Fatty Acid Composition of Milk. III. Variation with Stage of Lactation1

FattyAcid Composition of Milk. II!. Variation with Stage of Lactation 1 J. W. STULL, W. H. BROWN, CARLOS VALDEZ ~ Department of Dairy Science and HENRY TUCKER

Numerical Analysis Laboratory University of Arizona, Tucson Abstract

Variations in component fatty acids in milk of four Holstein cows were measured during their entire lactation (43-50 wk). Least-squares fits of the measured values were then made to a succession of polynomials and the added contribution to reducing the unexplained variation calculated to detennine the best-fitting function. Linear regression equations best describe the proportions of 6:0, 8:0, 16:0, and 16:1~ with only 16:0 having a negative slope. The proportion of 10:0, 12:0, 16 :iso, 17 :G 18:0, 18:iso, 18:1, and 18:2 were best described by quadratic regression equations. There were wide variations throughout the progress of lactation in the slopes of the curves in these latter cases. Cubic regression equations adequately described the proportions of 10:1, 12:1, 14:0, 14:1~ 15:0, and 18:3 fatty acids.

It has been known for some time that total daily milk produetion exhibits a characteristic rise for 3-4 wk after parturition, followed by a gradual decline until near the end of the lactation (5, 7, 18), that fat content generally varies inversely with nfilk produetion, although not necessarily in direct proportion (6, 14, 17), and that when the percentage of fat increases with advanced lactation, most of the other milk solids except laetose also increase (2, 3). Very little is known concerning variability of the fatty aeids of milk during progress of the lactation. One report (9) has shown that milk fat produced in the early stages of lactation was usually softer and has a lower melting point and higher iodine value that produced in later stages. What effects diet may have exerted on these results were not elearly defined.

This study was designed to deternfine the variation in the proportions of the various fatty acids of milk fat during the progress of lactation and to concurrently obselwe the variation in milk production, fat, and protein. Experimental Procedure

Four Holstein cows, all having calved within a ten-day period in July, were used as experimential animals. Two of the cows were identical twins; otherwise there was no close parental relationship among them. Morning and evening samples (ca. 12-hr intervals) were taken twice weekly throughout the entire lactation of each eow (range 43-50 wk). The end of the lactation was arbitrarily set at being 6 wk prior to the predicted date of the next ealving. During the experiment, the cows received a relatively uniform diet consisting of good-quality alfalfa hay and a concentrate (primarily barley, milo, and cottonseed meal). At no time were changes made in the feeding regimen, other than to routinely adjust energy consumption to meet the eow's requirements for maintenance and production according to ~lorrison (12). 5filk weights were recorded and per cent fat determined by the standard Babcock method. Per cent protein was determined by the Orange G method of Udy (19), but were not obtained during the first 7 wk of the experiment. Fatty acid components of the milk fat were determined, using gas-liquid ehromatography. Approximately 1 g of fat was separated from the milk by the TeSa fat test method ~ and dried under vacuum. The fatty acids were converted to their methyl esters by transmethylation, using sodium methoxide (10). The methyl esters were extracted in hexane and injeeted directly into the gas chromatograph. The gas chromatograph was a Perkin Ehner Model 154 with a flmne detector, using a 0.32 by 200-(qn colmlln (15% diethylene glycol suceinate on silanized Chromosorb W, 60/ 80-mesh) and an oven temperature of 200 C. Relative amounts of each acid were determined by comparison of the areas under the peaks drawn

Received for publication June 26, 1966. ~Arizona Agricultural Experiment Station Technical Paper no. 1152. -"Present address: University of Sonora, tIermo3Technical Industries, Fort Lauderdale, Florida. sillo, Sonora, Nexico. 1401

1402

J.w.

STULL ET AL

by the recorder. No adjustment was made for possible nonlinearity of detector response. C o m p a r i s o n s w e r e first m a d e b y u s e o f r e g r e s s i o n c o n s t a n t s ( 1 5 ) , to d e t e r m i n e w h e t h e r there was variation between morning and evenhlg samples. For each variable the measured values were plotted against time. Observations were made twice a week (average daily samples) for 50 wk. L e a s t - s q u a r e s fits w e r e t h e n m a d e to a succession of polynomials and the added con-

t r i b u t i o n to r e d u c i n g t h e u n e x p l a i n e d v a r i a t i o n c a l c u l a t e d to d e t e r m i n e t h e b e s t - f i t t i n g f u n c t i o n (1). Based on prior examination of the data, and knowledge of the change in the measured v a r i a b l e o v e r t i m e , p o l y n o m i a l s o n l y u p to t h e t h i r d d e g r e e w e r e fitted. E a c h o f t h e final p r e diction equations and their respective R ~ are g i v e n in T a b l e 1. T h i s m e t h o d u s e d in f i t t i n g is c o m m o n l y k n o w n as s t e p - w i s e r e g r e s s i o n a n d it is c a l c u l a t e d d i r e c t l y b y s t a n d a r d c o m p u t e r

TABLE ] :Regression e q u a t i o n s e x p r e s s i n g the v a r i a t i o n iu milk production, composition, anti c o m p o n e n t f a t t y acids with s t a g e of l a e t a a t i o n . Item

(y)

Regression e q u a t i o n

Daily milk Produetion (leg)

Y - - 16.6090 +

Fat

Y=

(ky)

.2701 T - - .007672 1 '~+ .00004365 T:* (.0561) ~ (.001292) (.00000840) .8919-- .0033 T

R?

N

rt,~

.8206

96

--.8477

.5911

78

--.7688

.0779

95

.2792

.7822

77

.3574

.6670

96

.8167

.6884

9(;

.8297

.7935

9~;

.8276

.671)7

92

.7387

.7604

96

.6351

.76,q9

81

.8205

.7818

90

.2896

.532l}

94

.3618

.7482

95

.7948

.6594

95

.7565

.6813

9{i

--.8254

.3913

94

.6255

(.0003) :Pat (9~()

Y--

Protein (%)

Y-

F a t t y Acid ( % ) 6:0

Y -

8:0 10:0 10:1 12:0 12:1 14:0 14:1 15:0 16 ~° 16:0 16:1

3.7662÷

.0(}33 T (.0/)12) 1 . 8 4 1 1 ÷ .1129 T (.0092)

.0326 T (.(1~124) Y. 1 5 6 9 + .0323 T (.0{}22) Y . 5 1 9 8 ÷ .O664 T - (.0059) Y=: .6138 .0359 T + (.0083) Y-.43(}4-+- .1097 T - (.0074) ~r-. 4 7 9 0 - - .0339 T + (.0081) Y 2 . 8 9 0 7 + .3469 T - (.0256) Y--- . 5 9 1 9 + .0504 T - (.0068) Y-.5201 + .0475 T - (.0070) Y= . 0 1 2 2 + .0210 T - (.0027) Y - - 2 7 . 7 8 5 2 - - .0808 T (.0057) Y - - 2 . 7 5 6 6 + .0118 T

.001803 T ~ + .0I)00I}839 T '~ (.000172) (.()0(}001198)

.0788 +

.000399 T" (.{}o0057) .001112 7'~ (.00{~0194) .000840 T ~ (.000071) .000991 7" (.000288) .006031 T~-t (.000590) .000870 7,~÷ (.000158) .000710 9"~q (.00(}16{~) .000125 T=' (.000026~

.o0000735 T :~ (.(1{){)(}0127)

.I)0(}IH}604 T:' (.IH}O{)(}J24) .(}(}003014 7'B (.0000o';83) .00000453 T :~ (.o0()o(}102) .0o000375 T:' (.{}0(}(}1(}4)

(.00]5) 17:0 18 is° 18:0 18:1 18:2 18:3

Y=

.0315 1 ' - .000166 (.006]) (.o00058) Iz = .1998 q- .0482 T - - .000276 (.0069) (.000066) Y--23.5689-.3062 2'q- .002004 (.0222) (.000214) Y=32.6360-.1693 T ÷ .001314 (.0235) (.000226)

T~

.5235

96

.6941

/,2

.6268

96

.7463

T"

.8245

96

--.8114

T2

.4399

96

--.4863

17 =

T ~

.5887

96

.7308

.4031

96

--.3244

Y :

.8727+

2.7492 +

.0645

T--

.000353

(.0105) (.000101) .4158 + .0585 T - - .001200 T'-'+ .00000635T ~ (.0113) (.000259) (.00000168)

" V a l u c s in p a r e n t h e s e s r c f e r to s t a n d a r d error of r e g r e s s i o n coefficient f o r value above. J. ])AIRY SCIEXCE VOIJ. 49, NO. 11

FATTY

ACIDS

programs. This gives the same results as the forward solution of the abbreviated Doolittle method, described by Anderson and Bancroft (1). While other types of funetionM forms (e.g., exponential logarithmie, fractional power, etc.) may have given better fits, there was no evidence that any of the alternatives were justified on theoretical grounds, and they would have been more difficult to interpret. Sinee the basic objective was to determine the general form of the change in the observed variable over time rather than a prediction equation, it was not considered to be of any real value to explore this question further. No attempt was made to adjust for correlation among successive observations in time.

IN

1403

?,:[ILK

~30

io ~o.g

~

o.8

oy

os STAGE OF LACTATION (WK$)

FIG. ~.° Fitted line for variation 5n daily fat produetion and per cents fat and protein with stage of lactation.

Results

I t was shown that there was no significant difference between milkings within days for individual animals for any of the variables studied. Table 1 shows the equation which best describes the data for each variable in the study. Graphs of the variables are shown in Figures 1-8. Total daily kilograms of milk produced during the lactation was best described by a cubic equation. P e r cent fat and daily kilograms of fat produced are both best described by a linear equation, with per cent fat having a positive slope and kilogrmus of fat a negative slope. During the observed period, the level of protein in milk rose slowly until about the 25th wk of the lactation. after whieh it decreased until near the end of' the lactation, then very slightly increased. Linear regression equations best describe the percentages of 6:0, 8:0, 16:0, and 16:1 appearing in the milk fat, with only 16:0 having a negative slope. The ratios of 10:0, 16 :iso, and 17:0, 18 :iso, and 18:2 were best described by quadratic regression equations. All five of the acids increased as the

3.C

2,,"

z.c

S T A ~ OF LACTATION [WK$|

FIO. 3. Fitted line fox" variation in per cents 6:0, 8:0, and 10:0 fatty acids with stage of laeta. tion.

i

i.o

o.5

2~

STAGE OF LACTATION (WKS)

Fro. 4. Fitted line for variation in per cents 10:1, 12:1, and 14:1 fatty acids with stage of lactation.

2o

STAGE OF LACTATfOt4 {WKS)

Fz¢~. 1. F i t t e d line fox" variation in daily milk production with stage of laetation. (For this and

succeeding figures see Table 1 for equation of line and reliability of fit.?

lactation progressed, but tended to level off at abo~t the halfway point. P er cent 12:0 was also described by the same type equation; however, it reached a peak toward the middle of the lactation, after which there was a decline equal to approximately one-third of the initial rise. The per cents 18:0 and 18:1 were also described by a quadratic equation; however, in both eases there was a negative initial slope, with a slight upturn in the last 15-20 wk of the lactation. J.

DAIRY

SOIENCE VOD. 49,

No.

11

1404

a.w.

STULL

ET

A cubic regression equation adequately describes the proportions of 10:1 and 12:1 appearing in the milk fat. Both acids began with a decline, rose to the original level, then again declined, giving the impression of being completely cyelic in nature. P e r cent 14:0, 14:1, 1.5:0, and 18:3 were also described by the same

~.I~

601

3.O

~o0 o

~.o B,O

1.0

ZO

oo

g

,~

,~

£

h

£

h

go

~,

£

STAGE OF LACTATION (WK$)

Fro. S. Fitted line for varlatim~ in per cents 17:0, 18:2, and 18:3 fatty acids with stage of lactation.

zo a0

°°

g

,~

,i

;o

",

io

h

"o

,',

2.

STIGE OF LACTATION {WK$]

Fro. ;5. Fitted line for vari:~tim~ i~ per cents 12:0, 14:0, and ]6:1 faatty acids with stage Of lactation.

type equation; however, all except 14:1 had an initial inerease followed by a, slight decline and finished the lactation rising again. Only one, 15:0, rose above the previous peak at the end. I n the ease of 14:0, there was an initial rise, followed by a decline followed by a leveling-off period at the end <)f the tactatiom Discussion

L~

~.0

g o o~

oo

5

Io

i~

20

25

~0

o

STAG~ OF LACTATION (WK$)

FZG. 6. Fitted line for var~glti(m in per eellts 15:0, 16:iso, and 18:iso fatty acids with stage of ]actatiom

,

'

o

~

,1:1

i0

2,

STAGE OF LACTATION {Wg$}

~'o

',

'

20

Fz(L 7. Fitted line for variation in pez'em~ts 16:0, 18:0, and 18:1 fatty acids with stage of lactation. J.

]DAIRY SOIE~rCE ~VOIS. 4 9 ,

NO. 1 1

The results here indicated that there were no differs.noes between milkings within days for individual animals emmected with any of the (-onstitmqlts ot' milk exambled or in tvtal prodm,tion ,fl' milk. Ttwse results are consistent with the' manage.,lent of the ('ows, in that they received identical rations twice a day and were milked twi(.(, daily at ]2-hr intervals. Total production .[' milk was consistent with the findings of others (5, 8, 117, 118). A t e(,ssat.ion of the experiment, all ()f the vows were (i wk away :from the predicted date of next calving. The p e r ('ent fat and kilograms fat produ(~ed during the ('ourse ()1' the lactation were also consistent with much earlier work (4, 6, 14, 17), in that p e r cent fat increased linearly throughout the lactation, whereas total fat, produced declined. Results <)f this experiment regarding the protein (,ontent of the milk are not in complete agreement with earlier work (3, 13). Azarme (3) has sh.wn that protein decreased very sig'nificmltly f o r the first 4 wk of the laetation~ then rises slowly until the end of lactation. I t is mffortunate that the protein production values for the first 7 wk of the experiment were not available. A linear equation describing the p e r cent protein of the milk produced during the course of this experiment would agree with A z a r m e (3). W h e t h e r the phenomena observed in this trial are due to seasonal variation is not known and no other explanation is evident at this time. The fact that all cows calved at ap-

FATTY ACIDS IN MILK p r o x i n m t e l y the same time does not allow f o r d i s t i n g u i s h i n g between seasonal a n d stage-ofl a c t a t i o n v a r i a t i o n w h e r e the a n s w e r is not a p p a r e n t , as in the ease of p e r cent p r o t e i n . Generally, the v a r i a t i o n in t h e f a t t y acid composition of the milk is not consistent with the much earlier findings of K u h h n a n a n d Gallup (9). E v e n t h o u g h the same analyses were n o t made in this study, the results would indicate h i g h e r melting p o i n t a n d a lower iodine value in the early stages of the lactation. This is ix* direct c o n t r a d i c t i o n to the earlier work (9). P e r cent 6:0, 8:0, a n d 10:0 quite obviously increase as the lactation progresses, w i t h 10:0 h a v i n g a completely cyclic a p p e a r a n c e . F l u c t u a tion of 12:0 a n d 14:0 is n o t so easily explained, as t h e r e is a g e n e r a l rise d u r i n g the first p a r t of the lactation, followed by a decrease. The level of 12:1 a n d 18:3 a p p e a r s to be s i m i l a r to 10:1, b e i n g cyclic in n a t u r e . S t a g e of lactation would a p p e a r to be the influencing f a c t o r in the g e n e r a l increase o2 14:1, 15:0, ] 6 :iso, 16:1, 17:0, 18:iso, a n d 18:2 t h r o u g h o u t the lactation. The 16:0, 18 :O, a n d 18:1 acids declined during' the course of the lactation. All evidence would indicate t h a t v a r i a t i o n in these acids u n d o u b t e d l y comes p r i n m r i l y f r o m v a r i a t i o n s in the a m o u n t s of the acids coming f r o n t body stores (11), as t y p e of feed was held c o n s t a n t a n d earlier work {15) has i n d i c a t e d t h a t only the s h o r t e r - c h a i n acids arc affected b y m a n u n a r y lipogenesis. I t is g e n e r a l l y accepted t h a t u n d e r so-called " n o r real feeding c o n d i t i o n s " cows lose weight d u r i n g the early stages of lactation. W h i l e body weight changes were not recorded d u r i n g this experiment, visual o b s e r v a t i o n s left little d o u h t t h a t the f o u r cows did lose weight during" t h e progress o f t h e i r lactation. I t could thus be hypothesized t h a t at the b e g i n n i n g of the lactation, there was a n initial surge of 16:0, 18:0, a n d 118:1 acids (the main c o m p o n e n t s of tallow) into the blood s t r e a m a n d thence into the udder. This mobilization of body f a t would t h e n theoretically decrease d u r i n g the lactation as body w e i g h t decreased a t a decreasing rate. E a r l i e r work (6) would give some credence to this theory. This r e p o r t showed t h a t the f e e d i n g of cottonseed oil or talh)w h a d no effect on the 16:0 level in the milk f a t and, thus, it m a y be derived d e p o t fat. I t m i g h t also be implied t h a t 18:0 a n d 1 8 : 1 f r a c t i o n s could be influenced b y b o t h d e p o t f a t and feed sources. References

(1) Andersou, R. L., and Bancroft, T. A. 1952. Statistical Theory in Research. McGrawHill Book Company, Inc., New York. (2) Asehaffenburg, R., and Temple, P. L. 194:1. The Freezing Point of Milk. I. The Freezing

(3) (4)

(5)

(6)

(7)

(8) (9)

(30)

(121)

(13) (14)

(15)

(16) (17) (18)

(19)

1405

Point and Solids-not-fat Content of the Milk of Individual Cows Throughout a Period of Lactation. J. Dairy Research, 12: 315. Azarme, E. ]938. Variations in the Protein Content of Milk During Lactation. J. Dairy Research, 9 : 121. Beaker, g. B., and Arnold, P. T. D. 1935. Influence o£ Season and Advancing Laetation on B u t t e r f a t Content of Jersey Milk. J. Dairy Sei., 18: 389. Brody, S., Ragsdale, A. C., and Turner, C. W. 1923. The l~ate of Decline 02 Milk Secretion with the Advance of the Period of Lactation. J. Gen. Physiol., 5: 441. Brown, W. H., StulI, J. W., and Stott, G. H. 1962. F a t t y Acid Composition of Milk. I. ;Effect of Roughage and Dietary Fat. J. Dairy Sol., 45: 191. Darkeley, T. J., and White, M. K. 1928. The J o i n t Iuffuence of the Period of Lactation and the Age of the Cow on the Yield and Quality of tile Milk. J. Agr. Sei.. 18 : 496. Gaines, W. L. 1926. Rate of Milk Secretion as Affected by Adwmce in Lactation and Gestation. Illinois Agr. Sta., Bull. 272. Kuhlman, A. H., and Gallup, W. D. 1939 The Effect of Ration on B u t t e r f a t Constants. J. Dairy Sci., ~.°')"424. Luddy, F. E., Barford, R. A., and Riemenschneider, R. W. 1960. Direct Conversion of Liquid Components to Their F a t t y Acid Methyl Esters. J. Am. Oil Chemists' See., 37 : 447. McCarthy, R. D., Patton, S., and :D]vm~s, Laura. 1960. Structm-e and Snythesis of Milk Fat. II. F a t t y Acid Distribution in the Triglycerides of Milk and Other Animal Fats. J. Dairy Sol., 43: 1196. Morrison, F. B. 1956. Feeds and Feeding. 22rid ed. Morrison Pub. Company, Ithaca, New York. Overman, O. I~., Stnnnann, F. P., and Wright, K. E. ]929. Studies of the Composition of Milk. Illinois Agr. Expt. Sta., Bull. 325. ]¢agsdale, A. C., and Turner, C. W. 1922. The Stage of Lactation as a Factor in the Variation of the Per Cent of F a t in Cow's Milk. J. Dairy Sei., 5: 22. Shaw, a. C., and Lakshmanau, S. 1957. La.etation and Hormones. Atomic Energy and Agrleulture. A A A S Sympos. Yol. 49. Washi~gton, D. C. Snedecor, G. W. 1956. Sta.tistieal Methods. 5tJ! ed. The Iowa State College Press, Ames. Turner, C. W. 1936. Factors Affecting tile Composltion of Milk. Missouri Agr. Expt. Sta., /Bull. 365. Turner, C. W., Ragsdale, A. C., and Brody, S. 7923. ttow the Advance of the Period of Lactation Affects the Milk Flow. J. Dairy Sci., 6: 527. Udy, D. C. 1956. A Rapid Method for Estimating Total Protein in Milk. Nature, 178: 314. J. DA[gY SCIENCI~I~]~f)L. 49. NO. 11