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Effect of Twinning and Supplemental Iron-saturated Lactoferrin on Iron Status of Newborn Calves
Effect of Twinning and Supplemental Iron-saturated Lactoferrin on Iron Status of Newborn Calves SHIN-ICHI KUME and SHINOBU TANABE Department of Animal Nutrition National Institute of Animal industry Tsukuba 305,Japan ABSTRACT
INTRODUCTION
Data from 53 calves by embryo transfer were collected to determine the effect of twinning and supplemental Fesaturated lactoferrin or FeSO4 for 5 d after parturition on iron status of calves. Blood hematocrit and hemoglobin of multiple calves born from multiparous cows at d 1 postpartum were lower than those of single calves born from multiparous cows, although those of their dams were not different. Plasma Ca, inorganic P, Mg, Zn, and Cu were similar for single and multiple calves or for calves born from primiparous and multiparous cows. Blood hematocrit and hemoglobin of untreated calves decreased from 1 to 6 d postpartum. Blood hematocrit of calves treated with 40 mg of Fe/d as FeSO4 increased from 1 to 10 d postpartum, and blood hemoglobin of calves treated with 20 or 40 mg of Fe/d as ferrous Fe increased from 1 to 10 d postpartum. Blood hematocrit and hemoglobin of calves treated with 20 mg of Fe/d as Fe-saturated lactofenin remained nearly constant for 10 d postpartum. Serum Fe of calves increased immediately after the treatment with 20 or 40 mg of Fe as FeS04, but serum Fe of calves treated with lactoferrin was not affected. Thus, ferrous Fe may be more effective than Fe-saturated lactofemn in elevating blood hematocrit and hemoglobin in newborn calves. (Key words: twinning, lactoferrin, iron, calves)
Iron-deficiency anemia can occur in calves at birth and can adversely affect calf growth rate and health (2, 3, 6, 9, 17). We (8) reported that lactation number of the dam was a factor for the alteration of Fe status, and calves born from primiparous cows developed low blood hematocrit (Hct) and hemoglobin (Hb). Twinning was another factor in the development of anemia, and the major differences between twin and single calves were that twins had lower Hct and Hb (1, 16). The use of embryo transfer to induce twinning in beef cattle has increased rapidly, but the lower survival of twins remains a problem (4, 5). Tennant et al. (16) reported that the mortality rate of anemic calves, including twins, was 22% during the first 2 wk of life, although most calves appeared to be clinically normal at birth. Thus, Fe supplementation after parturition may be essential for twin calves. Lactofemn (Lo, an Fe-binding protein, is abundant in colostrum at parturition and may act as an Fe source for newborn calves (18). The administration of Fe-saturated Lf can prevent anemia in rats (3, but whether Lf is a useful Fe source for calves is unclear. The objective of this study was to clarify the effect of twinning by embryo transfer on mineral status of calves and their dams and to compare the efficiency of supplemental Fe-saturated Lf and FeS04 on Fe status on calves during the 10 d after parturition. MATERIALS AND METHODS
Abbreviation key: Hb = hemoglobin, Hct = hematocrit, JBC = Japanese beef cattle, Lf = lactoferrin.
Received December 6, 1993. Accepted April 29. 1994. 1994 J Dairy Sci 77:3118-3123
Data from 53 calves and their dams were collected at the National Institute of Animal Industry from January 1990 to August 1993. The embryos of Japanese beef cattle (JBC), including Japanese Black, Japanese Brown, and Holstein x Japanese Black, were transferred into 35 Holstein and 9 JBC recipients. Recipients were managed in an environment relatively similar to that described by us previ-
31 18
TWINNING AND LACTOFERRIN
ously (8). Nine pairs of twins and 1 set of triplets were born from Holsteins, but 1 twin and 2 triplet calves died at parturition. Thus, calves born from Holsteins were assigned to three groups by the type of birth: 5 single calves born from primiparous cows, 20 single calves born from multiparous cows, and 18 multiple calves born from multiparous cows. Eight single and 1 pair of twin calves were born from JBC cows. Calves born from Holstein cows were separated from their dams at parturition and housed in individual pens. They received approximately 1 kg of colostrum at parturition and, thereafter, 2 kg of colostrum at 0900 and 1600 h daily from their dam’s colostrum for 1 wk postpartum. Dry feed was not offered for 1 wk postpartum. After 1 wk of age, calves were fed approximately 2 kg of whole milk twice a day, and calf starter pellets and mixed hay based on Italian ryegrass were offered; calf starter and mixed hay contained 159 and 213 ppm of Fe on a DM basis, respectively. Ten calves born from JBC cows were isolated with their dams in individual pens for 1 wk. Nineteen calves born from Holsteins were assigned to three treatments at d 1 postpartum by relatively similar hematological values and by sex. Eighteen calves born from Holsteins and 10 calves born from JBC were allocated to the untreated control group. Dietary treatments were 1) 20 mg of Fe/d as FeS04 (4 males and 2 females), 2) 40 mg of Fdd as FeS04 (4 males and 2 females), and 3) 20 mg of Fe/d as Fesaturated Lf (4 males and 3 females). Ironsaturated Lf (Morinaga Milk Industry Co., Ltd., Zama, Japan) was prepared according to the method of Mazurier and Spik (11) and contained 176 mg of Fe/100 g. The extent of Fe saturation was estimated to be about 132%. Treated calves were fed Fe sources mixed with morning colostrum from 1 to 5 d postpartum. Blood samples of Holstein recipients were obtained within 24 h after parturition. Blood samples of Fe-treated calves were collected at 0830 h of d 1, 2, 4, 6, and 10 after parturition, and those of untreated calves were collected at 0830 h on d 1 and 6 postpartum. Blood was collected via jugular puncture into heparinized and unheparinized vacuum tubes. Colostrum samples of Holstein were collected at parturition and at approximately 96 h postpartum. However, 4 blood samples of primiparous
3119
cows and 5 colostrum samples could not be taken at parturition. Blood Hct, Hb, and minerals of calves and their dams and colostrum composition were determined as previously (8). Serum samples were extracted from unheparinized blood and frozen at -20°C until they were analyzed for Fe. Serum Fe was determined by the method of Matsubara (10). The general linear models procedure of SAS (15) was used to analyze the effect of type of birth on blood and colostrum composition of cows at parturition and to analyze the effect of type of birth and sex on blood composition of calves at d 1 postpartum. Data from treated and untreated calves were analyzed by least squares ANOVA using the general linear models procedure of SAS (15). The model for treated and untreated calves was as follows:
where p = overall means, Ti = effect of treatment (or recipient for untreated calves), Cfij = random variable calf, nested in treatment (or recipient for untreated calves), Sk = effect of sampling time, TSk = interactions, and e i k = residuals.
Differences among type of birth or days postpartum were tested by least significant difference, and significance was declared at P e .05 unless otherwise noted. RESULTS AND DISCUSSION Mineral Status of Twin Calves
There was no significant difference in calf BW at birth, but BW of twin calves born from multiparous cows was slightly lower than that of single calves born from multiparous cows (Table 1). Blood Hct, Hb, serum Fe, and plasma minerals of dams were not significantly different among types of birth or between primiparous and multiparous cows. Blood Hct (Pe .01) and Hb (P< .00l) of twin calves born Journal of Dairy Science Vol. 77, No. 10, 1994
3120
KUME AND TANABE
TABLE 1. Least squares means of blood hematocrit (Hct), hemoglobin (Hb), serum iron, and plasma mineral concentrations of calves at d 1 postpartum and their dams within 24 h after parturition among type of birth.' Dams
Calves
Primiparous, Multiparous, Multiparous, single single multiple Animals, no. Age, mo. BW? kg Hct, % Hb, g!dl Serum Fe, ppm Plasma Ca. rng/dl PI> mg/dl Mg, mddl Zn, ppm Cu, ppm
5
SE
25.9b
16 54.58
10 48.1a
33.6 12.1 1.14
34.7 12.2 1.07
35.6 12.5 1.16
.6 .2 .07
9.5 5.8 2.08 1.09 .90
9.5 4.3 2.12
9.4 4.3 2.06
.2 .2
.80 .81
.88
.95
Primiparous, Multiparous, Multiparous, single single multiple
SE
20
18
31.1 35.8 11.8 .97
34.4 41.38 .84
30.8 32.6b 1o.w .69
1.2 1.1 .3 .06
11.7 7.1 1.95 .92 .40
11.3 7.7 1.85 .75 .45
11.3 7.7 1.72 .82 .37
.2 .2
5
1.8
.05 .08 .04
13.1C
.04
.07 .02
a.bMeans within dams or calves of same row with different superscript letters differ (f < .01). C.dMeans within dams or calves of same row with different superscript letters differ (f < ,001). ]Parity of dams and calving of single or multiple calves. *At birth. 3Inorganic P.
from multiparous cows were lower than those of single calves born from multiparous cows. Serum Fe and plasma Ca, inorganic P, Mg, Zn, and Cu of calves were not significantly different among types of birth or between calves born from primiparous and multiparous cows. Colostral specific gravity of primiparous cows at parturition was significantly lower
than that of multiparous cows (Table 2). Colostral Zn concentrations of multiparous cows that gave birth to twins were significantly higher than those of multiparous cows that gave birth to a single calf, although colostrum yields of multiparous cows that gave birth to twins were slightly higher. However, colostral Ca, P, Mg, Fe, and Cu at parturition were similar for the
TABLE 2. Least squares means of colostrum yield and colostrum composition of Holstein recipients among type of birth' immediately after parturition.
Type of birth
Cows, no. Colostrum yield, kg Specific gravity Total solids, 8 Protein, % Ca, mg/dl P, mddl Mg. mddl Fe, ppm Zn, ppm Cu. ppm
Primiparous, single
Multiparous, single
Multiparous, multiple
4 I .3 1.058b 31.6 15.7 263 207 34.9 1.4 21.9 .26
17 1.6 1.0698 30.8 18.1 220 189 32.4 1.2 18.7b .29
9 2.7 1,066a 32.2 17.6 230 193 32.3
a.bMeans within same row with different superscript letters differ (f < .05). 'Parity of dams and calving of single or multiple calves. Journal of Dairy Science Vol. 77, No. 10, 1994
1.5
25.7a .33
SE .2 ,001 1.o
.5
9 6 1.5
.1 1.3 .01
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TWINNING AND LACTOFERRIN
multiparous cows that gave birth to a single calf and twins. Anemia has adversely affected calf growth rate and health (2, 3, 6, 9, 17). Twinning and also lactation number were factors for the low blood Hct and Hb of newborn calves at parturition (1, 8, 16). Induction of twinning by embryo transfer drastically decreased blood Hct and Hb of twin calves born from multiparous cows in the present experiment. Additionally, blood Hct and Hb of twin calves born from primiparous cows (l),which were 27.5% and 8.8 g/dl at h 24 postpartum, were much lower than those of single calves born from primiparous cows. These results suggest that the risk of anemia development is high in twin calves. Hibbs et al. (6) reported that the dams of anemic calves had normal blood Hct and Hb,
even though their calves had low blood Hct and Hb at birth. Similarly, the twin calves had low blood Hct and Hb, but their dams had normal blood Hct and Hb (Table 1). Colostrum is the only source of Fe for newborn calves after parturition. However, colostral Fe concentrations, which were similar among dams that gave birth to a single or twin calves (Table 2), were much lower than the Fe requirement for the calves (13). Thus, further study is needed to clarify the lower hematological variables in twin calves at parturition. Although the use of embryo transfer to induce twinning in beef cattle has increased, calf mortality of twin calves was higher than that of singles by 1 wk postpartum (4,5). Adams et al. (1) suggested that the lower hematological variables of twins were associated with the increased mortality because
TABLE 3. Least squares means of blood hematocrit (Hct),hemoglobin (Hb), serum Fe, and plasma mineral concentrations of Fe-treated and untreated calves at d 1 and 6 after parturition. Time postpartum IBCI
Untreated
Fe-Treated
Holstein'
SE
FeSO4-202
FeS04403 Lf-204
18 31.7 40.l e 37.3' 12.38 11.7b .66 .70
1.2 .4 .4 .2 .2 .07 .07
6 38.2 36.4 36.7 11.7 12.3 .97 .78
6 29.0 34.9 36.4 10.7d 1 1 .7c .60 .89
7 33.4 36.6 36.1 11.2 11.3 1.02 .78
.I .I .2 .2 .03 .03
11.9 11.5 6.9f 8.7e 1.94 1.80
.06 .06 .03 .03
.90 1 .oo
11.6 11.3 8.5 8.6 1.65 I .60 .88 1.09 .496 .69c
11.2 11.3 7.4 8.8C 1.83 1.78 .77 .98 ,47f .74e
SE
(d) Calves, no. BW.5 kg Hct, 72
Hb, g/dl Serum Fe. ppm
1 6 1 6
10 32.5 37.6e 31.9' 11.9
1o.v
1
6 Plasma Ca, mg/dl
Pi,6 mg/dl Mg, mg/dl Zn. ppm Cu, pprn
1 6 1 6 1 6 1 6 1 6
11.8 11.3 9.6 9.7 2.06c 1.74d 1.12 1.30 .48'
.82e
11.5 11.5 8.0 8.5 1.83C 1.64d .75 .88 .4If .7oe
.44' .74e
2.2 .4 .4
.1 .1
.08
.08 .2 .2 .2 .2 .04 .04 .05 .05
.03 .03
a.bMeans between each component of same column with different superscript letters differ (P < ,051. CSdMeans between each component of same column with different superscript letters differ (P < .01). %leans between each component of same column with different superscript letters differ (P < ,001). 'Recipients. 2Treated 20 mg of Fe/d as FeSO4 from I to 5 d postpartum. 3Treated 40 mg of Fe/d as FeSO4 from 1 to 5 d postpartum. 4Treated 20 mg of Fe/d as Fe-saturated lactofemn from 1 to 5 d postpartum. 5At bixth. %organic P. Journal of Daiq Science Vol. 77, No. 10, 1994
3122
K U M E AND TANABE
of their difficulty in maintaining body temperature. Additionally, low colostral Fe was insufficient to maintain blood Hct and Hb of newborn calves, which decreased with time postpartum (8). Supplemental Fe Sources for Calves
Blood Hct (P < .OOl) and Hb of untreated calves born from Holstein or JBC recipients
40 39
~
T
i
351
10
T
L. '
3
1
"
5
"
7
I
"
9
11
I
16 I
'41
1
! l
4'
I
1
'
"
3
"
"
7 Days postpartum
5
"
9
11
Figure 1. Blood hematocrit (Ha), hemoglobin (Hb),and serum Fe of calves treated 20 mg of Fe/d as FeSO4 (a), 40 mg of Fdd as FeSO4 C).and 20 mg of Fdd as Fesaturated lactofemn (A) from 1 to 5 d postpartum. Vertical lines indicate standard errors. Journal of Dairy Science Vol. 77, No. 10, 1994
decreased from 1 to 6 d postpartum, although the decrease rate was greater for the calves born from JBC dams (Table 3). However, differences in serum Fe of untreated calves born from Holsteins were not significant at d 1 and 6 postpartum. Compared with the sampling time at d 1 and 6 postpartum, plasma Mg of untreated calves decreased (P < .Ol) with time postpartum, but plasma Cu (P < .001) increased. These results agree with the data of Holstein calves in our previous report (8). Blood Hct of calves treated with 40 mg of Fe as ferrous Fe significantly increased from 1 to 10 d postpartum, and blood Hb of calves treated with 20 (P < .01) and 40 (P < .OOl) mg of Fe as ferrous Fe increased from 1 to 10 d postpartum (Figure 1). However, blood Hct and Hb of Lf-treated calves remained nearly constant. Serum Fe of calves treated with 40 mg of Fe as ferrous Fe significantly increased (P < .001) at d 2 postpartum and then decreased, and the change in serum Fe of calves treated with 20 mg of Fe as ferrous Fe was similar. Although serum Fe of Lf-treated calves at d 2 was significantly higher than that at d 4, the increased rate of serum Fe from 1 to 2 d postpartum was less. The changes of plasma Ca, inorganic P, Mg, Zn, and Cu of treated calves from 1 to 6 d postpartum were similar to those of untreated calves born from Holstein recipients (Table 3). The mean colostral Fe of cows in the treatment groups were 1.5 ppm at parturition and .5 ppm at h 96 postpartum. Because calves received approximately 4 kg of colostrum daily and colostral Fe decreased rapidly by 24 h after parturition (8), Fe intakes from colostrum of treated calves were estimated to be about 2 to 4 mgld during 1 wk postpartum. The administration of 40 mg of Fe/d as FeS04, a level that has been reported to be the dietary requirement for calves (9, 12, 14), elevated blood Hct and Hb of calves for 10 d after birth. Miyata et al. (12) showed that blood Hct and Hb of calves that had been administered 40 mg of Fe/d as ferrous fumarate from d 3 to 22 of age increased immediately after treatment. Thus, the administration of 40 mg of Fe/d as ferrous Fe may be effective for the improvement of Fe status in newborn calves. Colostrum contains a high level of Lf, and colostral Lf functions as an Fe source for the neonate and is a potent antimicrobial factor in
TWINNING AND LACTOFERRIN
its alimentary tract (7, 18). The administration of Fe-saturated Lf enhanced blood Hct and Hb in anemic rats, but the same amount of Fe as FeS04 produced no improvement (7). In the present experiment, blood Hct and Hb of calves were not affected by the administration of 20 mg of Fe/d as Fe-saturated Lf, although the same amounts of Fe as FeS04 improved blood Hb. Serum Fe increased temporarily, and the peak occurred just after treatment with FeS04, but Fe-saturated Lf had no effect on serum Fe. These results suggest that ferrous Fe may be more effective in elevating blood Hct and Hb than Fe-saturated Lf in newborn calves. However, Tsuji et al. (18) reported that Lf in colostrum of cows varied by breed and lactation number. Thus, further study is needed to clarify the physiological functions of Lf for newborn calves because many biological functions have been attributed to Lf. CONCLUSIONS
Induction of twinning by means of embryo transfer drastically decreased blood Hct and Hb of twin calves at parturition. The administration of 40 mg of Fe/d as FeS04 elevated blood Hct and Hb of calves during 10 d after parturition. Blood Hct and Hb of calves were not affected by the administration of 20 mg of Fe/d as Fe-saturated Lf, although the same amounts of Fe as FeS04 improved blood Hb. Thus, ferrous Fe may be more effective than Fe-saturated Lf as an Fe source for newborn calves. ACKNOWLEDGMENTS
The authors thank T. Kojima for embryo transfer; Y. Fukuwatari and Morinaga Milk Industry Co., Ltd. for supply of Lf; and T. Shimada, T. Nishida, and the staff of National Institute of Animal Industry for technical help and assistance in sample collection. REFERENCES I Adams, R., F. B. Garry, B. M. Aldridge, M. D. Holland, and K. G . Odde. 1993. Physiologic differences between twin and single born beef calves in the first two days of life. Cornel1 Vet. 83:13. 2Baxter, K. L., G.A.M. Sharman. and A. M. MacDonald. 1957. Iron-deficiency anemia in calves. Br. J. Nutr. 11:234.
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3 Bremner, I., and A. C. Dalgamo. 1973. Iron metabolism in the veal calf. 2. Iron requirements and the effects of copper supplementation. Br. J. Nutr. 30:61. 4Gregory, K. E.. S. E. Echtemkamp, G. E. Dickerson, L. V. Cundiff, R. M. Koch, and L. D. Van Vleck. 1990. Twinning in cattle. 3. Effects of twinning on dystocia, reproductive traits, calf survival, calf growth and cow productivity. J. Anim. Sci. 68:3133. 5 Guerra-Martinez. P., G . E. Dickerson, G. B. Anderson, and R. D. Green. 1990. Embryo-transfer twinning and performance efficiency in beef production. J. h i m . Sci. 68:4039. 6Hibbs. J. W., H. R. Conrad, J. H. Vandersall, and C. Gale. 1963. Occurrence of iron deficiency anemia in dairy calves at birth and its alleviation by iron dextran injection. J. Dairy Sci. 46:1118. 7Kawakami. H.. M. Hiratsuka, and S. Dosilko. 1988. Effects of iron-saturated lactofemn on iron absorption. Agnc. Biol. Chem. 52:903. 8 Kume, S., and S . Tanabe. 1993. Effect of parity on colostral mineral concentrations of Holstein cows and value of colostrum as a mineral source for newborn calves. J. Dairy Sci. 76:1654. 9Matrone. G., C. Conley, G. H. Wise, and R. K. Waugh. 1957. A study of iron and copper requirement of dairy calves. J. Dairy Sci. 40:1437. 10Matsubara. T. 1961. Studies on the method for determination of iron in biological materials, especially serum and whole blood. A new proposal for the standardization of the method. Acta Haematol. Jpn. 24:434. (In Japanese.) 11 Mazurier, J., and G . Spik. 1980. Comparative study of the iron-binding properties of human transferrins. 1. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochim. Biophys. Acta 629:399. 12Miyata Y., K. Furugouri, and K. Shijimaya. 1984. Developmental changes in serum ferritin concentration of dairy calves. J. Dairy Sci. 67:1256. 13 National Research Council. 1988. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 14Roy, J.H.B., H. J. Gaston, K.W.G. Shillam, S. Y. Thompson, and I.J.F. Stobo. 1964. The nutrition of the veal calf. The effect of anemia and of iron and chlortetracycline supplementation on the performance of calves given large quantities of whole milk. Br. J. NUQ.18467. 15 SA@ User’s Guide: Statistics. Version 6.03 Edition. 1988. SAS Inst., Inc., Cary, NC. 16Tennant. B.. D. Harrold, M. Reina-Guerra, and J. J. Kaneko. 1975. Hematology of the neonatal calf. 3. Frequency of congenital iron deficiency anemia. Corne11 Vet. 65543. 17 Thomas, J. W., M. Okamoto, W. C. Jacobson! and L. A. Moore. 1954. A study of hemoglobin levels in the blood of young dairy calves and the alleviation of anemia by iron. J. Dairy Sci. 37:805. 18 Tsuji, S., Y. Hirata, F. Mukai, and S. Ohtagaki. 1990. Comparison of lactoferrin content in colostrum between different cattle breeds. J. Dairy Sci. 73:125.
Journal of Dairy Science Vol. 77, No. 10, 1994
INTRODUCTION
Data from 53 calves by embryo transfer were collected to determine the effect of twinning and supplemental Fesaturated lactoferrin or FeSO4 for 5 d after parturition on iron status of calves. Blood hematocrit and hemoglobin of multiple calves born from multiparous cows at d 1 postpartum were lower than those of single calves born from multiparous cows, although those of their dams were not different. Plasma Ca, inorganic P, Mg, Zn, and Cu were similar for single and multiple calves or for calves born from primiparous and multiparous cows. Blood hematocrit and hemoglobin of untreated calves decreased from 1 to 6 d postpartum. Blood hematocrit of calves treated with 40 mg of Fe/d as FeSO4 increased from 1 to 10 d postpartum, and blood hemoglobin of calves treated with 20 or 40 mg of Fe/d as ferrous Fe increased from 1 to 10 d postpartum. Blood hematocrit and hemoglobin of calves treated with 20 mg of Fe/d as Fe-saturated lactofenin remained nearly constant for 10 d postpartum. Serum Fe of calves increased immediately after the treatment with 20 or 40 mg of Fe as FeS04, but serum Fe of calves treated with lactoferrin was not affected. Thus, ferrous Fe may be more effective than Fe-saturated lactofemn in elevating blood hematocrit and hemoglobin in newborn calves. (Key words: twinning, lactoferrin, iron, calves)
Iron-deficiency anemia can occur in calves at birth and can adversely affect calf growth rate and health (2, 3, 6, 9, 17). We (8) reported that lactation number of the dam was a factor for the alteration of Fe status, and calves born from primiparous cows developed low blood hematocrit (Hct) and hemoglobin (Hb). Twinning was another factor in the development of anemia, and the major differences between twin and single calves were that twins had lower Hct and Hb (1, 16). The use of embryo transfer to induce twinning in beef cattle has increased rapidly, but the lower survival of twins remains a problem (4, 5). Tennant et al. (16) reported that the mortality rate of anemic calves, including twins, was 22% during the first 2 wk of life, although most calves appeared to be clinically normal at birth. Thus, Fe supplementation after parturition may be essential for twin calves. Lactofemn (Lo, an Fe-binding protein, is abundant in colostrum at parturition and may act as an Fe source for newborn calves (18). The administration of Fe-saturated Lf can prevent anemia in rats (3, but whether Lf is a useful Fe source for calves is unclear. The objective of this study was to clarify the effect of twinning by embryo transfer on mineral status of calves and their dams and to compare the efficiency of supplemental Fe-saturated Lf and FeS04 on Fe status on calves during the 10 d after parturition. MATERIALS AND METHODS
Abbreviation key: Hb = hemoglobin, Hct = hematocrit, JBC = Japanese beef cattle, Lf = lactoferrin.
Received December 6, 1993. Accepted April 29. 1994. 1994 J Dairy Sci 77:3118-3123
Data from 53 calves and their dams were collected at the National Institute of Animal Industry from January 1990 to August 1993. The embryos of Japanese beef cattle (JBC), including Japanese Black, Japanese Brown, and Holstein x Japanese Black, were transferred into 35 Holstein and 9 JBC recipients. Recipients were managed in an environment relatively similar to that described by us previ-
31 18
TWINNING AND LACTOFERRIN
ously (8). Nine pairs of twins and 1 set of triplets were born from Holsteins, but 1 twin and 2 triplet calves died at parturition. Thus, calves born from Holsteins were assigned to three groups by the type of birth: 5 single calves born from primiparous cows, 20 single calves born from multiparous cows, and 18 multiple calves born from multiparous cows. Eight single and 1 pair of twin calves were born from JBC cows. Calves born from Holstein cows were separated from their dams at parturition and housed in individual pens. They received approximately 1 kg of colostrum at parturition and, thereafter, 2 kg of colostrum at 0900 and 1600 h daily from their dam’s colostrum for 1 wk postpartum. Dry feed was not offered for 1 wk postpartum. After 1 wk of age, calves were fed approximately 2 kg of whole milk twice a day, and calf starter pellets and mixed hay based on Italian ryegrass were offered; calf starter and mixed hay contained 159 and 213 ppm of Fe on a DM basis, respectively. Ten calves born from JBC cows were isolated with their dams in individual pens for 1 wk. Nineteen calves born from Holsteins were assigned to three treatments at d 1 postpartum by relatively similar hematological values and by sex. Eighteen calves born from Holsteins and 10 calves born from JBC were allocated to the untreated control group. Dietary treatments were 1) 20 mg of Fe/d as FeS04 (4 males and 2 females), 2) 40 mg of Fdd as FeS04 (4 males and 2 females), and 3) 20 mg of Fe/d as Fesaturated Lf (4 males and 3 females). Ironsaturated Lf (Morinaga Milk Industry Co., Ltd., Zama, Japan) was prepared according to the method of Mazurier and Spik (11) and contained 176 mg of Fe/100 g. The extent of Fe saturation was estimated to be about 132%. Treated calves were fed Fe sources mixed with morning colostrum from 1 to 5 d postpartum. Blood samples of Holstein recipients were obtained within 24 h after parturition. Blood samples of Fe-treated calves were collected at 0830 h of d 1, 2, 4, 6, and 10 after parturition, and those of untreated calves were collected at 0830 h on d 1 and 6 postpartum. Blood was collected via jugular puncture into heparinized and unheparinized vacuum tubes. Colostrum samples of Holstein were collected at parturition and at approximately 96 h postpartum. However, 4 blood samples of primiparous
3119
cows and 5 colostrum samples could not be taken at parturition. Blood Hct, Hb, and minerals of calves and their dams and colostrum composition were determined as previously (8). Serum samples were extracted from unheparinized blood and frozen at -20°C until they were analyzed for Fe. Serum Fe was determined by the method of Matsubara (10). The general linear models procedure of SAS (15) was used to analyze the effect of type of birth on blood and colostrum composition of cows at parturition and to analyze the effect of type of birth and sex on blood composition of calves at d 1 postpartum. Data from treated and untreated calves were analyzed by least squares ANOVA using the general linear models procedure of SAS (15). The model for treated and untreated calves was as follows:
where p = overall means, Ti = effect of treatment (or recipient for untreated calves), Cfij = random variable calf, nested in treatment (or recipient for untreated calves), Sk = effect of sampling time, TSk = interactions, and e i k = residuals.
Differences among type of birth or days postpartum were tested by least significant difference, and significance was declared at P e .05 unless otherwise noted. RESULTS AND DISCUSSION Mineral Status of Twin Calves
There was no significant difference in calf BW at birth, but BW of twin calves born from multiparous cows was slightly lower than that of single calves born from multiparous cows (Table 1). Blood Hct, Hb, serum Fe, and plasma minerals of dams were not significantly different among types of birth or between primiparous and multiparous cows. Blood Hct (Pe .01) and Hb (P< .00l) of twin calves born Journal of Dairy Science Vol. 77, No. 10, 1994
3120
KUME AND TANABE
TABLE 1. Least squares means of blood hematocrit (Hct), hemoglobin (Hb), serum iron, and plasma mineral concentrations of calves at d 1 postpartum and their dams within 24 h after parturition among type of birth.' Dams
Calves
Primiparous, Multiparous, Multiparous, single single multiple Animals, no. Age, mo. BW? kg Hct, % Hb, g!dl Serum Fe, ppm Plasma Ca. rng/dl PI> mg/dl Mg, mddl Zn, ppm Cu, ppm
5
SE
25.9b
16 54.58
10 48.1a
33.6 12.1 1.14
34.7 12.2 1.07
35.6 12.5 1.16
.6 .2 .07
9.5 5.8 2.08 1.09 .90
9.5 4.3 2.12
9.4 4.3 2.06
.2 .2
.80 .81
.88
.95
Primiparous, Multiparous, Multiparous, single single multiple
SE
20
18
31.1 35.8 11.8 .97
34.4 41.38 .84
30.8 32.6b 1o.w .69
1.2 1.1 .3 .06
11.7 7.1 1.95 .92 .40
11.3 7.7 1.85 .75 .45
11.3 7.7 1.72 .82 .37
.2 .2
5
1.8
.05 .08 .04
13.1C
.04
.07 .02
a.bMeans within dams or calves of same row with different superscript letters differ (f < .01). C.dMeans within dams or calves of same row with different superscript letters differ (f < ,001). ]Parity of dams and calving of single or multiple calves. *At birth. 3Inorganic P.
from multiparous cows were lower than those of single calves born from multiparous cows. Serum Fe and plasma Ca, inorganic P, Mg, Zn, and Cu of calves were not significantly different among types of birth or between calves born from primiparous and multiparous cows. Colostral specific gravity of primiparous cows at parturition was significantly lower
than that of multiparous cows (Table 2). Colostral Zn concentrations of multiparous cows that gave birth to twins were significantly higher than those of multiparous cows that gave birth to a single calf, although colostrum yields of multiparous cows that gave birth to twins were slightly higher. However, colostral Ca, P, Mg, Fe, and Cu at parturition were similar for the
TABLE 2. Least squares means of colostrum yield and colostrum composition of Holstein recipients among type of birth' immediately after parturition.
Type of birth
Cows, no. Colostrum yield, kg Specific gravity Total solids, 8 Protein, % Ca, mg/dl P, mddl Mg. mddl Fe, ppm Zn, ppm Cu. ppm
Primiparous, single
Multiparous, single
Multiparous, multiple
4 I .3 1.058b 31.6 15.7 263 207 34.9 1.4 21.9 .26
17 1.6 1.0698 30.8 18.1 220 189 32.4 1.2 18.7b .29
9 2.7 1,066a 32.2 17.6 230 193 32.3
a.bMeans within same row with different superscript letters differ (f < .05). 'Parity of dams and calving of single or multiple calves. Journal of Dairy Science Vol. 77, No. 10, 1994
1.5
25.7a .33
SE .2 ,001 1.o
.5
9 6 1.5
.1 1.3 .01
3121
TWINNING AND LACTOFERRIN
multiparous cows that gave birth to a single calf and twins. Anemia has adversely affected calf growth rate and health (2, 3, 6, 9, 17). Twinning and also lactation number were factors for the low blood Hct and Hb of newborn calves at parturition (1, 8, 16). Induction of twinning by embryo transfer drastically decreased blood Hct and Hb of twin calves born from multiparous cows in the present experiment. Additionally, blood Hct and Hb of twin calves born from primiparous cows (l),which were 27.5% and 8.8 g/dl at h 24 postpartum, were much lower than those of single calves born from primiparous cows. These results suggest that the risk of anemia development is high in twin calves. Hibbs et al. (6) reported that the dams of anemic calves had normal blood Hct and Hb,
even though their calves had low blood Hct and Hb at birth. Similarly, the twin calves had low blood Hct and Hb, but their dams had normal blood Hct and Hb (Table 1). Colostrum is the only source of Fe for newborn calves after parturition. However, colostral Fe concentrations, which were similar among dams that gave birth to a single or twin calves (Table 2), were much lower than the Fe requirement for the calves (13). Thus, further study is needed to clarify the lower hematological variables in twin calves at parturition. Although the use of embryo transfer to induce twinning in beef cattle has increased, calf mortality of twin calves was higher than that of singles by 1 wk postpartum (4,5). Adams et al. (1) suggested that the lower hematological variables of twins were associated with the increased mortality because
TABLE 3. Least squares means of blood hematocrit (Hct),hemoglobin (Hb), serum Fe, and plasma mineral concentrations of Fe-treated and untreated calves at d 1 and 6 after parturition. Time postpartum IBCI
Untreated
Fe-Treated
Holstein'
SE
FeSO4-202
FeS04403 Lf-204
18 31.7 40.l e 37.3' 12.38 11.7b .66 .70
1.2 .4 .4 .2 .2 .07 .07
6 38.2 36.4 36.7 11.7 12.3 .97 .78
6 29.0 34.9 36.4 10.7d 1 1 .7c .60 .89
7 33.4 36.6 36.1 11.2 11.3 1.02 .78
.I .I .2 .2 .03 .03
11.9 11.5 6.9f 8.7e 1.94 1.80
.06 .06 .03 .03
.90 1 .oo
11.6 11.3 8.5 8.6 1.65 I .60 .88 1.09 .496 .69c
11.2 11.3 7.4 8.8C 1.83 1.78 .77 .98 ,47f .74e
SE
(d) Calves, no. BW.5 kg Hct, 72
Hb, g/dl Serum Fe. ppm
1 6 1 6
10 32.5 37.6e 31.9' 11.9
1o.v
1
6 Plasma Ca, mg/dl
Pi,6 mg/dl Mg, mg/dl Zn. ppm Cu, pprn
1 6 1 6 1 6 1 6 1 6
11.8 11.3 9.6 9.7 2.06c 1.74d 1.12 1.30 .48'
.82e
11.5 11.5 8.0 8.5 1.83C 1.64d .75 .88 .4If .7oe
.44' .74e
2.2 .4 .4
.1 .1
.08
.08 .2 .2 .2 .2 .04 .04 .05 .05
.03 .03
a.bMeans between each component of same column with different superscript letters differ (P < ,051. CSdMeans between each component of same column with different superscript letters differ (P < .01). %leans between each component of same column with different superscript letters differ (P < ,001). 'Recipients. 2Treated 20 mg of Fe/d as FeSO4 from I to 5 d postpartum. 3Treated 40 mg of Fe/d as FeSO4 from 1 to 5 d postpartum. 4Treated 20 mg of Fe/d as Fe-saturated lactofemn from 1 to 5 d postpartum. 5At bixth. %organic P. Journal of Daiq Science Vol. 77, No. 10, 1994
3122
K U M E AND TANABE
of their difficulty in maintaining body temperature. Additionally, low colostral Fe was insufficient to maintain blood Hct and Hb of newborn calves, which decreased with time postpartum (8). Supplemental Fe Sources for Calves
Blood Hct (P < .OOl) and Hb of untreated calves born from Holstein or JBC recipients
40 39
~
T
i
351
10
T
L. '
3
1
"
5
"
7
I
"
9
11
I
16 I
'41
1
! l
4'
I
1
'
"
3
"
"
7 Days postpartum
5
"
9
11
Figure 1. Blood hematocrit (Ha), hemoglobin (Hb),and serum Fe of calves treated 20 mg of Fe/d as FeSO4 (a), 40 mg of Fdd as FeSO4 C).and 20 mg of Fdd as Fesaturated lactofemn (A) from 1 to 5 d postpartum. Vertical lines indicate standard errors. Journal of Dairy Science Vol. 77, No. 10, 1994
decreased from 1 to 6 d postpartum, although the decrease rate was greater for the calves born from JBC dams (Table 3). However, differences in serum Fe of untreated calves born from Holsteins were not significant at d 1 and 6 postpartum. Compared with the sampling time at d 1 and 6 postpartum, plasma Mg of untreated calves decreased (P < .Ol) with time postpartum, but plasma Cu (P < .001) increased. These results agree with the data of Holstein calves in our previous report (8). Blood Hct of calves treated with 40 mg of Fe as ferrous Fe significantly increased from 1 to 10 d postpartum, and blood Hb of calves treated with 20 (P < .01) and 40 (P < .OOl) mg of Fe as ferrous Fe increased from 1 to 10 d postpartum (Figure 1). However, blood Hct and Hb of Lf-treated calves remained nearly constant. Serum Fe of calves treated with 40 mg of Fe as ferrous Fe significantly increased (P < .001) at d 2 postpartum and then decreased, and the change in serum Fe of calves treated with 20 mg of Fe as ferrous Fe was similar. Although serum Fe of Lf-treated calves at d 2 was significantly higher than that at d 4, the increased rate of serum Fe from 1 to 2 d postpartum was less. The changes of plasma Ca, inorganic P, Mg, Zn, and Cu of treated calves from 1 to 6 d postpartum were similar to those of untreated calves born from Holstein recipients (Table 3). The mean colostral Fe of cows in the treatment groups were 1.5 ppm at parturition and .5 ppm at h 96 postpartum. Because calves received approximately 4 kg of colostrum daily and colostral Fe decreased rapidly by 24 h after parturition (8), Fe intakes from colostrum of treated calves were estimated to be about 2 to 4 mgld during 1 wk postpartum. The administration of 40 mg of Fe/d as FeS04, a level that has been reported to be the dietary requirement for calves (9, 12, 14), elevated blood Hct and Hb of calves for 10 d after birth. Miyata et al. (12) showed that blood Hct and Hb of calves that had been administered 40 mg of Fe/d as ferrous fumarate from d 3 to 22 of age increased immediately after treatment. Thus, the administration of 40 mg of Fe/d as ferrous Fe may be effective for the improvement of Fe status in newborn calves. Colostrum contains a high level of Lf, and colostral Lf functions as an Fe source for the neonate and is a potent antimicrobial factor in
TWINNING AND LACTOFERRIN
its alimentary tract (7, 18). The administration of Fe-saturated Lf enhanced blood Hct and Hb in anemic rats, but the same amount of Fe as FeS04 produced no improvement (7). In the present experiment, blood Hct and Hb of calves were not affected by the administration of 20 mg of Fe/d as Fe-saturated Lf, although the same amounts of Fe as FeS04 improved blood Hb. Serum Fe increased temporarily, and the peak occurred just after treatment with FeS04, but Fe-saturated Lf had no effect on serum Fe. These results suggest that ferrous Fe may be more effective in elevating blood Hct and Hb than Fe-saturated Lf in newborn calves. However, Tsuji et al. (18) reported that Lf in colostrum of cows varied by breed and lactation number. Thus, further study is needed to clarify the physiological functions of Lf for newborn calves because many biological functions have been attributed to Lf. CONCLUSIONS
Induction of twinning by means of embryo transfer drastically decreased blood Hct and Hb of twin calves at parturition. The administration of 40 mg of Fe/d as FeS04 elevated blood Hct and Hb of calves during 10 d after parturition. Blood Hct and Hb of calves were not affected by the administration of 20 mg of Fe/d as Fe-saturated Lf, although the same amounts of Fe as FeS04 improved blood Hb. Thus, ferrous Fe may be more effective than Fe-saturated Lf as an Fe source for newborn calves. ACKNOWLEDGMENTS
The authors thank T. Kojima for embryo transfer; Y. Fukuwatari and Morinaga Milk Industry Co., Ltd. for supply of Lf; and T. Shimada, T. Nishida, and the staff of National Institute of Animal Industry for technical help and assistance in sample collection. REFERENCES I Adams, R., F. B. Garry, B. M. Aldridge, M. D. Holland, and K. G . Odde. 1993. Physiologic differences between twin and single born beef calves in the first two days of life. Cornel1 Vet. 83:13. 2Baxter, K. L., G.A.M. Sharman. and A. M. MacDonald. 1957. Iron-deficiency anemia in calves. Br. J. Nutr. 11:234.
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3 Bremner, I., and A. C. Dalgamo. 1973. Iron metabolism in the veal calf. 2. Iron requirements and the effects of copper supplementation. Br. J. Nutr. 30:61. 4Gregory, K. E.. S. E. Echtemkamp, G. E. Dickerson, L. V. Cundiff, R. M. Koch, and L. D. Van Vleck. 1990. Twinning in cattle. 3. Effects of twinning on dystocia, reproductive traits, calf survival, calf growth and cow productivity. J. Anim. Sci. 68:3133. 5 Guerra-Martinez. P., G . E. Dickerson, G. B. Anderson, and R. D. Green. 1990. Embryo-transfer twinning and performance efficiency in beef production. J. h i m . Sci. 68:4039. 6Hibbs. J. W., H. R. Conrad, J. H. Vandersall, and C. Gale. 1963. Occurrence of iron deficiency anemia in dairy calves at birth and its alleviation by iron dextran injection. J. Dairy Sci. 46:1118. 7Kawakami. H.. M. Hiratsuka, and S. Dosilko. 1988. Effects of iron-saturated lactofemn on iron absorption. Agnc. Biol. Chem. 52:903. 8 Kume, S., and S . Tanabe. 1993. Effect of parity on colostral mineral concentrations of Holstein cows and value of colostrum as a mineral source for newborn calves. J. Dairy Sci. 76:1654. 9Matrone. G., C. Conley, G. H. Wise, and R. K. Waugh. 1957. A study of iron and copper requirement of dairy calves. J. Dairy Sci. 40:1437. 10Matsubara. T. 1961. Studies on the method for determination of iron in biological materials, especially serum and whole blood. A new proposal for the standardization of the method. Acta Haematol. Jpn. 24:434. (In Japanese.) 11 Mazurier, J., and G . Spik. 1980. Comparative study of the iron-binding properties of human transferrins. 1. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochim. Biophys. Acta 629:399. 12Miyata Y., K. Furugouri, and K. Shijimaya. 1984. Developmental changes in serum ferritin concentration of dairy calves. J. Dairy Sci. 67:1256. 13 National Research Council. 1988. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 14Roy, J.H.B., H. J. Gaston, K.W.G. Shillam, S. Y. Thompson, and I.J.F. Stobo. 1964. The nutrition of the veal calf. The effect of anemia and of iron and chlortetracycline supplementation on the performance of calves given large quantities of whole milk. Br. J. NUQ.18467. 15 SA@ User’s Guide: Statistics. Version 6.03 Edition. 1988. SAS Inst., Inc., Cary, NC. 16Tennant. B.. D. Harrold, M. Reina-Guerra, and J. J. Kaneko. 1975. Hematology of the neonatal calf. 3. Frequency of congenital iron deficiency anemia. Corne11 Vet. 65543. 17 Thomas, J. W., M. Okamoto, W. C. Jacobson! and L. A. Moore. 1954. A study of hemoglobin levels in the blood of young dairy calves and the alleviation of anemia by iron. J. Dairy Sci. 37:805. 18 Tsuji, S., Y. Hirata, F. Mukai, and S. Ohtagaki. 1990. Comparison of lactoferrin content in colostrum between different cattle breeds. J. Dairy Sci. 73:125.
Journal of Dairy Science Vol. 77, No. 10, 1994