- Email: [email protected]
Effects of Lactation on Various Blood Parameters in Holstein Cows. I. Serum Protein and Protein Fractions1
Effects of Lactation on Various Blood • Parameters In Holstein Cows. I. Serum Protein and Protein Fractions1 J. D. ROUSSEL 2 and M. S. RAHMANIAN 3 Department of Dairy Science Louisi ana Agricu ltural Experiment Station Louisiana State University Agricultural Center Baton Rouge 70803 Summary
the nutritional and metabolic rates of the animal (Roussel and Sybt, 1984). By characterizing that composition it may then be possible to gain a better understanding of the complex interactions between maturation, environment, reproduction, and lactation. Proteins, being an integral part of the dietary and physiological status of an animal, provide an important pathway to study such interactions. Serum total protein, for example, is related both to protein metabolism and to the transportation of nutrients, hormones, and waste products to various organs for utilization, detoxification, or elimination (Laird, 1972). Blood urea nitrogen (BUN), being the main end product of protein metabolism, reflects both protein status and nutritional status. Protein is often monitored by albumin and globulin values. The primary function of albumin is to regulate colloid osmotic pressure at the capillary membrane in order to prevent the flu id portion of plasma from leaking into the interstitial spaces (Guyton, 1976). Albumin, therefore, plays an important role in the transport of sparingly solu ble products from one tissue to another. Globulins, on the other hand, are concerned with providing immunity to resist infection and toxicity (Guyton, 1976). As a result, the albumin to globulin ratio (A:G) will act to help identify physiological status while being independent of the diet. The objective of th is study was to investigate the effects of lactation on serum total protein, BUN, serum albumin, serum globulin, and A:G in Holstein cows. Lactational effects were broken down into lactational number, month of calving, month of lactation at conception, stage of lactation, milk yield, and milk fat yield.
Sixty -three Holstein cows were utilized to determine the effects of lactation on blood serum total protein, urea nitrogen, albumin, globulin, and the albumin to globulin ratio. Sampling consisted of monthly blood collections starting with the onset of lactation and continuing to the dry period. Blood values obtained were statistically analyzed by linear regression on lactation number, month of calving, month of lactation at conception, stage of lactation, milk yield, and milk fat yield. Serum total protein was influenced by all lactation parameters examined except milk fat yield. Circulating blood urea nitrogen levels varied with lactation number, month of calving, stage of lactation, and milk yield. Levels of serum albumin were influenced by month of calving. Serum globulin and the albumin to globulin ratio were influenced by all lactation parameters examined except milk and milk fat yield. Introduction
By assessing the metabolic profiles of a herd the farmer can have access to a complete health profile and helps to make decisions that would boost the production up. Metabolic profile testing is based on the physical properties of blood. Blood is composed of a number of different ingredients, each of which is intimately involved with overall health. Therefore, the chemical composition of the blood closely reflects
1 Approved for publication by the Director of Louisiana Agricultural Experiment Station as manuscript number 88-15-2295. 2 Professor. Person to whom reprint requests should be submitted. 3 Graduate student. Reviewed by Larry D. Guthrie and Stanley D. Musgrave.
9
10
ROUSSEL AND RAHMANIAN
Experimental Procedure
Results and Discussion
A total of 63 Holstein cows were randomly selected fro m the Louisiana State University Dairy Herd for blood analysis. All experimen t al animal s we re offered 100% of the National Research Council (N .R.C., 1978) estimated net energy requirements for maintenance, growth, and production. Feed selolrces-vafieEJ-witR-a€le-aAEiseasenef the year. Sourl,:es inc lu ded corn and sorghum silage, alfalfa and coastal bermuda hay, and pelletized 16% protein concentrate gra in mix. Water and trace minerals were available ad libitum . Routine management at the Louisiana State University Dairy Herd included the grouping of animals according to age, reproductive status, and production. Sampl ing was initiated with the onset of lactation utilizing a 30 (±1) d collection interval up to the compietion of that iactation . A ii sampies we re coi lected with minimum restra int by jugular venipuncture between 0500 and 0730 h on the middle of each month. Each co llection consisted of one 15-ml vacutainer tube. Samp les were allowed to coagulate after w hich the serum was isolated by centrifugation (20 min at 890 X g) and stored at -4° C until analysis. Blood serum total protein, BUN, and globulin were measured directly with a Hyce l Mark X autoanaly zer (Hyce l, Inc., Hou ston, TX). Data were analyzed using general linear model procedures (Steel and Torrie, 1980). The regression model included lactation number, month of calving, month of lactation at conception, stage of lactation, miik yieid, and miik fat yieid. Lactation number, month of calving, and month of lactat ion at concep t io n were considered as discreet variables while stage of lactation, milk y ield , and milk fat y ield were con si dered as continuous variables. The use of lactat ion number rather than age allowed for observations on matu ration, adaptation, and production potential. Month of calving served to define seasona l changes. Month of lactation at conception was used to identify variations due to postpartum infertility . Because of infertility problems, stage of pregnancy interactions could not be determined. Stage of lactation, milk yield, and m ilk fat yield served to identify influences from changing levels of milk production as well as f rom absolute level s of milk and milk fat.
The various influences of lactation on blood serum protein and protein fractions observed in th is study appear to confirm the general findings of previous works (Bjarghou et ai., 1976; Braun, 1946; Hewett, 1972,1974; Lane and Campbell, 1966; McNabbetal., 1974; Mylrea and Healy, 1968; Nakao et ai., 1976; ~aVHe -et-al~ ,-l-9+-3-~-R-eH-frieek---aflEJ-R-ay-,---1-9'7-2-:-R-0tJ-ssel
et ai., 1972 ; Roussel et ai., 1982; Shaffer et at., 1976). Significant patterns exhibited by total protein and BUN are located in odd numbered figures . Significant responses by albumin, globulin, and the A:G are located in even numbered figures.
Total Protein
Ci rculating total protein increased (P<.01) with iactation number to the fourth lactation, after which a decline was observed (Table 1, Figure 1). Si milar findings have been attributed to feeding (Hewett, 1972) or culling (Hewett, 1974; McNabb et al., 1974; Roussel et al., 1972) practices for milk production. Studies by Shaffer et ai., 1974 and Roussel et aI., 1972 have reported total protein means and standard error of 6.57 ± .92, and 7.80 ± .03 mg/ml, respectively. However, since lactation number represents age effects as well as adaptability to stress, results may be reflecting improved protein utilization with maturity (Roussel et aI., 1982) . The decrease in circulating total protei n on the fourth lactation could, therefore, be indicative of a breakdown in that efficiency follow ing the completion of maturation . Highly signif icant (P< .0 1) effects for month of calving were found; low total protein level s obse rved during the winter months generally increased thro ugh autumn (Table 2, F igure 2). Since low ambient temperatures have not been found to decrease blood total protein in con trolled-climate exp eriments (Brody, 1949; McNabb et aI., 1974; Murty and Mullick, 1960; Olbrich et al. , 1972), the values reported here m ight be representing influences from available dietary sources (Bjarghou et al ., 1986; Hewett, 1972; Roussel et ai., 1972) and water consumption (Weeth et ai. , 1970) more than seasonal temperatu re ch anges. The month of lactation in which cows conceived also exe rted a highly significant (P<.01) influence on
11
EFFECTS OF LACTATION ON BLOOD PARAMETERS
TABLE 1. Least squares means by lactation number for serum parameters corrected for month of calving, lactation month of conception, stage of lactation, mil k production and mil k fat production. Lactation number Parameter (unit) Total protein (g/100 ml)a Albumin (g/100 ml) Globulin (g/100 ml)a A:Gb BUN (mg/l00 ml)a
First
Second
Third
Fourth
Fifth
7.22 3.20 4 .02 0 .823 9.6
7.38 3.25 4.13 0.817 10.6
7.46 3.07 4.38 0.725 10.7
7.63 3.14 4.50 0.740 11.0
7.30 3.13 4.18 0.799 10.3
aMeans differ (P< .01). bMeans differ (P<.05) .
serum total protein (Table 3, Figure 3), Higher serum protein levels in cows failing to conceive agrees with reports by Hewett, 1972, and Nakao et aI., 1976; however, high levels were also found in cows that became pregnant early in lactation. Serum total protein levels for those animals that conceived may be expressing responses to lactational increases (Belyea et aI., 1975; Hewett, 1972, 1974; McNabb et aI., 1974; Roussel et aI., 1972), while those for cows failing to conceive may have been due, to some extent, to cystic ovarian conditions (Nakao et aI., 1976). A highly significant (P<.01) linear increase in serum total protein was observed with progressing lactation (Figure 4). Low periparturient levels may have resu Ited both from water retention in late pregnancy (Belyea et aI., 1975; Hewett, 1974; O'Kell and Elliot, 1970) and prepartum pasture diets (Belyea
7,7
E 0 0
7.6
0,
z
,_/J'~
0 a: a..
7.4
...J
7,3
~ 0
I--
0
.S-
j/
10.0
z
::::>
co
z
jjj
I--
0 a: a..
...J
~ 0
I--
11.5
7.9 7.8 7.7 7,6 7.5 7.4 7.3 7,2 7.1 7.0 6.9
11,0 10.5
E 0
2 10.0 0,
.S-
9.5
z
::::>
co
9.0
9,5
7.2 2
Mean BUN levels varied significantly (P<.01) with lactation number, increasing to the fourth lactation
E 0 0,
jjj
I--
Blood Urea Nitrogen
11.0
10.5
7.5
et aI., 1975). I ncreases through lactation, therefore, may have resulted from a diversion of body fluid to milk and from increased dietary protein in response to productive work, Serum total protein was found to increase linearly (P<.01) with milk yield (Figure 5). This observation has been associated with disproportionate feeding practices (Hewett, 1972); however, this relationship may also be indicative of genetic capabilities in terms of feed efficiency and the conversion of nutrients to milk. Serum total protein was not found to be influenced by milk fat yield, although Roussel et aI., 1982, has reported a negative correlation.
3
4
5
LACTATION NUMBER
Figure 1. Effects of lactation number on serum (e) total protein and (X) BUN through that lactation .
5
6
7
8
9
10 11 12
1
2
MONTH OF CALVING
Figure 2, Effects of month of calving on serum (e) total protein and (X) BUN through the subsequent lactation .
ROUSSEL AND RAHMANIAN
12
TABLE 2. Least squares means by month of calving for serum parameters corrected for lactation number, lactation month at conception, lactation stage, milk production and milk fat production . Month of calving Parameter (unit) Total protein (g/100 ml)a A lbumin (g/100 m l )a Globulin (g/100 ml)a A:Ga BUN (mg/l00 ml)a
July
June
May 7 .09 3 .21 3.88 _ 0_862 10.0
7 .54 3.18 4.36 - 0.756 9.4
7.31 3 .24 4 .07 0.847 10.4
Aug.
Sept.
7 .14 3 .30 3.83 0 .881 10.0
7.62 3.31 4.32 --0 ,809 9.9
Oct.
Nov.
Dec.
Jan.
Feb.
7.82 7.54 7.03 7.14 7.76 3 .10 3 .21 2.83 2.99 3.20 4.67 4.61 4.70 4.03 3.96 - 0.708 - -0.724- - 0.635 - - 0.765- - 0.823- 11 .6 10.8 1 1 .2 10 .9 10 .1
aMeans differ (P< .O1 ) .
TABLE 3 . Least squares means by lactation month at conception for serum parameters corrected for lactation number, month of calving , lactation stage, milk production, and milk fat production. Lactation month at conception Para m eter (unit)
Third
Fourth
Fif t l,
Sixth
Total protein (g/100 ml)a A lbumin (g/100 ml)a Globulin (g/100 ml)a A:Ga BUN (mg/l00 ml)a
7.69 3.01 4 .69 0.667 9.9
7.40 3.18 4.22 0 .804 10.3
7.45 3.19 4.26 0.785 10.8
6.83 3.40 3.43 0.958 11.8
Seven th
Eighth
Ninth
Tenth
7.44 3.12 4.32 0.747 10.5
7.53 3 .04 4.49 0.708 10.2
7.19 3.33 3.87 0.880 10.1
7.66 2.99 4.67 0.700 9.9
aMeans differ (P< .01).
and then decreasing somewhat (Table 1, Figure 1). These results appear to combine the find ings of Shaffer et aI., 1976, 1981, who found an increase in BUN to maturity, with those of Hewett, 1974, and
7.7 7.6
E 0 2
:9
7.5 7.4 7.3
z
7.2
I-
7.1
W
0
a:
0..
V
7.0
....J
~
0
I-
6.9 6.8 6 .7
3
4
5
6
7
8
9
10
MONTH OF LACTATION Figure 3. Effects of month of lactation in which cows become pregnant on serum total protein through that lactation.
Lane and Campbell, 1966, who observed declines after maturity . Roussel et aI., 1982, on the other hand, found no age effects. Shavver et al ., 1976 have reported BUN mean and standard error of 9.81 ± .11 mg/100 ml. Month of calving was found to have a highly significant (P<. 01 ) effect on BUN (T able 2, Figure 2). While the highest levels were observed during the winte r months, the majority of the literature (Lane and Campbell, 1966; Payne, 1972; Payne et aI., 1970; Payne et aI., 1973, 1974) has reported lowest levels to occur in winter , The pattern seen in this study may have been due to altered kidney function in response to interacting changes in both temperature and nutritional status. The months of lactation in which pregnancy occurred was not found to significantly influence BUN levels (data not shown). BUN was found to increase linearly (P<.01) as lactation progressed (Figure 4), Having closely paralleled levels for total protein, and due to the following re lationship with milk yield, it appears that this response was due to an overfeeding of the cows on this study late in the ir lactation.
13
EFFECTS OF LACTATION ON BLOOD PARAMETERS
..-.-
7.4
B.O
3.4
.-'-
E 7.3 0 0
.-'-
~
:§
/'
7.2
z
..-...-
E
.-.-.-.-'-
W 0 7.1
7.0
I-
....J
~ 0
7.0
.-.-
0.82
E
:9
~ 4.4
0.78
0 0
:J 4.2
z
0.74
~ 3.0
::l CO
::l CO
0.70
....J
« 2.9
0
....J ~
z
~
C, Z
..-.-
6.5
0.86
4.6
3.1
E
.-.-.-.-.-'-
cr: (L
E 0 0
0 0
7.5
.-.-
3.2
0.90
4.8
=>
2.8
11)
6.9
0.62
3.8
6
6.0 30
7
8
9 10 11 12
2
MONTH OF CALVING
Figure 6. Effects of month of ca lv ing on serum (0) albumin (e) globu li n and (X) A:G through the subsequen t lactat i on.
150 210 270 330 390
90
<{
0.66
4.0
5
I-
~
LACTATION DAY Figure 4. Effects of stage of lactation on serum (- -) tota l protein and ( - ) BUN.
Albumin
BUN levels were found to increase linearly (P<.01) with milk yield (Figure 5). Since total protein also increased with milk production, these results appear to be consistent with the commonly accepted correlation between BUN and nutritional status and between nutritional status and milk production. Milk fat yield was not found to significantly affect BUN levels.
7.9
.-,I" .-.-
7.8
E
7.6
9
7.5
z
[jJ I-
.-.-
7.7
0 0
.-'-
.-.-
7.3
.-" .-.-
7.2
0
.-.-"
.-.-'-
4.5
9.0
S
E
Z
0
::l
co
"
0.83 0.82 0.81 0.80 0.79 0.78 0.77 0.76 0.75 0.74 0.73 0.72
x____ x
4.4
S'
0, 4.3
z
.-'-
7.1 7.0
E 0
en
.-.-"
0
I-
12.0
.-
7.4
...J
~
.-." .-.-"
.-'.-'-
0
cr: (L
15.0
The only significant (P<.01) influence of lactation on serum albumin was found with month of calving (Table 2, Figure 6). Generally, serum albumin peaked in early autumn and then decreased to the winter months. Low winter calving levels of serum albumin may have been due to prolonged lactational periods through the warmer summer months. Shaffer et aI., 1974 have reported similar albumin means and standard errors of 3.03 ± .02 g/1 00 m I. Payne et aI., 1970, has characterized album in as the most important member of a metabolic profile test. Perhaps that belief stems from the hypothesis that only extremes in environment or animal health
::::;
::l
..-.-
4.2
co
6.0
6.9
0....J ~
4.1 4.0
10
20
30
40
50
2
3
4
~
<{
5
MILK (kg)
LACTATION NUMBER
Figure 5. Effects of sample day milk production on serum (- -) total protein and (-) BUN.
Figure 7. Effects of lactation number on serum (e) globu lin and (X) A:G through that lactation .
ROUSSEL AND RAHMANIAN
14 4.8 4.6
E o o
t~.::
'\
4.4
z
4.0
0.80
iii
3.8
0.76
3.6
0.72
3.4
0.68
O
--'
(')
E
0.88 0.84
:::::;
5.0
U.:J.£
4.2
:9
_~
t
0 0
(!:l
«
OJ z
4.9
~~~ ~~
;1'"" ..'" ~~~
- co 4.6 0 -.J CD
4.5 4.4
3
4
5
6
7
8
9
10
MONTH OF LACTATION Figure 8. Effects of month of lactation in which cows become pregnant on serum (e ) globulin and (X) A:G through that lactation.
will produce notable changes in circulating levels (Hewett, 1974; Nakao et aI., 1976; Payne et aI., 1970; Payne et aI., 1974). If so, significant changes would not be expected under the conditions of this study. Globulin
Globulin levels increased (P<.01) with lactation number to the fourth lactation and then decreased (Table 1, Figure7). Shafferet al. ( 1974) have reported similar globulin means and standard errors of 3.72 ± .02 gl100 ml. The general increase in globulin levels with productive age has been attributed to the development of immunologic systems (Braun, 1946; Hewett, 1974; Ivlyirea and Healy, i968; Roussei et al., 1982; Shaffer et aI., 1976), therefore, the decline observed for the fifth lactation may signify the acquisition of adaptation to production stress. High Iy sign ificant (P< .01) effects were found for month of calving on serum globulin (Table 2, Figure 6). Circulating globulin values peaked from October to December, then generally decreased to the summer months. While these results substantiate an inverse relationship between globulin and albumin noted by Payne et aI., 1973, and Roussel et aI., 1972, accounts vary as to a relationship with temperature. The inverse relationship with temperature found in this study is similar to that observed by McNabb et aI., 1974. Ross and Halliday 1974, and Roussel et aI., 1972, however, found a direct relationship with temperature. Bondarendo et aI., 1977 and Payne et al ., 1974, found no significant seasonal effects.
0.60
~~~
::J
t/ t
0.66
0.62
~~
4.7
0.68
0.64
~
4.8
:::i
_
~
/~t
CD
-0.58~ -
,.",,7~
0.56 0.54
I
I
30
90
I
I
I
I
I
150 210 270 330 390 LACTATION DAY
Figure 9. Effects of stage of lactation on serum (- -) globulin and ( - ) A:G.
Month of lactatio n at conception was found to exert a high Iy sign ificant (Table 3, P<.O 1) effect on circulating globulin levels (Figure 8). Highest levels were found both in those cows conceiving in the third month of lactation and in those that failed to conceive. The latter may have been due to chronic production-related infections (Payne et al., 1970; Roussel et aI., 1971) ovarian abnormalities (Nakao, 1977). A highly si gnificant (P<.01) linear relationship was observed between serum globulin and stage of lactation (Figure 9). Other reports (Hewett, 1974; Smith and Kesler, 1969), however, have indicated that circulating globulin levels peaked within lactations. While neither quadratic nor cubic effects were considered in the statistical model used here, such effects may have been significant. Significant effects were not found for milk or milk fat yield, although McNabb et aI., 1984, and Payne et aI., 1973, 1974, reported positive correlations with milk yield, and Roussel et aI., i 972, have noted negative correlations with both milk and milk fat production.
Albumin to Globulin Ratio
Lacta tion number exhibited a sign ificant (P< .01) relationship with the A:G as levels decreased to the third lactation and then increased (Table 1, Figure 7). Th is pattern was essentially opposite to that fou nd for globulin. Since albumin was not found to be af-
EFFECTS OF LACTATION ON BLOOD PARAMETERS
fected by lactation number, it can be concluded that globulin was the sole contributor. The month of calving exerted a significant (P< .01) effect on the A:G (Table 2, Figure 6). Lowest A:G values occurred during the winter months while the highest were observed during the summer. Roussel et aI., 1972, however, found values to decrease in the summer months. I n both cases, though, the A:G pattern followed that of serum albumin. A high Iy sign ificant (P<.O 1) relationsh ip was found between the A:G and the month of lactation in which cows conceived (Table 3, Figure 8). The A:G increased to the sixth month of lactation and then decreased. This pattern may have been a reflection of the response found between globulin and fertility. The A:G was found to decrease linearly (P<.01) as the stage of lactation progressed (Figure 9). As noted in the previous relationships, the A:G appears to have been influenced most by globulin. No significant effects were found between the A :G and milk or milk fat yield.
beneficial in prescribing the needed course of action. Th is wou Id provide a powerfu I management tool capable of saving both time and money . Literature Cited 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Conclusions
Lactationa l effects were fou nd to significantly influence levels of serum total protein, BUN, serum albumin, serum globulin, and the A:G. As a resu lt , when monitoring those blood parameters fo r a metabolic profile, such lactational effects would need to be taken into account. I n addition, due to the relationships found for serum total protein and BUN with stage of lactation and milk yield, it appears that those blood parameters wou Id be usefu I as a means of monitoring production. There are a number of factors such as: herd, breed, feeds, species, season, metabolism, health, etc., which contribute to the f luctuation of a given blood component change. The results from this study clearly shows that even the productive status of an animal can have a significant impact on various blood parameters. From the practical standpoint monitoring these parameters in a herd on a routine basis may become a useful diagnostic tool and can become
15
17. 18. 19. 20. 21. 22. 23. 24. 25 . 26. 27. 28. 29. 30 . 31. 32. 33 .
Belyea, R. L., C. E. Coppock, and G. B. Lake. 1975. J. Dairy Sci. 58: 1336. Bjarghou, R. S., P. Fjellheim, K. Hove, E . Jacobsen, S. Skjenneberg, and K. Try. 1976. Compo Biochem. Physiol. A55: 187. Bondarendo, G. A., N. I. Guseva, I. V. Ptashevskaya, N. V. Varnavskaya, T . A. Peryshkova, and E. W. Peryshkina. 1977. Genetika 13 :439. Braun, W. 1946. Am . J. Vet. Res. 7:450. Brody, S. 1949. Mo. Ag r . Exp. Stat. Res. Bull. No. 433. Guyton, A. C. 1976 . Textbook of medical physiology. 5th ed. W. B. Saunders Co., Philadelphia, PA. Hewett, C. D. 1972. Proc . XII Int. Congr. An im. Reprod. and A.I. 1 :707. Munich, Germany. Hewett, C. D. 1974 . Acta Vet. Scand. Suppl. 50:1 . Laird, C. W. 1972. Hycel, Inc., Houston, TX. Lane, A. G., and J. R. Campbell. 1966. J. Dairy Sci. 49:193. McNabb, L., J. D. Roussel, and L. F. Gomila. 1974. J. Dairy Sci. 57:41 . Murty, V. M., and D. N. Mullick. 1960. Ann. Biochem. Exp. Med.20:131. Mylrea, P. J., and P. J. Healy. 1968. Austr . Vet. J. 44:570. Nakao, T. 1977. Jpn. J. Vet . Sci. 39:93. Nakao, T., K. Sato, H. Ono, and M. Miyake. 1-976. Jpn. J. Vet. Sci. 38:207. National Research Council. 1978. Nat. Acad. Sci., Washington, DC. O'Kell, R. T., and J. R. Elliot . 1970. Clin. Chem. 16:161. Olbrich, S. E., F . A. Marty, M. E. Tumbleson, H. D. Johnson, and E. S. Hilderbraqcl. 1972. Compo Biochem. Physiol . 41A:255. Payne, J. M.1972. Royal Agr. Soc. Engl. J. 133:69. Payne, J . M ., S. M . Dew, R. Manston, and M. Faulks . 1970. Vet . Rec.87:150. Payne, J. M ., G. J. Rowlands, R . Manston, and S. M. Dew . 1973. Br. Vet. J. 129:370. Payne, J. M., G. J. Rowlands, R . Manston, S. M. Dew, and W. H. Parker. 1974. Br. Vet. J . 130:34. Ross, J. G., and W. G. Halliday. 1976. Br. Vet. J. 132:401. Roubicek, C. B., and D. E. Ray. 1972 . J. Anim. Sci. 34:931. Roussel, J. D., J. H. Gholson, M. A . Pinero, J. F. Beatty, and W. H. Waters. 1971. Am. Meteorol. Soc. 52:300. Roussel, J . D ., K . L . Koonce, and M . A. Pinero. 1972. J. Dairy Sci . 55: 1093. Roussel, J. D., S. H. Seybt, and G. Toups. 1982. Am. J. Vet. Res. 43:1075. Roussel , J. D., S . H. SeYbt. 1984 . The Louisiana Cattleman . Dec. page 11-12. Shaffer, L., J. D. Roussel, and K . L. Koonce . 1976. J. Dairy Sci . 59:16 . Shaffer, L., J. D. Roussel, and K. L . Koonce . 1981 . J. Dairy Sci. 64:62. Smith, J. W., and E . M. Kesler . 1969. J. Dairy Sci. 52:279. Steel, R .GD., and J . H. Torrie.1980. Princip les and Procedures of Statistics (2nd ed.) . McGraw-Hili Book Company, New York. Weeth, H . J. , R. Witton, C. F. Speth , and C . R. Blincoe. 1970. J. Anim. Sci. 30:219.
the nutritional and metabolic rates of the animal (Roussel and Sybt, 1984). By characterizing that composition it may then be possible to gain a better understanding of the complex interactions between maturation, environment, reproduction, and lactation. Proteins, being an integral part of the dietary and physiological status of an animal, provide an important pathway to study such interactions. Serum total protein, for example, is related both to protein metabolism and to the transportation of nutrients, hormones, and waste products to various organs for utilization, detoxification, or elimination (Laird, 1972). Blood urea nitrogen (BUN), being the main end product of protein metabolism, reflects both protein status and nutritional status. Protein is often monitored by albumin and globulin values. The primary function of albumin is to regulate colloid osmotic pressure at the capillary membrane in order to prevent the flu id portion of plasma from leaking into the interstitial spaces (Guyton, 1976). Albumin, therefore, plays an important role in the transport of sparingly solu ble products from one tissue to another. Globulins, on the other hand, are concerned with providing immunity to resist infection and toxicity (Guyton, 1976). As a result, the albumin to globulin ratio (A:G) will act to help identify physiological status while being independent of the diet. The objective of th is study was to investigate the effects of lactation on serum total protein, BUN, serum albumin, serum globulin, and A:G in Holstein cows. Lactational effects were broken down into lactational number, month of calving, month of lactation at conception, stage of lactation, milk yield, and milk fat yield.
Sixty -three Holstein cows were utilized to determine the effects of lactation on blood serum total protein, urea nitrogen, albumin, globulin, and the albumin to globulin ratio. Sampling consisted of monthly blood collections starting with the onset of lactation and continuing to the dry period. Blood values obtained were statistically analyzed by linear regression on lactation number, month of calving, month of lactation at conception, stage of lactation, milk yield, and milk fat yield. Serum total protein was influenced by all lactation parameters examined except milk fat yield. Circulating blood urea nitrogen levels varied with lactation number, month of calving, stage of lactation, and milk yield. Levels of serum albumin were influenced by month of calving. Serum globulin and the albumin to globulin ratio were influenced by all lactation parameters examined except milk and milk fat yield. Introduction
By assessing the metabolic profiles of a herd the farmer can have access to a complete health profile and helps to make decisions that would boost the production up. Metabolic profile testing is based on the physical properties of blood. Blood is composed of a number of different ingredients, each of which is intimately involved with overall health. Therefore, the chemical composition of the blood closely reflects
1 Approved for publication by the Director of Louisiana Agricultural Experiment Station as manuscript number 88-15-2295. 2 Professor. Person to whom reprint requests should be submitted. 3 Graduate student. Reviewed by Larry D. Guthrie and Stanley D. Musgrave.
9
10
ROUSSEL AND RAHMANIAN
Experimental Procedure
Results and Discussion
A total of 63 Holstein cows were randomly selected fro m the Louisiana State University Dairy Herd for blood analysis. All experimen t al animal s we re offered 100% of the National Research Council (N .R.C., 1978) estimated net energy requirements for maintenance, growth, and production. Feed selolrces-vafieEJ-witR-a€le-aAEiseasenef the year. Sourl,:es inc lu ded corn and sorghum silage, alfalfa and coastal bermuda hay, and pelletized 16% protein concentrate gra in mix. Water and trace minerals were available ad libitum . Routine management at the Louisiana State University Dairy Herd included the grouping of animals according to age, reproductive status, and production. Sampl ing was initiated with the onset of lactation utilizing a 30 (±1) d collection interval up to the compietion of that iactation . A ii sampies we re coi lected with minimum restra int by jugular venipuncture between 0500 and 0730 h on the middle of each month. Each co llection consisted of one 15-ml vacutainer tube. Samp les were allowed to coagulate after w hich the serum was isolated by centrifugation (20 min at 890 X g) and stored at -4° C until analysis. Blood serum total protein, BUN, and globulin were measured directly with a Hyce l Mark X autoanaly zer (Hyce l, Inc., Hou ston, TX). Data were analyzed using general linear model procedures (Steel and Torrie, 1980). The regression model included lactation number, month of calving, month of lactation at conception, stage of lactation, miik yieid, and miik fat yieid. Lactation number, month of calving, and month of lactat ion at concep t io n were considered as discreet variables while stage of lactation, milk y ield , and milk fat y ield were con si dered as continuous variables. The use of lactat ion number rather than age allowed for observations on matu ration, adaptation, and production potential. Month of calving served to define seasona l changes. Month of lactation at conception was used to identify variations due to postpartum infertility . Because of infertility problems, stage of pregnancy interactions could not be determined. Stage of lactation, milk yield, and m ilk fat yield served to identify influences from changing levels of milk production as well as f rom absolute level s of milk and milk fat.
The various influences of lactation on blood serum protein and protein fractions observed in th is study appear to confirm the general findings of previous works (Bjarghou et ai., 1976; Braun, 1946; Hewett, 1972,1974; Lane and Campbell, 1966; McNabbetal., 1974; Mylrea and Healy, 1968; Nakao et ai., 1976; ~aVHe -et-al~ ,-l-9+-3-~-R-eH-frieek---aflEJ-R-ay-,---1-9'7-2-:-R-0tJ-ssel
et ai., 1972 ; Roussel et ai., 1982; Shaffer et at., 1976). Significant patterns exhibited by total protein and BUN are located in odd numbered figures . Significant responses by albumin, globulin, and the A:G are located in even numbered figures.
Total Protein
Ci rculating total protein increased (P<.01) with iactation number to the fourth lactation, after which a decline was observed (Table 1, Figure 1). Si milar findings have been attributed to feeding (Hewett, 1972) or culling (Hewett, 1974; McNabb et al., 1974; Roussel et al., 1972) practices for milk production. Studies by Shaffer et ai., 1974 and Roussel et aI., 1972 have reported total protein means and standard error of 6.57 ± .92, and 7.80 ± .03 mg/ml, respectively. However, since lactation number represents age effects as well as adaptability to stress, results may be reflecting improved protein utilization with maturity (Roussel et aI., 1982) . The decrease in circulating total protei n on the fourth lactation could, therefore, be indicative of a breakdown in that efficiency follow ing the completion of maturation . Highly signif icant (P< .0 1) effects for month of calving were found; low total protein level s obse rved during the winter months generally increased thro ugh autumn (Table 2, F igure 2). Since low ambient temperatures have not been found to decrease blood total protein in con trolled-climate exp eriments (Brody, 1949; McNabb et aI., 1974; Murty and Mullick, 1960; Olbrich et al. , 1972), the values reported here m ight be representing influences from available dietary sources (Bjarghou et al ., 1986; Hewett, 1972; Roussel et ai., 1972) and water consumption (Weeth et ai. , 1970) more than seasonal temperatu re ch anges. The month of lactation in which cows conceived also exe rted a highly significant (P<.01) influence on
11
EFFECTS OF LACTATION ON BLOOD PARAMETERS
TABLE 1. Least squares means by lactation number for serum parameters corrected for month of calving, lactation month of conception, stage of lactation, mil k production and mil k fat production. Lactation number Parameter (unit) Total protein (g/100 ml)a Albumin (g/100 ml) Globulin (g/100 ml)a A:Gb BUN (mg/l00 ml)a
First
Second
Third
Fourth
Fifth
7.22 3.20 4 .02 0 .823 9.6
7.38 3.25 4.13 0.817 10.6
7.46 3.07 4.38 0.725 10.7
7.63 3.14 4.50 0.740 11.0
7.30 3.13 4.18 0.799 10.3
aMeans differ (P< .01). bMeans differ (P<.05) .
serum total protein (Table 3, Figure 3), Higher serum protein levels in cows failing to conceive agrees with reports by Hewett, 1972, and Nakao et aI., 1976; however, high levels were also found in cows that became pregnant early in lactation. Serum total protein levels for those animals that conceived may be expressing responses to lactational increases (Belyea et aI., 1975; Hewett, 1972, 1974; McNabb et aI., 1974; Roussel et aI., 1972), while those for cows failing to conceive may have been due, to some extent, to cystic ovarian conditions (Nakao et aI., 1976). A highly significant (P<.01) linear increase in serum total protein was observed with progressing lactation (Figure 4). Low periparturient levels may have resu Ited both from water retention in late pregnancy (Belyea et aI., 1975; Hewett, 1974; O'Kell and Elliot, 1970) and prepartum pasture diets (Belyea
7,7
E 0 0
7.6
0,
z
,_/J'~
0 a: a..
7.4
...J
7,3
~ 0
I--
0
.S-
j/
10.0
z
::::>
co
z
jjj
I--
0 a: a..
...J
~ 0
I--
11.5
7.9 7.8 7.7 7,6 7.5 7.4 7.3 7,2 7.1 7.0 6.9
11,0 10.5
E 0
2 10.0 0,
.S-
9.5
z
::::>
co
9.0
9,5
7.2 2
Mean BUN levels varied significantly (P<.01) with lactation number, increasing to the fourth lactation
E 0 0,
jjj
I--
Blood Urea Nitrogen
11.0
10.5
7.5
et aI., 1975). I ncreases through lactation, therefore, may have resulted from a diversion of body fluid to milk and from increased dietary protein in response to productive work, Serum total protein was found to increase linearly (P<.01) with milk yield (Figure 5). This observation has been associated with disproportionate feeding practices (Hewett, 1972); however, this relationship may also be indicative of genetic capabilities in terms of feed efficiency and the conversion of nutrients to milk. Serum total protein was not found to be influenced by milk fat yield, although Roussel et aI., 1982, has reported a negative correlation.
3
4
5
LACTATION NUMBER
Figure 1. Effects of lactation number on serum (e) total protein and (X) BUN through that lactation .
5
6
7
8
9
10 11 12
1
2
MONTH OF CALVING
Figure 2, Effects of month of calving on serum (e) total protein and (X) BUN through the subsequent lactation .
ROUSSEL AND RAHMANIAN
12
TABLE 2. Least squares means by month of calving for serum parameters corrected for lactation number, lactation month at conception, lactation stage, milk production and milk fat production . Month of calving Parameter (unit) Total protein (g/100 ml)a A lbumin (g/100 m l )a Globulin (g/100 ml)a A:Ga BUN (mg/l00 ml)a
July
June
May 7 .09 3 .21 3.88 _ 0_862 10.0
7 .54 3.18 4.36 - 0.756 9.4
7.31 3 .24 4 .07 0.847 10.4
Aug.
Sept.
7 .14 3 .30 3.83 0 .881 10.0
7.62 3.31 4.32 --0 ,809 9.9
Oct.
Nov.
Dec.
Jan.
Feb.
7.82 7.54 7.03 7.14 7.76 3 .10 3 .21 2.83 2.99 3.20 4.67 4.61 4.70 4.03 3.96 - 0.708 - -0.724- - 0.635 - - 0.765- - 0.823- 11 .6 10.8 1 1 .2 10 .9 10 .1
aMeans differ (P< .O1 ) .
TABLE 3 . Least squares means by lactation month at conception for serum parameters corrected for lactation number, month of calving , lactation stage, milk production, and milk fat production. Lactation month at conception Para m eter (unit)
Third
Fourth
Fif t l,
Sixth
Total protein (g/100 ml)a A lbumin (g/100 ml)a Globulin (g/100 ml)a A:Ga BUN (mg/l00 ml)a
7.69 3.01 4 .69 0.667 9.9
7.40 3.18 4.22 0 .804 10.3
7.45 3.19 4.26 0.785 10.8
6.83 3.40 3.43 0.958 11.8
Seven th
Eighth
Ninth
Tenth
7.44 3.12 4.32 0.747 10.5
7.53 3 .04 4.49 0.708 10.2
7.19 3.33 3.87 0.880 10.1
7.66 2.99 4.67 0.700 9.9
aMeans differ (P< .01).
and then decreasing somewhat (Table 1, Figure 1). These results appear to combine the find ings of Shaffer et aI., 1976, 1981, who found an increase in BUN to maturity, with those of Hewett, 1974, and
7.7 7.6
E 0 2
:9
7.5 7.4 7.3
z
7.2
I-
7.1
W
0
a:
0..
V
7.0
....J
~
0
I-
6.9 6.8 6 .7
3
4
5
6
7
8
9
10
MONTH OF LACTATION Figure 3. Effects of month of lactation in which cows become pregnant on serum total protein through that lactation.
Lane and Campbell, 1966, who observed declines after maturity . Roussel et aI., 1982, on the other hand, found no age effects. Shavver et al ., 1976 have reported BUN mean and standard error of 9.81 ± .11 mg/100 ml. Month of calving was found to have a highly significant (P<. 01 ) effect on BUN (T able 2, Figure 2). While the highest levels were observed during the winte r months, the majority of the literature (Lane and Campbell, 1966; Payne, 1972; Payne et aI., 1970; Payne et aI., 1973, 1974) has reported lowest levels to occur in winter , The pattern seen in this study may have been due to altered kidney function in response to interacting changes in both temperature and nutritional status. The months of lactation in which pregnancy occurred was not found to significantly influence BUN levels (data not shown). BUN was found to increase linearly (P<.01) as lactation progressed (Figure 4), Having closely paralleled levels for total protein, and due to the following re lationship with milk yield, it appears that this response was due to an overfeeding of the cows on this study late in the ir lactation.
13
EFFECTS OF LACTATION ON BLOOD PARAMETERS
..-.-
7.4
B.O
3.4
.-'-
E 7.3 0 0
.-'-
~
:§
/'
7.2
z
..-...-
E
.-.-.-.-'-
W 0 7.1
7.0
I-
....J
~ 0
7.0
.-.-
0.82
E
:9
~ 4.4
0.78
0 0
:J 4.2
z
0.74
~ 3.0
::l CO
::l CO
0.70
....J
« 2.9
0
....J ~
z
~
C, Z
..-.-
6.5
0.86
4.6
3.1
E
.-.-.-.-.-'-
cr: (L
E 0 0
0 0
7.5
.-.-
3.2
0.90
4.8
=>
2.8
11)
6.9
0.62
3.8
6
6.0 30
7
8
9 10 11 12
2
MONTH OF CALVING
Figure 6. Effects of month of ca lv ing on serum (0) albumin (e) globu li n and (X) A:G through the subsequen t lactat i on.
150 210 270 330 390
90
<{
0.66
4.0
5
I-
~
LACTATION DAY Figure 4. Effects of stage of lactation on serum (- -) tota l protein and ( - ) BUN.
Albumin
BUN levels were found to increase linearly (P<.01) with milk yield (Figure 5). Since total protein also increased with milk production, these results appear to be consistent with the commonly accepted correlation between BUN and nutritional status and between nutritional status and milk production. Milk fat yield was not found to significantly affect BUN levels.
7.9
.-,I" .-.-
7.8
E
7.6
9
7.5
z
[jJ I-
.-.-
7.7
0 0
.-'-
.-.-
7.3
.-" .-.-
7.2
0
.-.-"
.-.-'-
4.5
9.0
S
E
Z
0
::l
co
"
0.83 0.82 0.81 0.80 0.79 0.78 0.77 0.76 0.75 0.74 0.73 0.72
x____ x
4.4
S'
0, 4.3
z
.-'-
7.1 7.0
E 0
en
.-.-"
0
I-
12.0
.-
7.4
...J
~
.-." .-.-"
.-'.-'-
0
cr: (L
15.0
The only significant (P<.01) influence of lactation on serum albumin was found with month of calving (Table 2, Figure 6). Generally, serum albumin peaked in early autumn and then decreased to the winter months. Low winter calving levels of serum albumin may have been due to prolonged lactational periods through the warmer summer months. Shaffer et aI., 1974 have reported similar albumin means and standard errors of 3.03 ± .02 g/1 00 m I. Payne et aI., 1970, has characterized album in as the most important member of a metabolic profile test. Perhaps that belief stems from the hypothesis that only extremes in environment or animal health
::::;
::l
..-.-
4.2
co
6.0
6.9
0....J ~
4.1 4.0
10
20
30
40
50
2
3
4
~
<{
5
MILK (kg)
LACTATION NUMBER
Figure 5. Effects of sample day milk production on serum (- -) total protein and (-) BUN.
Figure 7. Effects of lactation number on serum (e) globu lin and (X) A:G through that lactation .
ROUSSEL AND RAHMANIAN
14 4.8 4.6
E o o
t~.::
'\
4.4
z
4.0
0.80
iii
3.8
0.76
3.6
0.72
3.4
0.68
O
--'
(')
E
0.88 0.84
:::::;
5.0
U.:J.£
4.2
:9
_~
t
0 0
(!:l
«
OJ z
4.9
~~~ ~~
;1'"" ..'" ~~~
- co 4.6 0 -.J CD
4.5 4.4
3
4
5
6
7
8
9
10
MONTH OF LACTATION Figure 8. Effects of month of lactation in which cows become pregnant on serum (e ) globulin and (X) A:G through that lactation.
will produce notable changes in circulating levels (Hewett, 1974; Nakao et aI., 1976; Payne et aI., 1970; Payne et aI., 1974). If so, significant changes would not be expected under the conditions of this study. Globulin
Globulin levels increased (P<.01) with lactation number to the fourth lactation and then decreased (Table 1, Figure7). Shafferet al. ( 1974) have reported similar globulin means and standard errors of 3.72 ± .02 gl100 ml. The general increase in globulin levels with productive age has been attributed to the development of immunologic systems (Braun, 1946; Hewett, 1974; Ivlyirea and Healy, i968; Roussei et al., 1982; Shaffer et aI., 1976), therefore, the decline observed for the fifth lactation may signify the acquisition of adaptation to production stress. High Iy sign ificant (P< .01) effects were found for month of calving on serum globulin (Table 2, Figure 6). Circulating globulin values peaked from October to December, then generally decreased to the summer months. While these results substantiate an inverse relationship between globulin and albumin noted by Payne et aI., 1973, and Roussel et aI., 1972, accounts vary as to a relationship with temperature. The inverse relationship with temperature found in this study is similar to that observed by McNabb et aI., 1974. Ross and Halliday 1974, and Roussel et aI., 1972, however, found a direct relationship with temperature. Bondarendo et aI., 1977 and Payne et al ., 1974, found no significant seasonal effects.
0.60
~~~
::J
t/ t
0.66
0.62
~~
4.7
0.68
0.64
~
4.8
:::i
_
~
/~t
CD
-0.58~ -
,.",,7~
0.56 0.54
I
I
30
90
I
I
I
I
I
150 210 270 330 390 LACTATION DAY
Figure 9. Effects of stage of lactation on serum (- -) globulin and ( - ) A:G.
Month of lactatio n at conception was found to exert a high Iy sign ificant (Table 3, P<.O 1) effect on circulating globulin levels (Figure 8). Highest levels were found both in those cows conceiving in the third month of lactation and in those that failed to conceive. The latter may have been due to chronic production-related infections (Payne et al., 1970; Roussel et aI., 1971) ovarian abnormalities (Nakao, 1977). A highly si gnificant (P<.01) linear relationship was observed between serum globulin and stage of lactation (Figure 9). Other reports (Hewett, 1974; Smith and Kesler, 1969), however, have indicated that circulating globulin levels peaked within lactations. While neither quadratic nor cubic effects were considered in the statistical model used here, such effects may have been significant. Significant effects were not found for milk or milk fat yield, although McNabb et aI., 1984, and Payne et aI., 1973, 1974, reported positive correlations with milk yield, and Roussel et aI., i 972, have noted negative correlations with both milk and milk fat production.
Albumin to Globulin Ratio
Lacta tion number exhibited a sign ificant (P< .01) relationship with the A:G as levels decreased to the third lactation and then increased (Table 1, Figure 7). Th is pattern was essentially opposite to that fou nd for globulin. Since albumin was not found to be af-
EFFECTS OF LACTATION ON BLOOD PARAMETERS
fected by lactation number, it can be concluded that globulin was the sole contributor. The month of calving exerted a significant (P< .01) effect on the A:G (Table 2, Figure 6). Lowest A:G values occurred during the winter months while the highest were observed during the summer. Roussel et aI., 1972, however, found values to decrease in the summer months. I n both cases, though, the A:G pattern followed that of serum albumin. A high Iy sign ificant (P<.O 1) relationsh ip was found between the A:G and the month of lactation in which cows conceived (Table 3, Figure 8). The A:G increased to the sixth month of lactation and then decreased. This pattern may have been a reflection of the response found between globulin and fertility. The A:G was found to decrease linearly (P<.01) as the stage of lactation progressed (Figure 9). As noted in the previous relationships, the A:G appears to have been influenced most by globulin. No significant effects were found between the A :G and milk or milk fat yield.
beneficial in prescribing the needed course of action. Th is wou Id provide a powerfu I management tool capable of saving both time and money . Literature Cited 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Conclusions
Lactationa l effects were fou nd to significantly influence levels of serum total protein, BUN, serum albumin, serum globulin, and the A:G. As a resu lt , when monitoring those blood parameters fo r a metabolic profile, such lactational effects would need to be taken into account. I n addition, due to the relationships found for serum total protein and BUN with stage of lactation and milk yield, it appears that those blood parameters wou Id be usefu I as a means of monitoring production. There are a number of factors such as: herd, breed, feeds, species, season, metabolism, health, etc., which contribute to the f luctuation of a given blood component change. The results from this study clearly shows that even the productive status of an animal can have a significant impact on various blood parameters. From the practical standpoint monitoring these parameters in a herd on a routine basis may become a useful diagnostic tool and can become
15
17. 18. 19. 20. 21. 22. 23. 24. 25 . 26. 27. 28. 29. 30 . 31. 32. 33 .
Belyea, R. L., C. E. Coppock, and G. B. Lake. 1975. J. Dairy Sci. 58: 1336. Bjarghou, R. S., P. Fjellheim, K. Hove, E . Jacobsen, S. Skjenneberg, and K. Try. 1976. Compo Biochem. Physiol. A55: 187. Bondarendo, G. A., N. I. Guseva, I. V. Ptashevskaya, N. V. Varnavskaya, T . A. Peryshkova, and E. W. Peryshkina. 1977. Genetika 13 :439. Braun, W. 1946. Am . J. Vet. Res. 7:450. Brody, S. 1949. Mo. Ag r . Exp. Stat. Res. Bull. No. 433. Guyton, A. C. 1976 . Textbook of medical physiology. 5th ed. W. B. Saunders Co., Philadelphia, PA. Hewett, C. D. 1972. Proc . XII Int. Congr. An im. Reprod. and A.I. 1 :707. Munich, Germany. Hewett, C. D. 1974 . Acta Vet. Scand. Suppl. 50:1 . Laird, C. W. 1972. Hycel, Inc., Houston, TX. Lane, A. G., and J. R. Campbell. 1966. J. Dairy Sci. 49:193. McNabb, L., J. D. Roussel, and L. F. Gomila. 1974. J. Dairy Sci. 57:41 . Murty, V. M., and D. N. Mullick. 1960. Ann. Biochem. Exp. Med.20:131. Mylrea, P. J., and P. J. Healy. 1968. Austr . Vet. J. 44:570. Nakao, T. 1977. Jpn. J. Vet . Sci. 39:93. Nakao, T., K. Sato, H. Ono, and M. Miyake. 1-976. Jpn. J. Vet. Sci. 38:207. National Research Council. 1978. Nat. Acad. Sci., Washington, DC. O'Kell, R. T., and J. R. Elliot . 1970. Clin. Chem. 16:161. Olbrich, S. E., F . A. Marty, M. E. Tumbleson, H. D. Johnson, and E. S. Hilderbraqcl. 1972. Compo Biochem. Physiol . 41A:255. Payne, J. M.1972. Royal Agr. Soc. Engl. J. 133:69. Payne, J . M ., S. M . Dew, R. Manston, and M. Faulks . 1970. Vet . Rec.87:150. Payne, J. M ., G. J. Rowlands, R . Manston, and S. M. Dew . 1973. Br. Vet. J. 129:370. Payne, J. M., G. J. Rowlands, R . Manston, S. M. Dew, and W. H. Parker. 1974. Br. Vet. J . 130:34. Ross, J. G., and W. G. Halliday. 1976. Br. Vet. J. 132:401. Roubicek, C. B., and D. E. Ray. 1972 . J. Anim. Sci. 34:931. Roussel, J. D., J. H. Gholson, M. A . Pinero, J. F. Beatty, and W. H. Waters. 1971. Am. Meteorol. Soc. 52:300. Roussel, J . D ., K . L . Koonce, and M . A. Pinero. 1972. J. Dairy Sci . 55: 1093. Roussel, J. D., S. H. Seybt, and G. Toups. 1982. Am. J. Vet. Res. 43:1075. Roussel , J. D., S . H. SeYbt. 1984 . The Louisiana Cattleman . Dec. page 11-12. Shaffer, L., J. D. Roussel, and K . L. Koonce . 1976. J. Dairy Sci . 59:16 . Shaffer, L., J. D. Roussel, and K. L . Koonce . 1981 . J. Dairy Sci. 64:62. Smith, J. W., and E . M. Kesler . 1969. J. Dairy Sci. 52:279. Steel, R .GD., and J . H. Torrie.1980. Princip les and Procedures of Statistics (2nd ed.) . McGraw-Hili Book Company, New York. Weeth, H . J. , R. Witton, C. F. Speth , and C . R. Blincoe. 1970. J. Anim. Sci. 30:219.