effect of a prolonged-release formulation of n-methionyl bovine somatotropin (sometribove) on milk...

Effect of a Prolonged-Release Formulation of N-Methionyl Bovine Somatotropin (Sometribove) on Milk Fat'

J. M. LYNCH: D. M. BARBAN0:n3 and D. E. BAUMAN4 Comell University Ithaca, NY 14853

G. F. HARTNELL5 and M. A. NEMETH5 Monsanto Agrlcultural Company

St. Louis, MO 63167

ABSTRACT

Nine Holstein cows were injected biweekly with a prolonged-release formulation of N-methionyl bST, and 9 cows were injected with excipient. Intramuscu- lar injections began at 60 f 3 d postpartum and continued at 14-d intervals for the full lactation. Adminis&tion of bST increased production of milk, total fat, and alI milk fat components measured. Average fatty acid composition of milk fat was not influenced by bST treatment. Stage of lactation had a large influence on production and percentage of individual fatty acids in milk fat from both bST- treated and control cows. The stage of lactation impact on the fatty acid composition of milk fat reflected changes in the relative contributions of body fat mobilization and de novo synthesis of milk fat components in response to changes in energy balance. Initiation of bST treatment caused some transient changes in milk fatty acid composition that were related to energy balance. These changes were small compared with the normal changes because of stage of lactation in all cows. Phospho-

Received October 8, 1991. Accepted March 6, 1992. lSuppo~ in part by Comell University Agricultural

Experiment Station. USDA (Hatch 1 2 7 4 7 and 143-444), and Monsanto Agricultural Company. Mention of m a of equipment or chemical suppliers is for scientifc accu- racy only and does not indicate any product endorsement by the authors or Comell University.

*Department of Food Science. 3Reprint requests. 4Department of ~nimal Science. 'Animal Science Division.

lipid and cholesterol content of milk also changed with stage of lactation but were not influenced by bST treatment. Melt- ing properties of milk fat were influenced greatly by stage of lactation. Bo- vine somatotropin did not cause any changes in composition or physical properties of milk fat that were outside the range of normal variation. (Key words: fatty acid composition, bovine somatotropin, stage of lactation)

Abbreviation key: DSC = differential scanning calorimetry.

INTRODUCTION

Triglycerides make up approximately 97 to 98% of bovine milk fat (9). In dairy cows, about 4% by weight of the fatty acids are synthesized de novo in the mammary gland (short- and mediumchain fatty acids), and the remainder (longchain fatty acids) enter the mammary cell preformed from the blood (6). In addition, substantial enzymatic desaturation of stearic acid to oleic acid can occur in the mammary cell (15).

The fatty acid composition of milk fat is of interest because it affects both the texture (19) and flavor (11) of dairy products. Factors such as season (9), breed (26), stage of lactation (10, 23, 27), and feeding practices (9) can influence the fatty acid composition of milk fat.

Administration of bST to lactating cows increases the production of milk and milk components, including fat (24). The fatty acid composition of milk fat has been examined in several studies involving short-term (10 to 14 d) treatment with bST. Increases occur in the production of fatty acids originating from both

1992 J Dairy Sci 75:1794-1809 1794

SOMATOTROPIN AND MILK FAlTY ACID COMPOSITION 1795

de novo synthesis and uptake from the circula- tion (8, 12, 16). However, the increase is pre- dominantly from the uptake of preformed fatty acids whenever treatment with bST causes animals to be in negative energy balance (8, 12).

A decrease in the percentage of short- and mediumchain fatty acids and increase in percentage of long-chain fatty acids (primarily c18:1) were observed in one long-term study involving daily bST injections beginning at 15 wk postpartum and continuing for the duration of the lactation (2). Differences in fatty acid composition between control and bST-treated cows were greatest during the first 8 wk of bST administration. These differences became smaller as bST treatment continued.

The phospholipid content of milk fat, although quantitatively minor compared with triglycerides, can influence the functionality of milk fat and the stability and integrity of the milk fat globule membrane. Cholesterol, an- other minor constituent of milk fat, is currently of nutritional interest to the consumer. The factors that influence phospholipid and cholesterol contents of milk fat are not well under- stood. Bitman et al. (8) reported a decrease in the percentage of phospholipids and cholesterol in milk fat with bST administration. However, the duration of bST treatment was short (14 d), and cows were in negative energy balance (8).

The objective of the present study was to determine the impact of biweekly injections of a prolonged-release formulation of bST on the synthesis and percentage of fatty acids, phospholipid, and cholesterol in milk fat and on the melting properties of the milk fat produced by Holstein cows. Additionally, the influence of stage of lactation on the synthesis and percentage of individual milk fatty acids and the melting properties of milk fat were characterized.

MATERIALS AND METHODS

Study Design and Milk Sampling

Eighteen lactating Holstein cows were uti- lized in this study. The 18 cows were a subgroup of a larger study involving 79 cows (7). Although the larger group of cows calved over a 7-mo period, the subgroup cows all calved within a 6-wk interval.

After parturition, cows were individually housed in a tie-stall barn with automatic venti- lation and a 24-h light regimen of c a 258 lx (14 h/d) to 54 lx (10 Wd) at shoulder height of the cows. More complete details of animal management are described elsewhere (7).

At 60 f 3 d postpartum, cows were ran- domly assigned to one of two treatments. There were 9 cows in each treatment group with an equal number of primiparous (n = 5) and multiparous (n = 4) cows among treatment groups. Cows received either 1.3 ml of a prolonged-release formulation containing 500 mg of n-methionyl bST [sometribove, pro- vided by Monsanto Agricultural Co., St. Louis, MO, sometribove is the nonproprietary name for n-methionyl somatotropin established by the USAN program in accordance with the Federal Food, Drug and Cosmetic Act (13)] or an equal volume of excipient (control). Treat- ments were administered at 14-d intervals (injection interval) by intramuscular injection in one of four alternating sites (upper or lower, right or left posterior semitendinosus). Treat- ment continued until dry-off or until the com- pletion of 25 injection intervals (410 f 3 d postpartum).

Cows were milked twice daily at 1200 and 2400 h. Milk weights were obtained using calibrated weigh jars. Consecutive p.m. and a.m. raw milk samples were taken 1 d each wk from each cow for 40 wk of lactation (wk 2 to 41 postpartum). During the treatment period, the sampling days represented d 5 and 12 of each 2-wk injection cycle. After milk weight was determined, milk in the weigh jar was air agitated, and the sample was taken. Milk samples were cooled to 4'C after collection. The cold p.m. and am. milk samples were mixed and composited in equal volumes on an individual cow basis. The percentage of milk fat was determined in duplicate on all weekly composite samples by infrared analysis (4). These determinations were independent of those previously reported by Bauman et al. (7).

Milk Fat isolation

Subsamples of the weekly composite milk samples were frozen in a blast freezer and stored at -2o'C until all cows completed 41 wk of lactation. For each sampling week, the frozen milk samples from each of the 9 control

Journal of Dairy Science Vol. 75, No. 7, 1992

1796 LYNCH ET AL..

cows were rapidly thawed in a microwave oven and composited in equal volumes. Sam- ple temperature was 4 o ' C after thawing. The same procedure was conducted for the milk from the 9 cows in the bST group. Thus, there were two weekly pooled milk samples (one per treatment group) for each of the 40 sampling wk (wk 2 to 41 postpartum).

Milk fat from each pooled milk sample was extracted in duplicate by the Mojonnier method (3). Ether was evaporated under a vacuum at W C , and the milk fat was stored at -2O'C as a 2.3% solution in chloroform. The solutions of chloroform containing fat were tempered to 23'C prior to removing aliquots for analysis. Chloroform was evaporated from the sample with nitrogen.

Determination of the Fatty Acid Composition of the Fat

Approximately .023 g of milk fat was used for fatty acid analysis. All analyses were conducted in duplicate. Milk fat was saponifkd using methanolic KOH followed by boron trifloride catalyzed methylation of the free fatty acids (1). Fatty acid methyl esters were extracted with hexane.

Fatty acid methyl esters were separated and quantitated using a Hewlett Packard (Avon- dale, PA) 5890A dual column gas chromatograph with dual flame ionization detectors and a Hewlett Packard 3392A integrator. Stainless steel columns (1.8 m x 2 mm i d ) were packed with 10% Silar 1OC on 100/120 mesh Gas Chrom Q II (Alltech Associates, Inc., Deer- field, E). After sample injection, column temperature was held for 2.5 min at 65'C and then increased at a rate of 8'C/min to a final temperature of 160'C (held for 45 min). Nitrogen carrier gas flow measured at 65'C at the inlet was 20 ml/min. Injector and detector temperatures were 250 and 350'C, respectively.

Fatty acid methyl esters were identified by comparison of their retention times with those of high purity fatty acid methyl ester standards (Alltech Associates, Inc.; Nu Chek Prep, Inc., Elysian, MN). A simulated milk fatty acid quantitative reference standard was prepared by combining known weights of individual free fatty acids (C4, c67 c8, CIO, C12, c14.

subjected to the same saponification, methyla- C14:lr C16r c16:1, ClSv C18:lr and c18:2) and

tion, and GLC analysis conditions as milk fat samples. A combined recovery and response factor for each individual fatty acid was calculated from the ratio of known weight percentage for each fatty acid methyl ester to measured area percentage. These factors were used to adjust the relative area percentages of the individual fatty acid methyl esters for the ex- perimental samples. The recovery and response factors for individual fatty acids were consistent throughout the study. Production of individual fatty acids (grams per day) was calculated, accounting for glycerol and assuming that all of the fatty acids present in the original milk fat were in the form of triglycerides.

Linolenic acid (C18:3) and other fatty acids greater than 18 carbons in length accounted for less that 1% of the fatty acids present in ex- perimental Samples and were not reported. Fatty acids normally present in milk in minor quantities, such as C15, C17, and branch- chained fatty acids, also were not reported.

Total Cholesterol Content of Milk Fat

Duplicate milk fat samples were weighed (ca. .075 g) into test tubes. Exactly .3 mg of an internal standard, 5-a-cholestane (Alltech As- sociates, Inc.), was added, followed by the addition of 3 ml of 10% potassium hydroxide in 70% ethanol and four drops of benzene. Contents of the sealed and capped test tube were heated in an 85'C water bath for 30 min, followed by cooling and the addition of 3 ml of distilled water. Cholesterol was extracted into 6 ml of hexane in a series of 2-ml extrac- tions. Approximately half of the hexane was evaporated with nitrogen in order to concen- trate the sample. Peak areas of cholesterol and 5-a-

cholestane were determined isothermally at 270'C using a Hewlett Packard 5890A gas chromatograph with a flame ionization detector, a single silanized glass column (1.8 m x 2 mm i d ) packed with 3% OV-17 on 100/120 mesh Gas &om Q, and a Hewlett Packard 3392A integrator. Nitrogen carrier gas flow at the inlet was 28 mumin. Injection and detector temperatures were 290 and 3WC, respective-

The peak area ratios of cholesterol:5-a- cholestane for a sample were compared with a standard curve of cholesterol:5-a-cholestane

ly.


SOMATOTROPJN AND MILK FATTY ACID COMPOSITION 1797

ratios generated from known amounts of cholesterol (Nu Chek Prep, Inc.) The standard curve was prepared daily by subjecting .3 mg of 5-acholestane, .075 g of tripalmitin (Nu Chek F'rep, Inc.), and a range of cholesterol concentrations (.075 to .524 mg) to the same conditions described for the sapodication, extraction, and concentration of the samples. The cholesterol content of the milk fat samples was calculated from the standard curve and the original amount of milk fat used for the analysis.

Total Phosphollpld Content of Mllk Fat

The phospholipid content of the milk fat samples was determined in duplicate using the method of Rahaja et al. (25). A standard curve was prepared daily with bovine lecithin (3.93% phosphorous; Matreya, Inc., Pleasant Gap, PA).

Thermal Properties of Mllk Fat

Milk fat samples from 5 wk during the pretreatment period (wk 2, 3, 5, 7, and 9 postpartum) and 8 wk during the treatment period (wk 13 to 14,21 to 22,29 to 30, and 37 to 38 postpartum) were selected for analysis. A higher frequency of analysis was used during the pretreatment because the greatest changes in the fatty acid composition of milk fat occur during early lactation. During the treatment period, the frequency of analysis represented two samples (consecutive 5 d and 12 d postinjection) within an injection interval every 6 W k

Melting characteristics of the milk fat samples were determined in duplicate by differential scanning calorimetry (DSC) using a Perkin-Elmer (Norwalk, CT) DSC-2 (range = 1 mcal/s). The milk fat was melted completely, and about 7 mg were sealed in an aluminum DSC sample pan for analysis. Milk fat samples were tempered at 52'C for 5 min, cooled (5'C/ min) to -53'C, and held for 5 min. The DSC was calibrated and monitored using benzoic acid (122'C), tristeatin (73"C), p-xylene (13"C), and o-xylene (-25'C) as melting point standards. Baseline thermograms with empty sample pans were run daily, and the slope was adjusted appropriately.

Onset and ending temperatures of melt were determined, and the relative percentages of low

(onset to 1.8"C), medium (1.8 to 24.8'C), and high (24.8'C to end) melting fat were determined using a planimeter to measure the area under each zone of the melting curve.

Statlstical Analysis

Data for milk and fat production for the 18cow subgroup were compared with the data reported elsewhere for the entire group of 79 cows (4) to determine whether the response of the 18-w subgroup to bST treatment was typical of that observed for the full group. For this comparison, means of the determinations were calculated for each cow for the 2 wk immediately prior to initiation of treatment (wk 8 and 9 postpartum) and for determinations obtained weekly for the first 32 wk of treatment (wk 10 through 41 postpartum). Milk and fat production during the treatment period were covariately adjusted for pretreatment levels. The pretreatment and treatment means were analyzed for the effect of treatment group, parity, and the interaction of treatment group and parity using ANOVA as described previously (4). Production responses of the bST and control groups for both the 1 8cow subgroup and 79-cow group were independent of parity. Thus, results are presented for the treatment group effect only.

RESULTS

Mllk and Mllk Fat Production

The 18 cows in this study (9 control and 9 bST) were selected from the larger group of 79 cows (39 control and 40 bST). A comparison of the milk and fat production for both groups is shown in Table 1. Milk production and fat production (kilograms per day) were increased similarly by bST administration for both the 79cow group and the 18-cow subgroup. For the subgroup, the increase averaged 13% for milk production and 15% for milk fat production, whereas the increases for the full group were 9 and 14%. respectively.

Average Mllk Fatty Acld Composttion and Production

Average fatty acid composition and average daily production of milk fatty acids for the

Journal of Dairy Science Vol. 75. No. 7. 1992

1798 LYNCH ET a. TABLE! 1. Comparison of the effect of biweekly injections of bST on milk production and fat production for the full group of 79 cows and the subgroup of 18 cows.

Means'

Period2 Roduction Groud Control bST SEM P

Pretreatment Milk 79 18

Fat 79 18

Treatment Milk 79 18

Fat 79 18

W d ) - 32.8 31.8 33.1 32.5

1.05 1.06 1.14 1.05

26.4 28.9 26.7 30.1

.94 1.07

.92 1.06

.7 1.3 .03 .05 .4 .7 .02 .03

.36

.76

.85 2 3

eo1 <01 <.01 <.01

~ ~~ ~~~

'Least squares means during the preweatment period and least squares means covariately adjusted for pretreatment

2Determinations made at 7 d intervals for 2 wk during the pretreatment period (wk 8 and 9 postpartum) and for 32

339 Control and 40 bST-treated cows for the 7 k o w group, and 9 control and 9 bST-treated cows in the

differences during the treatment period.

wk during the treatment period (wk 10 to 41 postpartum).

l8cow subgroup. Values for the entire group of 79 cows were reported elsewhere (4, 7).

pretreatment period (wk 2 to 9 postpartum) and for the treatment period (wk 10 to 41 postpartum) are presented in Tables 2 and 3, respectively. Average fatty acid composition and production of milk fat were similar for the control and bST groups during the pretreat-

ment period Fable 2). During the treatment period, the relative fatty acid composition of milk fat produced by the two groups was very comparable, but the bST group produced an average of 10.8% more milk fatty acids per day than the control group (Table 3). Produc-

TABLE 2. Average composition and production of milk total fatty acids, glycerol, cholesterol, and phospholipids during the pretreatment period.]

Composition Production

Lipid Control bST Control bST - (Relative weight %) - Wd)

Fatty acid c4 3.6 4.1 31.6 35.1 c6 2.4 2.4 21.8 21.8 c8 1.1 1.1 10.6 10.5 c10 2.5 2.5 24.6 23.2 c12 3.1 2.9 29.9 27.4

c141 2.2 2.1 21.8 20.4 c16 26.8 26.3 268.3 257.1 C161 4.5 4.4 45.1 43.1 c18 11.1 11.5 112.4 113.7 C181 28.4 28.7 287.1 283.1 c18:2 3.8 3.9 38.2 38.7 Total 100.0 100.0 994.1 970.5

c14 10.5 10.1 102.7 96.4

Wd) (% of fat) Glycerol 12.1 12.2 137.1 134.9 Cholesterol .319 .317 3.61 3.48 Phospholipid .639 .661 7.02 7.36

'Averages of determinations on pooled milk samples obtained and analyzed by week postpartum during the pretreatment period (wk 2 to 9). Nine cows for each group. Data represent actual unadjusted means.


SOMATOTROPIN AND MILK P A W ACID COMPOSITION 1799

TABLE 3. Average composition and production of milk total fatty acids, glycerol, cholesterol, and phospholipids during the treatment period.'

Production

Response Composition Lipid Control bST Control bST above control

- (Relative weight %) - Wd) (%I Fatty acid c4 2.9 2.8 20.7 22.5 8.7

2.2 2.2 16.9 18.3 8.3 8.8

c6 1.2 1.1 9.1 9.9

c 10 3.0 2.9 23.8 25.9 8.8 ClZ 3.9 3.8 31.4 34.2 8.9

C8

14 12.4 12.1 102.2 109.9 7.5 c141 3.1 3.2 25.8 28.9 12.0

C16:l 4.3 4.3 35.9 39.9 11.1 c16 32.7 33.2 271.5 305.4 12.5

c18 8.5 7.9 71.1 73.1 2.8 C18:l 23.0 23.7 192.3 219.3 14.0 C18:Z 2.8 2.8 23.7 25.8 8.9 Total 100.0 100.0 824.4 913.1 . . .

Glycerol 12.1 12.1 113.7 125.6 10.5 Cholesterol .388 .405 3.64 4.19 15.1 Phospholipid .743 .733 7.03 7.64 8.7

period (wk 10 to 41). Nine cows for each group. Data represeat actual unadjusted means.

(% of fat)

'Averages of determiuations on pooled milk samples obtained and analyzed by week postpartum during the treatment

tion increases for individual fatty acids in the bST group relative to the control group during the treatment period ranged from 7.5 to 14.0% with the exception of c18, which increased only 2.8% (Table 3).

Glycerol accounted for approximately 12% of the weight of the milk fat fraction (Tables 2 and 3). Thus, approximately 12% of the increase in milk fat production because of bST treatment was due to increased secretion of the glycerol portion of the triglyceride Fable 3).

Milk Fatty Acid Composltion and Production over the Lactation

The lactation curves for weight percentages of individual fatty acids were comparable for the control and bST-treated cows (Figures 1 and 2), although some trends in the temporal pattern of percentages of c 1 8 and c18:1 are noteworthy. Although the percentage of c 1 8 was similar for both groups during the first 13 wk of treatment (10 to 22 wk postpartum), the percentage of c l 8 was slightly but consistently lower for the bST-treated cows than for the control cows for the remainder of the treatment

period (7.6 vs. 8.6%). However, the percentage of c18:1 was comparable for the two groups during the Iatter part of the treatment period but tended to be higher for the bST group than for the control group during the first 13 wk of treatment (24.6 vs. 22.6%). Milk fat production (kilograms per day)

increased for the bST group relative to the control group approximately 8 wk after treatment began (Figure 3). As treatment continued, the difference in daily milk fat production between the two groups increased.

Changes during lactation in daily production of most fatty acids by the bST group relative to the control were very similar for the fist 13 wk of the treatment period (Figures 4 and 5). During the remainder of the treatment period, the daily production of short- and medium-chain fatty acids by the bST group was greater than that of the control group. There was little difference in daily production of c 1 8 between the control and bST groups during any part of the lactation (Figure 5D). Daily production of Clg:1 by the bST group was higher than that of the control group for the full treatment period (Figure SE).

Journal of Dairy Science Vol. 75. No. 7, 1992

LYNCH ET AL.. 1800

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2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 WEEK POSTPARTUM WEEK POSTPARTUM

Figure 1. Temporal pattern of relative weight percentage (g/100 g) of the milk fat fatty acids C4 (A), c6 (B), Cs (C), C1o @), Clz (E). and C14 0. Treatments began at wk 10 postpartum and involved biweekly injections of bST (.; n = 9 COWS) or excipient (0; n = 9 cows). For wk 10 to 41 postpartum, points on the grid line represent analysis of 5-d postinjection milk samples, and points between the grid lines represent analysis of 12-d postinjection milk samples.

Fatty Acid Composition and Production Wlthln an injection Interval

The percentages and production of some fatty acids in milk fat produced by the bST group during the treatment period cycled within each 146 injection interval. Beginning with the initiation of treatment at 10 wk ps t - partum, weekly milk sampling corresponded to either 5 d postinjection (even numbered wk 10 through 40 postpartum) or 12 d postinjection (odd numbered wk 11 through 41 p o s t p m ) .

Cycling was identified by subtracting each 12d postinjection observation from the previ- ous 5d postinjection observation (e.g., wk 10 minus wk 11, wk 12 minus wk 13, etc.) and by applying a binomial sign test to the 16 differences. To minimize the influence of stage of lactation on the identification of cycling, sig- nificance was set conservatively at P = .01.

Within a single injection interval, the percentages of cg, Clo, Cl2, and C14 were significantly lower in the 5d postinjection sample


SOMATOTROPIN AND MILK FATI"Y ACID COMPOSITION 1801

2 6 10 14 18 22 26 30 34 38 42

2 6 10 14 18 22 26 30 34 38 42

2 6 10 14 18 22 26 30 34 38 42 WEEK POSTPARTUM

Figure 2. Temporal pattern of relative weight percentage (g/lOO g) of the milk fat fatty acids C14:1 (A), c16 (B), c16:1 (c), cis P), C18:1 (E), and c1&2 0. Treatments began at wk 10 postpartum and involved biweekly injections of bST (q n = 9 cows) or excipient (0; n = 9 cows). For wk 10 to 41 postpartum, points on the grid line represent analysis o f 5 d postinjection milk samples, and points between the grid lines represent analysis of 12-d postinjection milk samples.

than in the 12-d postinjection sample (Figure 1, C to F). In contrast, the percentage of c18:1 was significantly greater at 5 d postinjection than at 12 d postinjection (Figure 2E). Relative percentages of individual fatty acids did not cycle for the control group (Figures 1 and 2).

Total milk fat production (Figure 3) and production of c16:1, c18:1, and c18:2 (Figure 5, C, E, and F) by the bST group were significantly greater at 5 d postinjection than at 12 d postinjection. Quantitatively, c18:1 made the major contribution to cycling of total milk fat

production. Production of other individual milk fatty acids at 5 and 12 d postinjection varied but did not form a consistent cycling pattern (Figure 4, A to F; Figure 5 , A, B, and D). No cycling of production of total fat Figure 3) or individual fatty acids was observed for the control group (Figures 4 and 5).

Influence of Stage of Lactation

Stage of lactation had a large and similar effect on fatty acid composition for both con-


1802 LYNCH ET AL.

trol and bST groups. Increases in percentages of c 6 through c 1 6 (Figure 1, B to F; Figure 2, A and B) and decreases in percentages of C4, CIS, c18:1, and c18:2 (Figure 1A; Figure 2, D to F> were observed in early lactation. In gen- eral, the rate at which the percentages of cfj to c 1 6 increased in early lactation was related to fatty acid chain length. As fatty acid chain length increased from c 6 to c16, the rate of increase of individual fatty acids decreased, and the postpartum week at which a relatively constant percentage was achieved also increased. Percentages of most fatty acids stabi- lized during midlactation (Figures 1 and 2). From mid to late lactation, the percentages of C, to c 1 6 and c 1 8 remained relatively constant (Figure 1, A to F; Figure 2, A, B, and D), c16:1 and c18:2 declined (Figure 2, C and F), and c18:1 increased slightly (Figure 2F). ‘

Production of milk fat (Figure 3) and individual fatty acids (Figures 4 and 5) over the lactation followed similar temporal patterns for both the bST and control groups, although the persistence of production in late lactation tended to be greater for the bST group. Milk fat production decreased over the lactation (Figure 3). During early lactation, production of c 6 to c 1 6 increased (Figure 4, B to F; Figure 5, A and B); production of C4, CIS, c18:1, and c18:2 decreased (Figure 4A; Figure 5, D to F); and production of C16. was steady Figure 5C). Production of most fatty acids was stable during midlactation, although production of c18:2 continued to decline (Figure 5F9. During late lactation, production of all fatty acids except c 1 6 and c18:1 gradually declined (Figures 4 and 5). The production of c18:1 was relatively stable in late lactation (Figure 5E), whereas production of c 1 6 decreased for the control group but remained steady for the bST-treated cows (Figure 5B).

Cholesterol and Phosphollplds

The average percentages of total cholesterol and phospholipids in milk fat were similar for the control and bST groups both prior to (Ta- ble 2) and during treatment (Table 3). Average daily production of cholesterol and phospholipids increased 15.1 and 8.7%, respectively, with bST administration. Temporal patterns of percentage and production of cholesterol and phospholipids are shown in Figures 6 and 7. Within an injection interval, a cycling pattern

Journal of Dairy Science Vol. 75, No. 7. 1992

was significant (P < .01) for the percentage (Figure 6), but not production (Figure 7), of cholesterol and phospholipids in the milk fat from the bST-treated cows. Percentages were lower at 5 d than at 12 d postinjection (P < .Ol). No cycling within an injection interval was observed for the control group. For both the control and bST groups, the effect of stage of lactation was most apparent for cholesterol; the largest increases in percentage (Figure 6) and production (Figure 7) were observed in early lactation.

Thermal Properties of Milk Fat

Mean temperatures for the onset and end of melting for milk fat produced by the control and bST groups during the pretreatment and treatment periods are shown in Table 4. Tem- perature differences between the two groups for both onset and end of melt during either the pretreatment or treatment periods were negligible. Stage of lactation, as reflected by comparisons of pretreatment and treatment period data within a group, had little influence on onset and end of melt.

The relative percentages of low, medium, and high melting fractions for control and bST groups with time during lactation are shown in Figure 8. The medium melting fraction accounted for about 55 to 60% of the total melting area and was similar for both groups. The relative percentage of the high melting fraction was greater in the control group than

6 10 14 18 22 26 30 34 38 1

WEEK POSTPARTUM

Figure 3. Temporal pattern of milk fat production Treatments began at wk 10 postpartum and involved biweekly injections of bST (.; n = 9 cows) or excipient (0; n = 9 cows). For wk 10 to 41 postpartum, points on the grid line represent 5-d postinjection production, and points between the grid lines represent 1 2 4 postinjection pmduc- tion

SOMATOTROPIN AND MILK FATTY ACID COMPOSITION

8 50

B 0

n

40

t- 2 30

2 20

0" 10

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2 6 10

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22 26

1803

30 34 38 42

2 6 10 14 18 22 26 30 34 38 42

2 6 10 14 18 22 26 30 34 38 42 WEEK POSTPARTUM

figure 4. Taporal pattern of production of the milk fat fatty acids C4 (A), C6 (B), Cs (0, Clo @), C12 (E!), and 1214 0. Treatments began at wk 10 postpartum and involved biweekly injections of bST (.; n = 9 cows) or excipient (0; n = 9 COWS). For wk 10 to 41 postpartum, points on the grid line represent 5-d postinjection production. and points between the grid lines represent 124 postinjection production.

in the bST group for the first half of the treatment period. However, this appeared to be the result of inherent differences between the two groups of cows, because this same difference was also apparent during the pretreatment period. Variation in the relative proportions of the melting fractions between consecutive ob- servations within an injection intervd was observed for both the control and bST groups, and no cycling was evident (Figure 8).

Stage of lactation caused the greatest shift in relative proportions of the melting fractions of milk fat @igure 8). The largest effect of stage of lactation occurred for the high and low melting fractions during the pretreatment period. At the onset of lactation, the lower melting fraction represented a greater proportion of the total melting area than the high melting fraction, but, by wk 13 postpartum, this relationship was reversed.

Journal of Dairy Science Vol. 75, NO. 7, 1992

1804 LYNCH ET AL.

Y .

Z 50- 0 - I- 45:

35:

2 40-

30: 7- .. 25- w . 0- 20,

. . . . . . . . . . . . . . . . . .

2 6 10 14 18 22 26 30 34 1 42 WEEK POSTPARTUM

2 6 10 14 18 22 26 30 34 38 42

WEEK POSTPARTUM

Figure 5. Temporal pattern of production of the milk fat fatty acids C14. (A), c16 (B), C16. (C), c18 @), c18:1 @), and c18:2 0. Treatments began at wk 10 postpartum and involved biweekly injections of bST (.; n = 9 cows) or excipient (0; n = 9 cows). Por wk 10 to 41 postpartum, points on the grid line represent 5-d postinjection production, and points between the grid lines represent 12-d postinjection production.

DISCUSSION

Milk Fatty Acids and bST Administration

Total milk fat production increased with bST administration (Table 1) because of an increased output of both de novo and preformed fatty acids (Table 3). There was little difference (5 .7% absolute for any individual fatty acid) between the control and bST- treated cows in the fatty acid composition of the milk fat averaged across the 32 wk of

treatment (Table 3). However, some transient shifts in fatty acid composition were observed at the beginning of bST treatment. The percentage of c18:1 (Figure 2E) tended to be slightly higher (ca 2.1%) in the milk from the bST-treated cows during the first 13 wk of treatment (wk 10 to 22 postpartum). Bauman et al. (7) reported, for the full group of 79 cows, that the control cows returned to positive energy balance at about wk 12 postpartum, whereas the bST-treated cows achieved positive energy balance on about wk 17. Assuming

J o d of Dairy Science Vol. 75, No. 7, 1992

SOMAToTRopIN AND MILK FATTY ACTD COMPOSITION 1805

TABLE 4. Effect of biweekly injections of bST on the onset and end of melting temperatures of milk fat.l

Group PaiOd Meltbg Control bST Difference

Pretreatment onset -33.7 -35.1 1.4 EQd 36.4 36.2 .2

Treatment onset -32.4 -31.8 -.6 End 37.4 37.1 .3

‘Averages of determinations on pooled milk samples obtained and analyzed during the pretreatment period (wk 2.3. 5, 7, and 9 postpamun) and treatment period (wk 13, 14, 21,22, 29, 30, 37, and 38 postpartum). Nine cows for each group. Data represent actual unadjasted means.

that the energy balance data (7) for the 79-cow group are representative of the l8-cow subgroup, the smal l increase in concentration of C1gZ1 during early bST treatment would be expected, because it took the bST- treated cows longer to achieve positive energy balance. Others (2, 8, 12) have observed increases in the relative percentage of c18:1 in milk fat at the initiation of bST treatment whenever cows are in negative energy balance and are mobilizing body fat stores.

In contrast to C18:lr the percentages of Clg in the milk from bST-treated and control cows were similar during the first weeks of bST administration (Figure 2D). As bST treatment progressed, the percentage of Clg in the milk fat from the bST-treated cows was consistently

1 0 0 L z 9

6 8

0 5 5

$ g 7

p .3

0 8 ” 6

z * 4

2 6 10 14 18 22 26 30 34 38 42 0 2

WEEK POSTPARTUM

Figure 6. Temporal pattern of percentage of total cholesterol (CHOL) (bST, .; control, a) and phospholipid (bST, e; control, 0) in milk fat. Treatments began at wk 10 postpartum and involved biweekly injections of bST (n = 9 cows) or excipient (n = 9 cows). For wk 10 to 41 postpartum, points on the grid line represent analysis of 5-d postinjection milk samples, and points between the grid lines represent analysis of 12-d postinjection milk samples. Phospholipid data from the bST group were not available for wk 37 postpartum.

lower (ca. 1.1%) than that in the milk fat produced by control cows. Averaged over the treatment period, the relative increase in c18 production with bST administration was con- siderably less than increases in production of the other fatty acids (Table 3). A slightly lower percentage of C1g with bST administration was observed in all of the other investigations that measured milk fatty acid composition during bST administration (2, 8, 12, 16).

The primary fatty acids available in the blood for milk fat synthesis are c16, Clg, and c18:1 (18). Although the relative increase in percentage of C18:1 during periods of energy deficit is logically the result of increased entry of preformed fatty acids into the mammary gland from the blood, it is interesting that the

WEEK POSTPARTUM

Figure 7. Temporal pattern of production of total cholesterol (CHOL) (bST, .; control, 0) and phospholipid @ST, .; control, 0) in milk fat. Treatments began at wk 10 postpartum and involved biweekly injections of bST (n = 9 cows) or excipient (n = 9 cows). For wk 10 to 41 postpartum, points on the grid line represent 5d postiqjeo tion production, and points between the grid lines represent 124 postinjection production. phospholipid data from the bST group were not available for wk 37 postpartum.


1806 LYNCH ET A L

I I U +! $ 30 2 !j 20 E 10

0 5 1.0 1 5 20 25 30 35 40 WEEK POSTPARTUM

Figure 8. Temporal pattern of the relative percentage of low (bST, .; control, o), medium (bST, 4 control, 0) and high (bST, A; control, A) melting dyceride fractions of milk fat. Treatments began at wk 10 postpartum and involved biweekly injections of bST (n = 9 cows) or excipient (n = 9 cows).

percentage of c16 and CIS did not also increase (Figure 2, B and D). The percentage of C18 apparently was slightly lower in the milk from bST-treated cows than in the milk from control cows during the latter part of the treatment period when cows were in positive energy balance (Figure 2D). A possible explana- tion for a differential effect of bST administration on c18 versus c18:1 is that bST treatment enhanced the activity of mammary stearyl-coenzyme A desaturase. Desaturase activity in the Nminant mammary gland is normally high (15). The increase in C14:. and c16:1 production with bST treatment (Figure 5, A and C) supports the idea that mammary desaturase activity may increase with bST administration. The percentage of c16 may not have been influenced by bST treatment (F@re 2B), because c16 originates from two sources: the blood and by de novo synthesis in the mammary gland (22). It may be possible that, as the amount of c16 contributed by one source increases, the amount contributed by the other source decreases, resulting in no net change in t O t d c16.

Cycling Within 14-d Injection interval

Greater production at 5 d than at 12 d postinjection was reported for milk volume and production of milk components @e., fat, protein, lactose, total solids, and SNF) by the bST-treated cows in the full group of 79 cows

(4, 7). Bauman et al. (7) characterized the change in daily milk production within a bST injection interval as parabolic; production maxima occurred at approximately 7 to 9 d postinjection.

The cycling of total fat production (Figure 3) within each injection interval was primarily because of changes in production of c18:1, because the contribution of c16:1 and c18:2 to total milk fat production is relatively small. Although c16 (22) and, presumably, c16:1 production can be either de novo or preformed, cycling was observed in production of C16.. c18:1, and c18:2 and not in the production of the short- or medium-chain fatty acids, sug- gesting that the cycling over the injection interval was related to changes in the uptake of preformed fatty acids and, possibly, fatty acyl- coenzyme A desaturase activity.

Changes in the production of individual fatty acids were reflected in the relative percentages of fatty acids in milk fat. Because the production of C16:., C18:1. and CIS. cycled @gh on d 5 postinjection), and because the production of the other fatty acids did not cycle (Figures 4 and 5), the relative percentages of these three fatty acids were higher at 5 d than at 12 d postinjection (Figure 2, C, E, and F). As a result of the dilutional effect of higher production of C16:., C181, and c18:2 on d 5, the relative percentages (but not production) of other fatty acids, such as c8 through C14, were significantly lower at 5 d postinjection.

Overall, the primary effect of bST administration was to increase both the de novo synthesis of fatty acids in the mammary gland and uptake of preformed fatty acids from the blood. The cyclic pattern of production of c18:1 within the injection interval explains most of the variations in relative percentages of individual fatty acids within an injection interval.

Stage of Lactatlon and Fatty Acid Synthesis

The translactational patterns of fatty acid percentage (Figures 1 and 2) and production (Figures 4 and 5) were similar for both the control and bST-treated cows. These profiles also clearly illustrate that the effects of stage of lactation on milk fat composition are much


SOMATOTROPIN AND MLLK PATTY ACID COMPOSITION 1807

larger than the small transient changes associ- ated with bST administration. The temporal pattern of percentage and production of individual fatty acids appears to be related to their metabolic origin. Percentages and production of the preformed fatty acids, as reflected by the c18 series (Figure 2, D to F; Figure 5, D to F), were relatively high at the onset of lactation, when cows were in negative energy balance, and gradually declined for the first third of lactation as energy balance became less negative (7). The high production of preformed fatty acids at the onset of lactation are thought to reflect the mobilization of body adipose stores (10, 23, 27). As lactation progresses and energy balance becomes more positive, lipid mobilization decreases, and, consequently, the proportion of preformed fatty acids in milk fat decreases (10, 23, 27).

The percentages (Figure 1, B to F; Figure 2A) and production (Figure 4, B to F; Figure 5A) of most short- and medium-chain fatty acids (c6 to C14.) were relatively low at parturition and increased over the first third of the lactation, reflecting increasing de novo synthesis of fatty acids by the mammary gland. An increase in percentages of c6 to C14:1 over the first third of lactation has been observed previously (10, 23, 27). However, it appeared that the rate at which percentage and production of each of the de novo fatty acids (c6 to C14.) increased during early lactation was related to fatty acid chain length: the longer the carbon chain, the longer production and percentage took to reach a stable level.

There are at least two possible mechanisms that could explain limited de novo synthesis of fatty acids in early lactation. First, the bio- chemical machinery may not be optimize& For example, acetyl-coenzyme A carboxylase, which is thought to be a ratelimiting enzyme in fatty acid synthesis (17), is inhibited by both long-chain free fatty acids and fatty acyl- coenzyme A esters (20, 21). MelIenberger et al. (17) demonstrated that acetyl-coenzyme A carboxylase activity in the mammary tissue increased over the first 40 d of lactation postpartum and closely paralleled in vitro rates of de novo fatty acid synthesis. A second possi- bility is that the availability of substrates may be limited in early lactation. The supply of acetate (carbon source of fatty acids), glucose (source of NADPH and triglyceride-glycerol),

or both may be insufficient to allow maximum de novo fatty acid synthesis. In this scenario, supply of substrate gradually increases as voluntary feed intake increases over the first weeks postpartum.

The c16 series fatty acids in milk represent a unique situation because they can originate both preformed from the blood and by de novo synthesis in the secretory cells (22). In early lactation, the relative contribution of preformed c16 likely starts out high and decreases, whereas de novo synthesis of c16 starts out low and increases. The decrease in the relative amount of preformed c16 may be more than offset by the increase in de novo synthesis of c16, because total production of c16 increases during early lactation (Figwe 5B). This could impact the thermal properties of milk fat, because the stereospecific distribution of c16 on milk triglycerides may depend on whether the c16 originates from the blood or is made de novo in the mammary gland (5).

The translactational changes in butyric acid (C4) percentage pigure 1A) and production ( F i p 4A) differed from the other de novo fatty acids and resembled that of the preformed fatty acids c18 and C181. The lactation c w e s for the percentage and production of C4 are similar to those reported by others (10.23) and suggest that the primary substrate or synthetic pathway of C4 production differs from that of the other de novo fatty acids. Indeed, Palm- quist et al. (22) demonstrated that approximately 50% of the butyrate in milk fat was synthesized from acetate and that approximately 50% came directly from p- hydroxybutyrate (absorbed from the blood). Butyrate synthesized from p-hydroxybutyrate is independent of acetyl-coenzyme A carboxylase activity. In early lactation, an increase in p-hydroxybutyrate in the blood and an increase in uptake by the mammary cells could result in relatively high C4 production, especially because synthesis of other de novo fatty acids was limited during this time (Figure 4, B to F; Figure 5A).

Mllk Cholesterol and Phosphollplds

The cholesterol and phospholipid contents of milk fat produced by bST-treated cows and untreated cows during the pretreatment (Table 2) and treatment period (Table 3) were similar.


1808 LYNCH ET AL.

Daily production of both cholesterol and phospholipids increased because of bST treatment by amounts similar to the other components of the milk fat fraction (Table 3). Cholesterol content of milk is of concern for human nutri- tion. The USDA indicates that the cholesterol content of milk with 3.3% fat is about 33 mg/ 244-g (8-02) glass (28). During the treatment period, cholesterol content (calculated from Table 3, assuming 3.3% fat) of milk from the control and bST groups was similar to the USDA value: 31.2 and 32.6 mg/244-g (842) glass, respectively. Phospholipids are an im- portant component of the milk fat globule membrane and may influence the stability of milk fat. The concentration of phospholipid in milk was not influenced by treatment of cows with bST (Table 3).

Cholesterol and phospholipid contents of milk changed with stage of lactation for both treatment groups (Figure 6). Effects of stage of lactation were larger than any differences be tween treatment groups. Cholesterol and phospholipid content of milk were lower during early lactation than in late lactation for both groups (Figure 6). Nutritionally, the change in cholesterol content of milk with stage of lactation would represent a change from about 26 mg (prior to wk 10) to about 32 mg of cholesterol/244-g (8-02) glass (after wk 10). The temporal pattern of change in the cholesterol content of milk with stage of lactation is similar to that observed for the de novo fatty acids with stage of lactation.

Thermal Properties of Milk Fat

Changes in the thermal characteristics of milk fat can influence the texture and meltabil- ity of high fat dairy products. Thermal properties of milk fat are influenced by both fatty acid composition and positional distribution of fatty acids on triglycerides (14). Changes in the diet of dairy cows can markedly influence the fatty acid composition and thermal properties of milk fat (14). The beginning and ending melting temperatures for milk fat were similar for bST-treated and control cows (Table 4). There were large and similar changes in the relative proportions of low and high melting fractions of milk fat with stage of lactation for both bST-treated and control cows (Figure 8). These changes reflect the substantial changes

in fatty acid composition of milk fat that occurred with stage of lactation for both bST- treated and control cows. Changes in the melting characteristics of milk fat because of stage of lactation were large enough to be of practi- cal importance in the manufacturing of dairy products, but bST had no impact on the thermal properties of milk fat outside of these normal variations. The change in thermal properties of milk fat with stage of lactation is probably one of the major factors (in addition to diet) that contributes to seasonal variation (14) of the thermal properties of milk fat.

CONCLUSIONS

Treatment of lactating dairy cows with bST did not cause any changes in the chemical composition or physical properties of the milk fat fraction that were outside of the range of normal variation. The primary effect of bST administration was to increase both the de novo synthesis of fatty acids in the mammary gland and the uptake of preformed fatty acids from the blood. Composition of milk fat was influenced greatly by stage of lactation. The basis for this influence is the change in energy status of the cow and the relative contribution of preformed fat components entering the mammary gland from the blood and the contribution of de novo synthesis of milk fat components.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Patricia Nelson, Barbara B. Frederick, and Vivian Smith for their technical assistance with sample analysis.

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