rutanen et al., 1988; Gopinath et al., 1989). Although an inhibition in the mitogenic activity of IGF-I by IGFBP has been reported (De Vroede et al., 1986; Rutanen et al., 1988), others have indicated an enhancement in IGFI-stimulated DNA synthesis (Elgin et al., 1987; Blum et al., 1989; Busby et al., 1989) and glucose and aminoisobutyric acid uptake (Bar et al., 1989). At the present time, the physiological importance of IGFBP in prepartum mammary secretions remains unknown. Additionally, the large quantities of IGF-I,


Concentrations of immunoreactive IGF-I and IGF-11 in the mammary secretions of dairy cows peaked 2 wk before parturition, followed by a constant decline. Activity of IGFBP followed the same pattern of IGF-I and IGF-11, with peak concentration 2 wk before parturition. McCusker et al. (1989) reported that the relative amounts of serum IGF binding in pregnant sows did not change during the last trimester of pregnancy, although no samples were collected at parturition. Serum IGFBP in dairy cows have been reported to be affected by physiological status. A decline in the ability of a 140- to 160-kDa fraction to bind [1251]IGF-I was obsewed after parturition (Ronge and Blum, 1989). Pyke (1988) has shown changes in IGFBP profiles in prepartum and postpartum mammary secretions in dajr cows. An overall decrease in concentration associated with a change in the profile of the IGFBP occurs at parturition (Pyke and Baumrucker, 1988). The nature of such changes is unknown at the moment. The IGFBP present in mammary secretions may be transported from the blood and also result from local production by the mammary gland. De novo synthesis of IGFBP by bovine mammary acini and explant cultures has been reported (Campbell et al., 1990). A reduction in the synthesis of an insulin- and IGF-Iresponsive 42- to 46-kDa IGFBP in response to lactogenic hormones (prolactin plus cortisol) in a cloned bovine mammary epithelial cell line has been documented (Skaar et al., 1990). Induction of lactogenesis before parturition by regular prepartum milking causes a decrease in IGF-I, IGF-11, and immunoglobulins in the prepartum milk (Malven et al., 1987). In our study, the highest concentration of IGFBP and IGF in mammary secretions occurred at the time of maximal immunoglobulin G1 (IgG1) transport (Brandon et al., 1971; Sasaki et al., 1976). Temporal patterns of IGFI, IGF-11, and IGFBP in pre and postpartum secretions reported here are virtually superimposable on that reported for immunoglobulins, which suggests the significance of the contribution from the serum IGFBP and IGF to mammary secretions. In four nonpregnant dairy cows, serum IGFI concentrations rose 1.5-fold in the 1st wk after cessation of milking, whereas IGF-I in mammary secretions at up to 3 wk after cessation of milking remained less than half of that found in circulation (Hurni, Bruckmier,

2545

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VEGA ET AI.,.

lrnpllcations

Baxter, R C., 2. Zaltsman and J. R Turtle. 1984. Immunoreactive somatomedin-C/lnsulin-likegrowth factor I and its binding protein in human milk. J. Clin. Endocrinol. & Metab. 58:955. Binoux, M., P. Hossenlopp, S. Hardouin, D. Seurin, C. Lassarre and C. Gonrmelen. 1986. Somatomedin (insulin-likegrowth factors)-bindii proteins: Molecular forms and regulation. Horm. Res. (Basel) 24: 141. Blum, W. F.,E.W. Jenne, F. Reppin, K.Kietzmann, M.B. Ranke and J. R. Bierich. 1989. Insulin-like growth factor I (IGF-I)-binding protein complex is a better than frm IGF-I. Endocrinology 125~766. Bornfeldt, K. E., H. J. knqvst, B. Enberg, L. S. Mathews and G. Norstedt 1989. Regulation of insulin-like growth factor-I and growth hormone receptor gene expression by diabetes and nutritional state in rat tissue. J. Endocrinol. 122651. Brandon, M.R, D. L. Watson and A. K. Lascelles. 1971. "he mechanism of transfer of immunoglobulin into mammary secretion of cows. Aust J. Exp. Biol. Med. Sci. 49:613. Breier, B.H.,P. D. Gluckman and J. J. Bass. 1988. Influence of nutritional status and oestradiol-l7a on plasma growth hormom, insulin-likt growth factors-I Md -11 and the response to exogenous growth hormone in young steers. I. Endocrinol. 118243. Busby, W. H., P. Hossenlopp, M. Binom and D. R. Clemmons. 1989. Purified preparations of the amniotic fluid-derived insalin-like growth faCtOr-binding protein contain multimdc forms that are biologically active. Endocrinology 125773. Campbell. P. G. and C. R. Baumrucker. 1989. Insulin-like growth factor-I and its association with binding proteins in bovine milk. J. Endocrinol. 120:21. Campbell, P. G., T. C. Skaar, J. R. Vega and C. R. Ba-cker. 1990. Secretion of insulin-like growth factor I (IGF-I) and IGF binding proteins from bovine mammary tissue in vitro. J. EndocrinoL (In press). Collier, R J., S.Gangulj P. T.Menke, P. C. Buonomo, M. F. McGrath, C. E. Koas and G. G. Krivi. 1989. Changes in insulin and somatomedin receptors and uptake of hdh,IGF-I and IGF-II during I I I I L D ~ D ~growth, ~T~ lactogenesis and lactation. In: R B. Heap, C. G. Prosser a d G. E. Lamming (Ed.) The Biotechnology in Growth Regulation. pp 153-163. Bntterworths, London. Daugbaday, W. H.1987. Radioligand assay for insulin-like

The increase in serum insulin-like growth factor-I (IGF-I) after the cessation of millcing and the decline just before onset of lactation show that lactation events affect blood IGF-I concentrations. These could be influenced by nutritional status, transport by the mammary gland, or other unknown factors. Furthermore, IGF and IGF binding proteins (IGFBP) followed similar patterns in both serum and mammary secretions, suggesting that their synthesis or transport into the mammary gland may be associated with each other. Studies growthfactorII.In:D.BamesandD.A.Sirbaskn(Ed.) Me$hods in Enzymology. 248-259. Academic Press, addressing the hormonal regulation of systemic New York. IGF-I and IGFBP synthesis by mammary epithelial cells and their involvement in a Davis, S. R., P. D. Gluckman, I. C. Hart and H. V. Henderson. 1987. Effects of injecting growth hormone potential transcytosis during the prepartum or thyroxine on milk production and blood plasma period in the mammary gland are needed. concentrationof insulin-like.growth factors I and II in dairy cows. J. Endocrinol. 11417. Literature Cited

De Vroede, M. A., L. Y.-H. Tseng, P.G. Katsoyannis, S. P. Nissley and M. M. Rechler. 1986. Mcdulation of

Bar, R. S., B. A. Booth, M.Boes and B. L. Dake. 1989. Insulin-like growth factor-binding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities. Endocrinology 125: 1910. Baumrucker, C. R. and B. H. Stemberger. 1989. Insulin and insulin-like growth factor-I stimulate DNA synthesis in bovine mammary tissue in vitro. J. h i m . Sci. 67: 3503.

insulin-likegrowthfactor1bindingtohumanfibroblast monolayer cultures by insulin-like growth factor carrier proteins released into the incubation media. I. Clin. Invest. 77:602. Dehoff, M. H., R G. Elgin, R. J. Collier and D. R. Clemmon~.1988. Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mammary tissue. Endocrinology 1222412.


IGF-11, and IGFBP present in colostrum might be important as maternal mediators for growth and development of the gastrointestinal tract or overall systemic effect of the neonate. Schober et al. (1990) reported that type I IGF receptors are abundant in the small intestine of the neonatal pig and that the increase in tissue immunoreactive IGF-I was not due to an increase in IGF-I mRNA. This suggests a receptor-mediated uptake of milk-borne IGF-I. In neonatal rats, IGF-I treatment increased organ and body weights (Philipps et al., 1988). The data presented in this study demonstrate that IGF-I, IGF-IC, and IGFBP in prepartum mammary secretions followed the same profile previously described for IgGl (Brandon et al., 1971; Sasaki et al., 1976), suggesting a selective transfer by the mammary epithelial cells. However, bovine mammary organ cultures (Campbell et al., 1990) and a cloned bovine mammary epithelial cell line (Gibson et al., 1989; Skaar et al., 1990) synthesize IGFBP. Thus, the relative contributions of mammary synthesis versus uptake from the blood of IGF-I and IGFBP remain unknown.


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the goat. J. Endocrinol. 126437. Elgin, R G., W. H. Busby and D. R. Clemmons. 1987. An insulin-like growth factor (IGF) binding protein Prosser, C. G., I. R. Fleet and R. B. Heap. 1989. Action of IGF-I on mammary function. In: R B. Heap, C. G. enhances the biological response to IGF-I. Proc. NatI. Prosser and G. E. Lamming (Ed.) The Biotechnology Acad. Sci. USA W3254. in Growth RegUlati011. p~ 141-151. B~tterwOrths, Etherton, T. D.. J. P. Wiggins, C. M Evock, C. S. Chung, J. London. F. Rebhun, P. E. Walton and N. C. Steele. 1987. Stimulation of pig growth performance by porcine Pyke, S. N. 1988. Analysis of insulin-like growth factors (IGFs) and IGF bindmg proteins in bovine mammary growth hormone: Determiuation of the dose-response relationship. J. Anim. Sci. W433. secretionsand serum. M. S. Dissertation.The PennsylFrancis, G. L., F. M. Upton, F.J. Ballard, K. A. McNeil and vania State University, University Park J. C. Wallace. 1988. Insulin-like growth factors 1 and 2 F'yke. S. N. and C. R BZUXDIU~~CX. 1988.Analof insulinin bovine colostrum: Sequences and biological activlike growth factor (IGF) binding protein in bovine ities compared to a potent truncated form. Biochem. J. serum, colostrum and milk. Endocr. Soc. p 152 2501. (Abstr.). Gibson, C. A., J. R. Vega and C. R Baummcker. 1989. Rihros, O., T. Ran@ J. Jalkanen, A. M. Sukkari, R. Establishment of a bovine mammary epithelial cell Voutilainen, H. Bohn and E. M. Rutanen. 1988. line. Am. SOC. Cell Biol. p 329 (Abstr.). Insulin-like growthfactor (IGF) binding protein from Glimm, D. R, V. E. Baracos and J. I. Kennelly. 1988. Effect human decidua inhibits the b h d q and biological of bovine growth hormone on the distribution of action of IGF-I in cultured choriocarcinm cells. immnmnectve insulin-like growth factor-I in lactating Endocrinology 122:2150. bovine 1mamma1-y tissue. J. Dairy Sci. 71:2923. Ronge, H. and J. W. Blum. 1988. Somatomedin C and other Gopith, R, P. E. Walton and T. D. Etherton. 1989. An hormones in dairy cows around parturition, in newborn acid-stable insulin-like growth factor (IGF>binding calves and in mille J. Anim. physiol. Anim. Nutr. 60: protein fiom pig serum inhibits binding of IGPI and 168. IGF-II to vascular endothelialcells. J. Endocrinol. 120 Ronge,H.andJ. W.Blum. 1989.Insulin-likegrowthfactorI 231. biuding proteins in dairy cows,calves and bulls. Acta Hadsell, D. L., P. G. Campbell and C. R Baumrucker. Endocrinol. 121:153. 1%. Characterization of the change in type I and 11 Ronge. H., I. Blum, C. Clemenc F. Jans,H. Leuenberger and insulin-like growth factor receptors of bovine mamH. Binder. 1988. Somatomedin C in dairy cows related mary tissue during the pre- and postpartum periods. to energy and protein supply and to milk production. Endocrinology 126637. Anim. Prod. 47165. Hadsell, D. L..I. R. Vega, T. C. Skaar,C. A. Gibson and C. R u m E. M.,K. Pekonen and T. Makinen. 1988. Soluble R Baummcker. 199Ob. Regulation of type I insulin34K binding protein inhibits the binding of insulin-like like growth factor receptors in a clonal population of growthfactor I to its cell receptors in human secretory bovine mammary epithelial cells. J. Dairy Sci. phase endometrim evidence for autocrinc/paracriue 73(Suppl. 1):215 (Abstr.). regulationof growth factor action. J. CLia EndocrinOl. Ken. D. E. 1989. Studies on the role of insUlin-like growth & Metab. 66:173. factor-I in growth and lactation. Ph.D. Dissertation. SAS. 1985. SAS User's Guide: Statistics. SAS Inst, Inc., univ. of Saskatchewan$ saskatoon. Gary, NC. Malven, P. V., H. H. Head and R. J. Collier. 1987. Sasaki, M.,C. L. Davis and B. L. Larson. 1976. Production Periparturient changes in secretion an mammary and turnover of IgGl and IgG2 immunoglobulins in uptake of insulin and in collcentratiOn of insulin and the bovine around parturition. J. Dairy Sci. 592046. insulin-like growth factors in milk of dairy cows.J. Schober, D. A, F. A. Simmen, D. L. Hadsell and C. R. Dairy Sci. 59889. Baumruck. 1990. Perinatal expression of type I IGF McCusker, R H., D. R. Campion, W. Kelly and D. R. receptors in porcine small intestine. Endocrinology. Clemmons. 1989. The insulin-like growth factor126 1125. bmding proteins of porcine serum: endocrine and Shamay, A., N. Cohen, M. Niwa and A. Wer. 1988.Effect nutritional regulation. Endocrinology 125501. of insulin-like growth factor I on deoxyribonucleic McGrath, M. F.and R. J. Collier. 1988. Effect of epidermal acid synthesisand galactopoiesisin bovine uodifferengrowth factor @GF) and insulin-likegrowth factor-I tiated and lactating mammary tissue in vitro. Endocri(JGF-I) on ruminant mammary development. J. Dairy nology 123:804. Sci. 71(Suppl. 1):229 (Abstr.) Moats-Staats. B. M., J. L. Brady Jr. and L. E. Underwood. Simmen, F. A., R C. Simmm and G. Reinhart. 1988. Maternal and w o ~ t a lsomatomedin C/insulin-Uce 1989. Dietary protein restriction in artificiaUy reared growth factor-I (IGF-I) and IGF binding proteins neonatal rats causes a reduction of insulin-like growth during early lactation in the pig. Dev. Biol. 13016. factor-I gene expression Endocrinology 125:2368. Philipps. A. F., B. Persson, K. Hall,M.Lake, A. Skotlner,T. Skaar,T. C., C. A. Gibson, J. R Vega and C. R. Baummcker. 1990. Insulin-like growth factor binding proteins are Sanengen and V. R. Sara. 1988. The effects of hormonally regulated in a bovine mamma- epithelial biosynthetic insulin-like growth factor-I supplementacell line. Endocr. Soc. p 307 (Abstr.). tion on somaticgrowth,maturation, and erythropoiesis Thissen. J. P., S. Triest, L. E. Underwood,M. Maes and J. M. in the neonatal rat. EWiatr. Res. 23298. Ketelslegers. 1990. Divergent responses of sem Prosser, C. G., I. R.Fleet,A. N. Corps, E. R. Froesch and R. insulin-& growth factor-I and liver growth hormone B. Heap. 1990. Increase in milk secretion and (GH) receptors to exogenous GH in protein-restricted mammary blood flow by intra-arterial infusion of rates. Endocrinology 126:908. insulin-likegrowth factor-I into the mammarygland of

Associations of cytological endometritis with energy metabolism and inflammation during the periparturient period and early lactation in dairy cows.

Evaluation of interference by insulin-like growth factor I (IGF-I) binding proteins in a radioimmunoassay for IGF-I in serum from dairy cows.

Changes of Adipose Tissue Morphology and Composition during Late Pregnancy and Early Lactation in Dairy Cows.

Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in porcine serum during fetal and postnatal development.

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Regulation of serum insulin-like growth factor-I (IGF-I) and IGF binding proteins during rat pregnancy.

Opsonic activity in mammary secretion and serum of gilts during the lactation period.

Resistin in dairy cows: plasma concentrations during early lactation, expression and potential role in adipose tissue.

Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines.

The insulin-like growth factor (IGF)-binding proteins and IGF bioactivity in Laron-type dwarfism.

Effects of dry period length and dietary energy source on metabolic status and hepatic gene expression of dairy cows in early lactation.

Pten regulates development and lactation in the mammary glands of dairy cows.

Total and differential somatic cell counts and N-acetyl-beta-D-glucosaminidase activity in mammary secretions during dry period.

Clinical significance of serum insulin-like growth factor-1 (IGF-1) and insulinlike growth factor binding protein-3 (IGFBP-3) in patients with epithelial ovarian cancer.

Radioimmunoassay for insulin-like growth factor (IGF) II: interference by pure IGF-binding proteins.

Insulin-like growth factor (IGF)-I and IGF binding proteins axis in diabetes mellitus.

Choline and choline metabolite patterns and associations in blood and milk during lactation in dairy cows.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Altered affinity of insulin-like growth factor II (IGF-II) for receptors and IGF-binding proteins, resulting from limited modifications of the IGF-II molecule.

The Impact of Intramammary Escherichia coli Challenge on Liver and Mammary Transcriptome and Cross-Talk in Dairy Cows during Early Lactation Using RNAseq.

Prolactin binding in rat mammary gland during pregnancy and lactation.

Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows.

Circadian patterns of serum insulin-like growth factor (IGF) II and IGF binding protein 3 in growth hormone-deficient patients and age- and sex-matched normal subjects.

Supplementation with conjugated linoleic acids extends the adiponectin deficit during early lactation in dairy cows.