DOMESTIC

ELSEVIER

ANIMAL ENDOCRINOLOGY Vol. 13(4):351-359, 1996

PLASMA BOVINE PLACENTAL LACTOGEN CONCENTRATION THROUGHOUT PREGNANCY IN THE COW; RELATIONSHIP TO STAGE OF PREGNANCY, FETAL MASS, NUMBER AND POSTPARTUM MILK YIELD
O.V. Patel,* M. Hirako,** T. Takahashi,** *Department N. Sasaki,*s and I. Domeki***

of Veterinary Surgery and Obstetrics, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan **Department of Animal Reproduction, National Institute of Animal Industry, P.O. Box 5, Norindanchi, Tsukuba, lbaraki 305, Japan ***Department of Animal Reproduction, Faculty of Agriculture, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156, Japan
Received July 6, 1995

This study characterized the peripheral plasma placental lactogen (bPL) profile throughout gestation and examined the relationship between the stage of gestation, fetal mass, number, and postpartum lactation with circulating levels of bPL in Holstein cows after nonsurgical embryo transfer. Cows (n = 12) were divided into two groups: Group 1 = single embryo recipient cows (n = 5); Group 2 = twin embryo recipient cows (n = 7). Blood was collected about every third day from Day 0 (Day 0 was defined as the first day of standing e&us), then daily for the last 10 d of gestation, and sampling was stopped 1 d postpartum. The cows were milked twice daily at 0800 and 1800 hr. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from that of the group and reported separately. The time trend concentrations of plasma bPL were significantly affected by the stage of gestation (P < 0.01) but not fetal number (P < 0.21). In both groups bPL levels remained low during the first two trimesters, then increased rapidly (P < 0.01) to peak concentrations between Days 200 and 220, and stabilized at this elevated level until parturition. Postpartum milk yields were indistinguishable between the singleton and twin-bearing cows. Calf birth weight and postpartum lactation were both correlated (P c 0.01) to peripheral bPL concentration in singleton cows, however, this relationship decreased with a subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited a deviating bPL profile. These results indicate that peripheral bPL levels are positively associated with the stage of gestation but not with fetal number. Otherwise, the peripheral pattern of bPL is a valuable index for predicting feto-placental viability.

INTRODUCTION
A number of species secrete a proteinaceous hormone of placental origin that shares structural and functional homology to growth hormone and prolactin. This hormone was initially labeled as chorionic somatomammotropin but is more commonly called placental lactogen (PL) (1,2). The function of PL probably varies according to species, however, it has been suggested that it influences mammogenesis, lactogenesis, and ovarian and placental steroidogenesis, and that it alters the maternal metabolism to accommodate the growth and development of the fetus (1,2). Among the domestic ruminants, Buttle et al. (3) were the first to determine its presence in goats, sheep (4), and cows (5). Placental lactogen has been purified to homogeneity in these species and its biochemical structure
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PATEL ET AL.

characterized (6-8). Earlier methods for quantifying the peripheral levels of this hormone in ruminants included bioassays or radioreceptor assays for lactogenic hormones (3-5). The drawback of these assay systems was that they measured combined lactogenic activity of pituitary and placental sources. However, with the availability of purified material, specific and sensitive radioimmunoassay systems have been established (9-l 1). This has allowed the changes in the peripheral levels of PL to be characterized more accurately in the ruminants, notably the cow. Recent studies employing radioimmunoassays against highly purified bPL have reported concentrations of several magnitudes lower in the cow than those found in the goat and the sheep (1612). In addition, in these latter species PL levels have been shown to be related to the stage of gestation, fetal number, and subsequent milk production (9,13). In humans, PL concentrations are also correlated with the number and weight of the neonate (14,15). So far, there is evidence in the cow that PL is related to the stage of pregnancy (12), but definitive fetal influence on the circulating levels of PL remains uncharacterized. In addition, in cattle there are no data on the association between PL levels during pregnancy and postpartum lactation. Therefore, the objectives of this study were to characterize the PL profiles in the cow and determine whether there is any relationship between peripheral PL levels, stage of pregnancy, mass of the fetus, number of fetuses being carried and postpartum lactation. MATERIALS AND METHODS

Animals and Embryo Transfer. Regularly cycling multiparous Holstein cows (n = 12), housed and managed in the National Institute of Animal Industry were used as recipient animals. The animals were divided into two groups: single embryo recipient cows (Group 1; n = 5) and twin embryo recipient cows (Group 2; n = 7). Both groups were fed the same total mixed ration. The cows were machine milked twice daily at 0800 and 1800 hr. Daily milk production for each cow was recorded electronically at each milking. The Japanese Black cattle embryos used in this study were generated by in vitro fertilization, were obtained from a commercial supplier (Tokyo Bio-tech Center, Tokyo, Japan), and details of their production are given elsewhere (16). In all cows, the embryos were non-surgically transferred on Day 7 of the estrous cycle (Day 0 was defined as the first day of standing estrus. A singleton pregnancy was established by transfer of a single embryo into the uterine horn ipsilateral to the corpus luteum-bearing ovary. A twin pregnancy was established by transfer of individual embryos into each uterine horn. Pregnancy was diagnosed at Day 30 by ultrasonography and confirmed by the birth of the calf or calves. In the present investigation, the results of cows that maintained their pregnancies and gave birth to a calf or calves are reported. Blood Collection. Blood was collected via jugular venipuncture into a 50-ml heparinized polypropylene tube, placed immediately on ice, and centrifuged (1800 g at 4 C for 1 hr) within 1 hr of collection. The harvested plasma was stored at -20 C until assayed. Blood was collected about every third day from Day 0, then daily for the last 10 d of pregnancy, and sampling was stopped 1 d postpartum. bPL Radioimmunoassay. Bovine placental lactogen assay and validation criteria have been previously described (11). Briefly, plasma samples (300 ~1, neat) in duplicate and appropriately diluted working standards (0.3 to 19.2 ng/ml) were incubated overnight at room temperature after the addition of primary antibody (200 l_~.l, diluted to 1: 10,000, U.S. Department of Agriculture-F56). The following day, 100 p,l of iodinated bPL [1251]-bPL, lot 9205 14-10, Monsanto Co., St. Louis, MO) was added to all tubes and further incubated at room temperature for an additional 48 hr. Radioiodinated bPL and bPL standards were

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recombinant material (Lot 920514-10, Monsanto Co.). Appropriate amounts of control plasma were added to the standards. Free and bound [ 1251]-bPL were separated after the addition of 200 pl of second antibody (anti-rabbit gamma globulin, diluted to 1:40), 500 pl of 6% (w/v) polyethylene glycol and incubation for 24 hr at 4 C by centrifugation (1,800 g, 25 min). The supematant was aspirated and discarded while the precipitate was counted in an auto-gamma counter. Plasma samples (enriched with bPL) were assayed at dilutions of 10, 20, 40, and 80 JJJ to check for parallelism with bPL standards in buffer. The recovery of bPL added to control plasma was 98 + 7.0%. Assay sensitivity was 0.1 r&ml. Irma- and inter-assay coefficients of variation were 10.5 and 16%, respectively. Statistical Analysis. The plasma bPL concentrations for the last 10 d of gestation in each cow were averaged (including the parturition day) before analyzing the relationship between bPL levels, calf birth weight, and the first 100 d of postpartum lactation by Pearson s correlation method (17). Single point means were compared by paired Student s r-test. Differences in plasma bPL concentrations within a group and between groups were subjected to analysis of variance and analyzed by the general linear models procedure of SAS (17). For analysis, plasma bPL concentrations between the day of embryo transfer and Day 10 prepartum were approximated to the nearest tenth day with the following model: Yijk = c~ + Ti + Co)j + S, + TS,, + Eijk Where = overall mean = effect of fetal number cow nested in fetal number = = effect of day of pregnancy = interaction between fetal number = error term associated with y RESULTS Mean f SD length of pregnancy in singleton and twin-bearing cows were 290.2 + 5.0 and 283 f 3.9 d, respectively. The mean live birth weight of calves in the singleton group was 31.5 + 3.5 kg (n = 5, all males) and in the twin-bearing group were 32.9 + 6.6 and 28.2 + 8.0 kg for males (n = 7) and females (n = 3), respectively. The correlation coefficients (r) between bPL levels and calf birth weight in singleton and twin-bearing groups (total weight) were 0.94 (P < 0.01) and 0.44 (P = 0.06), respectively. The correlation coefficients (r) between bPL levels and the first 100 d of postpartum lactation in singleton and twin-bearing cows were 0.93 (P < 0.01) and 0.52 (P = 0.5), respectively. Two twin-embryo recipient cows had abnormal pregnancies, and therefore their data were excluded from those of the group reported separately. Plasma bPL Concentration Throughout Gestation in Singleton and Twin-Bearing Cows. The time trend concentrations of plasma bPL in both groups of cows are shown in Figure 1 and their relationship to different stages of gestation is shown in Table 1. bPL was detectable in both groups from about Day 60 postestrus to postpartum. Mean plasma bPL concentrations were identical in both groups of cows up to the end of first trimester (i.e., 0.2 f 0.1 ng/ml in singleton and twin-bearing cows, Figure 1). Thereafter, the mean levels increased gradually in the twin-bearing group and reached a concentration of 0.5 f 0.1 r&ml by mid-gestation (Day 140 postconception). On the other hand, the mean concentration in the singleton group undulated markedly during the same period of observation to reach a concentration of 0.4 f 0.2 ng/ml by midgestation. The mean concen-

(&j

sk

T&k Eijk

and day of pregnancy

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0 0 so 100 150

DAYS

0.5:;'7 0 2su -30

-20

-10

DAYS

Figure 1. Mean (+SEM) plasma bPL concentration throughout gestation in singleton (n = 5) (m) and twinbearing cows (n = 5) (A). Day 0 in (a) and (b) are referenced to the day of standing estrus and the day of parturition, respectively.

tration rapidly doubled in both groups of cows between Days 200 and 220 (0.7 f 0.1 to 1.3 + 0.1 ng/ml and 0.7 f 0.2 to 1.3 f 0.2 ng/ml in singleton (P < 0.01) and twin-bearing cows (P < O.Ol), respectively). The concentration stabilized at this peak level in both groups of cows up to the day of parturition (1.2 f 0.2 q/ml for both singleton and twin-bearing cows). The mean plasma bPL concentration in both groups of cows declined precipitously 1 d postpartum. Plasma bPL Concentration Throughout Gestation in Cows Giving Birth Prematurely or to Malformed Calves. During the course of this study, one cow (cow P) prematurely delivered stillborn calves (male weighing 27 kg, female weighing 18 kg) on Day 254 of pregnancy. Another twin-embryo recipient cow (Cow S) gave birth to a monstrous schistosomus reflexus calf (67.5 kg) on approximately Day 274 of pregnancy. The plasma bPL profiles of these two cows during gestation are shown in Figures 2 and 3, respectively. Cow F expelled a grossly discolored placenta. Cow F s plasma bPL profile was indistinguishable from the mean profile of the twin-bearing group up to about Day 200 (0.6 ng/ml vs. 0.7 + 0.2 ng/ml, respectively). Thereafter, the bPL levels in Cow F progressively declined between Days 210 and 250 by about 50% (0.3 ng/ml at Day 250) as opposed to the rapidly peaking and plateauing pattern observed during the same period in the twin-bearing group (1.2 + 0.2 ng/ml at Day 250 : Day -30 prepartum). Cow S had double the plasma bPL concentration on Day 100 when compared to the mean of the twin-bearing group (0.6 vs. 0.3 + 0.2 ng/ml, respectively). By Day 150, Cow S had tenfold higher bPL levels (3.4 vs. 0.35 f 0.1 ng/ml, respectively) compared to the

TABLE ~.LEASTSQLJAREANALYSIS OFPLASMABPLCONCENTRATIONSIN (USING COW(F) TO TESTTHE SIGNIFICANCE OF P AND ERROR

SINGLETON ANDTWWBEAIUNGCOWS TO TEST THE SIGNIFICANCE OF

DAY AND DAY*F). Source F COW(F) DAY DAY*F ERROR df 1 8 31 31 219 MS 0.6 0.3 1.I1 0.03 0.02 P< 0.21 0.01 0.05

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2c

50

100

150

200

250
(Cow F) to stillborn

Figure 2. Plasma bPL concentration throughout gestation in a cow giving birth prematurely calves. Day 0 is referenced to day of standing estrus. (J) indicates day of parturition.

mean concentration of the twin-bearing group. bPL concentration around Day 200 was sevenfold larger in Cow S than in the twin-bearing group (5.4 vs. 0.7 f 0.3 ng/ml, respectively). By Day 250, the bPL levels in Cow S were identical to the mean of the twin-bearing group (1.2 vs. 1.2 f 0.3 ng/ml, respectively). Cow S had relatively higher bPL levels in the last month of pregnancy compared to the twin-bearing group. Milk Yield in Singleton and Twin-Bearing Cows. Mean (*SE) weekly milk yields (Figure 4) increased to peak levels by the 5th wk of lactation in both groups of cows (101.8 + 8.4 to 188.4 f 13.6 kg and 97.2 f 10.8 to 181.4 f 6.1 kg in singleton and twin-bearing cows, respectively). This peak production was maintained in singleton and twin-bearing cows up to about the 15th wk of lactation (184.4 f 20.4 and 168.9 f 15.2 kg in singleton and twin-bearing cows, respectively). Thereafter, the mean weekly milk yields declined gradually in both groups to reach by the 30th wk of lactation, 97.2 + 15.9 and 99.7 f 12.4 kg in singleton and twin-bearing cows, respectively. Although the mean weekly milk yield was higher in the singleton group, it was not statistically significant.

(4

0u
-30
DAYS

-50 -19 DAYS

Figure 3. Plasma bPL concentration throughout gestation in a cow giving birth to a schistosomus (Cow S). Day 0 in (a) and (b) are referenced to the day of standing estrus and the day of parturition,

reflexus calf respectively.

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01

(,

,,

1
30

10
WEEKS

15

20

25

POST PARTUM (n = 5; N) and twin-bearing cows

Figure 4. Mean (n = 5; 0).

(+SEM)

weekly

postpartum

milk yield in singleton

DISCUSSION The time of detection and biphasic profile of bPL in singleton cows is consistent with recent reports (10-12). The significant interaction between the stage of pregnancy and bPL levels confirms Wallace s (12) findings. There are no reports in the literature on the influence of the fetus on the peripheral levels of bPL. This study is the first to show that peripheral bPL levels are not related to fetal number in cattle as opposed to the findings in other ruminant species (9,13). Interestingly, our results indicate that the trend of plasma bPL throughout gestation is indistinguishable between singleton and twin-bearing cows. Notwithstanding this, bPL has been suggested to be lactogenic and somatotrophic (1820). Correspondingly, the rapid elevation observed during the third trimester of the present study coincides with the period of accelerated growth of the fetus (21) and the period of more rapid mammary growth in the cow (22). Therefore, our results confirm that there is a maximal secretion of bPL into the maternal compartment concurrent with these two complementary physiological phases. A number of studies have compared yearly milk yields between single and multiple bearing cows with conflicting conclusions (23-26). Disagreements between these studies are partly attributable to the heterogeneity of their experimental population. This study is the first to describe the postpartum milk yield during a lactation period in a homogenous group of cows giving birth to singletons or twins. Our findings indicate that the pattern of the lactation in the twin-bearing cows is identical to that of the singleton group. The present findings in the cow contrast with those of the goat, sheep, rodents, and humans in which postpartum lactation is related to the number of offspring delivered (9,13,14,27). The positive association between neonate birth weight, postpartum lactation, and bPL levels in singleton cows further supports the conjectured role for bPL in modulating mammary growth. However, this relationship in the cow was not maintained with subsequent increase in fetal number. Identical findings in the cow have been reported for a pregnancy-specific steroid (28). Notwithstanding this, the positive association between offspring number, postpartum lactation, and PLs reported in a number of species is directly attributed to an increase in their placental mass (9,13,14,27). On the other hand, in the cow placental mass per fetus is reduced in multiple gestation although total calf weight increases by 50-100% (28). In addition, there is no difference between the number

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of cotyledons and overall placental weight between cows bearing singlets or twins (28). This suggests that in this species the placental dimension concurrent with its physiological output do not change proportionally to the number of fetuses being carried. Calf birth weight and milk yield are positively correlated in singleton pregnancies (29). Therefore, it is plausible that a relationship exists between circulating bPL levels and the weight of the placenta in singleton cows. However, the latter parameter was not investigated in the present study. Subsequently, additional studies are advocated to substantiate this. Present findings indicate that peripheral bPL levels could be used as an index for predicting anticipated calf size concurrent with the lactational capacity of singleton cows, but their use in the management of multiple pregnancies is limited. The peripheral pattern of serially characterized PL in humans (hPL) is clinically useful (30). Abnormalities of both the fetus and the placenta have been found to influence the peripheral profiles of hPL (31,32). Cow S lost one of the fetuses after Day 30, presumably due to the nature of competition induced by the developing monstrosity. Echtemkamp (28) found that most fetal losses in polytocous cows occurred after Day 35. Additionally, the loss of one fetus in a cow with multiple pregnancies does not significantly alter the peripheral pattern of the pregnancy-specific protein B (33). Similarly, the loss of a single fetus in this particular cow indicated no considerable influence on the peripheral concentration of bPL. The fetus directly regulates the physiological output of the placenta in the ruminants (34). Subsequently, the abnormal profile indicates either a relay of mutated signals or an abnormal fetal growth pattern. The overall elevated bPL levels reflect the gigantic size of the calf and further affirms the positive association between calf size and peripheral bPL levels in the singleton cows, as noted above. Our findings, although based on an individual case, indicate that bPL is a potent indicator of fetal confirmity in cows, as is hPL in humans. hPL is a practical gauge of placental viability (31,32). Abrupt deviations in the hPL are frequently linked to placental dysfunction. The etiology responsible for the premature birth concurrent with fetal death in Cow F was not exhaustively investigated. However, the deviation in the bPL profile together with the presence of gross changes in the placenta accompanied with no gross findings in the fetus suggest that fetal death was secondary to an abnormal placenta. Reports in the literature indicate that some opportunistic abortificient agents have the capability of invading the placenta and altering the binucleate cell morphology without producing any characteristic gross changes in the fetus or the placenta (35). Definite proof is lacking to link an opportunistic agent to the findings in Cow F, however, it cannot be ruled out. Our results indicate that bPL is an indicator of placental viability in the cow and that deviations from a progressively increasing pattern during serial sampling suggest placental compromise. To conclude, our findings indicate that peripheral bPL levels are positively associated with the stage of pregnancy but not fetal number. Otherwise, the peripheral pattern of bPL is a valuable index for predicting fetoplacental viability. ACKNOWLEDGMENTS/FOOTNOTES
We thank Drs. N. Takenouchi, Chugoku National Agricultural Experiment Station, K. Iwama, Kyoritsu Shoji Co., Ltd., M. Kobayashi, Morinaga Milk Industry Co., Ltd., N. Kusakari, Takikawa Animal Husbandry Experimental Station and T. Iwahori, Shizuoka Prefectual Animal Husbandry Experimental Station for their assistance with sample collection. T. Tomizuka T. Kojima and K. Hashizume of the National Institute of Animal Husbandry for their expert help. H. Kamomae of Tokyo University of Agriculture and Technology for valuable advice. We also express our profound gratitude to Dr. D.J. Bolt, USDA Animal Hormone Program, for providing the bPL antisera and Dr. J.C. Byatt, Monsanto Co., for the recombinant bPL used in the radioiodination and standards, as well as critically reading the manuscript. O.V. Pate1 is currently on a Ph.D. fellowship from the Japanese Ministry of Education (Monbusho). Corresponding author: N. Sasaki, Dept. Of Vet Surgery and Obstetrics, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Tel. 81-3-3812-2111, Fax 81-3-5800-6866.

PATEL ET AL.

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